The Challenge of Human Mesenchymal Stromal Cell Expansion: Current and Prospective Answers

In the field of cell therapy, allogenic human mesenchymal stromal cells (hMSCs) are often used in clinical trials, creating a demand for cell mass production using efficient dynamic bioreactor systems. As an advanced therapy medicinal product (ATMP), such cells should meet certain special requirements, including product specifications requiring a production process compatible with good manufacturing practice (GMP). The development of processes in which the cells are the product therefore remains a significant challenge. This chapter describes the requirements at different steps in the upstream and downstream phases of such dynamic processes. Potential solutions are presented and future prospects are discussed, including the selection of media and carriers for the strictly adherent growing cells, allowing efficient cell adhesion and detachment. Strategies for dynamic cultivation in bioreactors are described in detail for fixed‐bed and stirred‐tank reactors based on GMP requirements and the integration of process analytical technology (PAT). Following cell harvest, separation and purification, the formulation and storage of the product are also described. Finally, the chapter covers important cell quality characteristics necessary for the approval of ATMPs.

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title: Regenerative Medicine - from Protocol to Patient
abstract: Generation and regeneration as an answer to disease are far from being a new idea. Philosophers, naturalists and scientists were intrigued by the marvels of regeneration seen in nature. By the middle of the nineties life scientists thought we were only a few years away from bioartifi cial organs grown in a Petri dish. However, by the dawn of the new millennium it became clear that the mechanistic approach dictated by tissue engineering so far, had neglected issues of vascularization. Processes of angiogenesis were central to homeostasis, bioassimilation and biointegration of tissue engineered constructs. Furthermore, the fi eld of tissue engineering had evolved into something vast, encompassing satellite technologies that were becoming separate science sectors. Advances in genetical engineering, stem cell biology, cloning, biomaterials and biomedical devices to name a few, would come to play a major role of their own – tissue engineering had become a part of a bigger whole. Regenerative medicine is the collective fi eld to shelter these technologies “... that seeks to develop functional cell, tissue, and organ substitutes to repair, replace or enhance biological function that has been lost due to congenital abnormalities, injury, disease, or aging”.

title: Clinical applications of mesenchymal stem cells
abstract: Mesenchymal stem cells (MSC) have generated a great amount of enthusiasm over the past decade as a novel therapeutic paradigm for a variety of diseases. Currently, MSC based clinical trials have been conducted for at least 12 kinds of pathological conditions, with many completed trials demonstrating the safety and efficacy. This review provides an overview of the recent clinical findings related to MSC therapeutic effects. Roles of MSCs in clinical trials conducted to treat graft-versus-host-disease (GVHD) and cardiovascular diseases are highlighted. Clinical application of MSC are mainly attributed to their important four biological properties- the ability to home to sites of inflammation following tissue injury when injected intravenously; to differentiate into various cell types; to secrete multiple bioactive molecules capable of stimulating recovery of injured cells and inhibiting inflammation and to perform immunomodulatory functions. Here, we will discuss these four properties. Moreover, the issues surrounding clinical grade MSCs and principles for MSC therapeutic approaches are also addressed on the transition of MSCs therapy from bench side to bedside.

title: Expanded allogeneic adipose-derived mesenchymal stem cells (Cx601) for complex perianal fistulas in Crohn's disease: a phase 3 randomised, double-blind controlled trial
abstract: Complex perianal fistulas in Crohn's disease are challenging to treat. Allogeneic, expanded, adipose-derived stem cells (Cx601) are a promising new therapeutic approach. We aimed to assess the safety and efficacy of Cx601 for treatment-refractory complex perianal fistulas in patients with Crohn's disease.



We did this randomised, double-blind, parallel-group, placebo-controlled study at 49 hospitals in seven European countries and Israel from July 6, 2012, to July 27, 2015. Adult patients (≥18 years) with Crohn's disease and treatment-refractory, draining complex perianal fistulas were randomly assigned (1:1) using a pre-established randomisation list to a single intralesional injection of 120 million Cx601 cells or 24 mL saline solution (placebo), with stratification according to concomitant baseline treatment. Treatment was administered by an unmasked surgeon, with a masked gastroenterologist and radiologist assessing the therapeutic effect. The primary endpoint was combined remission at week 24 (ie, clinical assessment of closure of all treated external openings that were draining at baseline, and absence of collections >2 cm of the treated perianal fistulas confirmed by masked central MRI). Efficacy was assessed in the intention-to-treat (ITT) and modified ITT populations; safety was assessed in the safety population. 
212 patients were randomly assigned: 107 to Cx601 and 105 to placebo. A significantly greater proportion of patients treated with Cx601 versus placebo achieved combined remission in the ITT (53 of 107 [50%] vs 36 of 105 [34%]; difference 15·2%, 97·5% CI 0·2-30·3; p=0·024) and modified ITT populations (53 of 103 [51%] vs 36 of 101 [36%]; 15·8%, 0·5-31·2; p=0·021). 18 (17%) of 103 patients in the Cx601 group versus 30 (29%) of 103 in the placebo group experienced treatment-related adverse events, the most common of which were anal abscess (six in the Cx601 group vs nine in the placebo group) and proctalgia (five vs nine).



Cx601 is an effective and safe treatment for complex perianal fistulas in patients with Crohn's disease who did not respond to conventional or biological treatments, or both.

title: Maintenance of differentiation potential of human bone marrow mesenchymal stem cells immortalized by human telomerase reverse transcriptase gene despite [corrected] extensive proliferation.
abstract: Human bone marrow mesenchymal stem cells (hMSC) represent a population of stem cells that are capable of differentiation into multiple lineages. However, these cells exhibit senescence-associated growth arrest and phenotypic changes during long-term in vitro culture. We have recently demonstrated that overexpression of human telomerase reverse transcriptase (hTERT) in hMSC reconstitutes telomerase activity and extends life span of the cells [ (2002) 592]. In the present study, we have performed extensive characterization of three independent cell lines derived from the parental hMSC-TERT cell line based on different plating densities during expansion in culture: 1:2 (hMSC-TERT2), 1:4 (hMSC-TERT4), and 1:20 (hMSC-TERT20). The 3 cell lines exhibited differences in morphology and growth rates but they all maintained the characteristics of self-renewing stem cells and the ability to differentiate into multiple mesoderm-type cell lineages: osteoblasts, adipocytes, chondrocytes, and endothelial-like cells over a 3-year period in culture. Also, surface marker studies using flow cytometry showed a pattern similar to that known from normal hMSC. Thus, telomerization of hMSC by hTERT overexpression maintains the stem cell phenotype of hMSC and it may be a useful tool for obtaining enough number of cells with a stable phenotype for mechanistic studies of cell differentiation and for tissue engineering protocols.

title: Telomerase expression extends the proliferative life-span and maintains the osteogenic potential of human bone marrow stromal cells
abstract: Human bone marrow stromal cells (hMSCs) were stably transduced by a retroviral vector containing the gene for the catalytic subunit of human telomerase (hTERT). Transduced cells (hMSC-TERTs) had telomerase activity, and the mean telomere length was increased as compared with that of control cells. The transduced cells have now undergone more than 260 population doublings (PD) and continue to proliferate, whereas control cells underwent senescence-associated proliferation arrest after 26 PD. The cells maintained production of osteoblastic markers and differentiation potential during continuous subculturing, did not form tumors, and had a normal karyotype. When implanted subcutaneously in immunodeficient mice, the transduced cells formed more bone than did normal cells. These results suggest that ectopic expression of telomerase in hMSCs prevents senescence-associated impairment of osteoblast functions.

title: The translation of cell-based therapies: clinical landscape and manufacturing challenges.
abstract: Cell-based therapies have the potential to make a large contribution toward currently unmet patient need and thus effective manufacture of these products is essential. Many challenges must be overcome before this can become a reality and a better definition of the manufacturing requirements for cell-based products must be obtained. The aim of this study is to inform industry and academia of current cell-based therapy clinical development and to identify gaps in their manufacturing requirements. A total of 1342 active cell-based therapy clinical trials have been identified and characterized based on cell type, target indication and trial phase. Multiple technologies have been assessed for the manufacture of these cell types in order to facilitate product translation and future process development.

title: Large‐scale production of human mesenchymal stem cells for clinical applications
abstract: Human mesenchymal stem cells (hMSCs) have many potential applications in tissue engineering and regenerative medicine. Currently, hMSCs are generated through conventional static adherent cultures in the presence of fetal bovine serum (FBS) for clinical applications (e.g., multiple sclerosis). However, these methods are not appropriate to meet the expected future demand for quality‐assured hMSCs for human therapeutic use. Hence, it is imperative to develop an effective hMSC production system, which should be controllable, reproducible, and scalable. To this end, efforts have been made by several international research groups to develop (i) alternative media either by replacing FBS with human‐sourced supplements (such as human serum or platelet lysate) or by identifying defined serum‐free formulations consisting of key growth/attachment factors, and (ii) controlled bioreactor protocols. In this regard, we review here current hMSC production technologies and future perspectives toward efficient methods for the generation of clinically relevant numbers of hMSC therapeutics.

title: Application of human mesenchymal and pluripotent stem cell microcarrier cultures in cellular therapy: achievements and future direction.
abstract: Mesenchymal stem cells (MSCs) have recently made significant progress with multiple clinical trials targeting modulation of immune responses, regeneration of bone, cartilage, myocardia, and diseases like Metachromatic leukodystrophy and Hurler syndrome. On the other hand, the use of human embryonic and induced pluripotent stem cells (hPSCs) in clinical trials is rather limited mainly due to safety issues. Only two clinical trials, retinal pigment epithelial transplantation and treatment of spinal cord injury were reported. Cell doses per treatment can range between 50,000 and 6 billion cells. The current 2-dimensional tissue culture platform can be used when low cell doses are needed and it becomes impractical when doses above 50 million are needed. This demand for future cell therapy has reinvigorated interests in the use of the microcarrier platform for generating stem cells in a scalable 3-dimensional manner. Microcarriers developed for culturing adherent cell lines in suspension have been used mainly in vaccine production and research purposes. Since MSCs grow as monolayers similar to conventional adherent cell lines, adapting MSCs to a microcarrier based expansion platform has been progressing rapidly. On the other hand, establishing a robust microcarrier platform for hPSCs is more challenging as these cells grow in multilayer colonies on extracellular matrices and are more susceptible to shear stress. This review describes properties of commercially available microcarriers developed for cultivation of anchorage dependent cells and present current achievements for expansion and differentiation of stem cells. Key issues such as microcarrier properties and coatings, cell seeding conditions, medium development and improved bioprocess parameters needed for optimal stem cell systems are discussed.

title: Accreditation and regulations in cell therapy
abstract: Advances in stem cell biology and immunology raise hopes for new developments in regenerative medicine as well as cell‐based immune intervention, with potential applications in many medical disciplines. Emerging from the medical practices of organ, tissue and cell transplantations over several decades, the development of cellular therapies nowadays enter a new era in which the industry may take cell manufacturing activities away from academic facilities, using complex procedures for cell engineering and large‐scale production at centralized facilities. Recent changes in regulations push towards this historical transition; however, a pragmatic analysis of met and unmet clinical needs, as well as of specific procedural and organizational aspects that govern human cell procurement and processing suggest that a significant role will remain for academic facilities in the near future. Taking advantage of the large experience in the field of haematopoietic stem cell transplantation, we here review actual and potential consequences of regulatory changes in these medical practices, with a focus on the unique system for quality management that has been established in Europe and the United States of America to address specific risks associated with these therapeutic procedures.

title: Regulation for Gene and Cell Therapy Medicinal Products in Europe
abstract: An important step in the regulation of cell and gene therapy medicinal products, which are classified as advanced therapy medicinal products (ATMPs) in the European Union, has been made with Regulation 1394/2007/EC. By this regulation a new committee, the Committee for Advanced Therapies, has been established to ensure appropriate coverage of scientific and regulatory aspects of ATMPs. In addition, novel regulatory tools specific for ATMPs such as the classification, certification, and hospital exemption were introduced to support the development of this product class. By nature, ATMPs are a special class of medicinal products with characteristics different to conventional drugs and even other biologicals. Hence, regulatory requirements for manufacturing and clinical evaluation need to be tailored to the type and design of the ATMP, as well as to its manufacturing process and clinical indication. This chapter summarizes the general regulatory pathway for ATMPs as well as the currently applicable ATMP-specific procedures. In addition, the regulatory requirements regarding manufacturing, quality, and nonclinical and clinical testing for cell and gene therapy medicinal products are discussed. Important aspects of the environmental risk assessment, a provision for ATMPs containing genetically modified organisms, are also reviewed.

title: Good manufacturing practice and clinical-grade human embryonic stem cell lines.
abstract: Human embryonic stem cell (hESC) lines, after directed differentiation, hold the greatest potential for cell transplantation treatment in many severe diseases. Good manufacturing practice (GMP) quality, defined by both the European Medicines Agency and the Food and Drug Administration, is a requirement for clinical-grade cells, offering optimal defined quality and safety in cell transplantation. Using animal substance-free culture media, feeder cells or feeder-free matrix in derivation, passaging, expansion and cryopreservation procedures, immune reactions against animal proteins in the cells, and infection risk caused by animal microbes can be avoided. It is also possible to apply GMP to animal components if no better options are available. In recent production of GMP-quality hESC lines, feeder cells had been cultured in fetal bovine serum, and the medium supplemented with an animal protein containing a serum replacement component. Using embryos cultured in a GMP laboratory, isolating the inner cell mass mechanically, deriving lines on human feeder cells originally cultured in xeno-free medium in a GMP laboratory, and using xeno-free media for derivation and culture of hESC lines themselves, GMP-quality xeno-free hESC lines could be established today. Human serum is a xeno-free component available today, but many chemically defined media are under development.

title: Ex vivo expanded mesenchymal stromal cell minimal quality requirements for clinical application.
abstract: Mesenchymal stromal cells (MSCs), as advanced therapy products, must satisfy all the requirements for human use of medicinal products, aiming to maintain the quality and safety of the cells. The MSC manufacturing process for clinical use should comply with the principles of Good Manufacturing Practice (GMP). This ensures that cell preparations are produced and controlled, from the collection and manipulation of raw materials, through the processing of intermediate products, to the quality controls, storage, labeling and packaging, and release. The objective of this document is to provide the minimal quality requirements for the MSC production and its delivery for clinical use, so that the safety of the final cell therapy product will not be compromised. For this purpose, the document evaluates the most important steps of GMP-compliant MSC production: the isolation and expansion process; the validation phase of the process, including all quality controls for the characterization, functionality, potency, and safety of MSCs; and the quality control at the batch release to guarantee the safety of patient infusion.

title: Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement.
abstract: The considerable therapeutic potential of human multipotent mesenchymal stromal cells (MSC) has generated markedly increasing interest in a wide variety of biomedical disciplines. However, investigators report studies of MSC using different methods of isolation and expansion, and different approaches to characterizing the cells. Thus it is increasingly difficult to compare and contrast study outcomes, which hinders progress in the field. To begin to address this issue, the Mesenchymal and Tissue Stem Cell Committee of the International Society for Cellular Therapy proposes minimal criteria to define human MSC. First, MSC must be plastic-adherent when maintained in standard culture conditions. Second, MSC must express CD105, CD73 and CD90, and lack expression of CD45, CD34, CD14 or CD11b, CD79alpha or CD19 and HLA-DR surface molecules. Third, MSC must differentiate to osteoblasts, adipocytes and chondroblasts in vitro. While these criteria will probably require modification as new knowledge unfolds, we believe this minimal set of standard criteria will foster a more uniform characterization of MSC and facilitate the exchange of data among investigators.

title: Standardization of Good Manufacturing Practice-compliant production of bone marrow-derived human mesenchymal stromal cells for immunotherapeutic applications.
abstract: Human mesenchymal stem or stromal cells (MSCs) represent a potential resource not only for regenerative medicine but also for immunomodulatory cell therapies. The application of different MSC culture protocols has significantly hampered the comparability of experimental and clinical data from different laboratories and has posed a major obstacle for multicenter clinical trials. Manufacturing of cell products for clinical application in the European Community must be conducted in compliance with Good Manufacturing Practice and requires a manufacturing license. In Germany, the Paul-Ehrlich-Institut as the Federal Authority for Vaccines and Biomedicines is critically involved in the approval process.



This report summarizes a consensus meeting between researchers, clinicians and regulatory experts on standard quality requirements for MSC production.



The strategy for quality control testing depends on the product's cell composition, the manufacturing process and the indication and target patient population. Important quality criteria in this sense are, among others, the immunophenotype of the cells, composition of the culture medium and the risk for malignant transformation, as well as aging and the immunosuppressive potential of the manufactured MSCs.



This position paper intends to provide relevant information to interested parties regarding these criteria to foster the development of scientifically valid and harmonized quality standards and to support approval of MSC-based investigational medicinal products.

title: Process control in cell culture technology using dielectric spectroscopy.
abstract: In the biopharmaceutical industry, mammalian and insect cells as well as plant cell cultures are gaining worldwide importance to produce biopharmaceuticals and as products themselves, for example in stem cell therapy. These highly sophisticated cell-based production processes need to be monitored and controlled to guarantee product quality and to satisfy GMP requirements. With the process analytical technology (PAT) initiative, requirements regarding process monitoring and control have changed and real-time in-line monitoring tools are now recommended. Dielectric spectroscopy (DS) can serve as a tool to satisfy some PAT requirements. DS has been used in the medical field for quite some time and it may allow real-time process monitoring of biological cell culture parameters. DS has the potential to enable process optimization, automation, cost reduction, and a more consistent product quality. Dielectric spectroscopy is reviewed here as a tool to monitor biochemical processes. Commercially available dielectric sensing systems are discussed. The potential of this technology is demonstrated through examples of current and potential future applications in research and industry for mammalian and insect cell culture.

title: Mass Production of Mesenchymal Stem Cells — Impact of Bioreactor Design and Flow Conditions on Proliferation and Differentiation
abstract: Stem cells have enormous potential in health and medical research due to their ability to differentiate into specialized cells and to self-renew. Applications can be seen in cell-based therapies, e.g. for the treatment of Parkinson's disease, type I diabetes, arthritis, burn victims, and cardiovascular diseases, as well as in tissue engineering of artificial organs, development and testing of drugs, and in vitro toxicity tests [1-9].

title: Media formulation options and manufacturing process controls to safeguard against introduction of animal origin contaminants in animal cell culture
abstract: Technical limitations and evolution of therapeuticapplications for cell culture-derived products haveaccelerated elimination of animal-derived constituentsto minimize inadvertent introduction of adventitiousviral or prion agents. Practical considerationsdemand adequate emphasis both on design of theserum-free/protein-free culture environment and onnutrient media manufacturing process controls. Protein components may be acceptable, given adequateattention to synthetic process, sourcing (e.g.,geographic location and endemicity, species andtissue/organ) and validated treatment method. Variousoptions exist for re-engineering of traditionalserum-free formulations (containing insulin,transferrin and other protein factors) withnon-protein substitutes. Caution must also beexercised with sourcing of non-protein additives,particularly amino acids and lipids, to avoidintroducing adventitious contaminants. Simpleguidelines facilitate adaptation, cryopreservation andrecovery of many cell types within a protein-freeculture environment. Scrupulous maintenance offacility and equipment and monitoring of processwater, air handling systems and technical personnelare required to ensure that approved raw materials arecorrectly formulated and dispensed. Validatedsanitization processes provide additional assuranceagainst cross-contamination from previous batches ina multi-use facility.

title: Ex Vivo Expansion of Human Mesenchymal Stem Cells in Defined Serum-Free Media
abstract: Human mesenchymal stem cells (hMSCs) are presently being evaluated for their therapeutic potential in clinical studies to treat various diseases, disorders, and injuries. To date, early-phase studies have indicated that the use of both autologous and allogeneic hMSCs appear to be safe; however, efficacy has not been demonstrated in recent late-stage clinical trials. Optimized cell bioprocessing protocols may enhance the efficacy as well as safety of hMSC therapeutics. Classical media used for generating hMSCs are typically supplemented with ill-defined supplements such as fetal bovine serum (FBS) or human-sourced alternatives. Ideally, culture media are desired to have well-defined serum-free formulations that support the efficient production of hMSCs while maintaining their therapeutic and differentiation capacity. Towards this objective, we review here current cell culture media for hMSCs and discuss medium development strategies.

title: Development of a serum‐free system to expand dental‐derived stem cells: PDLSCs and SHEDs
abstract: Recently, extracted teeth have been identified as a viable source of stem cells for tissue regenerative approaches. Current expansion of these cells requires incorporation of animal sera; yet, a fundamental issue underlying cell cultivation methods for cell therapy regards concerns in using animal sera. In this study, we investigated the development of a chemically defined, serum‐free media (K‐M) for the expansion of human periodontal ligament stem cells (PDLSCs) and human stem cells from exfoliated deciduous teeth (SHEDs). Proliferation assays were performed comparing cells in serum‐containing media (FBS‐M) with cells cultured in four different serum‐free medium and these demonstrated that in these medium, the cell proliferation of both cell types was significantly less than the proliferation of cells in FBS‐M. Additional proliferation assays were performed using pre‐coated fibronectin (FN) tissue culture plates and of the four serum‐free medium, only K‐M enabled PDLSCs and SHEDs to proliferate at higher rates than cells cultured in FBS‐M. Next, alkaline phosphatase activity showed that PDLSCs and SHEDs exhibited similar osteogenic potential whether cultured in K‐M or FBS‐M, and, additionally, cells retained their multipotency in K‐M as seen by expression of chondrogenic and adipogenic genes, and positive Von Kossa, Alcian blue, and Oil Red O staining. Finally, differential expression of 84 stem cell associated genes revealed that for most genes, PDLSCs and SHEDs did not differ in their expression regardless of whether cultured in K‐M or FBS‐M. Taken together, the data suggest that K‐M can support the expansion of PDLSCs and SHEDs and maintenance of their multipotency.

title: A defined medium and substrate for expansion of human mesenchymal stromal cell progenitors that enriches for osteo- and chondrogenic precursors.
abstract: Human mesenchymal stromal cells (hMSCs) have generated significant interest due to their potential use in clinical applications. hMSCs are present at low frequency in vivo, but after isolation can be expanded considerably, generating clinically useful numbers of cells. In this study, we demonstrate the use of a defined embryonic stem cell expansion medium, mTeSR (Stem Cell Technologies), for the expansion of bone-marrow-derived hMSCs. The hMSCs grow at comparable rates, demonstrate tri-lineage differentiation potential, and show similar surface marker profiles (CD29(+), CD44(+), CD49a(+), CD73(+), CD90(+), CD105(+), CD146(+), CD166(+), CD34(-), and CD45(-)) in both the fetal bovine serum (FBS)-supplemented medium and mTeSR. However, expression of early differentiation transcription factors runt-related transcription factor 2, sex-determining region Y box 9, and peroxisome proliferator-activated receptor gamma changed significantly. Both runt-related transcription factor 2 and sex-determining region Y box 9 were upregulated, whereas peroxisome proliferator-activated receptor gamma was downregulated in mTeSR compared with FBS. Although osteogenic and chondrogenic differentiation was comparable in cells grown in mTeSR compared to FBS, adipogenic differentiation was significantly decreased in mTeSR-expanded cells, both in terms of gene expression and absolute numbers of adipocytes. The removal of the FBS from the medium and the provision of a defined medium with disclosed composition make mTeSR a superior study platform for hMSC biology in a controlled environment. Further, this provides a key step toward generating a clinical-grade medium for expansion of hMSCs for clinical applications that rely on osteo- and chondroinduction of MSCs, such as bone repair and cartilage generation.

title: Growth factor-defined culture medium for human mesenchymal stem cells.
abstract: Human bone marrow-derived mesenchymal stem cells (hMSCs) are potential cellular sources of therapeutic stem cells as they have the ability to proliferate and differentiate into a wide array of mesenchymal cell types such as osteoblasts, chondroblasts and adipocytes. hMSCs have been used clinically to treat patients with graft vs. host disease, osteogenesis imperfect, or alveolar cleft, suggesting that transplantation of hMSCs is comparatively safe as a stem cell-based therapy. However, conventional culture medium for hMSCs contains fetal bovine serum (FBS). In the present study, we developed a growth factor-defined, serum-free medium for culturing hMSCs. Under these conditions, TGF-beta1 promoted proliferation of hMSCs. The expanded hMSC population expressed the human pluripotency markers SSEA-3, -4, NANOG, OCT3/4 and SOX2. Furthermore, double positive cells for SSEA-3 and a mesenchymal cell marker, CD105, were detected in the population. The potential to differentiate into osteoblasts and adipocytes was confirmed. This work provides a useful tool to understand the basic biological properties of hMSCs in culture.

title: Defined serum- and xeno-free cryopreservation of mesenchymal stem cells
abstract: Mesenchymal stem cells (MSCs) have vast potential in cell therapy, and are experimentally used in the clinic. Therefore, it is critical to find a serum- and xeno-free cryopreservation method. The aim of this study was to compare two serum- and xeno-free cryoprotectants for MSCs. Adipose tissue MSCs (Ad-MSCs) and bone marrow MSCs (BM-MSCs) were cryopreserved in two cryoprotectants: the defined serum- and xeno-free STEM-CELLBANKER™ (CB) and 10 % dimethyl sulfoxide (DMSO) in a xeno-free serum replacement cell culture medium and compared to non-cryopreserved MSCs. MSCs cryopreserved in CB or DMSO had similar morphology and surface marker expression compared to their respective non-cryopreserved MSC. Ad-MSCs and BM-MSC in both cryoprotectant media exhibited reduced mean fluorescence intensity (MFI) for CD105, BM-MSCs for CD73 and Ad-MSC increased MFI for HLA class I compared to non-cryopreserved MSCs. Population doubling time of CB cryopreserved and non-cryopreserved Ad-MSCs was similar (38.1 ± 13.6 and 36.8 ± 12.1 h), but somewhat higher when cryopreserved in DMSO (42.2 ± 10.8 h). BM-MSCs had higher population doubling time (CB 47.7 ± 11.4 and DMSO 62.3 ± 32.9 h respectively, p < 0.05) compared to Ad-MSCs. The viability of Ad-MSCs was significantly higher after cryopreservation in CB compared to DMSO (90.4 ± 4.5 % vs. 79.9 ± 3.8 % respectively). Ad-MSCs and BM-MSCs retained their mesodermal differentiation potential when cryopreserved in both cryoprotectants. The characteristics of Ad-MSCs post-thawing are better preserved by CB than by DMSO in serum- and xeno-free medium. Furthermore, Ad-MSCs and BM-MSCs are differently affected by the cryoprotectants, which may have implications for cell therapy.

title: Serum‐free media for the production of human mesenchymal stromal cells: a review
abstract: The regenerative potential of mesenchymal stromal cells (MSC) holds great promise in using them for treatment of a wide range of debilitating diseases. Several types of culture media and systems have been used for large‐scale expansion of MSCs in vitro; however, the majority of them rely heavily on using foetal bovine serum (FBS)‐supplement for optimal cell proliferation. FBS‐based cultures pose the potential threat of spread of transmissible spongiform encephalopathy and bovine spongiform encephalopathy to MSCs and then to their recipients. A recent trend in cell culture is to change from serum‐use to serum‐free media (SFM). In this context, the current review focuses specifically on employment of various SFM for MSCs and discusses existences of various options with which to substitute FBS. In addition, we analyse MSC population growth kinetic patterns using various SFM for large‐scale production of MSCs.

title: Serum-free cell culture: the serum-free media interactive online database.
abstract: Fetal bovine serum (FBS) is a ubiquitously used essential supplement in cell culture media. However, there are serious scientific and ethical concerns about the use of FBS regarding its harvest and production. During the last three decades, FBS could be substituted by other supplements or by the use of defined chemical components in serum-free cell culture. A number of serum-free medium formulations have been described for mammalian and insect cell lines as well as for primary cultures. However, the switch to serum-free media still demands a time-consuming literature survey and a manufacturer search for appropriate medium formulations, respectively. Here we present the second collection of commercially available serum-free media in an updated, freely accessible interactive online database. Searches for serum-free media and continuous cell lines already adapted to serum-free culture can be performed according to various criteria. These include the degree of chemical definition, e.g. serum-free (SF), animal-derived component free (ADCF) or chemically defined (CD), and the type of medium, e.g. basal media, medium supplements, or full replacement media. In order to specify the cell lines that are adapted to serum-free media, search terms like species, organ, tissue, cell type and disease can be used. All commercially available serum-free media and adapted cell lines currently available from major distributors (e.g. ATCC, ECACC and DMSZ) are included in the database. Despite an extensive search for serum-free media and adapted cell lines, detailed information from certain companies and suppliers is still lacking and is specifically highlighted. It is intended to create a platform for the interactive exchange of information and experience by experts in the field in order to continuously improve and extend the serum-free online database. The database is accessible at

title: Mesenchymal Stem Cells and Their Cell Surface Receptors
abstract: Daily increasing evidence indicates that stem cells can be found in nearly every tissue. Mesenchymal stem cells (MSCs) are adult stem cells, which reside in the bone marrow and other mesenchymal tissues. MSCs can be expanded to large numbers and can be driven into diverse mesenchymal cell lineages, including chondrocytes. Therefore, MSCs have attracted the attention of the biomedical community as very promising tools for repair of joint tissues, such as articular cartilage. This review will outline the MSC surface receptors and will focus on receptors that deliver important signals for chondrogenic differentiation of MSCs. Finally, the role of receptors in the progression of cartilage degeneration disorders, such as osteoarthritis (OA), will be discussed.

title: Control of stem cell fate by physical interactions with the extracellular matrix.
abstract: A diverse array of environmental factors contributes to the overall control of stem cell activity. In particular, new data continue to mount on the influence of the extracellular matrix (ECM) on stem cell fate through physical interactions with cells, such as the control of cell geometry, ECM geometry/topography at the nanoscale, ECM mechanical properties, and the transmission of mechanical or other biophysical factors to the cell. Here, we review some of the physical processes by which cues from the ECM can influence stem cell fate, with particular relevance to the use of stem cells in tissue engineering and regenerative medicine.

title: Advances in cell culture: anchorage dependence
abstract: Anchorage-dependent cells are of great interest for various biotechnological applications. (i) They represent a formidable production means of viruses for vaccination purposes at very large scales (in 1000–6000 l reactors) using microcarriers, and in the last decade many more novel viral vaccines have been developed using this production technology. (ii) With the advent of stem cells and their use/potential use in clinics for cell therapy and regenerative medicine purposes, the development of novel culture devices and technologies for adherent cells has accelerated greatly with a view to the large-scale expansion of these cells. Presently, the really scalable systems—microcarrier/microcarrier-clump cultures using stirred-tank reactors—for the expansion of stem cells are still in their infancy. Only laboratory scale reactors of maximally 2.5 l working volume have been evaluated because thorough knowledge and basic understanding of critical issues with respect to cell expansion while retaining pluripotency and differentiation potential, and the impact of the culture environment on stem cell fate, etc., are still lacking and require further studies. This article gives an overview on critical issues common to all cell culture systems for adherent cells as well as specifics for different types of stem cells in view of small- and large-scale cell expansion and production processes.

title: Attachment, Growth, and Detachment of Human Mesenchymal Stem Cells in a Chemically Defined Medium
abstract: The manufacture of human mesenchymal stem cells (hMSCs) for clinical applications requires an appropriate growth surface and an optimized, preferably chemically defined medium (CDM) for expansion. We investigated a new protein/peptide-free CDM that supports the adhesion, growth, and detachment of an immortalized hMSC line (hMSC-TERT) as well as primary cells derived from bone marrow (bm-hMSCs) and adipose tissue (ad-hMSCs). We observed the rapid attachment and spreading of hMSC-TERT cells and ad-hMSCs in CDM concomitant with the expression of integrin and actin fibers. Cell spreading was promoted by coating the growth surface with collagen type IV and fibronectin. The growth of hMSC-TERT cells was similar in CDM and serum-containing medium whereas the lag phase of bm-hMSCs was prolonged in CDM. FGF-2 or surface coating with collagen type IV promoted the growth of bm-hMSCs, but laminin had no effect. All three cell types retained their trilineage differentiation capability in CDM and were detached by several enzymes (but not collagenase in the case of hMSC-TERT cells). The medium and coating did not affect detachment efficiency but influenced cell survival after detachment. CDM combined with cell-specific surface coatings and/or FGF-2 supplements is therefore as effective as serum-containing medium for the manufacture of different hMSC types.

title: Expansion and Harvesting of hMSC-TERT
abstract: The expansion of human mesenchymal stem cells as suspension culture by means of spinner flasks and microcarriers, compared to the cultivation in tissue culture flasks, offers the advantage of reducing the requirements of large incubator capacities as well as reducing the handling effort during cultivation and harvesting. Nonporous microcarriers are preferable when the cells need to be kept in viable condition for further applications like tissue engineering or cell therapy. In this study, the qualification of Biosilon, Cytodex 1, Cytodex 3, RapidCell and P102-L for expansion of hMSC-TERT with an associated harvesting process using either trypsin, accutase, collagenase or a trypsin-accutase mixture was investigated. A subsequent adipogenic differentiation of harvested hMSC-TERT was performed in order to observe possible negative effects on their (adipogenic) differentiation potential as a result of the cultivation and harvesting method. The cultivated cells showed an average growth rate of 0.52 d-1. The cells cultivated on Biosilon, RapidCell and P102-L were harvested succesfully achieving high cell yield and vitalities near 100%. This was not the case for cells on Cytodex 1 and Cytodex 3. The trypsin-accutase mix was most effective. After spinner expansion and harvesting the cells were successfully differentiated to adipocytes.

title: Good manufacturing practices production of mesenchymal stem/stromal cells.
abstract: Because of their multi/pluripotency and immunosuppressive properties mesenchymal stem/stromal cells (MSCs) are important tools for treating immune disorders and for tissue repair. The increasing use of MSCs has led to production processes that need to be in accordance with Good Manufacturing Practice (GMP). In cellular therapy, safety remains one of the main concerns and refers to donor validation, choice of starting material, processes, and the controls used, not only at the batch release level but also during the development of processes. The culture processes should be reproducible, robust, and efficient. Moreover, they should be adapted to closed systems that are easy to use. Implementing controls during the manufacturing of clinical-grade MSCs is essential. The controls should ensure microbiological safety but also avoid potential side effects linked to genomic instability driving transformation and senescence or decrease of cell functions (immunoregulation, differentiation potential). In this rapidly evolving field, a new approach to controls is needed.

title: Closed system isolation and scalable expansion of human placental mesenchymal stem cells
abstract: Mesenchymal stem cells (MSC) are emerging as a leading cellular therapy for a number of diseases. However, for such treatments to become available as a routine therapeutic option, efficient and cost‐effective means for industrial manufacture of MSC are required. At present, clinical grade MSC are manufactured through a process of manual cell culture in specialized cGMP facilities. This process is open, extremely labor intensive, costly, and impractical for anything more than a small number of patients. While it has been shown that MSC can be cultivated in stirred bioreactor systems using microcarriers, providing a route to process scale‐up, the degree of numerical expansion achieved has generally been limited. Furthermore, little attention has been given to the issue of primary cell isolation from complex tissues such as placenta. In this article we describe the initial development of a closed process for bulk isolation of MSC from human placenta, and subsequent cultivation on microcarriers in scalable single‐use bioreactor systems. Based on our initial data, we estimate that a single placenta may be sufficient to produce over 7,000 doses of therapeutic MSC using a large‐scale process. Biotechnol. Bioeng. 2012;

title: Bioprocessing of human mesenchymal stem/stromal cells for therapeutic use: Current technologies and challenges
abstract: The long-term outlook for regenerative medicine predicts an increased need for scalable cell expansion technologies that utilize non-animal derived materials and are compatible with the limited number of downstream processing steps required for cell-based therapies. As more stem cell therapeutics progress through clinical testing, current in vitro culture methods using planar vessels are proving cumbersome to scale. Therefore, alternative processes are under investigation. Many human mesenchymal stem/stromal cell (hMSC) bioreactor-based manufacturing processes, in particular, are complicated by the requirement to separate cells from microcarriers with high cell yield and viability whilst maintaining target phenotypic and functional characteristics. Here we review currently available technologies and ongoing development for the expansion of cellular therapeutics, with focus on allogeneic hMSCs and microcarrier-based processes. Upstream challenges include the interplay between the cell culture substrate and media formulation, sourcing of high quality animal-free reagents, and considerations for the use of microcarriers in stirred-tank systems. Complications in downstream processes include harvest approaches for separation of cells from microcarriers and volume reduction.

title: Stirred tank bioreactor culture combined with serum-/xenogeneic-free culture medium enables an efficient expansion of umbilical cord-derived mesenchymal stem/stromal cells.
abstract: Mesenchymal stem/stromal cells (MSC) are being widely explored as promising candidates for cell-based therapies. Among the different human MSC origins exploited, umbilical cord represents an attractive and readily available source of MSC that involves a non-invasive collection procedure. In order to achieve relevant cell numbers of human MSC for clinical applications, it is crucial to develop scalable culture systems that allow bioprocess control and monitoring, combined with the use of serum/xenogeneic (xeno)-free culture media. In the present study, we firstly established a spinner flask culture system combining gelatin-based Cultispher(®) S microcarriers and xeno-free culture medium for the expansion of umbilical cord matrix (UCM)-derived MSC. This system enabled the production of 2.4 (±1.1) x10(5) cells/mL (n = 4) after 5 days of culture, corresponding to a 5.3 (±1.6)-fold increase in cell number. The established protocol was then implemented in a stirred-tank bioreactor (800 mL working volume) (n = 3) yielding 115 million cells after 4 days. Upon expansion under stirred conditions, cells retained their differentiation ability and immunomodulatory potential. The development of a scalable microcarrier-based stirred culture system, using xeno-free culture medium that suits the intrinsic features of UCM-derived MSC represents an important step towards a GMP compliant large-scale production platform for these promising cell therapy candidates.

title: Production of oncolytic adenovirus and human mesenchymal stem cells in a single‐use, Vertical‐Wheel bioreactor system: Impact of bioreactor design on performance of microcarrier‐based cell culture processes
abstract: Anchorage‐dependent cell cultures are used for the production of viruses, viral vectors, and vaccines, as well as for various cell therapies and tissue engineering applications. Most of these applications currently rely on planar technologies for the generation of biological products. However, as new cell therapy product candidates move from clinical trials towards potential commercialization, planar platforms have proven to be inadequate to meet large‐scale manufacturing demand. Therefore, a new scalable platform for culturing anchorage‐dependent cells at high cell volumetric concentrations is urgently needed. One promising solution is to grow cells on microcarriers suspended in single‐use bioreactors. Toward this goal, a novel bioreactor system utilizing an innovative Vertical‐Wheel™ technology was evaluated for its potential to support scalable cell culture process development. Two anchorage‐dependent human cell types were used: human lung carcinoma cells (A549 cell line) and human bone marrow‐derived mesenchymal stem cells (hMSC). Key hydrodynamic parameters such as power input, mixing time, Kolmogorov length scale, and shear stress were estimated. The performance of Vertical‐Wheel bioreactors (PBS‐VW) was then evaluated for A549 cell growth and oncolytic adenovirus type 5 production as well as for hMSC expansion. Regarding the first cell model, higher cell growth and number of infectious viruses per cell were achieved when compared with stirred tank (ST) bioreactors. For the hMSC model, although higher percentages of proliferative cells could be reached in the PBS‐VW compared with ST bioreactors, no significant differences in the cell volumetric concentration and expansion factor were observed. Noteworthy, the hMSC population generated in the PBS‐VW showed a significantly lower percentage of apoptotic cells as well as reduced levels of HLA‐DR positive cells. Overall, these results showed that process transfer from ST bioreactor to PBS‐VW, and scale‐up was successfully carried out for two different microcarrier‐based cell cultures. Ultimately, the data herein generated demonstrate the potential of Vertical‐Wheel bioreactors as a new scalable biomanufacturing platform for microcarrier‐based cell cultures of complex biopharmaceuticals.

title: Expansion of Human Mesenchymal Stem Cells in a Fixed-Bed Bioreactor System Based on Non-Porous Glass Carrier – Part B: Modeling and Scale-up of the System
abstract: Human mesenchymal stem cells (hMSC) are a promising cell source for the manufacturing of cell therapy or tissue-engineered implants. In part A of this publication a fixed-bed bioreactor system based on non-porous borosilicate glass spheres and procedures for the automated expansion of hMSC with high yield and vitality was introduced. Part B of this study deals with the modeling of the process in order to transfer the bioreactor system from the laboratory to the production scale. Relevant model parameters were obtained by fitting them to the experimental data of hMSC-TERT cultivations in scales up to 300 cm3. Scale-up calculations were carried out exemplarily for a target cell number of twenty billion cells.

title: Expansion of human mesenchymal stem cells in fibrous bed bioreactor
abstract: Expansion of human mesenchymal stem cells (hMSCs) in bioreactor while preserving their innate properties is important in translation of hMSC-based therapy to clinical applications. The present study investigates the feasibility of hMSC expansion in a 2.5 L CelliGen ® 310 Bioreactor packed with Fibra-Cel ® disks. After 9 days of expansion, a 9.2-fold increase in cell number with the population doubling (PD) time of 2.8 days (67.2 h) was achieved and that the specific glucose consumption and lactate production were measured to be 12.48 pmol/cell/day and 20.95 pmol/cell/day, respectively. hMSCs harvested from the bioreactor maintained their properties based on the analysis of phenotypic surface markers, colony forming unit-fibroblasts (CFU-F) number, and multilineage differentiation ability. The results demonstrate the feasibility and the potential of the fibrous bed bioreactor for large scale hMSC expansion.

title: hMSC Production in Disposable Bioreactors with Regards to GMP and PAT
abstract: Reactor concepts for human mesenchymal stem cell (hMSC) production are introduced. Thereby, special interest is laid on the realization of these concepts as disposables fulfilling the GMP and PAT requirements. The specialty of the hMSC production process is the cell itself being the product. This results in completely different process requirements compared to e.g. protein production in mammalian cells. Thus, great attention has to be given to the shear sensitivity of the cells. The cultivation and the harvest of the cells have to be very gentle to neither influence cell viability nor cell differentiability. Further, the production process should not cause any undesirable cell changes. For hMSC production, cell harvest is the main challenging process step. The reactor concepts should be suitable for hMSC production for clinical trials as ATMPs. Therefore, disposable systems are especially applicable. The review describes more detailed bone marrow-derived hMSC production in a disposable stirred tank reactor as promising reactor concept.

title: Optimising Human Mesenchymal Stem Cell Numbers for Clinical Application: A Literature Review
abstract: Adult mesenchymal stem cells (MSCs) are being investigated further for their use in stem cell therapies. However, as they are found in very low numbers in adult tissue, expansion in vitro is required to produce desired MSC numbers for clinical application. The need for effective cell-based therapies is increasing due to a rise in the ageing population, increasing the prevalence of musculoskeletal disorders. This review investigates how factors, age and gender of donor, as well as seeding density can affect MSC expansion. Age and gender of donor have received mixed results from studies, whereas seeding density studies have produced consistent results for numerous MSC sources, favouring lower seeding densities. Further research is required to reduce the risk of infection, loss of cell characterisation in cell culture, and making cell-based therapies more cost effective through creating rapid expansion of MSCs regardless of patient factors.

title: Expansion of human mesenchymal stem cells on microcarriers
abstract: The effects on human mesenchymal stem cell growth of choosing either of two spinner flask impeller geometries, two microcarrier concentrations and two cell concentrations (seeding densities) were investigated. Cytodex 3 microcarriers were not damaged when held at the minimum speed, NJS, for their suspension, using either impeller, nor was there any observable damage to the cells. The maximum cell density was achieved after 8–10 days of culture with up to a 20-fold expansion in terms of cells per microcarrier. An increase in microcarrier concentration or seeding density generally had a deleterious or neutral effect, as previously observed for human fibroblast cultures. The choice of impeller was significant, as was incorporation of a 1 day delay before agitation to allow initial attachment of cells. The best conditions for cell expansion on the microcarriers in the flasks were 3,000 microcarriers ml−1 (ca. 1 g dry weight l−1), a seeding density of 5 cells per microcarrier with a 1 day delay before agitation began at NJS (30 rpm), using a horizontally suspended flea impeller with an added vertical paddle. These findings were interpreted using Kolmogorov’s theory of isotropic turbulence.

title: A Microcarrier‐Based Cultivation System for Expansion of Primary Mesenchymal Stem Cells
abstract: Microcarrier cultures have been shown to allow extensive cell expansion of tissue engineering relevant cells, such as chondrocytes, while maintaining their phenotype. Our aim was to investigate the in vitro three‐dimensional expansion of porcine bone‐marrow‐derived primary mesenchymal stem cells (MSC) using commercially available Cytodex type 1, type 2, and type 3 microcarriers. In comparison, the Cytodex type 1 microcarriers showed the best results for adherence with over 80% adherent cells after 3 h of incubation, analyzed by the Poisson distribution. Different start cell densities ranging from 1 to 3 × 106 cells per 100 cm2 had only a minor influence on adhesion. The proliferation was examined on Cytodex type 1 microcarriers over a cultivation time of 28 days, which could reveal cell growth and proof of cells recolonizing freshly added microcarriers. Scanning electron microscopy displayed appropriate cell morphology and confirmed cell proliferation. After enzymatic harvest from microcarriers, the osteogenic and chondrogenic differentiation of these cells was induced and shown by relevant histochemistry, such as von Kossa and Alcian blue staining. Totaling the results, we have shown that the three‐dimensional expansion of MSC on microcarriers represents a beneficial alternative to the conventional two‐dimensional monolayer cultivation method.

title: Expansion of human mesenchymal stromal cells on microcarriers: growth and metabolism
abstract: Adult stem cells, or mesenchymal stromal cells (MSCs), are of great potential for cell therapy and tissue‐engineering applications. However, for therapeutic use, these cells need to be isolated from tissue or a biopsy and efficiently expanded, as they cannot be harvested in sufficient quantities from the body. In our opinion, efficient expansion of MSCs can be achieved in a microcarrier‐based cultivation system. This study selected a suitable microcarrier for human bone marrow‐derived stromal cells (HBMSCs), optimized cell‐seeding strategies by varying serum concentrations, and optimized dynamic expansion of the HBMSCs in a microcarrier‐based spinner flask cultivation system by applying various feeding regimes. Cytodex 1 microcarriers in combination with a low‐serum concentration (0–5%) in the medium resulted in the highest seeding efficiency for the HBMSCs. Subsequently, significant expansion of the HBMSCs on these carriers has been observed. The highest number of HBMSCs population doublings (4.8 doublings) was obtained by a combination of 50% medium refreshment combined with addition of 30% medium containing microcarriers every 3 days. Exponential cell growth was observed for at least 9 days after seeding, provided that sufficient nutrients (such as glucose) were present, metabolite concentrations (such as ammonia) were kept below growth‐inhibitory concentrations and adequate surface area was present for the cells. After dynamic expansion of the HBMSCs, the cells retained their differentiation potential and their cell surface markers, indicating that HBMSCs expansion on Cytodex 1 microcarriers did not alter the phenotypic properties of the cells.

title: Microcarrier-based Expansion Process for hMSCs with High Vitality and Undifferentiated Characteristics
abstract: For cell therapy, a high biomass of human mesenchymal stem cells (hMSCs) is required for clinical applications, such as in the form of encapsulated implants. An easy and reproducible microcarrier-based stirred tank reactor cultivation process for hMSCs in 1.68 L scale is described. To avoid medium changes, studies comparing high-glucose DMEM (DMEM-HG) with low-glucose EMEM were performed showing that high-glucose medium has positive effects on cell proliferation and that cell differentiability remains. Studies on the inoculation strategy and cell density, carrier concentration, volume, and stirrer speed were performed and resulted in a set of optimized parameters, inoculation strategy was found to be 45 minutes of static state and 2 minutes of stirring repeated in 4 cycles. The inoculation density was chosen to be 7×103 cells/cm2, and the carrier concentration of glass surface carrier was 25 g/L. For the described reactor system, a stirrer speed of 120 rpm for the inoculation process and a daily increase of 10 rpm up to 160 rpm were found to be suitable. Process reproducibility was shown by 3 repeated cultivations at the determined set of parameters allowing high biomass values of up to 7×108 cells per batch. With DMEM-HG, no limitation of glucose was found, and lactate and ammonia remained lower than critical inhibitory concentrations. Comparison of the static (T-flask) and dynamic cultures in the stirred tank reactor showed for both cases, that cells were of high vitality and both maintained differentiability. In both cases, encapsulation of the cells resulted in high bead vitality, a basic requirement for cell therapy application.

title: Biomanufacturing of human mesenchymal stem cells in cell therapy: Influence of microenvironment on scalable expansion in bioreactors
abstract: Human mesenchymal stem cells (hMSCs) are the primary candidate in cell therapy and have demonstrated significant potential in a wide range of diseases. The clinical translation of hMSC therapy requires robust and scalable expansion technology for the biomanufacturing of therapeutically competent cells. By nature, hMSCs are highly sensitive and responsive to the microenvironments and their therapeutic potency is significantly influenced by the culture conditions during expansion. Here, we discuss the emerging roles of microenvironments in regulating hMSC fate and the implication of regulating hMSC microenvironment to achieve scalable hMSC expansion in bioreactors.

title: OPTIMIZING CULTURE CONDITIONS FOR THE PRODUCTION OF ANIMAL CELLS IN MICROCARRIER CULTURE
abstract: Cytodex 1 microcarriers have been used for the successful culture of more than 80 different types of animal cells--including primary cells, normal diploid cell strains and established or transformed cell lines. culture volumes have ranged from a few milliliters for diagnostic studies to over several hundred liters for vaccine production. Experience with this wide variety of cell types and culture volumes has enabled the identification of several parameters critical for obtaining maximum cell yields from microcarrier cultures. The most vital stage for the successful microcarrier culture of many cell types was the initial stage of the culture cycle. To achieve high cell yields, it was necessary to use culture procedures which maximized plating efficiency and final cell yield could be further increased by ensuring that the inoculation cell density exceeded a critical viable cell/microcarrier ratio. Modifications of the standard microcarrier culture procedures included reducing initial culture volume, reducing the initial stirring speed and/or supplementing the medium during the early stages of the culture cycle. Control of pH, nutrient supply, and gas tension were all critical throughout the culture cycle. Results with low-serum and serum-free media indicate that the requirement for fetal calf serum in the microcarrier culture of Vero and MRC-5 cells can be reduced or even eliminated. Large scale microcarrier culture equipment should take into account the modified culture procedures which are often required to achieve the full potential of this culture method. The design of a new flexible culture system suitable for pilot and production scale cultures is presented. This system accomodates a wide variety of culture and production procedures and through a recirculation system permits: (a) "in-line" monitoring and control of culture parameters; (b) provides an efficient gas exchange capacity which obviates the need for fermenter headspace and sparging; and (c) allows for maximal utilization of medium components and rapid harvesting of medium or cell products.

title: Scale‐up of adipose tissue‐derived mesenchymal stem cell production in stirred single‐use bioreactors under low‐serum conditions
abstract: Suspension cultures, in which human mesenchymal stem cells are cultivated on microcarriers in scalable single‐use stirred bioreactor types, have been shown to be a promising alternative to planar flask cultures. However, stirred single‐use bioreactors were originally developed for production processes with robust, permanent cell lines. Human mesenchymal stem cells are adherent primary cells and thus expanding them in such bioreactor systems imposes more stringent requirements on bioreactor systems. For low‐serum conditions (5%) and different types of stirred single‐use bioreactors, a suspension criteria‐based approach for expanding human adipose tissue‐derived mesenchymal stem cells (hASCs) from milliliter to pilot scale was successfully developed. For process scale‐up, experimental and numerical investigations were performed to (i) predict optimum impeller speeds, (ii) determine the main engineering parameters (local shear stress, turbulent dissipation rate, Kolmogorov microscale), and (iii) verify suspension criteria NS1 and NS1u for rapid process transfer from 100 mL to 2 L and 35 L cultures. Using optimized medium‐microcarrier combinations as well as NS1 and NS1u as scale‐up factors, total hASC quantities between 3 × 107 (100 mL scale) and 1 × 1010 (35 L scale) were obtained. The cell quantities obtained are the highest reported to date for scalable single‐use bioreactors under low‐serum conditions.

title: Multiphase mixing characteristics in a microcarrier-based stirred tank bioreactor suitable for human mesenchymal stem cell expansion
abstract: Large-scale human mesenchymal stem cell expansion calls for a bioreaction system, that provides a sufficient growth surface. An alternative to static cultivations systems like cell factories are disposable stirred tank reactors. Here, microcarriers provide the required growth surface, but these make it difficult to achieve a complete homogenization in the bioreactor, while avoiding shear stress. To gain insight into this process, we investigated the impact of different power inputs (0.02–2.6 W m −3 ) on the mixing time (t m ). Whereas t m was inversely proportional to agitation in a one-phase-system, aeration resulted in a constant mixing time at 30–70 rpm. A high microcarrier concentration (30 g L −1 ) and low stirrer speed (30 rpm) in the liquid-solid system caused a 50-fold increase in t m and the formation of a discrete non-mixed upper zone. The effect of the microcarrier concentration on t m became negligible at higher stirrer speeds. In the three-phase system, microcarrier settling was prevented by aeration and a minimal specific power input of 0.6 W m −3 was sufficient for complete homogenization. We confirmed that a low power input during stem cell expansion leads to inhomogeneity, which has not been investigated in the three-phase system up to date.

title: Maximizing the ex vivo expansion of human mesenchymal stem cells using a microcarrier-based stirred culture system.
abstract: Bioreactor systems have been developed as alternatives to standard culture flasks due to their homogeneous nature, easiness of monitoring and increased cell production. Here we investigated the in vitro expansion of bone marrow (BM) mesenchymal stem cells (MSC) in spinner flasks, using gelatin microcarriers (Cultispher S) to support cell adhesion and proliferation. MSC expansion was performed using a low-serum containing medium (2% of fetal bovine serum, FBS). A strategy was defined for the maximization of cell expansion: microcarriers were pre-coated with FBS in order to increase cell seeding efficiency and an adequate feeding regime was established (25% medium exchange everyday). The maximum cell density, 4.2 x 10(5)cells/mL, was obtained at day 8, corresponding to a fold increase in total cell number of 8.4+/-0.8. Expanded MSC retained their differentiation potential into adipogenic and osteogenic lineages, as well as their clonogenic ability. Harvested cells expressed >90% of CD73, CD90 and CD105 markers. These results demonstrated that a microcarrier-based stirred culture system is adequate for human MSC expansion, using a low-serum containing medium, allowing the generation of significant cell numbers for potential applications in regenerative medicine.

title: The catalytic triad of serine peptidases
abstract: The catalytic action of serine peptidases depends on the interplay of a nucleophile, a general base and an acid. In the classic trypsin and subtilisin families this catalytic triad is composed of serine, histidine and aspartic acid residues and exhibits similar spatial arrangements, but the order of the residues in the amino acid sequence is different. By now several new families have been discovered, in which the nucleophile-base-acid pattern is generally conserved, but the individual components can vary. The variations illustrate how different groups and different protein structures achieve the same reaction.

title: CELL DETACHMENT BY PROLYL-SPECIFIC ENDOPEPTIDASE FROM WOLFIPORIA COCOS
abstract: As requirements for Advanced Therapy Medicinal Product (ATMP) production differ from other production processes (e.g., therapeutic protein production), cell detachment is often a crucial step for the process success. In most cases, cell detachment is done enzymatically. Although many peptidases are established in cell culture in R&D, e.g., Trypsin as gold standard, many of them seem to be unsuitable in ATMP production processes. Therefore, the present study investigated a novel endopeptidase used in food biotechnology for its applicability in ATMP processes where cell detachment is needed. The Prolyl-specific Peptidase (PsP) is of non-mammalian origin and considered as safe for humans. PsP was purified from the supernatant of the fungus Wolfiporia cocos. The isolation and purification resulted in an enzyme solution with 0.19 U mg-1 prolyl-specific activity. By in silico analysis it was confirmed that attachment-promoting proteins can be cleaved by PsP in a similar amount than with Trypsin. Further the proteolytic activity was determined for PsP and Trypsin by using the same enzymatic assay. Detachment with both enzymes was compared for cells used in typical therapeutic production processes namely a mesenchymal stem cell line (hMSC-TERT) as a model for a cell therapeutic, Vero and MA104 cells used for viral therapeutic or vaccine production. The cell detachment experiments were performed with comparable enzyme activities (1.6 U mL-1). hMSC-TERT detachment was faster with PsP than with Trypsin. For Vero cells the detachment with PsP was not only faster but also more efficient. For MA104 cells the detachment rate with PsP was similar to Trypsin. For all cell types, detachment with PsP showed less influence on cell growth and metabolism compared to standard Trypsin.Thus, three cell types used in ATMP, viral therapeutics or vaccine production can be detached efficiently and gently with PsP. Therefore, PsP shows potential for cell detachment in ATMP and viral/vaccine production processes.

title: Process engineering of human pluripotent stem cells for clinical application.
abstract: Human pluripotent stem cells (hPSCs), including embryonic and induced pluripotent stem cells, constitute an extremely attractive tool for cell therapy. However, flexible platforms for the large-scale production and storage of hPSCs in tightly controlled conditions are necessary to deliver high-quality cells in relevant quantities to satisfy clinical demands. Here we discuss the main principles for the bioprocessing of hPSCs, highlighting the impact of environmental factors, novel 3D culturing approaches and integrated bioreactor strategies for controlling hPSC culture outcome. Knowledge on hPSC bioprocessing accumulated during recent years provides important insights for the establishment of more robust production platforms and should potentiate the implementation of novel hPSC-based therapies.

title: Allogeneic cell therapy bioprocess economics and optimization: downstream processing decisions.
abstract: To develop a decisional tool to identify the most cost effective process flowsheets for allogeneic cell therapies across a range of production scales.



A bioprocess economics and optimization tool was built to assess competing cell expansion and downstream processing (DSP) technologies.



Tangential flow filtration was generally more cost-effective for the lower cells/lot achieved in planar technologies and fluidized bed centrifugation became the only feasible option for handling large bioreactor outputs. DSP bottlenecks were observed at large commercial lot sizes requiring multiple large bioreactors. The DSP contribution to the cost of goods/dose ranged between 20-55%, and 50-80% for planar and bioreactor flowsheets, respectively.



This analysis can facilitate early decision-making during process development.

title: Filtration methodologies for the clarification and concentration of human mesenchymal stem cells
abstract: Currently human mesenchymal stem cells (hMSC) are expanded using microcarrier-based stirred culture systems from one to hundreds of liters of culture volume to guarantee the required cell numbers to be delivered to the clinic. Such culture volumes need to be clarified, ensuring efficient removal of microcarriers, and concentrated without compromising the cells׳ characteristics. The aim of this work was to evaluate the applicability of filtration methodologies, as dead end filtration and tangential flow filtration, for the clarification and concentration of hMSC, respectively. Different process variables and their impact on hMSC quality were evaluated, showing that polypropylene filters with pore sizes higher than 75 μm can ensure the removal of microcarriers from the cell suspension bulk, without compromising cells׳ recovery or viability. Furthermore, hMSC could be successfully concentrated up to a factor of ten while maintaining their identity, potency and high cell viability, allowing for the recovery of over 80% of viable cells; an initial cell concentration higher than 2×10 5 cell/mL, and polysulfone membranes with pore sizes higher than 0.45 μm were identified to be key conditions to obtain such concentration factors; shear rate and permeate flux were also shown to impact the cells׳ recovery yields, viability and quality.

title: Industrial scale harvest of proteins from mammalian cell culture by tangential flow filtration
abstract: An industrial‐scale methods for harvest of biologically active proteins form mammalian cell culture has been developed using tangential flow filtration. A robust and economical process capable of processing approximately 5000 L conditioned media/h with protein yields in excess of 99% has been achieved. A completely contained system has been designed in which total cell number and viability are maintained throughout the process. The process has successfully been implemented at 1.25 × 104 L scale for the recovery of kilogram quantities of pharmaceutical proteins such as recombinant tissue type plasminogen activator (rt‐PA).

title: Influence of stress on adherent cells.
abstract: Stress is a broad term often used with animal cells. Frequently mechanical forces are meant using this term but chemical stress is also important cultivating animal cells. The chemical environment of the cell in a reactor have to be considered very carefully. The complexity of the medium requirements and the metabolic pathway cause very often growth limitations. Studying these limitations in order to find the reasons showed to be difficulty because of the complexity of the system. Nevertheless, glucose, glutamine, lactate and ammonia are found to be critical parameter as well as the osmotic pressure. The influence of mechanical forces on cell viability is of great importance when growing the cells in agitated systems. By far the greatest amount of work reported in the literature has been done on suspension cells but adherent cells also experience shear forces not only in bioreactors also in vivo. Therefore, most research has be done on endothelial cells but studies exists done on non-endothelial cells. The influence of shear forces on cell growth, morphology and productivity will be discussed as well as possibilities of making the cells more resistant.

title: Response of mesenchymal stem cells to shear stress in tissue-engineered vascular grafts
abstract: Recent studies have demonstrated that mesenchymal stem cells (MSCs) can differentiate into endothelial cells. The effect of shear stress on MSC differentiation is incompletely understood, and most studies have been based on two-dimensional systems. We used a model of tissue-engineered vascular grafts (TEVGs) to investigate the effects of shear stress on MSC differentiation.MSCs were isolated from canine bone marrow. The TEVG was constructed by seeding MSCs onto poly-ɛ-caprolactone and lactic acid (PCLA) scaffolds and subjecting them to shear stress provided by a pulsatile bioreactor for four days (two days at 1 dyne/cm2 to 15 dyne/cm2 and two days at 15 dyne/cm2).Results:Shear stress significantly increased the expression of endothelial cell markers, such as platelet-endothelial cell adhesion molecule-1 (PECAM-1), VE-cadherin, and CD34, at both the mRNA and protein levels as compared with static control cells. Protein levels of alpha-smooth muscle actin (α-SMA) and calponin were substantially reduced in shear stress-cultured cells. There was no significant change in the expression of α-SMA, smooth muscle myosin heavy chain (SMMHC) or calponin at the mRNA level.Shear stress upregulated the expression of endothelial cell-related markers and downregulated smooth muscle-related markers in canine MSCs. This study may serve as a basis for further investigation of the effects of shear stress on MSC differentiation in TEVGs.

title: Additional techniques to improve microfiltration
abstract: Concentration polarisation, cake formation and pore fouling lead to a performance loss of microfiltration membranes in terms of reduced permeate flux. Causes of these phenomena are varied and depend largely on components and properties of the feed suspension, the most common causes are summarised in this paper. Techniques to limit this reduction or retard fouling are available and are reviewed, the range of methods covered include feed pre-treatment, choice of membrane material, flow manipulation, the use of high shear, gas sparging and additional force fields.

title: Mechanisms of cryoinjury in living cells.
abstract: Biological metabolism in living cells dramatically diminishes at low temperatures, a fact that permits the long-term preservation of living cells and tissues for either scientific research or many medical and industrial applications (e.g., blood transfusion, bone marrow transplantation, artificial insemination, in vitro fertilization, food storage). However, there is an apparent contradiction between the concept of preservation and experimental findings that living cells can be damaged by the cryopreservation process itself. The challenge to cells during freezing is not their ability to endure storage at very low temperatures (less than -180 degrees C); rather, it is the lethality of an intermediate zone of temperature (-15 to -60 degrees C) that a cell must traverse twice--once during cooling and once during warming. Cryobiological research studies the underlying physical and biological factors affecting survival of cells at low temperatures (during the cooling and warming processes). These factors and mechanisms (or hypotheses) of cryoinjury and its prevention are reviewed and discussed, including the most famous two-factor hypothesis theory of Peter Mazur, concepts of cold shock, vitrification, cryoprotective agens (CPAs), lethal intracellular ice formation, osmotic injury during the addition/removal of CPAs and during the cooling/warming process, as well as modeling/methods in the cryobiological research.

title: Long-term storage of tissues by cryopreservation: critical issues.
abstract: The technique of cryopreservation (maintenance of biological samples in a state of 'suspended animation' at cryogenic temperatures), its potential use in tissue engineering applications and current obstacles to the development of effective cryopreservation methods for tissues are reviewed. A didactic overview of the principles of cryobiology and the methodology of cryopreservation is given, with emphasis on the processes of injury to cells during freezing and thawing, and how these are related to the physicochemical and biophysical changes occurring during cryopreservation. Critical issues relevant to the application of cryopreservation methods to tissues are then addressed, including heat and mass transfer limitations in these bulk systems, intrinsic differences between isolated and cultured cells, and mechanisms of freezing injury unique to tissue systems.

title: Cryopreservation of rabbit spermatozoa using acetamide in combination with trehalose and methyl cellulose.
abstract: Glycerol is not an effective cryoprotectant for rabbit spermatozoa; therefore, rabbit spermatozoa were used as a model for developing cryopreservation procedures for other cell types which also freeze poorly when glycerol is used as the cryoprotectant. Experiments were conducted to 1) compare several published protocols for cryopreserving rabbit spermatozoa; 2) determine if removal of seminal granules, required for flow cytometry analysis, affects the motility of rabbit spermatozoa; and 3) determine if using a combination of cell permeating cryoprotectants (acetamide) with cell nonpermeating cryoprotectants (trehalose and methyl cellulose; MC), can increase the recovery of viable rabbit spermatozoa after cryopreservation. Media containing acetamide as a cryoprotectant were found to be most effective for rabbit spermatozoa. The cryoprotectants ethylene glycol, dimethylsulfoxide and glycerol were not effective for cryopreserving rabbit spermatozoa. Second, rabbit spermatozoa could be centrifuged through a Percoll gradient composed of equal volumes of Prcoll and a HEPES-buffered sperm medium. This centrifugation removed all seminal granules without affecting the percentage of motile spermatozoa after initial sperm dilution (85 vs 74%) or after cryopreservation (35 vs 30%), when sperm were either centrifuged or not centrifuged, respectively. The substitution of trehalose in the cryopreservation medium for raffinose did not improve recovery of motile cells following cryopreservation (P > 0.05). However, addition of MC resulted in higher percentages of motile sperm after cryopreservation (43 vs 31%; P < 0.05). In addition, sperm viability and acrosomal integrity were simultaneously evaluated using flow cytometry. The addition of both trehalose and MC to media containing acetamide resulted in higher percentages of live acrosome-intact cells than acetamide alone (53 vs 37%; P < 0.05). These results indicate that a combination of permeating and nonpermeating cryoprotectants (acetamide, trehalose and MC) were more effective in preserving rabbit spermatozoa than acetamide alone and that analyzing multiple sperm characteristics, by flow cytometry, can assess sperm damage not detected by analyzing sperm motion characteristics.

title: Mesenchymal stromal cells derived from various tissues: Biological, clinical and cryopreservation aspects.
abstract: Originally isolated from bone marrow, mesenchymal stromal cells (MSCs) have since been obtained from various fetal and post-natal tissues and are the focus of an increasing number of clinical trials. Because of their tremendous potential for cellular therapy, regenerative medicine and tissue engineering, it is desirable to cryopreserve and bank MSCs to increase their access and availability. A remarkable amount of research and resources have been expended towards optimizing the protocols, freezing media composition, cooling devices and storage containers, as well as developing good manufacturing practices in order to ensure that MSCs retain their therapeutic characteristics following cryopreservation and that they are safe for clinical use. Here, we first present an overview of the identification of MSCs, their tissue sources and the properties that render them suitable as a cellular therapeutic. Next, we discuss the responses of cells during freezing and focus on the traditional and novel approaches used to cryopreserve MSCs. We conclude that viable MSCs from diverse tissues can be recovered after cryopreservation using a variety of freezing protocols, cryoprotectants, storage periods and temperatures. However, alterations in certain functions of MSCs following cryopreservation warrant future investigations on the recovery of cells post-thaw followed by expansion of functional cells in order to achieve their full therapeutic potential.

title: Preserving human cells for regenerative, reproductive, and transfusion medicine
abstract: Cell cryopreservation maintains cellular life at sub‐zero temperatures by slowing down biochemical processes. Various cell types are routinely cryopreserved in modern reproductive, regenerative, and transfusion medicine. Current cell cryopreservation methods involve freezing (slow/rapid) or vitrifying cells in the presence of a cryoprotective agent (CPA). Although these methods are clinically utilized, cryo‐injury due to ice crystals, osmotic shock, and CPA toxicity cause loss of cell viability and function. Recent approaches using minimum volume vitrification provide alternatives to the conventional cryopreservation methods. Minimum volume vitrification provides ultra‐high cooling and rewarming rates that enable preserving cells without ice crystal formation. Herein, we review recent advances in cell cryopreservation technology and provide examples of techniques that are utilized in oocyte, stem cell, and red blood cell cryopreservation.

title: Recent advances in serum-free microcarrier expansion of mesenchymal stromal cells: Parameters to be optimized.
abstract: Mesenchymal stromal cells (MSCs) are being investigated for a variety of therapeutic indications. However, current 2D planar technology cannot meet the anticipated demand and a shift to serum-free microcarrier cultures is needed in order to meet the quality and quantity of cells required. Here we summarize several recent attempts to grow cells in such conditions, and identify several variables that affect cell expansion, including tissue source, serum-free medium formulation, microcarrier type and matrix, and agitation regime (continuous versus intermittent). Optimization of these culture conditions will be necessary to ensure success in bioreactor-scale production of MSCs for cell therapies.

title: Manufacturing of Human Umbilical Cord Mesenchymal Stromal Cells on Microcarriers in a Dynamic System for Clinical Use
abstract: The great properties of human mesenchymal stromal cells (hMSCs) make these cells an important tool in regenerative medicine. Because of the limitations of hMSCs derived from the bone marrow during isolation and expansion, hMSCs derived from the umbilical cord stroma are a great alternative to overcome these issues. For a large expansion of these cells, we performed a process transfer from static culture to a dynamic system. For this reason, a microcarrier selection out of five microcarrier types was made to achieve a suitable growth surface for the cells. The growth characteristics and metabolite consumption and production were used to compare the cells growth in 12-well plate and spinner flask. The goal to determine relevant process parameters to transfer the expansion process into a stirred tank bioreactor was achieved.

title: A xenogeneic‐free bioreactor system for the clinical‐scale expansion of human mesenchymal stem/stromal cells
abstract: The large cell doses (>1 × 106 cells/kg) used in clinical trials with mesenchymal stem/stromal cells (MSC) will require an efficient production process. Moreover, monitoring and control of MSC ex‐vivo expansion is critical to provide a safe and reliable cell product. Bioprocess engineering approaches, such as bioreactor technology, offer the adequate tools to develop and optimize a cost‐effective culture system for the rapid expansion of human MSC for cellular therapy. Herein, a xenogeneic (xeno)‐free microcarrier‐based culture system was successfully established for bone marrow (BM) MSC and adipose tissue‐derived stem/stromal cell (ASC) cultivation using a 1L‐scale controlled stirred‐tank bioreactor, allowing the production of (1.1 ± 0.1) × 108 and (4.5 ± 0.2) × 107 cells for BM MSC and ASC, respectively, after 7 days. Additionally, the effect of different percent air saturation values (%Airsat) and feeding regime on the proliferation and metabolism of BM MSC was evaluated. No significant differences in cell growth and metabolic patterns were observed under 20% and 9%Airsat. Also, the three different feeding regimes studied—(i) 25% daily medium renewal, (ii) 25% medium renewal every 2 days, and (iii) fed‐batch addition of concentrated nutrients and growth factors every 2 days—yielded similar cell numbers, and only slight metabolic differences were observed. Moreover, the immunophenotype (positive for CD73, CD90 and CD105 and negative for CD31, CD80 and HLA‐DR) and multilineage differentiative potential of expanded cells were not affected upon bioreactor culture. These results demonstrated the feasibility of expanding human MSC from different sources in a clinically relevant expansion configuration in a controlled microcarrier‐based stirred culture system under xeno‐free conditions. The further optimization of this bioreactor culture system will represent a crucial step towards an efficient GMP‐compliant clinical‐scale MSC production system. Biotechnol. Bioeng.

title: Long Term Expansion of Bone Marrow-Derived hMSCs on Novel Synthetic Microcarriers in Xeno-Free, Defined Conditions
abstract: Human mesenchymal stem cells (hMSCs) present an attractive target for cell therapy given their wide availability, immunomodulatory properties, and multipotent nature for differentiation into chondrocytes, osteocytes, and adipocytes. With the progression of hMSC clinical studies, there is an increasing demand for development of technologies that enable efficient cell scale-up into clinically relevant quantities. Commercial scale manufacturing of hMSCs will require a large surface area which is not cost effective with available two-dimensional culture vessels. Recent studies showed that microcarriers provide a three-dimensional culture environment suitable for hMSC expansion. Traditionally, biological coatings and/or serum-containing medium are required to facilitate hMSC attachment and expansion in dynamic conditions. These limitations may hinder the use of microcarriers as a scale-up technology for hMSC therapeutics, where cell products, and therefore patient safety, are more controlled with the use of xeno-free, defined culture conditions. Here we report the long term culture of hMSCs on novel synthetic Synthemax II microcarriers in two different xeno-free media. Cells were maintained over 40 days on sterile, ready-to-use microcarriers in spinner flasks with programmed agitation. hMSC expansion was obtained by addition of fresh beads without the need for enzymatic dissociation. We achieved a cumulative cell expansion of >10,000 fold, and cells retained normal hMSC phenotype, karyotype, and tri-lineage differentiation potential. To our knowledge, this report is the first example of long term culture of hMSCs on synthetic microcarriers in xeno-free, defined conditions.

title: Culture of human mesenchymal stem cells on microcarriers in a 5 l stirred-tank bioreactor
abstract: For the first time, fully functional human mesenchymal stem cells (hMSCs) have been cultured at the litre-scale on microcarriers in a stirred-tank 5 l bioreactor, (2.5 l working volume) and were harvested via a potentially scalable detachment protocol that allowed for the successful detachment of hMSCs from the cell-microcarrier suspension. Over 12 days, the dissolved O2 concentration was >45 % of saturation and the pH between 7.2 and 6.7 giving a maximum cell density in the 5 l bioreactor of 1.7 × 105 cells/ml; this represents >sixfold expansion of the hMSCs, equivalent to that achievable from 65 fully-confluent T-175 flasks. During this time, the average specific O2 uptake of the cells in the 5 l bioreactor was 8.1 fmol/cell h and, in all cases, the 5 l bioreactors outperformed the equivalent 100 ml spinner-flasks run in parallel with respect to cell yields and growth rates. In addition, yield coefficients, specific growth rates and doubling times were calculated for all systems. Neither the upstream nor downstream bioprocessing unit operations had a discernible effect on cell quality with the harvested cells retaining their immunophenotypic markers, key morphological features and differentiation capacity.

title: Growth of three established cell lines on glass microcarriers
abstract: Three established cell lines were examined for growth on a newly developed microcarrier which consists of glass beads. The cells were simultaneously exmined for growth on commercially available microcarriers made from DEAE‐dextran and from plastic. Cell yields on the glass microcarriers were comparble to the cell yields on the commercially available products. Cells grown on the glass microcarriers were easily separated from the substratum by trypsinization (as were the cells grown on the plastic substratum) while the cells grown on the DEAE‐dextran particles were much more trypsin resistant. After removal of cells from the glass microcarriers, the cells reattached and spread out in plastic flasks as readily as cells harvested from monolayer. Scanning electron microscopy revealed dramatic differences in the appearence of the cell grown on the glass microcarriers and cells grown on the DEAE‐dextran microcarriers. On the glass microcarriers, cells attached to the substratum through lond, slender filopodia while on the DEAE‐dextran microcarriers, the entire edge of the cell appeared to be in contact with the substratum. This dissimilarity in attachment could underly the difference in sensitivity to trypsin‐mediated detachment. Finally, the glass microcarriers were washed after being used once and retested for their ability to support cell growth a second time. Nearly identical results were obtained with the reprocessed beads as with previously unused ones.

title: Enzymatic Detachment of Therapeutic Mesenchymal Stromal Cells Grown on Glass Carriers in a Bioreactor
abstract: Cell therapies require the in vitro expansion of adherent cells such as mesenchymal stromal cells (hMSCs) in bioreactor systems or other culture environments, followed by cell harvest. As hMSCs are strictly adherent cells, cell harvest requires cell detachment. The use of hMSCs for cell therapy requires GMP production in accordance with the guidelines for advanced therapeutic medical products. Therefore, several GMP-conform available proteolytic enzymes were investigated for their ability to promote hMSC detachment. An allogeneic hMSC cell line (hMSC-TERT) that is used in clinical trials in the form of alginate cell capsules was chosen as a model. This study investigated the influence of several factors on the outcome of proteolytic hMSC-TERT detachment. Therefore, hMSC-TERT detachment was analyzed in different cultivation systems (static, dynamic) and in combination with further cell processing including encapsulation. Only two of the commercially available enzymes (AccutaseTM, TrypZeanTM) that fulfill all process requirements (commercial availability, cost, GMP conditions during manufacturing and non-animal origin) are found to be generally suitable for detaching hMSC-TERT. Combining cell detachment with encapsulation demonstrated a high impact of the experimental set up on cell damage. It was preferable to reduce the temperature during detachment and limit the detachment time to a maximum of 20 minutes. Cell detachment in static systems was not comparable with detachment in dynamic systems. Detachment yields in dynamic systems were lower and cell damage was higher for the same experimental conditions. Finally, only TrypZeanTM seemed to be suitable for the detachment of hMSC-TERT from dynamic reactor systems.

title: Systematic microcarrier screening and agitated culture conditions improves human mesenchymal stem cell yield in bioreactors
abstract: Production of human mesenchymal stem cells for allogeneic cell therapies requires scalable, cost‐effective manufacturing processes. Microcarriers enable the culture of anchorage‐dependent cells in stirred‐tank bioreactors. However, no robust, transferable methodology for microcarrier selection exists, with studies providing little or no reason explaining why a microcarrier was employed. We systematically evaluated 13 microcarriers for human bone marrow‐derived MSC (hBM‐MSCs) expansion from three donors to establish a reproducible and transferable methodology for microcarrier selection. Monolayer studies demonstrated input cell line variability with respect to growth kinetics and metabolite flux. HBM‐MSC1 underwent more cumulative population doublings over three passages in comparison to hBM‐MSC2 and hBM‐MSC3. In 100 mL spinner flasks, agitated conditions were significantly better than static conditions, irrespective of donor, and relative microcarrier performance was identical where the same microcarriers outperformed others with respect to growth kinetics and metabolite flux. Relative growth kinetics between donor cells on the microcarriers were the same as the monolayer study. Plastic microcarriers were selected as the optimal microcarrier for hBM‐MSC expansion. HBM‐MSCs were successfully harvested and characterised, demonstrating hBM‐MSC immunophenotype and differentiation capacity. This approach provides a systematic method for microcarrier selection, and the findings identify potentially significant bioprocessing implications for microcarrier‐based allogeneic cell therapy manufacture.

title: Online- and offline- monitoring of stem cell expansion on microcarrier
abstract: In the biopharmaceutical industry, adherent growing stem cell cultures gain worldwide importance as cell products. The cultivation process of these cells, such as in stirred tank reactors or in fixed bed reactors, is highly sophisticated. Cultivations need to be monitored and controlled to guarantee product quality and to satisfy GMP requirements. With the process analytical technology (PAT) initiative, requirements regarding process monitoring and control have changed and real-time on-line monitoring tools are recommended. A tool meeting the new requirements may be the dielectric spectroscopy for online viable cell mass determination by measurement of the permittivity. To establish these tools, proper offline methods for data correlation are required. The cell number determination of adherent cells on microcarrier is difficult, as it requires cell detachment from the carrier, which highly increases the statistical error. As an offline method, a fluorescence assay based on SYBR®GreenI was developed allowing fast and easy total cell concentration determination without the need to detach the cells from the carrier. The assay is suitable for glass carriers used in stirred tank reactor systems or in fixed bed systems, may be suitable for different cell lines and can be applied to high sample numbers easily. The linear dependency of permittivity to cell concentration of suspended stem cells with the dielectric spectroscopy is shown for even very small cell concentrations. With this offline-method, a correlation of the cell concentration grown on carrier to the permittivity data measured by the dielectric spectroscopy was done successfully.

title: Process analytical technology (PAT) in insect and mammalian cell culture processes: dielectric spectroscopy and focused beam reflectance measurement (FBRM).
abstract: Modern bioprocesses demand for a careful definition of the critical process parameters (CPPs) already during the early stages of process development in order to ensure high-quality products and satisfactory yields. In this context, online monitoring tools can be applied to recognize unfavorable changes of CPPs during the production processes and to allow for early interventions in order to prevent losses of production batches due to quality issues. Process analytical technologies such as the dielectric spectroscopy or focused beam reflectance measurement (FBRM) are possible online monitoring tools, which can be applied to monitor cell growth as well as morphological changes. Since the dielectric spectroscopy only captures cells with intact cell membranes, even information about dead cells with ruptured or leaking cell membranes can be derived. The following chapter describes the application of dielectric spectroscopy on various virus-infected and non-infected cell lines with respect to adherent as well as suspension cultures in common stirred tank reactors. The adherent mammalian cell lines Vero (African green monkey kidney cells) and hMSC-TERT (telomerase-immortalized human mesenchymal stem cells) are thereby cultured on microcarrier, which provide the required growth surface and allow the cultivation of these cells even in dynamic culture systems. In turn, the insect-derived cell lines S2 and Sf21 are used as examples for cells typically cultured in suspension. Moreover, the FBRM technology as a further monitoring tool for cell culture applications has been included in this chapter using the example of Drosophila S2 insect cells.

title: Monitoring the ex‐vivo expansion of human mesenchymal stem/stromal cells in xeno‐free microcarrier‐based reactor systems by MIR spectroscopy
abstract: Human mesenchymal stem/stromal cells (MSCs) have received considerable attention in the field of cell‐based therapies due to their high differentiation potential and ability to modulate immune responses. However, since these cells can only be isolated in very low quantities, successful realization of these therapies requires MSCs ex‐vivo expansion to achieve relevant cell doses. The metabolic activity is one of the parameters often monitored during MSCs cultivation by using expensive multi‐analytical methods, some of them time‐consuming. The present work evaluates the use of mid‐infrared (MIR) spectroscopy, through rapid and economic high‐throughput analyses associated to multivariate data analysis, to monitor three different MSCs cultivation runs conducted in spinner flasks, under xeno‐free culture conditions, which differ in the type of microcarriers used and the culture feeding strategy applied. After evaluating diverse spectral preprocessing techniques, the optimized partial least square (PLS) regression models based on the MIR spectra to estimate the glucose, lactate and ammonia concentrations yielded high coefficients of determination (R2 ≥ 0.98, ≥0.98, and ≥0.94, respectively) and low prediction errors (RMSECV ≤ 4.7%, ≤4.4% and ≤5.7%, respectively). Besides PLS models valid for specific expansion protocols, a robust model simultaneously valid for the three processes was also built for predicting glucose, lactate and ammonia, yielding a R2 of 0.95, 0.97 and 0.86, and a RMSECV of 0.33, 0.57, and 0.09 mM, respectively. Therefore, MIR spectroscopy combined with multivariate data analysis represents a promising tool for both optimization and control of MSCs expansion processes.

title: Cultivation and Differentiation of Encapsulated hMSC-TERT in a Disposable Small-Scale Syringe-Like Fixed Bed Reactor
abstract: The use of commercially available plastic syringes is introduced as disposable small-scale fixed bed bioreactors for the cultivation of implantable therapeutic cell systems on the basis of an alginate-encapsulated human mesenchymal stem cell line. The system introduced is fitted with a noninvasive oxygen sensor for the continuous monitoring of the cultivation process. Fixed bed bioreactors offer advantages in comparison to other systems due to their ease of automation and online monitoring capability during the cultivation process. These benefits combined with the advantage of single-use make the fixed bed reactor an interesting option for GMP processes. The cultivation of the encapsulated cells in the fixed bed bioreactor system offered vitalities and adipogenic differentiation similar to well-mixed suspension cultures.

title: Packed Bed Bioreactor for the Isolation and Expansion of Placental-Derived Mesenchymal Stromal Cells
abstract: Large numbers of Mesenchymal stem/stromal cells (MSCs) are required for clinical relevant doses to treat a number of diseases. To economically manufacture these MSCs, an automated bioreactor system will be required. Herein we describe the development of a scalable closed-system, packed bed bioreactor suitable for large-scale MSCs expansion. The packed bed was formed from fused polystyrene pellets that were air plasma treated to endow them with a surface chemistry similar to traditional tissue culture plastic. The packed bed was encased within a gas permeable shell to decouple the medium nutrient supply and gas exchange. This enabled a significant reduction in medium flow rates, thus reducing shear and even facilitating single pass medium exchange. The system was optimised in a small-scale bioreactor format (160 cm2) with murine-derived green fluorescent protein-expressing MSCs, and then scaled-up to a 2800 cm2 format. We demonstrated that placental derived MSCs could be isolated directly within the bioreactor and subsequently expanded. Our results demonstrate that the closed system large-scale packed bed bioreactor is an effective and scalable tool for large-scale isolation and expansion of MSCs.

title: Bioreactor expansion of human mesenchymal stem cells according to GMP requirements.
abstract: In cell therapy, the use of autologous and allogenic human mesenchymal stem cells is rising. Accordingly, the supply of cells for clinical applications in highest quality is required. As hMSCs are considered as an advanced therapy medicinal products (ATMP), they underlie the requirements of GMP and PAT according to the authorities (FDA and EMA). The production process of these cells must therefore be documented according to GMP, which is usually performed via a GMP protocol based on standard operating procedures. This chapter provides an example of such a GMP protocol for hMSC, here a genetically modified allogenic cell line, based on a production process in a microcarrier-based stirred tank reactor including process monitoring according to PAT and final product quality assurance.

title: Scalable ex vivo expansion of human mesenchymal stem/stromal cells in microcarrier-based stirred culture systems.
abstract: The clinical demand for human mesenchymal stem/stromal cells (MSC) drives the need for reproducible, cost-effective, and good manufacturing practices (GMP)-compliant ex vivo expansion protocols. Bioprocess engineering strategies, namely controlled stirred bioreactor systems combined with the use of xenogeneic(xeno)-free materials, provide proper tools to develop and optimize cell manufacturing for the rapid expansion of human MSC for cellular therapies. Herein we describe a microcarrier-based stirred culture system operating under xeno-free conditions using a controlled stirred-tank bioreactor for an efficient and controlled ex vivo expansion of human MSC. This culture platform can be applied to MSC from different human sources, as well as different microcarriers and xeno-free medium formulations.

title: Exploring continuous and integrated strategies for the up- and downstream processing of human mesenchymal stem cells.
abstract: The integration of up- and downstream unit operations can result in the elimination of hold steps, thus decreasing the footprint, and ultimately can create robust closed system operations. This type of design is desirable for the bioprocess of human mesenchymal stem cells (hMSC), where high numbers of pure cells, at low volumes, need to be delivered for therapy applications. This study reports a proof of concept of the integration of a continuous perfusion culture in bioreactors with a tangential flow filtration (TFF) system for the concentration and washing of hMSC. Moreover, we have also explored a continuous alternative for concentrating hMSC. Results show that expanding cells in a continuous perfusion operation mode provided a higher expansion ratio, and led to a shift in cells' metabolism. TFF operated either in continuous or discontinuous allowed to concentrate cells, with high cell recovery (>80%) and viability (>95%); furthermore, continuous TFF permitted to operate longer with higher cell concentrations. Continuous diafiltration led to higher protein clearance (98%) with lower cell death, when comparing to discontinuous diafiltration. Overall, an integrated process allowed for a shorter process time, recovering 70% of viable hMSC (>95%), with no changes in terms of morphology, immunophenotype, proliferation capacity and multipotent differentiation potential.

title: A potentially scalable method for the harvesting of hMSCs from microcarriers
abstract: The use of hMSCs for allogeneic therapies requiring lot sizes of billions of cells will necessitate large-scale culture techniques such as the expansion of cells on microcarriers in bioreactors. Whilst much research investigating hMSC culture on microcarriers has focused on growth, much less involves their harvesting for passaging or as a step towards cryopreservation and storage. A successful new harvesting method has recently been outlined for cells grown on SoloHill microcarriers in a 5L bioreactor . Here, this new method is set out in detail, harvesting being defined as a two-step process involving cell 'detachment' from the microcarriers' surface followed by the 'separation' of the two entities. The new detachment method is based on theoretical concepts originally developed for secondary nucleation due to agitation. Based on this theory, it is suggested that a short period (here 7min) of intense agitation in the presence of a suitable enzyme should detach the cells from the relatively large microcarriers. In addition, once detached, the cells should not be damaged because they are smaller than the Kolmogorov microscale. Detachment was then successfully achieved for hMSCs from two different donors using microcarrier/cell suspensions up to 100mL in a spinner flask. In both cases, harvesting was completed by separating cells from microcarriers using a Steriflip® vacuum filter. The overall harvesting efficiency was >95% and after harvesting, the cells maintained all the attributes expected of hMSC cells. The underlying theoretical concepts suggest that the method is scalable and this aspect is discussed too.

title: Expansion, harvest and cryopreservation of human mesenchymal stem cells in a serum‐free microcarrier process
abstract: Human mesenchymal stem cell (hMSC) therapies are currently progressing through clinical development, driving the need for consistent, and cost effective manufacturing processes to meet the lot‐sizes required for commercial production. The use of animal‐derived serum is common in hMSC culture but has many drawbacks such as limited supply, lot‐to‐lot variability, increased regulatory burden, possibility of pathogen transmission, and reduced scope for process optimization. These constraints may impact the development of a consistent large‐scale process and therefore must be addressed. The aim of this work was therefore to run a pilot study in the systematic development of serum‐free hMSC manufacturing process. Human bone‐marrow derived hMSCs were expanded on fibronectin‐coated, non‐porous plastic microcarriers in 100 mL stirred spinner flasks at a density of 3 × 105 cells.mL−1 in serum‐free medium. The hMSCs were successfully harvested by our recently‐developed technique using animal‐free enzymatic cell detachment accompanied by agitation followed by filtration to separate the hMSCs from microcarriers, with a post‐harvest viability of 99.63 ± 0.03%. The hMSCs were found to be in accordance with the ISCT characterization criteria and maintained hMSC outgrowth and colony‐forming potential. The hMSCs were held in suspension post‐harvest to simulate a typical pooling time for a scaled expansion process and cryopreserved in a serum‐free vehicle solution using a controlled‐rate freezing process. Post‐thaw viability was 75.8 ± 1.4% with a similar 3 h attachment efficiency also observed, indicating successful hMSC recovery, and attachment. This approach therefore demonstrates that once an hMSC line and appropriate medium have been selected for production, multiple unit operations can be integrated to generate an animal component‐free hMSC production process from expansion through to cryopreservation. Biotechnol. Bioeng. 2015;112: 1696–1707.

title: Continuous harvest of stem cells via partial detachment from thermoresponsive nanobrush surfaces.
abstract: Stem cell culture is typically based on batch-type culture, which is laborious and expensive. Here, we propose a continuous harvest method for stem cells cultured on thermoresponsive nanobrush surfaces. In this method, stem cells are partially detached from the nanobrush surface by reducing the temperature of the culture medium below the critical solution temperature needed for thermoresponse. The detached stem cells are harvested by exchange into fresh culture medium. Following this, the remaining cells are continuously cultured by expansion in fresh culture medium at 37 °C. Thermoresponsive nanobrush surfaces were prepared by coating block copolymers containing polystyrene (for hydrophobic anchoring onto culture dishes) with three types of polymers: (a) polyacrylic acid with cell-binding oligopeptides, (b) thermoresponsive poly-N-isopropylacrylamide, and (c) hydrophilic poly(ethyleneglycol)methacrylate. The optimal coating durations and compositions for these copolymers to facilitate adequate attachment and detachment of human adipose-derived stem cells (hADSCs) and embryonic stem cells (hESCs) were determined. hADSCs and hESCs were continuously harvested for 5 and 3 cycles, respectively, via the partial detachment of cells from thermoresponsive nanobrush surfaces.

title: Suspension Culture of Mammalian Cells Using Thermosensitive Microcarrier that Allows Cell Detachment without Proteolytic Enzyme Treatment
abstract: Microcarriers are used to expand anchorage-dependent cells in large-scale suspension bioreactors. Proteolytic enzyme treatment is necessary to detach cells cultured on microcarriers for cell harvest or scale-up, but the enzyme treatment damages the cells and extracellular matrices and complicates the culture process. Here, we fabricated thermosensitive microcarriers from which cells can be detached by temperature change without proteolytic enzyme treatment. A thermosensitive polymer, poly-N-isopropylacrylamide (pNIPAAm), was incorporated on the surface of Cytodex-3® microcarriers. pNIPAAm-grafted microcarriers allowed human bone marrow-derived mesenchymal stem cells (hBMMSCs) to adhere, spread, and grow successfully on the microcarriers as nongrafted microcarriers did. By dropping temperature below 32°C, more than 82.5% of hBMMSCs were detached from pNIPAAm-grafted microcarriers. The trypsin treatment for cell detachment induced apoptosis and death of some of the detached cells, but cell detachment from pNIPAAm-grafted microcarriers by temperature change significantly reduced the apoptosis and cell death. pNIPAAm-grafted microcarriers can significantly reduce cell extracellular matrix damage in the cell detachment process and simplify the cell detachment process by avoiding proteolytic enzyme treatment. pNIPAAm-grafted microcarriers would be valuable to a variety of potential fields demanding a large amount of cells without cell damage, such as cell therapy, tissue engineering, and other biological and clinical applications.

title: In vitro culture and harvest of BMMSCs on the surface of a novel thermosensitive glass microcarrier.
abstract: Traditional two-dimensional (2D) static culture environment for stem cells followed by enzymatic cell detachment or mechanical treatment is routinely used in research laboratories. However, this method is not ideal as stem cells expand slowly, with cell damage and partial loss of specific stemness. For this reason, a better culture condition is urgently needed to improve stem cell recovery. A novel thermosensitive P(NIPAAm-co-HPM)-g-TMSPM-g-microcarrier was prepared here as a three-dimensional (3D) culture substitute. This novel microcarrier was prepared by grafting NIPAAm and HPM to the surface of glass microcarrier using TMSPM through surface free radical copolymerization. The prepared material was tested in cell culture and via cooling harvest method. We found that NIPAAm was successfully grafted on to the surface of the microcarriers, providing an excellent biocompatible environment for BMMSC adhesion and growth. More importantly, BMMSCs could be fully removed from the thermosensitive glass microcarriers with remained cell viability.

title: Derivation, characterization and expansion of fetal chondrocytes on different microcarriers
abstract: Fetal chondrocytes (FCs) have recently been identified as an alternative cell source for cartilage tissue engineering applications because of their partially chondrogenically differentiated phenotype and developmental plasticity. In this study, chondrocytes derived from fetal bovine cartilage were characterized and then cultured on commercially available Cytodex-1 and Biosilon microcarriers and thermosensitive poly(hydroxyethylmethacrylate)-poly(N-isopropylacrylamide) (PHEMA-PNIPAAm) beads produced by us. Growth kinetics of FCs were estimated by means of specific growth rate and metabolic activity assay. Cell detachment from thermosensitive microcarriers was induced by cold treatment at 4 °C for 20 min or enzymatic treatment was applied for the detachment of cells from Cytodex-1 and Biosilon. Although attachment efficiency and proliferation of FCs on PHEMA-PNIPAAm beads were lower than that of commercial Cytodex-1 and Biosilon microcarriers, these beads also supported growth of FCs. Detached cells from thermosensitive beads by cold induction exhibited a normal proliferative activity. Our results indicated that Cytodex-1 microcarrier was the most suitable material for the production of FCs in high capacity, however, ‘thermosensitive microcarrier model’ could be considered as an attractive solution to the process scale up for cartilage tissue engineering by improving surface characteristics of PHEMA-PNIPAAm beads.

title: A novel filtration device for point of care preparation of cellular therapies
abstract: Many biological samples are stored at cryogenic temperatures (i.e. temperatures below -1500C) to preserve their viability. Typically, these samples are stored in liquid nitrogen vapor-phase freezers. The underlying assumption is that biological samples show highly reduced degradation and metabolic activity while below Tg (the glass transition temperature). On the other hand, every time a sample is manipulated or temporarily removed from an LN2 freezer, the sample will experience thermal excursions marked by warm-up rates of several degrees per second. In these cases, the risk of harming a sample by inadvertently crossing the Tg threshold is very likely. This paper’s objective is to provide supporting data to fully characterize the thermal excursions of cryogenically frozen single vials filled with H2O during typical transient temperature events. Specifically, we focus our attention on the cold-chain steps that involve transferring a single vial from an LN2 vapor environment to either an ambient temperature or transportable dry ice (-800C) environment. In general, the warm-up rates experienced by the biological sample correlate to the thermal energy exchanged with the warmer environment via convective and conductive heat transfer. The magnitude of the heat transfer is driven by multiple factors: the warmer environment temperature and the overall time of exposure, the size and shape of the vial, its placement in a cryobox, the type of handling (manual or automated), the handling speed, etc. In this paper, we rationalize all the above factors and present experimental measurements supported by calibrated finite elements simulations showing typical expected warm-up rates. As a result, best-practice time constants for handling vials of biological samples without risking excessive thermal excursions above Tg are suggested.

title: Improving washing strategies of human mesenchymal stem cells using negative mode expanded bed chromatography.
abstract: The use of human mesenchymal stem cells (hMSC) in clinical applications has been increasing over the last decade. However, to be applied in a clinical setting hMSC need to comply with specific requirements in terms of identity, potency and purity. This study reports the improvement of established tangential flow filtration (TFF)-based washing strategies, further increasing hMSC purity, using negative mode expanded bed adsorption (EBA) chromatography with a new multimodal prototype matrix based on core-shell bead technology. The matrix was characterized and a stable, expanded bed could be obtained using standard equipment adapted from what is used for conventional packed bed chromatography processes. The effect of different expansion rates on cell recovery yield and protein removal capacity was assessed. The best trade-off between cell recovery (89%) and protein clearance (67%) was achieved using an intermediate expansion bed rate (1.4). Furthermore, we also showed that EBA chromatography can be efficiently integrated on the already established process for the downstream processing (DSP) of hMSC, where it improved the washing efficiency more than 10-fold, recovering approximately 70% of cells after global processing. This strategy showed not to impact cell viability (>95%), neither hMSC's characteristics in terms of morphology, immunophenotype, proliferation, adhesion capacity and multipotent differentiation potential.

title: Systematic parameter optimization of a Me(2)SO- and serum-free cryopreservation protocol for human mesenchymal stem cells.
abstract: Human mesenchymal stem cells (hMSCs) have great potential for clinical therapy and regenerative medicine. One major challenge concerning their application is the development of an efficient cryopreservation protocol since current methods result in a poor viability and high differentiation rates. A high survival rate of cryopreserved cells requires an optimal cooling rate and the presence of cryoprotective agents (CPA) in sufficient concentrations. The most widely used CPA, dimethylsulfoxide (Me(2)SO), is toxic at high concentrations at temperatures >4°C and has harmful effects on the biological functionality of stem cell as well as on treated patients. Thus, this study investigates different combinations of non-cytotoxic biocompatible substances, such as ectoin and proline, as potential CPAs in a systematic parametric optimization study in comparison to Me(2)SO as control and a commercial freezing medium (Biofreeze®, Biochrom). Using a freezing medium containing a low proline (1%, w/v) and higher ectoin (10%, w/v) amount revealed promising results although the highest survival rate was achieved with the Biofreeze® medium. Cryomicroscopic experiments of hMSCs revealed nucleation temperatures ranging from -16 to -25°C. The CPAs, beside Me(2)SO, did not affect the nucleation temperature. In most cases, cryomicroscopy revealed intracellular ice formation (IIF) during the cryopreservation cycle for all cryoprotocols. The occurence of IIF during thawing increased with the cooling rate. In case of hMSC there was no correlation between the rate of IIF and the post-thaw cell survival. After thawing adipogenic differentiation of the stem cells demonstrated cell functionality.

title: Frozen adipose-derived mesenchymal stem cells maintain high capability to grow and differentiate.
abstract: In recent years, there has been a shift toward tissue-engineering strategies using stem cells for plastic and reconstructive surgical procedures. Therefore, it is important to develop safe and reproducible protocols for the extraction of adipose-derived stromal cells (ASCs) to allow cells to be stored in liquid nitrogen for future needs. The aspirated liposuction obtained from healthy donors were immediately processed after the suction using a protocol developed in our laboratory. The resulting stromal vascular fraction (SVF) was then characterized by the presence of adipose-derived stromal cells, at later stage frozen in liquid nitrogen. After that, cells were thawed and again characterized by adipose-derived stromal cells, cellular survival, differentiation ability and Colony Forming Unit-Fibroblast like colonies (CFU-F). Extraction and freezing of cells contained in the stromal vascular fraction demonstrate that thawed cells maintain the full capability to grow and differentiate in culture. The advent of adipose-derived stromal cells use in tissue engineering will assume a wide role in esthetic restoration in plastic surgery. It is thus important to develop clinically translatable protocols for the preparation and storage of adipose-derived stromal cells. Our results show that adipose-derived stromal cells in serum free can easily be frozen and stored in liquid nitrogen with retention of 85% of cell viability and 180,890 cell/g yield plus normal proliferative capacity and differentiation potential compared with fresh controls. These observations set the basis for adipose-derived stromal cells banking.

title: Boron increases the cell viability of mesenchymal stem cells after long-term cryopreservation.
abstract: The field of stem-cell biology has emerged as a key technology for the treatment of various disorders and tissue regeneration applications. However, a major problem remains in clinical practice, which is the question of whether stem cells preserve their self-renewal and differentiation potential in the culture conditions or not. In the current study, effects of boron on the cryopreservation of human tooth germ stem cells (hTGSCs) were evaluated for the first time. The impacts of various boron concentrations (sodium pentaborate pentahydrate (NaB)) were tested on characterized hTGSCs viability for different time intervals (24, 48, and 72 h). 20 μg/ml NaB with lower Me(2)SO concentration was found to display positive effects on hTGSCs during repeated freezing and defrosting cycles, and long-term cryopreservation. After thawing, cells were analyzed for their surface antigens and differentiation capacity. hTGSCs were successfully cryopreserved without any change in their mesenchymal stem cell characteristics as they were treated with boron containing freezing medium. In addition, fatty acid composition was examined to demonstrate membrane fatty acid profiles after freeze-thawing. Besides, NaB treatment extended osteogenic and chondrogenic differentiation of hTGSCs remarkably after long-term cryopreservation with respect to control groups. The study clearly suggests that NaB has a protective role on the survival of hTGSCs in short- and long-term cryopreservation. Due to the possible storage of hTGSCs at early ages, development of a functional and reliable cryopreservation media can be designed as a future solution to the dental stem cell banking.

title: Biological and Physicochemical Characterization of a Serum-and Xeno-Free Chemically Defined Cryopreservation Procedure for Adult Human Progenitor Cells
abstract: While therapeutic cell transplantations using progenitor cells are increasingly evolving towards phase I and II clinical trials and chemically defined cell culture is established, standardization in biobanking is still in the stage of infancy. In this study, the EU FP6-funded CRYSTAL (CRYo-banking of Stem cells for human Therapeutic AppLication) consortium aimed to validate novel Standard Operating Procedures (SOPs) to perform and validate xeno-free and chemically defined cryopreservation of human progenitor cells and to reduce the amount of the potentially toxic cryoprotectant additive (CPA) dimethyl sulfoxide (DMSO). To achieve this goal, three human adult progenitor and stem cell populations—umbilical cord blood (UCB)-derived erythroid cells (UCB-ECs), UCB-derived endothelial colony forming cells (UCB-ECFCs), and adipose tissue (AT)-derived mesenchymal stromal cells (AT-MSCs)—were cryopreserved in chemically defined medium supplemented with 10% or 5% DMSO. Cell recovery, cell repopulation, and functionality were evaluated postthaw in comparison to cryopreservation in standard fetal bovine serum (FBS)-containing freezing medium. Even with a reduction of the DMSO CPA to 5%, postthaw cell count and viability assays indicated no overall significant difference versus standard cryomedium. Additionally, to compare cellular morphology/membrane integrity and ice crystal formation during cryopreservation, multiphoton laser-scanning cryomicroscopy (cryo-MPLSM) and scanning electron microscopy (SEM) were used. Neither cryo-MPLSM nor SEM indicated differences in membrane integrity for the tested cell populations under various conditions. Moreover, no influence was observed on functional properties of the cells following cryopreservation in chemically defined freezing medium, except for UCB-ECs, which showed a significantly reduced differentiation capacity after cryopreservation in chemically defined medium supplemented with 5% DMSO. In summary, these results demonstrate the feasibility and robustness of standardized xeno-free cryopreservation of different human progenitor cells and encourage their use even more in the field of tissue-engineering and regenerative medicine.

title: Clinical grade adult stem cell banking
abstract: There has been a great deal of scientific interest recently generated by the potential therapeutic applications of adult stem cells in human care but there are several challenges regarding quality and safety in clinical applications and a number of these challenges relate to the processing and banking of these cells ex-vivo. As the number of clinical trials and the variety of adult cells used in regenerative therapy increases, safety remains a primary concern. This has inspired many nations to formulate guidelines and standards for the quality of stem cell collection, processing, testing, banking, packaging and distribution. Clinically applicable cryopreservation and banking of adult stem cells offers unique opportunities to advance the potential uses and widespread implementation of these cells in clinical applications. Most current cryopreservation protocols include animal serum proteins and potentially toxic cryoprotectant additives (CPAs) which prevent direct use of these cells in human therapeutic applications. Long term cryopreservation of adult stem cells under good manufacturing conditions using animal product free solutions is critical to the widespread clinical implementation of ex-vivo adult stem cell therapies. Furthermore, to avoid any potential cryoprotectant related complications, reduced CPA concentrations and efficient post-thaw washing to remove CPA are also desirable. The present review focuses on the current strategies and important aspects of adult stem cell banking for clinical applications. These include current good manufacturing practices (cGMPs), animal protein free freezing solutions, cryoprotectants, freezing & thawing protocols, viability assays, packaging and distribution. The importance and benefits of banking clinical grade adult stem cells are also discussed.

title: Advances in cell encapsulation technology and its application in drug delivery
abstract: Cell encapsulation technology has improved enormously since it was proposed 50 years ago. The advantages offered over other alternative systems, such as the prevention of repetitive drug administration, have triggered the use of this technology in multiple therapeutic applications.  In this article, improvements in cell encapsulation technology and strategies to overcome the drawbacks that prevent its use in the clinic have been summarized and discussed. Different studies and clinical trials that have been performed in several therapeutic applications have also been described.  The authors believe that the future translation of this technology from bench to bedside requires the optimization of diverse aspects: i) biosafety, controlling and monitoring cell viability; ii) biocompatibility, reducing pericapsular fibrotic growth and hypoxia suffered by the graft; iii) control over drug delivery; iv) and the final scale up. On the other hand, an area that deserves more attention is the cryopreservation of encapsulated cells as this will facilitate the arrival of these biosystems to the clinic.

title: Cell-based delivery of glucagon-like peptide-1 using encapsulated mesenchymal stem cells
abstract: Glucagon-like peptide-1 (GLP-1) CellBeads are cell-based implants for the sustained local delivery of bioactive factors. They consist of GLP-1 secreting mesenchymal stem cells encapsulated in a spherically shaped immuno-isolating alginate matrix. A highly standardized and reproducible encapsulation method is described for the manufacturing of homogeneous CellBeads. Viability and sustained secretion was shown for the recombinant GLP-1 and the cell endogenous bioactive factors like vascular endothelial growth factor, neurotrophin 3 (NT-3) and glial cell line-derived neurotrophic factor. Manufacturing and quality control is performed in compliance with good manufacturing practice and fulfils all regulatory requirements for human clinical use. GLP-1 CellBeads combine the neuro- and cardioprotective properties of both GLP-1 and mesenchymal stem cells. First promising results were obtained from preclinical studies and an ongoing safety trial in humans but further studies have to prove the overall potential of CellBead technology in cell-based regenerative medicine.

title: Alginate micro-encapsulation of mesenchymal stromal cells enhances modulation of the neuro-inflammatory response.
abstract: Modulation of inflammation after brain trauma is a key therapeutic goal aimed at limiting the consequences of the subsequent injury cascade. Mesenchymal stromal cells (MSCs) have been demonstrated to dynamically regulate the inflammatory environment in several tissue systems, including the central nervous system. There has been limited success, however, with the use of direct implantation of cells in the brain caused by low viability and engraftment at the injury site. To circumvent this, we encapsulated MSCs in alginate microspheres and evaluated the ability of these encapsulated MSCs to attenuate inflammation in rat organotypic hippocampal slice cultures (OHSC).



OHSC were administered lipopolysaccharide to induce inflammation and immediately co-cultured with encapsulated or monolayer human MSCs. After 24 h, culture media was assayed for the pro-inflammatory cytokine tumor necrosis factor-alpha (TNF-α) produced by OHSC, as well as MSC-produced trophic mediators.



Encapsulated MSCs reduced TNF-α more effectively than did monolayer MSCs. Additionally, there was a strong correlation between increased prostaglandin E2 (PGE2) and reduction of TNF-α. In contrast to monolayer MSCs, inflammatory signals were not required to stimulate PGE2 production by encapsulated MSCs. Further encapsulation-stimulated changes were revealed in a multiplex panel analyzing 27 MSC-produced cytokines and growth factors, from which additional mediators with strong correlations to TNF-α levels were identified.



These results suggest that alginate encapsulation of MSCs may not only provide an improved delivery vehicle for transplantation but may also enhance MSC therapeutic benefit for treating neuro-inflammation.

title: Process engineering of high voltage alginate encapsulation of mesenchymal stem cells.
abstract: Encapsulation of stem cells in alginate beads is promising as a sophisticated drug delivery system in treatment of a wide range of acute and chronic diseases. However, common use of air flow encapsulation of cells in alginate beads fails to produce beads with narrow size distribution, intact spherical structure and controllable sizes that can be scaled up. Here we show that high voltage encapsulation (≥ 15 kV) can be used to reproducibly generate spherical alginate beads (200-400 μm) with narrow size distribution (± 5-7%) in a controlled manner under optimized process parameters. Flow rate of alginate solution ranged from 0.5 to 10 ml/h allowed producing alginate beads with a size of 320 and 350 μm respectively, suggesting that this approach can be scaled up. Moreover, we found that applied voltages (15-25 kV) did not alter the viability and proliferation of encapsulated mesenchymal stem cells post-encapsulation and cryopreservation as compared to air flow. We are the first who employed a comparative analysis of electro-spraying and air flow encapsulation to study the effect of high voltage on alginate encapsulated cells. This report provides background in application of high voltage to encapsulate living cells for further medical purposes. Long-term comparison and work on alginate-cell interaction within these structures will be forthcoming.

title: Use of Encapsulated Stem Cells to Overcome the Bottleneck of Cell Availability for Cell Therapy Approaches
abstract: Nowadays cell-based therapy is rarely in clinical practice because of the limited availability of appropriate cells. To apply cells therapeutically, they must not cause any immune response wherefore mainly autologous cells have been used up to now. The amount of vital cells in patients is limited, and under certain circumstances in highly degenerated tissues no vital cells are left. Moreover, the extraction of these cells is connected with additional surgery; also the expansion in vitro is difficult. Other approaches avoid these problems by using allo-or even xenogenic cells. These cells are more stable concerning their therapeutic behavior and can be produced in stock. To prevent an immune response caused by these cells, cell encapsulation (e.g. with alginate) can be performed. Certain studies showed that encapsulated allo-and xenogenic cells achieve promising results in treatment of several diseases. For such cell therapy approaches, stem cells, particularly mesenchymal stem cells, are an interesting cell source. This review deals on the one hand with the use of encapsulated cells, especially stem cells, in cell therapy and on the other hand with bioreactor systems for the expansion and differentiation of mesenchymal stem cells in reproducible and sufficient amounts for potential clinical use.

title: Microencapsulation Technology: A Powerful Tool for Integrating Expansion and Cryopreservation of Human Embryonic Stem Cells
abstract: The successful implementation of human embryonic stem cells (hESCs)-based technologies requires the production of relevant numbers of well-characterized cells and their efficient long-term storage. In this study, cells were microencapsulated in alginate to develop an integrated bioprocess for expansion and cryopreservation of pluripotent hESCs. Different three-dimensional (3D) culture strategies were evaluated and compared, specifically, microencapsulation of hESCs as: i) single cells, ii) aggregates and iii) immobilized on microcarriers. In order to establish a scalable bioprocess, hESC-microcapsules were cultured in stirred tank bioreactors. The combination of microencapsulation and microcarrier technology resulted in a highly efficient protocol for the production and storage of pluripotent hESCs. This strategy ensured high expansion ratios (an approximately twenty-fold increase in cell concentration) and high cell recovery yields (>70%) after cryopreservation. When compared with non-encapsulated cells, cell survival post-thawing demonstrated a three-fold improvement without compromising hESC characteristics. Microencapsulation also improved the culture of hESC aggregates by protecting cells from hydrodynamic shear stress, controlling aggregate size and maintaining cell pluripotency for two weeks. This work establishes that microencapsulation technology may prove a powerful tool for integrating the expansion and cryopreservation of pluripotent hESCs. The 3D culture strategy developed herein represents a significant breakthrough towards the implementation of hESCs in clinical and industrial applications.

title: Cryopreservation of alginate encapsulated mesenchymal stromal cells.
abstract: Human mesenchymal stromal cells (MSCs) can differentiate into various cell types, which makes them attractive for regenerative medicine and tissue engineering. Encapsulation of MSCs in alginate microspheres (AMS) is a novel and promising approach of tissue engineering. Application and research of such cell-hydrogel systems require selection of adequate cryopreservation protocols. In this study we investigated the response of MSCs encapsulated in AMS to different cryopreservation protocols. Bone marrow MSCs either encapsulated in AMS and or as cells in suspension, were cryopreserved with 5% and 10% of dimethyl sulfoxide (ME₂SO) using conventional 2-step slow cooling (protocol 1). The viability and metabolism of MSCs in AMS following cryopreservation with 5% Me₂SO were lower than in the group cryopreserved with 10% Me₂SO. MSCs in suspension were more resistant to cryopreservation than cells in AMS when cryopreserved with 5% Me₂SO, although when using a concentration of 10% Me₂SO, no differences were detected. Comparisons of the viability and metabolic activity of MSC cryopreserved either in AMS or as cell suspensions with 10% ME₂SO using protocol 1 (2-step cooling), protocol 2 (3-step slow cooling with induced ice nucleation) or protocol 3 (rapid 1-step freezing), showed that the highest viabilities and metabolic rates were obtained following cryopreservation of MSCs in AMS by protocol 2 (with controlled ice nucleation). Cryopreservation with protocol 3 resulted in critical damage of the encapsulated MSCs. After cryopreservation by protocol 2, AMS encapsulated MSCs were capable of achieving multilineage differentiation directed towards osteogenic, adipogenic and chondrogenic lineages. The data obtained indicate that cryo-banking of AMS encapsulated MSCs is feasible for future regenerative medicine projects.

title: Cryopreservation of microencapsulated murine mesenchymal stem cells genetically engineered to secrete erythropoietin.
abstract: The ability to cryopreserve and store for long term the structure and function of therapeutic cells and tissues plays a pivotal role in clinical medicine. In fact, it is an essential pre-requisite for the commercial and clinical application of stem cells since preserves cells at low temperature and creates a reserve for future uses. This requisite may also affect the encapsulated stem cells. Several parameters should be considered on encapsulated cell cryopreservation such as the time and temperature during the cryopreservation process, or the cryoprotectant solutions used. In this study, we have compared the influence of penetrating and nonpenetrating cryoprotectants on the viability and functionality of encapsulated mesenchymal stem cells genetically modified to secrete erythropoeitin. Several cryoprotectant solutions combining DMSO, glycerol and trehalose at different concentrations were studied. Although almost no differences among the studied cryoprotectant solutions were observed on the differentiation potential of encapsulated mesenchymal stem cells, the penetrating cryoprotectant DMSO at a concentration of 10% displayed the best viability and erythropoietin secretion profile compared to the other cryoprotectant solutions. These results were confirmed after subcutaneous implantation of thawed encapsulated mesenchymal stem cells secreting erythropoeitin on Balb/c mice. The hematocrit levels of these animals increased to similar levels of those detected on animals transplanted with noncryopreserved encapsulated cells. Therefore, DMSO 10% represents the most suitable cryoprotectant solution among the solutions here studied, for encapsulated mesenchymal stem cells cryopreservation and its translation into the clinic. Similar studies should be performed for the encapsulation of other cell types before they can be translated into the clinic.

title: A novel alternative to cryopreservation for the short-term storage of stem cells for use in cell therapy using alginate encapsulation.
abstract: Efficient transport of stem/progenitor cells without affecting their survival and function is a key factor in any practical cell-based therapy. However, the current approach using liquid nitrogen for the transfer of stem cells requires a short delivery time window is technically challenging and financially expensive. The present study aims to use semipermeable alginate hydrogels (crosslinked by strontium) to encapsulate, store, and release stem cells, to replace the conventional cryopreservation method for the transport of therapeutic cells within world-wide distribution time frame. Human mesenchymal stem cell (hMSC) and mouse embryonic stem cells (mESCs) were successfully stored inside alginate hydrogels for 5 days under ambient conditions in an air-tight environment (sealed cryovial). Cell viability, of the cells extracted from alginate gel, gave 74% (mESC) and 80% (hMSC) survival rates, which compared favorably to cryopreservation. More importantly, the subsequent proliferation rate and detection of common stem cell markers (both in mRNA and protein level) from hMSCs and mESCs retrieved from alginate hydrogels were also comparable to (if not better than) results gained following cryopreservation. In conclusion, this new and simple application of alginate hydrogel encapsulation may offer a cheap and robust alternative to cryopreservation for the transport and storage of stem cells for both clinical and research purposes.

title: Alginate‐Encapsulation for the Improved Hypothermic Preservation of Human Adipose‐Derived Stem Cells
abstract: Despite considerable progress within the cell therapy industry, unmet bioprocessing and logistical challenges associated with the storage and distribution of cells between sites of manufacture and the clinic exist. We examined whether hypothermic (4°C–23°C) preservation of human adipose‐derived stem cells could be improved through their encapsulation in 1.2% calcium alginate. Alginate encapsulation improved the recovery of viable cells after 72 hours of storage. Viable cell recovery was highly temperature‐dependent, with an optimum temperature of 15°C. At this temperature, alginate encapsulation preserved the ability for recovered cells to attach to tissue culture plastic on rewarming, further increasing its effect on total cell recovery. On attachment, the cells were phenotypically normal, displayed normal growth kinetics, and maintained their capacity for trilineage differentiation. The number of cells encapsulated (up to 2 × 106 cells per milliliter) did not affect viable cell recovery nor did storage of encapsulated cells in a xeno‐free, serum‐free,current Good Manufacturing Practice‐grade medium. We present a simple, low‐cost system capable of enhancing the preservation of human adipose‐derived stem cells stored at hypothermic temperatures, while maintaining their normal function. The storage of cells in this manner has great potential for extending the time windows for quality assurance and efficacy testing, distribution between the sites of manufacture and the clinic, and reducing the wastage associated with the limited shelf life of cells stored in their liquid state.

title: Assays of osteogenic differentiation by cultured human mesenchymal stem cells.
abstract: One of the most noteworthy characteristics of mesenchymal stem cells (MSCs) is their ability to differentiate into osteoblasts in vitro and in vivo. In vitro, this is easily achieved by culturing in the appropriate induction medium. It is because of the reliability and ease of this process that osteogenic differentiation has become a popular assay for the demonstration of MSC plasticity. Although the conditions required for inducing osteogenic differentiation by MSCs typically do not vary particularly between investigators, many methods are employed to measure the extent of differentiation. These methods include, but are not limited to, reverse transcriptase PCR (RT-PCR) for detection of osteogenic transcripts, enzyme linked immunosorbent assay (ELISA) for secreted protein markers, colorimetric assays for osteogenic enzymes, and direct staining of matrix components. This chapter reviews the protocols most commonly utilized for the evaluation of osteogenic differentiation for cultured MSCs.

title: Adipogenic differentiation of human mesenchymal stem cells.
abstract: Mesenchymal stem cells have the capability to differentiate into a number of cell types including adipocytes. The adipocytic phenotype is characterized by intracellular accumulation of lipid droplets as well as transcription of adipocyte-specific genes. This paper details a basic protocol for adipogenic induction of bone marrow and adipose tissue-derived stem cells, as well as protocols for staining lipid accumulation and the transcriptional analysis of PPAR-γ and aP2 by real-time RT-PCR.

title: Chondrogenic differentiation of bone marrow-derived mesenchymal stem cells: tips and tricks.
abstract: It is well known that adult cartilage lacks the ability to repair itself; this makes articular cartilage a very attractive target for tissue engineering. The majority of articular cartilage repair models attempt to deliver or recruit reparative cells to the site of injury. A number of efforts are directed to the characterization of progenitor cells and the understanding of the mechanisms involved in their chondrogenic differentiation. Our laboratory has focused on cartilage repair using mesenchymal stem cells and studied their differentiation into cartilage. Mesenchymal stem cells are attractive candidates for cartilage repair due to their osteogenic and chondrogenic potential, ease of harvest, and ease of expansion in culture. However, the need for chondrogenic differentiation is superposed on other technical issues associated with cartilage repair; this adds a level of complexity over using mature chondrocytes. This chapter will focus on the methods involved in the isolation and expansion of human mesenchymal stem cells, their differentiation along the chondrogenic lineage, and the qualitative and quantitative assessment of chondrogenic differentiation.

title: Single-Step RNA Extraction from Different Hydrogel-Embedded Mesenchymal Stem Cells for Quantitative Reverse Transcription-Polymerase Chain Reaction Analysis.
abstract: For many tissue engineering applications, cells such as human mesenchymal stem cells (hMSCs) must be embedded in hydrogels. The analysis of embedded hMSCs requires RNA extraction, but common extraction procedures often produce low yields and/or poor quality RNA. We systematically investigated four homogenization methods combined with eight RNA extraction protocols for hMSCs embedded in three common hydrogel types (alginate, agarose, and gelatin). We found for all three hydrogel types that using liquid nitrogen or a rotor-stator produced low RNA yields, whereas using a microhomogenizer or enzymatic/chemical hydrogel digestion achieved better yields regardless of which extraction protocol was subsequently applied. The hot phenol extraction protocol generally achieved the highest A260 values (representing up to 40.8 μg RNA per 10(6) cells), but the cetyltrimethylammonium bromide (CTAB) method produced RNA of better quality, with A260/A280 and A260/A230 ratios and UV spectra similar to the pure RNA control. The RNA produced by this method was also suitable as a template for endpoint and quantitative reverse transcription-PCR (qRT-PCR), achieving low Ct values of ∼20. The prudent choice of hydrogel homogenization and RNA extraction methods can ensure the preparation of high-quality RNA that generates reliable endpoint and quantitative RT-PCR data. We therefore propose a universal method that is suitable for the extraction of RNA from cells embedded in all three hydrogel types commonly used for tissue engineering.

title: Risk of tumorigenicity in mesenchymal stromal cell-based therapies--bridging scientific observations and regulatory viewpoints.
abstract: In the past decade, the therapeutic value of mesenchymal stromal cells (MSCs) has been studied in various indications, thereby taking advantage of their immunosuppressive properties. Easy procurement from bone marrow, adipose tissue or other sources and conventional in vitro expansion culture have made their clinical use attractive. Bridging the gap between current scientific knowledge and regulatory prospects on the transformation potential and possible tumorigenicity of MSCs, the Cell Products Working Party and the Committee for Advanced Therapies organized a meeting with leading European experts in the field of MSCs. This meeting elucidated the risk of potential tumorigenicity related to MSC-based therapies from two angles: the scientific perspective and the regulatory point of view. The conclusions of this meeting, including the current regulatory thinking on quality, nonclinical and clinical aspects for MSCs, are presented in this review, leading to a clearer way forward for the development of such products.

title: Defining the risks of mesenchymal stromal cell therapy.
abstract: We address the issue of the potential for malignant transformation of cultured mesenchymal stromal cells (MSC) commonly used in clinical cell-therapy protocols and describe the culture conditions under which tumorigenesis is likely to be an extremely uncommon event.

title: Frequent occurrence of non-malignant genetic alterations in clinical grade mesenchymal stromal cells expanded for cell therapy protocols
abstract: Human bone marrow mesenchymal stromal cells (BM-MSC) represent one of the most investigated “advanced therapeutic medicinal products”.[1][1] Recent safety concerns have focused attention on the possible malignant transformation due to mutations acquired during their large-scale in vitro