Enhancing our understanding of lymphatic anatomy from the microscopic to the anatomical scale is essential to discern how the structure and function of the lymphatic system interacts with different tissues and organs within the body and contributes to health and disease. The knowledge of molecular aspects of the lymphatic network is fundamental to understand the mechanisms of disease progression and prevention. Recent advances in mapping components of the lymphatic system using state of the art single cell technologies, the identification of novel biomarkers, new clinical imaging efforts, and computational tools which attempt to identify connections between these diverse technologies hold the potential to catalyze new strategies to address lymphatic diseases such as lymphedema and lipedema. This manuscript summarizes current knowledge of the lymphatic system and identifies prevailing challenges and opportunities to advance the field of lymphatic research as discussed by the experts in the workshop. The lymphatic vasculature is comprised of a vast network of vessels in all tissues of the body that converge to transport lymph away from tissues to the blood in order to maintain extracellular fluid homeostasis and provide critical immunologic trafficking. This traditional paradigm of passive transport of lymph fluid has been updated by cellular and molecular characterization of lymphatic vascular development. Lymph production and flow is critical for removal of interstitial fluid from tissues to prevent tissue edema. Our greatly improved understanding of the function of the lymphatic vasculature has revealed new roles in health and disease.The scope of lymphatic disease research is broad and includes, but is not limited to, investigating lymphedema, lipedema, chylous leak disorders, lymphatic malformations, and protein losing enteropathies . While sYet to be Charted: Mapping the Lymphatic System Across Body Scales and Expertise Domains at the Boston Lymphatic Symposium identified major knowledge gaps in lymphatic anatomy and lymphatic biomolecular signatures as major barriers needing to be addressed to accelerate advancement of medical management of lymphatic diseases. Research opportunities and ongoing efforts to address these gaps were discussed and are presented in this report.The 2021 National Heart, Lung, and Blood Institute Workshop The lymphatic vasculature consists of a network of thin-walled, blind-ended, highly permeable initial lymphatics or lymphatic capillaries which first drain into pre-collecting lymphatic vessels, merging into larger secondary collecting lymphatics. The valves of the collecting lymphatics control the unidirectional transport of lymph back to the blood circulation. Lymph is transferred to pre-nodal collecting lymphatics, also called afferent lymphatics, leading to lymph nodes. The lymph exits lymph nodes through post-nodal collecting lymphatics, eventually draining into the thoracic duct and the right lymphatic duct, which, in turn, discharge lymph into the large veins at the base of the neck. More in-depth reviews provide a detailed understanding of the structure, anatomy, development, and embryogenic origins of the lymphatic system . DespiteThe lack of clinically relevant knowledge of the human lymphatic system, in comparison to the remainder of the vascular system, is due to several reasons. First, while lymphatic capillaries are significantly larger than blood capillaries, the remainder of the lymphatic vasculature is significantly smaller than the major arteries and thus are challenging to appreciate with the naked eye during surgery or even with existing clinical imaging methods . Second,While actual translation of omic data into significant clinical interventions is in the nascent phase, the promise of multi-omic data in driving diagnosis, prognosis, and providing targets for intervention is an area of intense study. Therefore, it is only to be expected that a tremendous effort has been placed on obtaining the biomolecular signatures of the human cardiovascular system . SurprisIn a self-perpetuating manner, the challenges of visualizing the lymphatic system either grossly or with imaging and gaps in biomolecular signatures has been further exacerbated by insufficient coverage of this topic in medical education and training . Such knTraditionally, our understanding of clinical anatomy begins in the gross anatomy laboratory. As the challenges of gross anatomic dissection of the lymphatic system have been previously described, new techniques in anatomic dissection have been recently introduced. Specifically, a microinjection technique utilizing hydrogen peroxide now allows for reliable simultaneous identification and dilation of the lymphatic channels which is accomplished when oxygen bubbles are absorbed into the lymphatic vessels . OvercomAdvances in understanding of structural and physiological characteristics of the lymphatic vasculature have guided improvements in the clinical imaging of the lymphatic system. The two main clinical lymphatic imaging techniques for the last 70 years were pedal lymphangiography and lymphoscintigraphy. However, these techniques have significant drawbacks. In order to overcome the drawbacks, a newer approach has been developed by introducing contrast (oil and water based iodinated as well as gadolinium) through a needle into the lymphatic system by accessing organs rich with lymphatic vessels such as lymph nodes or the liver. This technique is often referred to as an interstitial injection. This approach has led to the development of innovative imaging techniques utilizing fluoroscopy such as intranodal lymphangiography, liver lymphangiography, mesenteric lymphangiography as well as dynamic contrast enhanced magnetic resonance lymphangiography and computed tomography lymphangiography . These mLymphedema is the most common of all the lymphatic disorders and is considered one of the most significant cancer survivorship issues in the United States. Despite its relative prevalence, it remains unclear why certain cancer patients develop this condition following treatment while others are spared. Focusing specifically on breast cancer related lymphedema (BCRL), the top 3 risk factors include an axillary lymph node dissection, regional lymph node radiation, and a Body Mass Index (BMI) > 30. However, despite these known risk factors, only 1 in 3 women with all three risk factors will develop lymphedema. Utilizing the modern imaging technique of indocyanine green (ICG) lymphography to obtain real-time scans of the upper extremity peripheral lymphatic systems prior to axillary lymph node dissection, significant variations in the superficial lymphatic anatomy have been recently demonstrated. Anatomists had previously postulated that collateral lymphatic pathways of the upper extremity, or a pathway that avoids the axilla, may be protective against BCRL as the lymphatic flow would divert away from the axilla following dissection. Modern ICG imaging has recently confirmed the presence of two variants of a specific collateral pathway, the Mascagni–Sappey (M-S) pathway, which is hypothesized to decrease an individual’s risk of BCRL development . IdentifCentrally, lymphatic variants have been discovered to be the root cause of certain pulmonary disorders. Specifically, pulmonary lymphatic disorders (PLD) are a diverse group of conditions characterized by the presence of abnormal lymphatic tissue in the chest and are often accompanied by chylous leaks, such as non-traumatic chylothorax, plastic bronchitis and chyloptysis. PLD is associated with a variety of conditions including congenital heart disease, Noonan syndrome, and lymphatic malformations. The understanding of PLD has been limited due to lack of robust central lymphatic imaging. Recent development of the intranodal lymphangiography and Dynamic Contrast Enhanced MR lymphangiography demonstrated the presence of abnormal lymphatic pathways from the thoracic duct and/or retroperitoneum into lung parenchyma, termed abnormal pulmonary lymphatic flow (PLF) . The abnin vivo imaging, and knowledge management modalities.It is incumbent on the lymphatic community to update our current compendium of normal lymphatic anatomy utilizing the most modern dissection, The lymphatic system has an intricate superficial and deep vasculature. Our inability to reliably image the deep lymphatic system, and importantly, potential connections between the deep and superficial systems, remains a significant limitation in creating a map of the peripheral lymphatic system. Even the most modern imaging methods such as ICG lymphography that allows precise mapping of the superficial peripheral lymphatic system still have significant limitations such as its shallow depth of penetration of less than 2 cm . The grePromising advances in magnetic resonance lymphography and photoacoustic images may soon help bridge this crucial gap. For advancing the understanding of the lymphatic vasculature, further research not only on the anatomy, but also on lymphatic flow will be critical to understand how abnormal central lymphatic system flow, such as PLF may play a role in serious conditions, including congestive heart failure and pulmonary alveolar proteinosis.Additionally, all the information will need to be systematically incorporated and organized into a comprehensive, publicly available, updatable, online reference library of the normal lymphatic vasculature anatomy. As an initial step towards creating such a map, more than 900 entries related to lymphatic vessels and nodal stations have been extracted from the 1938 textbook by Rouvière and conceptually organized using a present-day approach, the “Miro Map”. The lymphatic vasculature map is available at a publicly accessible websiteOne of the most important parts of the efforts to map the normal human lymphatic vasculature across the scales of the human body will be to discover, catalogue, and explain the commonly shared clinical level features and the variants of the lymphatic anatomy in normal individuals. The mapping of anatomical variants remains a priority in order to better understand the foundation of several lymphatic disease pathophysiology. Taking a page from the current efforts to map the human blood vasculatureHigher resolution investigations of cellular composition, organization, and biomolecular markers of the lymphatic system are also needed to understand the basis for normal lymphatic anatomy and variants, as well for the clinical management of lymphatic conditions.While the etiology of primary lymphedema can often be clearly ascertained to genetic mutations, such as in Milroy’s disease where hereditary inactivating mutations in vascular endothelial growth factor (VEGF) receptor-3 (VEGFR-3) have been implicated, the underlying factors of secondary lymphedema development are less well understood. A variety of experimental models have been employed to study lymphedema development, including surgically disconnecting the superficial and deep lymphatic vessels leading to oedema formation in the tail extremity, or in the mouse paw after popliteal lymph node dissection. While both represent acute lymphedema models that spontaneously resolve over time with little fibrosis or adipose tissue deposition, they help point to cellular and molecular mechanisms that may be at play. For instance, researchers were able to identify that prolonged T helper two biased immune responses regulates the pathology of the response by promoting tissue fibrosis, inhibiting formation of collateral lymphatics, decreasing lymphatic vessel pumping capacity, and increasing lymphatic leakiness .Lipedema is a chronic adipose tissue disorder characterized by the disproportional subcutaneous deposition of fat and is commonly misdiagnosed as lymphedema or obesity. Compared to age and BMI matched patients, lipedema patients have increased systemic levels of VEGF-C, cholesterol, and triglycerides . HoweverWe have gained an increased appreciation of the important contributions of the lymphatic vasculature to the normal function of various organs, precipitating or helping with recovery from other medical conditions. Lymphatic vessels show remarkable plasticity and heterogeneity, reflecting their functional specialization to control the tissue microenvironment . For insOver the last 10 years, the identification and characterization of venous sinus-associated lymphatic vessels embedded within the brain’s dura mater that drain solutes from the cerebrospinal fluid (CSF) and central nervous system (CNS) has allowed a dramatic revision of our understanding of brain fluid dynamics and waste clearance . An inteSnta1) gene eliminates the perivascular AQP4 localization, impairment of glymphatic function and more rapid Aβ deposition was observed (AQP4 gene modified risk of cognitive decline in a cohort of AD patients (Glymphatic exchange contributes to the clearance of amyloid ß (Aß) and tau , two proobserved . Lastly,patients . Althougpatients . These dSkin, the largest human organ, is the setting for a peripheral lymphatic system superhighway with highly abundant lymphatic vessel network that is spatially separated into superficial and deep vascular plexuses. Much of our understanding of the anatomic and molecular details of the dermal lymphatic vasculature comes from studies of the mouse skin where the three-dimensional architecture can be visualized using fluorescence microscopy methods. These studies have revealed a hierarchy of functionally specialized vessels; the blind-ended lymphatic capillaries that take up excess interstitial fluid, cells and macromolecules, and fluid-transporting collecting vessels. The importance of functional specialization is reflected in the unique morphological features of the different lymphatic vessel types. For example, the two types of vessels display distinct organization of cell-cell junctions . Other vin situ  by ectopic VEGF-C delivery to the infarcted heart, improves cardiac function and prevents adverse cardiac remodeling in rodent acute MI models . HoweverUnderstanding the role of lymphatics in the central nervous system is a burgeoning field with endless opportunities. Certain areas for future research would include understanding the anatomical and function linkage between distributed perivascular pathways and meningeal routes of lymphatic drainage from the cranium particularly in the human brain. Moreover, further delineation of the common or distinct signaling and physiological processes regulating sleep-active glymphatic function and meningeal lymphatic drainage is needed. The relative contribution of glymphatic-lymphatic function to the clearance of different pathogenic proteins in humans is unknown, as well as how these processes change in the setting of neurodegenerative diseases like AD. Finally, the contribution of lymphatic biology and its dysfunction to neurological and psychiatric conditions beyond neurovascular disorders and neurodegenerative diseases remains almost entirely unexplored.Notably, genes identified through single cell level analyses are likely not only markers of cell subtype identity, but functionally important and thus pointing to potential biomarkers and targets for future studies and interventions. For instance, changes in the LEC transcriptome during pathological changes, or differences between the normal LEC transcriptome from various tissues are likely to reveal state and organ-specific molecular features that could be used for targeting various parts of the lymphatic system.3. Remaining challenges for the field include accurate segmentation of irregularly shaped cells and deep knowledge of ill-defined cell types such as fibroblastic reticular cells. Fortunately, recent progress has been made on quantifying stromal cell types in human LNs by single cell RNA-sequencing and multiplexed imaging  is seeking to turn discovery into health. Many open resources are available to help advance the knowledge of normal human biology and function through the application of new technologies and to identify the underpinnings of various diseases, including lymphatic disease .3 As oA draft table of anatomical structures, cell types, and biomarkers of several lymphatic organs, but not including all of the components of the lymphatic system, has been published  and is uAdvancement of lymphatic research requires refinement of existing knowledge and application of advanced spatial mapping approaches for a greater understanding of the normal lymphatic system and connections with development of lymphatic dysfunction and disease.Working together across specialty domains and across the human body scales we hope to further expand this knowledge base for the betterment of the human condition.


In this report, we also build on the workshop discussion and highlight and discuss the full range of costs and unexpected challenges on resources for the delivery of stocks of hPSCs suitable for use as starting materials in the manufacture of stem cell-based medicines. The experiences of global leaders from different national resource centers highlight issues to consider in cost management and the possibilities for reducing costs while moving into the clinical application stage.This report summarizes key issues contributing to the cost of preparing human pluripotent stem cell lines for use in cell therapy manufacturing based on discussion between stem cell banking experts from ten countries at a workshop session on ‘cost of goods’ for human pluripotent stem cell banking organized by the International Stem Cell Banking Initiative (ISCBI) held at the Korea National Institutes of Health in Korea (25 The strategies adopted for the expansion and processing of cells can impact significantly on the ‘cost of goods’ (COGs) for the manufacturing process and control of these costs is a crucial element to facilitate an economically viable supply chain for advanced cell-based medicines3. Efforts to address COGs for cell-based medicines may start with increased efficiencies in the cell culture process 4. Evaluation of cost-effective manufacturing with human pluripotent stem cells (hPSCs)5 as starting materials will need to include expansion of the stem cells, their processing, and testing6. However, whilst the broad and significant challenges which arise at the creation of these crucial starting materials have been addressed by others7, the variation in COGs between different sources of hPSCs in different jurisdictions have not yet been explored in detail. It is important to note that the definitions of raw materials may vary between jurisdictions and in some cases the term ‘starting materials’ is used for components that will persist in the final product such as the hPSC line used in manufacturing or vectors that are not eliminated after an iPSC line is established.The manufacture of advanced cell-based medicines is often complex and costly and prone to the unexpected impacts on the manufacturing process and this is especially relevant for products derived from cultured cellswww.iscbi.org). The ISCBI works to find consensus on issues and best practices in pluripotent stem cell biobanking and applications9. In 2019, an ISCBI workshop was held in Korea National Institutes of Health, Osong, Korea that included a session on COGs considerations for the production of hPSCs for clinical use. ISCBI experts at this workshop also discussed the management of stem cell data and genetic testing of human pluripotent stem cell lines (hPSCs) and a summary can be obtained request from admin@iscbi.org.The International Stem Cell Banking Initiative (ISCBI), initiated in 2007, brings together a global community of around 300 professionals from 28 countries, including directors of the major pluripotent stem cell banks and experts in stem cell research, biobanking, regulation, and public policy development , indirect costs of organization overheads, non-recurring costs , and wasted batches. Here, we report case-studies of key issues arising for COGs hPSC-biobanking in five different institutions from four countries and summarize the key points considered in workshop discussion. We then go on to identify important common issues and potential solutions relating to COGs which hPSC-based product developers should include in their considerations to manage and reduce costs. Furthermore, we identify certain impacts on costs which may not be clear at the outset of cell line development, but which may influence cell development strategies.activities for which it may be most difficult to ascertain the cost in advance.the generation of cell lines that will meet regulatory requirements.the full requirements for construction of appropriate facilities, their validation, and maintenance.the demands of progression to new drug application (NDA) status.the additional and extensive regulatory and patient costs of clinical trials.During the development of stem cells to the clinical stage, researchers, clinicians, and manufacturers often face unexpected costs and hurdles to bring the products to clinical trials. Workshop delegates discussed in detail the most significant cost elements and identified a number of key cost issues not always fully addressed by early product developers including:On the basis of these discussion topics, experts including leaders in hPSC banking, cell-based therapy product development and regulation, drew on the benefit of the combined perspectives from 27 different institutes in 10 countries represented at the workshop, to highlight issues to be included in considerations for the management of the COGs of hPSC starting materials both allogenic and “autologous”. The workshop was led by five case-studies from four countries.10 and is supplying them as starting materials in two ways: firstly, for a number of clinical applications and secondly, by partnering with other researchers and contract manufacturing organizations (CMOs) since 2018. The total area of clean rooms and the QC area of production facilities are ~1600 m2, and the maintenance cost of is ~$2.2 million (USD)/year). This cost does not include raw materials and product QC costs. KNIH experience is that the facility maintenance costs, such as the temperature, humidity, pressure, gas, and equipment monitoring, validation systems, and environmental monitoring for 24 h/7 d etc., are much higher than any other costs, and the percentage of the labor cost is high .Other significant costs were associated with obtaining all the necessary documentation and traceability for donor cells, but KNIH had not yet identified generic methods to minimize the associated costs as the ease of obtaining adequate documentation varies considerably depending on the source of donated cells. However, it is important to select and fully document donor cells to ensure that they will meet the applicable regulatory requirements.In addition to these specific issues a number of learning points which enabled KNIH to address cost reduction in development of iPSC lines for clinical application were shared.A standardized and well-controlled manufacturing process will reduce the unexpected ‘dead time’ of cleanroom manufacturing facilities. The manufacturing scale of production is established from lab-scale production methods and thus, there are numerous hurdles and risks which may not be feasible to predict. However, it is vital to address the challenge of developing a scale-up system that will enable production to clinically relevant cell numbers and quality. Having achieved this, KNIH has experienced a further challenge of transferring the developed technologies into a GMP manufacturing quality management system and the requirement to translate the method into the hands of a Contract Manufacturing Organization (CMO). The latter issue in particular adds a further and significant level of complexity. Translation time for academic protocols for use in KNIH facilities had varied depending on product type, but had typically taken a minimum of 3 months. Difficulties and delays at this translational stage are very costly and the CMO staff typically require specialist training in the cell culture process. In the KNIH experience of iPSC generation the development of lab-scale processing using culture-ware and raw materials that are known to be suitable for GMP manufacture, has shortened the technology transfer time as it was not necessary to adapt the cultures to new reagents during the product development phase.7. Finding suitable donors for cells with a defined homozygous HLA type can be challenging12. In such cases, cell sources from the cord blood banks would be useful to find homozygous lines, however, their utility depends on the suitability of donor selection procedures used13. Nevertheless, staff time must still be allocated to carefully review donor traceability, documentation of donor eligibility testing and contents of the individual informed consents.Donor eligibility tests conducted to find suitable donors may take time, which can lead to unexpected delays in manufacturing, however, formal donor selection criteria can help to make this process more efficientContacting the regulatory authority at an early stage, certainly before commitment of significant resources on manufacturing facilities and the product development stage, has significantly helped KNIH to avoid delays to clinical application. For example, the regulatory authority is likely to request full and documented traceability, including the certificates of origin and certificates of analysis (CoA) of all raw- and starting-materials including the Sendai virus that is used for iPSC generation. If the iPSC lines have been generated where such traceability was not established, it will be much more difficult to demonstrate suitability of the iPSC to the regulator’s satisfaction and KNIH has found that frequent discussion with the Korean regulatory authority is important before starting the manufacturing process in a GMP facility to avoid the need for costly changes to procedures and even facility adaptation, before manufacturing can commence.Qualification of the seed stock or master cell bank (MCB) has been an important step for assurance of KNIH product manufacturing. KNIH staff have focused on performance and documentation of critical quality control (QC) including cell line identity and adventitious agent testing of the MCB. This has reduced the risk of product failure and can limit delays and unexpected costs by ensuring MCB QC information is sufficient prior to initiation of the GMP manufacturing process. Currently, Korea’s Regulatory Authority (MFDS) requires, amongst other testing, the submission of the results of short tandem repeat (STR) tests of the seed stock (primary cells) for both the MCB and working cell bank (WCB) for manufactured cell therapy products.Human errors in cell therapy product manufacturing and GMP-compliant seed-stock banking processes often lead to extra costs and delay product outcomes . Although such errors may be rare, they can have serious consequences, such as discarding of product batches, closure, and emergency clean-down of production areas, followed by requalification of facilities. KNIH has therefore focused on avoidance of the likelihood of human errors by ensuring that written instructions and procedures are clear and use unambiguous language. However, even more crucial is staff training which is mandatory under Korean national regulations, and only qualified staff can be involved in the manufacturing process. Natural disasters such as violent weather systems and earth-quakes can damage the integrity of facilities and back-up systems. Human viral epidemics and pandemics can also threaten adequate staffing and have incurred additional unexpected costs which have had to be managed by KNIH on a number of occasions. Electrical power failure may well cause shut-down during the manufacturing process leading to very high unexpected costs because product may be lost and instruments and environmental controls may need to be requalified and disinfected prior to restarting manufacturing processes. Therefore, risk assessment, implementing of risk management plans and establishing disaster recovery procedures are recommended by KNIH in order to prepare for expected natural disasters. Such procedures will also help to protect manufacturing from other kinds of risk to the raw materials supply chain and product safety and quality.14. In addition, Dr. Karnielli reported on a new automated and controlled CAR- T platform which can deal with the autologous manufacturing challenge. He presented a case study in which a facility wished to manufacture 20,000 doses a year. This challenge required a platform capacity to carry out 500 manipulations per day under clean room conditions for 720 patients in parallel with 55 new patients per day. Only an automated and fully controlled platform can enable such capacity and in turn assure the quality.Dr. Ohad Karnielli described attempts to make mesenchymal stromal cell (MSC) manufacture more efficient using a semi-automated bioreactor with automated real time monitoring of glucose levelsDr. Karnielli went on to describe the translation costs from R&D to GMP. Compared with the R&D development time, translation to GMP manufacturing had taken around 3x longer which meant costs for developing a GMP process were $3M minimum in the presented case. Translation of the process for GMP manufacture to a CMO could then take ~9 months and the uncertainty of the time-lines for these aspects increases costs. Cleanroom consumables could be a major cost element for cell culture processed and in Adva’s experience could represent 50% of total manufacturing costs.Dr Eihachiro Kawase pointed out that it was important to consider future projected need for the number of vials and cells per vial required, and costs of validation including preservation and storage. Failure to address these aspects could lead to the necessity for repeated banking campaigns, causing delays and additional production and testing costs.At the stage of clinical studies, the development costs for the product implantation method and also for patient monitoring and financial reimbursement systems for implementation warranty could be very significant and need to be considered.Cell culture related consumables and notably media suitable for manufacture of clinical products , matrix , and ROCKi .Outsourced testing including biological safety evaluation including endotoxin testing, pathogenic microorganism contamination tests and antibiotic residues testing ).Staff costs including training which is especially challenging under Japanese law (i.e. the “5 year rule”), can make it difficult to retain long-serving employees.Most significant costs experienced in Dr. Kawase’s hESC manufacturing facility in Kyoto had been:It had been difficult to accurately cost QC, validation, and ICH Q5A requirements for testing, however, no significant costs had emerged that had not been expected. A significant cost over time in Japanese industry　had also been the requirement for maintenance fees to get governmental permission, as in Japan a special cell processing facilities were required to be replaced every 5 years.Patient viral testing: $500/patient (in-house).Plasmid for reprogramming: $2000 per round of reprogramming (Sendai vectors <$1500).Media and reagents: $25–30 K (bulk purchase important to keep costs down).Karyology: $450/clone.Proving plasmid loss from each cell line: $1000/clone (this could be controlled to <$250 using Nanodrop technology).Oncogene screen: $1600/clone (use of non-CLIA regulated testing could keep costs to below $500)STR analysis: $220/clone.Flow cytometry: $200/clone (in-house).Sterility assays: $200–500/clone (in-house).Dr. Kapil Bharti had found that the largest cost in hPSC-derived RPE was the time in GMP manufacturing and he believed that anything to reduce that would improve COGs. For Dr. Bharti’s group, contracting out GMP manufacture had proven more costly than in-house provision. Validation was also a major part of the cost but QC, whilst not cheap, was not their major cost. Dr. Bharti summarized key NEI-NIH costs in the RPE manufacturing and some possible cost reductions as follows:He reflected on certain costs of patient bespoke  iPSCs that were difficult to calculate including manufacturing cell lines at a rate of only one or two at a time in early trial phases. This requires maintenance of all staff even when there is limited demand and whilst it was anticipated that this would become more consistent at fuller capacity operation, it would only be realized when moving into phase III clinical trials.Cost saving where adventitious agent testing had not been required for autologous donors.Limited cell passaging was needed for autologous cells compared to allogenic product which required cells to be expanded to greater scale for multiple patients.Dr. Bharti also discussed the costs of this kind of “autologous” versus allogenic approach. Autologous lines required replicated banking and testing costs for each cell line compared to a single process for an allogenic product. However, there were some benefits for autologous cells that included:Dr. Bharti explained that the current challenges for the NEI-NIH facility were in obtaining sufficient GMP cleanroom occupancy and down-time for maintenance which was a crucial issue causing delays and increased cost. He also described possible means to reduce this for example by making maintenance more efficient and less invasive and running multiple manufacturing batches together in the same facility. The latter possibility could be particularly beneficial, but would require validation. Such parallel  manufacturing would require procedural and physical protection mechanisms to keep products separate will be essential to meet GMP requirements.Another way Dr. Bharti envisaged streamlining production was to make quality system SOPs consistent for different products where possible and at the NIH facility there were 25 common protocols used for different patient products.A solution also considered beneficial by Dr. Bharti for reduced cost was to rent cleanroom space and avoid long-term costs re dead-time when there is no work to do and down-time required for maintenance.www.wicell.org), UK Stem Cell Bank (www.ukscb.org), and EBISC (www.ebisc.eu) that supply cell lines in the range of $1000 to $1500 depending on the application, however, some other banking centers such as the stem cell facility at the Korea National Institute of Health provide cell lines at no charge.Dr. Yuji Arakawa reported that manpower costs were ~50% of total cost in the CiRA facility and other significant costs were due to quality assurance (QA), maintenance, and servicing. These can be high per banking activity if outsourced, but in-house provision meant ongoing continuous costs. CiRA also found that outsourced virus testing and whole genome sequencing were costly. Cell banks were produced at 300 vials per bank which took 1 month to produce and cost $300,000 overall per batch. Dr. Arakawa noted that different sources of iPSCs varied in charge per vial and some charges varied depending on the intended use. He reported that the CiRA foundation was planning to sell iPSC stocks at $1000/vial. This was broadly consistent with other hPSC banking centers such as Wicell  the balance of cost of validation across manufacturing was as follows: 45% staff, 23–25% testing and 25% materials.The discussion also turned to the issue of false claims of “GMP” quality of equipment. This had also been covered in the discussion at previous ISCBI meetings and had been a clear concern for regulators (see notes from ISCBI Los Angeles June 2020 available on request from admin@iscbi.org).Delegates reflected on the fact that molecular interventions in manufacturing can also raise complexities regarding safety and quality control. The development of gene-edited hPSCs for therapeutics was also likely to incur additional costs for quality controls which may include as yet to be determined safety testing as already discussed in ongoing ISCBI workshops . Furthermore, the preparation of cell substrates may have impacts for downstream costs as experienced for CART cells where the viral load (i.e. copy number per cell) may need to be monitored in patients post treatment.15, that will be particularly important given the increasing implementation of new analytical methods for cell-based medicines including genome-wide clinical genetic testing.Costs for supply of vials of cells for use in manufacture were discussed for different centers and it was clear that as discussed in the case studies, there was significant variation from $1000 to 10,000 per vial and in some cases special contract conditions were understood to incur additional charges depending on factors such as stage of product development. Delegates also agreed that the cost assessment for cells intended for future manufacture should also include implementation of clinical laboratory standards. These would now include the recently established standard for clinical testing ISO151896. The history of animal cell biotechnology has shown that translation from lab scale research procedures to manufacturing is not simply a matter of increasing the size of the culture system. Many factors in the cell culture system which affect cell growth such as changes in culture vessel materials, different liquid/atmosphere surface areas for gas exchange, sheer stress from bubbles or impellers and altered mass or heat transfer dynamics in the culture medium18, all potentially impact on the efficiency of large-scale systems compared to the researcher’s original protocols. In addition, the biomarkers and analytical approaches may need to be developed during product development20 so that it is difficult to calculate cost of goods for manufacturing on the basis of a research protocol yet to be translated into a GMP manufacturing process. Furthermore, the comparison of COGs in different national and institutional settings is challenging and figures for the cost of production of hPSC lines has been difficult to assess21. However, in this paper experiences from a number of centers making hPSCs for manufacturing cell-based medicines have consistently indicated that the key direct and most obvious manufacturing costs arose from the need for experienced staff, use of qualified clean-room facilities, specialist cell culture materials and testing for quality control and safety. One example of autologous CART manufacture has shown that the largest component of labor costs could be over 70% of the total manufacturing cost22, comprising mostly manufacturing processing (48%), quality control (16%), and quality assurance (16%). In this case materials were the next highest COGs element (18%) and costs of facilities (8%) and equipment (4%) seemed to represent a relatively minor element of the COGs. it is vitally important too. However, another study of a different T-cell adoptive therapy differed in that facility costs were highest , with labor and materials both significantly less (i.e. ~20% each)23. It is important to recognize that these costs arise within the manufacture of an autologous cell product with bioprocessing far less complex than for an hPSC-derivative. In one comparison of an adoptive T-cell therapy product and hPSC COGs for each appeared to have a similar profile. the COGs for the hPSC system simply assessed costs for development of a master cell bank of an hPSC line  and not an actual product23. However, this study calculated average costs for very early and late stages of preclinical development and revealed that both in the case of the hPSC bank and adoptive T cell therapy, facility costs decreased compared to labor costs as each process progressed towards clinical trial stage. Thus, the ability to directly translate labor costs and COGs in general, from reports of one product type to another and between somatic cell and pluripotent stem cell manufacturing are not straightforward even where the products are nominally very similar. For manufacturing of hPSC-based products the cell culture systems are multifaceted, generate complex mixed cell populations, are often much more prolonged (up to 300 days for batch cultures of certain hPSC derivatives) and also based on complex raw materials often not available under appropriately qualified conditions. For the hPSC banking process alone, whilst the challenge of creating a reproducible cell differentiation process is not an issue, the stability of the system during cell expansion and qualification of novel raw materials and assays can still create high COGs.COGs is a crucial element in enabling uptake of advanced hPSC-based medicines or cell therapies22, but again translation of such COGs elements between somatic and pluripotent stem cell manufacturing processes can be problematic for the reasons already stated. Furthermore, COGs evaluation between regional jurisdictions may also vary depending on the predicted equipment and facility life-span  and the specific frequency of facility and equipment requalification required. Validation is also required for various aspects of the manufacturing process and includes, but is not limited to, facilities , reagents, equipment, analytical methods, storage, and shipment. Thus, early assurance that raw materials, vectors and production cell lines would all meet regulatory requirements was crucial to avoid costly changes at a later stage. Finally, the importance and costs of qualification of the cell source  will be important to consider, especially if multiple donors are required as in haplo-banking or generation of autologous cell lines. The ISCBI workshop speakers and delegates also considered opportunities where the direct costs could be cut and concluded that avoiding commitment to ongoing manufacturing facilities by rental of clean room space or outsourcing manufacture to a CMO was attractive. However, this could also increase timelines and incur new costs due to the need to either wait for access to manufacturing slots and/or train CMO staff. Also, in relation to efficient use of cleanroom time the need to plan and prospectively manage down-time for maintenance was considered important to avoid costs due to unplanned down-time. A number of workshop presentations reflected on the use of CMOs or facility rental and some of the key pros and cons to be considered are summarized as described in Table Validation of the manufacturing process was also considered a major and necessary cost, but one which was often not obvious to early-stage product developers. As commented on by Dr. Bharti (NEI-NIH) (Case study 3 above), validation procedures for GMP manufacturing and could cost almost as much as the item being validated i.e., cost of validation of vector removal in some cases had exceeded the cost of vector manufacture. Indeed, other areas of cell manufacture such as CART therapy facility validation costs in might make up 20% of the total facility costsApproaches used under GMP to address this include planned preventative maintenance  and design features that enable maintenance from outside of the cleanroom without compromising cleanroom integrity and air quality. Examples typically used in GMP manufacturing include ceiling or wall embedded fittings permitting maintenance without lab entry and gas supplies piped and filtered through the cleanroom wall.Processing of multiple cell lines or products in the same cleanroom at the same time were also considered attractive to enhance cost efficiencies in terms of campaign progress, staff time, and reducing cleanroom occupancy time. However, this approach would also require rigorous process controls to assure separation of different cultures and products to avoid switching of process materials and cross-contamination.7 is typically most readily accommodated in-house.Outsourced testing continues to be a cost which is difficult to reduce. Care is needed to assure the quality of testing and reporting and a key prerequisite should be use of testing services subject to formal accreditation by appropriate professional bodies. Attempts to carry out in-house testing, while bringing timelines within greater control of the manufacturer, could also incur significant staff-time and training commitment. In addition, they represent an additional quality assurance burden . The decision to out-source versus in-house provision will be influenced by a number of local factors including ease with which the staff can accommodate the testing, level of quality assurance support and availability of specialist equipment and facilities. Testing which is carried out for information and is not used for cell bank or product release25. Sealed (“closed”) automation systems offer both advantages of avoiding the need for high air quality cleanroom conditions and the limitations of ‘isolator’ operation. Much of the technology for aseptic connection of these devices for media supply, quality control sampling, and harvesting have been developed in industry production systems for cell culture-based vaccine and biotherapeutics. Challenges for automation may include unexpected reaction of stem cells to new environments and surfaces, altered mass transfer effects  within the bioreactors. Microcarriers and suspension systems are well advanced for manufacture of cell culture derived vaccines and biotherapeutics and are under development for culture and differentiation of pluripotent stem cells28. Furthermore, an IMI (Innovative Medicine Initiatives) - a partnership between the European Union (represented by the European Commission) and the European pharmaceutical industry  - a funded hPSC-biobanking project  is using a 4 × 50 ml volume stand-alone bioreactor system for biobanking cell expansion and differentiation a funded hPSC-biobanking project  is using a 50-ml volume stand-alone bioreactor system for biobanking cell expansion with cells in suspension or on alginate beads29. Automation systems may also have the benefit of permitting reduction in size of equipment and thus, reducing the highly expensive manufacturing space needed. In cell-based medicines the use of standard commercially available culture expansion units such as described by Dr. Karnielli (see case studies above) also provide ready scalability without the significant technology transfer and validation costs often associated with the switch to full-scale manufacturing30. Thus, combined isolator and automated culture could in principle help to optimize cost reduction. Isolator systems for standard culture modalities had been tried in many labs and had become popular. They can reduce facility costs of a cleanroom but still require the same consumables, significant staff time and quality control costs.Automation was an area where considerable benefit is perceived for the future with positive experience in the manufacture of medical devices31. Beyond quality control methods, the overall efficiency of quality assurance can in principle32 and in practice be transformed by streamlining and switching to electronic operating systems.It is also important not to forget the potential impact of automated quality control and substantial improvements have already been experienced for QC of autologous CART therapies  and in molecular analysis33. It addresses a key element of COGs reduction for cell therapies which is to optimize utility of capacity22.Typically, the cell banking process involves preparation of a large master cell bank (MCB) and then following completion of quality control and characterization, a single MCB vial is thawed and re-expanded to generate a working cell bank (WCB). The WCB is then subject to a focused set of quality control tests. However, the operational and quality control costs can be made in adjusting the fundamental approach to cell banking which might be termed a “continuous banking” process. This involves retaining some cultured cells from the batch of cultures used for a MCB and expanding them immediately without cryopreservation to the WCB passage level. This avoids time consumed in recovery of cryopreserved MCB cells and potential loss of cells and increased risk of expansion of genetic variants. However, it also means that WCB may be created before the MCB testing is completed so potentially the cost of WCB could be wasted if cells at MCB level do not pass QC. However, implementation of rapid screening for genetic stability could enable early abandonment of WCB manufacture due to the most likely QC problem of expansion of genetic variants. This approach has been tested in a number of centers including the UK Stem Cell Bank (UKSCB) and has been published by the Hadassah Medical Centre34, This involves careful screening of candidate manufacturing cell lines and/or donor tissues to ensure the cells have the appropriate ethical provenance and biological characteristics, are free from evident microbial contamination and allow the user freedom to act without adverse intellectual property issues.A further element that is crucial to avoiding wasted resources on failed batches is the implementation of a due diligence process during procurement of stem cell lines36 and particularly where bespoke genotypes are required such as homozygous HLA haplotypes12, this is an issue that will significantly impact on the cost of generating hPSC-derived medicines. In order to reduce such costs, it is wise to consider using existing donor selection systems such as health services or sharing resources with collaborators with similar needs. In conclusion, a significant number of iPSC and hESC lines are available from professional hPSC biobanks established to assure all the correct procedures for donor selection have been documented and these make a valuable first point of contact for hPSC-based product developers seeking suitable production cell lines7.Another means to reduce early commitment of resources to hPSCs is to generate a large MCB and allocate a certain proportion of vials as an early Distribution Cell Bank. Such a system has been used at the UKSCB to substantially cut early investment in cell culture  and quality control , when popularity of individual hPSC lines has yet to be determined and thus overtime, allows the biobank to focus resources on those lines that become more popular with biobank users. The KNIH case-study above reflected on the significant costs that may be associated with donor procurement and selection and this is often forgotten in discussions about costs of biobanking as it was typically a historical activity which did not involve the biobank or is not such a key issue for suitability as in the case of “autologous” iPSC lines used by the KNIH and NIH facilities. However, as biobanks move towards development of cell lines specifically intended for manufacture of cell-based medicinesConsideration of most costly elements such as facilities, services, and staff, depend significantly on the local methods for measuring costs. Many centers in academic institutions may only figure in additional costs of consumables, external testing etc. and do not include core staff, general use equipment and facilities  provided for other projects and overheads, which can be very different between different institutions or academia and industry. All of the latter items may not be included in published estimations of cost per vial or bank of cells and so need to be born in mind when comparing published cost of goods for use of hPSC lines.In order to proactively tackle the issue of COGs reduction it is also an important consideration to identify the various sources of influence on COGs and score the impact that these might have overall. This can enable issues with more significant potential to raise costs to be prioritized and appropriate action taken to limit or reduce COGs. Some important examples of these variables in the hPSC manufacturing chain, including cell sourcing and banking, are given in Table www.iscbi.org) who are supplying hPSCs specifically for manufacture of cell-based medicines. These centers will have already addressed the key issues for assuring the suitability of their hPSC stocks and can save significant investment of time and resources for the early stage of product development. Furthermore, the attention applied by stem cell resource centers to assure the suitability of cell lines7 will reduce the risks of failure in product development.In conclusion, figures quoted in the literature for costs of hPSC lines are influenced by many factors. We have identified some that represent significant costs common to most centers making hPSCs for manufacture of cell-based medicines. However, other factors are significantly variable depending on the nature of the stem cell bank’s host organization including its core objectives, the expertize and services available in-house and how the organization manages facility costs and especially how it establishes overhead costs. Furthermore, there are costs which are less obvious to early developers , and others that are much less predictable including donor recruitment and technology transfer where CMOs are used for manufacturing. Any organization wishing to derive its own hPSC production cell lines, should consider the utility of existing resources such as those engaged in the ISCBI community (


Recent reports demonstrate that SARS-CoV-2 utilizes cell surface heparan sulfate as an attachment factor to facilitate the initial interaction with host cells. Heparan sulfate interacts with the receptor binding domain of SARS-CoV-2 spike glycoprotein, and blocking this interaction can decrease cell infection. We and others reported recently that the family of compounds of 2,5-dihydroxyphenylic acid interferes with the binding of the positively charged groove in growth factor molecules to negatively charged cell surface heparan sulfate. We hypothesized that Calcium Dobesilate (CaD)—calcium salt of 2,5-dihydroxyphenylic acid—may also interfere with the binding of SARS-CoV-2 spike protein to heparan sulfate. Using lentiviral SARS-CoV-2 spike protein pseudotyped particles we show that CaD could significantly reduce pseudovirus uptake into endothelial cells. On the contrary, CaD did not affect cell infection with VSVG-expressing lentivirus. CaD could also prevent retention of SARS-CoV-2 spike protein in ex vivo perfused mouse kidney. Using microfluidic culture of endothelial cells under flow, we show that CaD prevents spike protein interaction with heparan sulfate glycocalyx. Since CaD has no adverse side effects and is approved in humans for other medical indications, our findings can rapidly translate into clinical studies. This form of infection is self-limiting and causes no serious threat to the infected person. However, in 20% of patients, infection with SARS-CoV-2 leads to life-threatening respiratory, cardiac, renal, and cerebral injury3. The underlying mechanisms of this severe form of the disease are not fully understood. The clinical picture in these patients resembles the systemic inflammatory response syndrome (SIRS) with a strong microvascular component7. It seems that an inflammatory endothelial response that may involve the coagulation cascade and/or the complement pathway plays a vital role9. Endothelial activation results into elevated levels of pro-inflammatory cytokines—interleukin-1, interleukin-6 (IL-6) and tumor necrosis factor-α (TNFα), chemokines and antithrombotic factors—von Willebrand factor and factor VIII in these critically endangered patients10. Higher levels of acute phase reactants  are also associated with severe SARS-CoV-2 infection. Therefore, it is reasonable to assume that endothelial dysfunction contributes to COVID-19-associated vascular inflammation. Pro-inflammatory cytokines, elevated in patients with COVID-19, induce the loss of the normal antithrombotic and anti-inflammatory functions of endothelial cells, leading to coagulation dysregulation, complement and platelet activation, and leukocyte recruitment in the microvasculature9. Furthermore, recent report suggested that proteolytically released S1 domain of SARS-CoV-2 spike protein induces endothelial damage, vascular permeability and expression of PAI-1 and VEGF by lung microvascular endothelial cells12.Infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is in most patients limited to bronchoalveolar epithelial cells and associated with local bronchopulmonary symptoms14. There is still controversy existing whether SARS-CoV-2 infects the endothelium directly. Multiple reports demonstrate infection of endothelium both, in vivo and in vitro18. Whereas other investigation find no evidence for endothelial infection20. Organ-specific and regulated expression of ACE2 is important for cell infection with SARS-CoV-221. Expression of ACE2 by endothelial cells has been documented22. Some organ-specific endothelial cells such as from brain show high ACE2 levels even in normal conditions23. Another important factor that could potentially explain this controversy is the abundance of endothelial glycocalyx both in vivo and in vitro experimental systems24.These microvascular complications of SARS-CoV-2 infection are most likely responsible for the high rate of morbidity and mortality of infected patients27, influenza virus, immunodeficiency virus, and different coronaviruses (SARS-CoV-1 and MERS-CoV)29. Several viruses interact with sialic acids located on the ends of glycans found in glycolipids and glycoproteins30. Other viruses interact with heparan sulfate (HS)32. This initial attachment of the virus to HS can lead to the engagement of protein receptors on the host plasma membrane, such as ACE2, that facilitate membrane fusion or engulfment and internalization.The glycocalyx is a complex mixture of glycans and glycoproteins covering the endothelial cell surface. Viruses and other infectious organisms must transgress the glycocalyx to engage receptors that mediate viral entry into host cells. Many viral pathogens have evolved to utilize glycans as attachment factors, which facilitate the initial interaction with host cells, including alpha-, beta- and gammaherpesviruses33. These findings identify cellular HS as a necessary co-factor for SARS-CoV-2 infection and emphasize the potential for targeting spike protein–HS interactions to attenuate virus infection. Further reports confirmed this observation34. Yue and colleagues35 investigated the role of heparin and HS sulfation for the binding of spike protein in details. They have also demonstrated that spike protein of highly infective G614 SARS-CoV-2 variant has higher binding to heparin and HS. Application of heparin in COVID-19 patients exerted beneficial effects via anti-coagulant and non-anticoagulant mechanisms37. On the contrary, Targosz-Korezka and colleagues have reported recently that HS glycocalyx shields endothelial cell surface and prevents SARS-CoV-2 interaction with ACE2 receptor38. Even more complexity was hypothesized by Buqaileh et al., who suggested that primary ciliary pockets—membranous invagination around the primary ciliary of endothelial cells—can serve as specific entry points for SARS-CoV-2 as ACE2 was concentrated at those area39. When interpreting experimental data on the role of the glycocalyx in the virus entry mechanisms, it should be kept in mind that correct physiological models must be applied.Esko and his group have recently shown that the ectodomain of the SARS-CoV-2 spike protein interacts with cell surface HS through the receptor-binding domain. Surface binding to cells and virus uptake requires the engagement of both cellular HS and ACE2, suggesting that HS acts as a co-receptor priming the virus envelope for ACE2 interaction41. We hypothesized that 2,5-DHPA might also interfere with the binding of SARS-CoV-2-HS binding.We and others recently identified a family of small molecules, 2,5-dihydroxyphenylic acid , which interfere with the binding of the positively charged groove in growth factors to negatively charged HS44, diabetic kidney damage45, and venous insufficiency46. Structure of CaD is shown in Supplementary Fig. 47. However, the mechanism of action of CaD is not yet fully understood. 2,5-DHPA compound class interferes with growth factor signaling. For example, CaD binds to the heparin-binding domain of Fibroblast Growth Factor-148. We have recently shown that CaD could function similarly as a VEGF antagonist without adverse side effects41. Recent meta-analyses further demonstrated the clinical safety of CaD52.Calcium dobesilate (CaD)—calcium salt of 2,5-dihydroxyphenylic acid CaD is a vasoprotective drug that demonstrated beneficial effects on vascular leakage and inflammation in diabetic retinopathyWe have developed a lentiviral expression system of luciferase- and GFP-expressing SARS-CoV-2 spike pseudotyped viral particles and studied uptake in cultured microvascular endothelial cells. We showed that CaD interferes with the binding of the spike protein to heparan sulfate and reduces pseudovirus uptake into endothelial cells. 2,5-DHPA (and possible derivatives) are potent inhibitors of SARS-CoV-2-HS binding and have the potential to prevent viral uptake and endothelial dysfunction in infected patients. Since CaD has been approved in humans for other medical indications, our findings can rapidly translate into clinical studies.First, we assessed whether kidney epithelial (HK-2) and human microvascular endothelial cells HMEC-1) can bind recombinant SARS-CoV-2 spike protein by dot blot assay  or GFP was used as reporter to quantify cell infection rate. The expression of SARS-CoV-2 spike protein in pseudoviral particles has been verified by Western blotting . Our data confirmed that CaD did not induce any cell toxicity under our experimental conditions Fig. A. SecondE. coli, respectively. CaD exerted no significant effects on these processes receptor to increase local virus concentration and facilitate the search for higher affinity protein receptor for cell entry. Recent reports showed that various viruses use HS as the initial cell attachment site56. It is still not well understood how different spatial compositions of HS, its occupancy by ligands  and shedding can affect virus attachment and cell infection in vivo. Increased plasma content of soluble HS fragments and proteoglycans can bind viral particles and decrease the probability of finding and interaction with cell surface receptors57.Endothelial cells develop especially pronounced glycocalyx on the luminal side. We have recently demonstrated that endothelial glycocalyx thickness decreases in covid-19 critically ill patients and serum of covid-19 patients induces shedding of endothelial cell glycocalyx in vitro37. CaD has many advantages over heparin and HS mimetics. It is an approved vasoprotective drug without the adverse effects of, for example, increased coagulation; it can be applied as a nasal spray to prevent upper airways infections, or systemically through the oral route. Moreover, a recent case report of covid-19 demonstrated improvement on clinical parameters in a patient with covid-19 after CaD application58.Inhibitory effects of heparin and other HS-mimetics on the cell infection by different viral types are known and the strategy was confirmed to be efficient against experimental SARS-CoV-2 infectionOur in vitro binding assay confirmed that CaD can interfere with spike protein binding to HS. The kinetics were similar in the case of high and low molecular weight heparin. Binding of spike protein to HS was less efficient suggesting that sulfation is important for the interaction. However, we cannot exclude that lower binding was observed due to less efficient plate coating with HS.Taken together, these data inferns that CaD can be potentially used to prevent cell infection with SARS-CoV-2 and urge further research towards clinical testing of this substance for the therapy of covid-19 patients.62. Several models of possible interactions has been proposed by the model confirming our hypothesis . The following settings were used: excitation wavelength filter was 485 nm, emission wavelength filter was 535 nm. Integration time was 1000 ms. Duolink proximity ligation assay kit was purchased from Sigma  and used accordingly to the manufacturer’s instructions. CY3-conjugated Sambucus Nigra Lectin  (CL-1303-1) was from Vector Laboratories, Inc. .Recombinant SARS-CoV-2 spike S1 subunit (40589-V08B1) was purchased from , full length spike protein (10549-CV) and nucleocapsid (10474-CV) proteins were purchased from R&D Systems. Antibody against spike protein (NBP2-41058) was from Novus Biologicals; antibody against spike S1 subunit was from Sino Biological (40150-R007); HS antibody (10E4) was from Amsbio. Luciferase assay was performed using Pierce Gaussia Luciferase Glow Assay Kit . CaD was purchased from Sigma . Heparin, Heparan sulfate, LMW heparin Enoxaparin, Heparinase III were from Sigma. Toxicity of CaD was assessed using Cell Counting Kit-8 . Cell proliferation was measured by using BrdU incorporation assay . The effect of CaD on pinocytosis was assessed using FITC-Dextran . Effect of CaD on phagocytosis was measured using FITC-labelled E.coli as described recentlyQiagen RNAeasy kit was used for RNA purification from the cells. TaqMan RT-PCR was performed using TaqMan Master Mix and Light Cycler96 (Roche). TaqMan assays oligonucleotides were from ThermoFisher Scientific, Waltham, MA, USA.Human dermal microvascular endothelial cells HMEC-1 were acquired from ATCC and cultured as recommended by the supplier. HUVECs were purchased from Lonza and cultured as recommended. THP-1 cells were from ATCC. HEK-Blue-hTLR4 were from Invivogene.https://molview.org/).Chemical structure of CaD was drawn using MolView online tool (E. coli DH5α). The products were verified by sequencing .cDNA of GAPDH promoter and Gaussia Luciferase (GLuc) were derived from pEZX-LvPG04 vector  used as a template. A full-length cDNA of GAPDH promoter and GLuc were amplified using Phire Hot Start II DNA Polymerase . The sequences of all primers used are given in Supplementary Table https://www.invivogen.com/wuhan-spike-d614g-pseudotyping-vector). Second-generation lentiviral packaging construct plasmid psPAX2 (expressing HIV-gag) was acquired from Addgene (http://www.addgene.org/12260/). The work was performed under S2 biosafety level. Pseudoviral particles were produced by transforming packaging cell line HEK293T using Perfectin transfection reagent from Genlantis  as established69. HEK293T conditioned medium containing lentiviral particles was centrifuged at low speed, filtered through 45 µm pore size filter, and stored at + 4° C for up to 1 week before cell infection. Expression of spike protein of SARS-CoV-2 in lentiviral pseudoparticles was confirmed by Western blotting using SARS-CoV-2 (COVID-19) spike antibody [1A9], IgG1 from Genetex . Lentivirus titer was determined using Quick Titer kit from Cell Biolabs . Lentivirus was added at the MOI 50 for 2 h where not indicated otherwise. For infection, inhibitors were added to the lentivirus containing supernatant, and the cocktail was added to the cells for 2 h. After the incubation, the medium containing the remaining lentivirus was removed, cells were washed once with a warm growth medium, a fresh medium was added and cells were incubated for 24 h before measurement of Gluc activity or microscopy.To generate pseudotyped viral particles with the SARS-CoV-2 spike protein on a lentiviral core, spike glycoprotein expression plasmid pLV-Spike V1 (SARS-CoV-2 Wuhan-Hu-1 isolate with the D614G mutation) was used (Gaussia luciferase activity was measured using Pierce™ Gaussia Luciferase Glow Assay Kit (Thermofisher Scientific).33. Plates were blocked with 0.1% BSA on PBS, washed and then recombinant His-tagged spike protein (R&D Systems) was added at the concentration of 30 nM along with the indicated concentrations of CaD. Spike protein detection was performed using anti-His antibody (R&D Systems).High binding ELISA plates were coated with heparin, enoxaparin and HS as described by Clausen et al.69. Briefly, cells were seeded in the channels of polydimethylsiloxane (PDMS) microfluidic chips and allowed to adhere for 6 h. Then, medium perfusion was initiated using peristaltic pump (Watson Marlow) and teflon tubing (Diba Industries). Cells were perfused with shear stress of 16 dyn/cm2 for 5 days before further experiments. To exclude absorption of lentiviruses to the teflon tubing and PDMS, control experiments were performed. Lentivirus containing medium was perfused through the tubing and empty microfluidic chips. Then, this medium was used for cell infection and virus quantification. These experiments showed that there were no absorption of the lentivirus to chips and tubes.We applied developed by us microfluidic system described elsewhere53. Briefly, after electrophoresis the proteins were transferred to PVDF membrane using semi-dry western blotting apparatus . Chemiluminescence pictures were taken using VersaDoc gel documentation system and quantified by QuantityOne software .Western blotting was performed as described previouslyhttps://imagej.nih.gov/ij/).Cells were fixed with 2% PFA and blocked with 1% of BSA on PBS. Duolink assay was performed using kits from Sigma following the protocol suggested by the company. Confocal microscopy was then performed using Leica TCS-SP2 AOBS confocal microscope (Leica Microsystems) at the Research Core Unit for Laser Microscopy at Hannover Medical School. Quantification of the images was performed using ImageJ analysis software . Animals were euthanized humanely accordingly to Paragraph 7, Section 3 of German Animal Welfare Act. All experiments were approved by the animal protection committee of the Phenos GmbH (Tierschutzkomission der Phenos GmbH). All animals were handled in compliance with the guidelines for the care and use of animals at our institution and in accordance with the EU Directive 2010/63/EU for animal experiments. Methods are reported in accordance with ARRIVE guidelines. C57BL6N wild type male mice were purchased from Charles River Laboratories . The following groups were used with three animals per group: PBS perfusion; spike protein perfusion; spike protein with CaD perfusion. The mice were euthanized humanly and both kidneys, abdominal aorta and inferior vena cava were exposed via abdominal incision. Small vessels were cauterized with the electrosurgical pencil and cut. Proximal ends of the aorta and vena cava and the distal aorta and vena cava near bifurcation were also clotted and cut. Ureters were cut to separate both kidneys. A 7/0 ligature was preset around the aorta. The preset ligature was closed after inserting 27G catheter without core into the aorta. Kidneys were perfused with sterile saline to remove the blood.70, perfusion rate was set to 1.3 ml/min. Kidneys were perfused with PBS (Group 1); 15 µg/ml spike protein (Group 2); 15 µg/ml spike protein with 100 µM CaD (Group 3) for 30 min. Where indicated, kidneys were perfused with heparinase III 50 mU/ml for 30 min prior perfusion with spike protein or lentiviruses. Fluid flowing from the vein was absorbed back into the perfusion solution tube by another catheter connected to the peristaltic pump. Then, the kidneys were perfused with saline for 5 min, washing solution was collected in 1 ml fractions for western blotting analysis. After perfusion kidneys were cut into three parts; upper and lower parts were frozen in liquid nitrogen for biochemical experiments, the middle part was fixed in formalin for immunohistochemical staining. For His Mag sepharose pull down assay, the tissue were homogenated in 10:1 (v:w) ratio in RIPA lysis buffer and incubated for 15 min on ice. Lysates were cleared by centrifugation and used for the pull down assay.Both kidneys with catheter were moved to a dry dish, the catheter was connected to the peristaltic pump (Watson Marlow) and perfusion of the kidneys with saline was continued for 5 min. Based on reported blood flow rate in the mouse kidneyhttps://www.graphpad.com/) was used for data analysis.All data were obtained with at least three biological replications. Data are presented as mean ± standard deviation (SD). Multiple comparisons were analyzed by ANOVA with Tukey post hoc test. P-values < 0.05 were considered statistically significant. GraphPad Prism 8.3.0 (Supplementary Information.