A Bioactive 3D Encapsulation Culture System For Cell Expansion

Systems and methods for growing cells are provided. A capsule for growing or storing cells includes a shell defining an interior compartment and a substrate for cell attachment located within the compartment. The substrate comprises a polymer and one or more adhesion molecules. The substrate for cell attachment can be an inner surface of the shell and/or a hydrogel disposed within the interior compartment. The capsule can further include a cell, such as a stem cell, adhered to the substrate

BACKGROUND

Cells are found in many industrial applications ranging from production of vaccines to use as cell therapies. Suspension of cells in bioreactor systems has allowed for the automation and scale up of cell cultures for such industrial applications. Of particular interest and challenge, anchorage-dependent cells have been cultured as monolayers on two-dimensional (2D) tissue-culture treated surfaces, such as T-flasks, multilayer cell factories [3, 4], and roller bottles or hollow-fiber based bioreactor systems [5, 6]. More recently, a transition to culturing anchorage-dependent cells in suspension cultures has emerged, as suspension cultures have the capability of providing high yields in a more spatially efficient format than traditional 2D cultures, which require logistically impractical planar growth surface areas, particularly for commercial production scales.

Microcarriers, most commonly made of polystyrene and coated with collagen or laminin for cell attachment, have enabled suspension culture of anchorage-dependent cells in stirred-tank bioreactors [7-10]. Mesenchymal stem cells (MSCs) adhere to microcarriers and are cultured in stir tank bioreactors that range from 300 mL to 1000 L for large scale expansion [11]. Microcarrier-based bioreactor technology provides significant advantages, such as large surface area to volume ratio, process control, closed loop sampling and homogeneous culture conditions [12-16]. Despite the advantages of microcarrier technology, there remain a number of challenges to overcome, and there exists a need for improved systems and methods for culturing anchorage-dependent cells.

SUMMARY

Methods and systems of the present invention provide for encapsulated cell culture systems that can be used for expansion and/or storage of cells.

In one embodiment, the invention relates to a capsule for growing or storing cells, such as adherent cells. The capsule includes a shell defining an interior compartment and a substrate for cell attachment located within the interior compartment. The substrate comprises a polymer that may contain one or more adhesion molecules.

In another embodiment, the invention relates to a method of storing cells that includes encapsulating cells in a capsule having a shell defining an interior compartment and a substrate for cell attachment located within the interior compartment. The cells adhere to the substrate within the interior compartment of the capsule.

In further embodiments, the adhesion molecule of the substrate can be an adhesion molecule that affects cell attachment to the material, cell viability, cell proliferation, cell survival, growth, and/or differentiation of the cell. For example, the adhesion molecule can be an adhesion peptide, such as RGDS (SEQ ID NO:1), YIGSR (SEQ ID NO:2), IKVAV (SEQ ID NO:3), REDV (SEQ ID NO:4), GKKQRFRHRNRKG (SEQ ID NO:5), RNIAEIIKDI (SEQ ID NO:6), KTRWYSMKKTTMKIIPFNR (SEQ ID NO:7), or any combination thereof. In addition, or alternatively, the adhesion molecule can be a partial or full-length protein, such as, for example, collagen type I, fibronectin, laminin, denatured collagen (also known as gelatin), collagen type IV, Matrigel® (Corning Life Sciences, Bedford, Mass.), poly-L-lysine (PLL), poly-D-lysine (PDL), or any combination thereof. Antibodies that engage with cell surface receptors (e.g., CD3 and CD28) and/or small molecules (e.g., nonpeptide small molecules such as stemregulin or reversine), for example, small molecules that activate a differentiation program in cells to enhance adhesion, can also be an adhesion molecule included on the substrate for cell attachment.

In further embodiments, the substrate further includes a growth factor, which can be located in the interior compartment, such as conjugated to the polymer, embedded within a hydrogel, and/or located within a liquid (e.g., a culture medium). The growth factor can be, for example, FGF, TGF-β1, VEGF, PDGF-BB, PDGF, IGF1, stem cell factor (SCF), thrombopoeitin (TPO), FMS-like tyrosine kinase 3 ligand (Flt-3L), erythropoietin, DL-1 notch ligand, Wnt, stromal derived factor (SDF)-1, interleukin (IL)-2, IL-3, IL-4, IL-6, IL-7, IL-15, IL-15R, CD40L, G-CSF, GM-CSF, 4-1BB and BMP superfamily members, or any combination thereof.

In yet further embodiments, the shell of the capsule is porous, having a pore size, for example, of about 10 nm to about 35 nm, or of about 20 nm. The shell can further include an enzyme-sensitive peptide, such as a protease-sensitive peptide, or other dissolvable material and conjugation moiety.

In yet further embodiments, the capsule includes a DNA-containing or RNA-containing molecule. The DNA-containing or RNA-containing molecule can be located in the interior compartment, such as conjugated to a polymer, embedded within a hydrogel, and/or located within a liquid (e.g., a culture medium) contained within the shell. The DNA or RNA-containing molecules can be, for example, cDNA, plasmid DNA, transposable DNA (e.g., using sleeping beauty transposons), viral vectors (e.g., adeno-associated virus, retrovirus, lentivirus, Sendai virus), modified RNA, siRNA, miRNA, antisense oligonucleotides, gene editing molecules, such as CRISPR/gRNA, zinc finger nucleases (e.g., TALENs), and meganucleases, as well as lipid vesicles containing these molecules, or any combination thereof.

In some embodiments, the capsule includes a cell adhered to a substrate, such as a stem cell. The cell can be, for example, a Mesenchymal Stem Cell (MSC), a Chinese Hamster Ovary (CHO) cell, a Madin-Darby Canine Kidney Epithelial (MDCK) cell, a Vero cell, a pancreatic islet, a peripheral blood mononuclear cell, an endothelial progenitor cell, a blood fibrocyte, a bone marrow cell, a T cell, a B cell, a dendritic cell, a CD34+ cell, an NK cell, a monocytes, a hepatocyte, a neural stem cell, a gastrointestinal cell, a skin cell, a skin cell progenitor cell, a cancer cell, a hybridoma cell, a prokaryotic cell, a HEK293T packaging cell line, a yeast cell, a pancreatic precursor cell, an embryonic stem cell, or an induced pluripotent stem cell. The cells may be encapsulated by exposing a porous shell of the capsule to the cells, the cells translocating through the pores into the interior compartment of the shell. Alternatively, the cells may be encapsulated during polymerization of the capsule. The capsule can include a culture medium in the interior compartment, thereby producing a suspension culture of encapsulated cells. The cells can grow and/or expand in a suspension culture. The suspension culture can be a stirred-tank suspension culture. Upon completion of a cell culturing or storage process, the shell of the capsule can be degraded and the cells harvested.

In another embodiment, the invention relates to a cell culture kit that includes first and second compositions. The first composition comprises a polymer precursor material and an adhesion peptide. The second composition comprises reagents for polymerizing the polymer precursor material to form a capsule having a shell that comprises a polymer produced by polymerization of the precursor material and the adhesion peptide. The adhesion peptide is present on an inner surface of the shell. The first composition can, optionally, further include a growth factor and/or a protease-sensitive peptide. The second composition can, optionally, further include a cross-linking reagent for forming a hydrogel within the interior compartment of the shell. The first composition may be lyophilized.

In another embodiment, the invention relates to a system for producing encapsulated cells that includes first and second compositions. The system comprises of a pumping apparatus, tubing sets, a specified light source, and liquid handling/collections bags. The first composition comprises a polymer precursor material and one or more substrates conjugated to the polymer. The second composition comprises reagents for polymerizing the polymer precursor material to form a capsule having a shell that comprises a polymer produced by polymerization of the precursor material and the conjugated substrate(s). The substrate(s) is present on an inner surface of the shell. The first composition can, optionally, further include a growth factor, DNA/RNA containing structure, and/or a protease-sensitive peptide. The second composition can, optionally, further include a cross-linking reagent for forming a hydrogel within the interior compartment of the shell. The first composition may be lyophilized.

DETAILED DESCRIPTION

A description of example embodiments follows.

Methods and systems of the present invention provide for encapsulated cell culture systems that can be used for expansion and/or storage of adherent cells. Such encapsulated cell culture systems and methods provide several advantages over prior art microcarriers.

As shown inFIG. 1, prior art microcarriers provide for cell attachment on the outside of the carriers, which exposes adhered cells to fluid shear stresses in suspension cultures. Such exposure can lead to cell detachment from the microcarrier and cell damage. While cell quality is important for many bioprocessing applications, it is of particular importance for applications involving the growth and harvesting of cells for cell therapies. For such applications, the cells are the therapeutic/medicinal agent to be collected upon completion of the bioprocess, as opposed to, for example, applications in which the cells are a source for protein production and are discarded after the bioprocess.

Prior art microcarriers, while providing several advantages over 2D cell cultures for large scale bioprocessing applications, have several disadvantages, and there exists a number of challenges for their use, as summarized in Table 1 with regard to, for example, mesenchymal stem cells (MSCs). MSCs are an example of an adherent cell type, the bioprocessing of which could benefit from methods and systems of the present invention. However, it should be understood that the same or similar comparisons may be drawn with regard to other cell types, and methods and systems of the present invention may provide for or include the culturing and/or storage of a variety of different cell types, as described further. Examples of other cell types that could be input into capsules with adherent populations include Chinese Hamster Ovary (CHO) cells, Madin-Darby Canine Kidney Epithelial (MDCK) cells, HeLa cells, PC9 cells, Vero cells, pancreatic islets, peripheral blood mononuclear cells, endothelial progenitor cells, blood fibrocytes, bone marrow, T cells, B cells, dendritic cells, NK cells, monocytes, CD34+ cells, iPSC cells, EPCs, hepatocytes, neural stem cells, gastrointestinal cells, skin cells and progenitors, cancer cells, hybridoma cells, microorganisms, HEK293T packaging cell lines, yeast cells, pancreatic precursor cells, embryonic stem cells, or an induced pluripotent stem cells.

TABLE 1Current challenges associated with the integration of microcarriers into MSCmanufacturing.BenefitChallengePotential MitigationProductReduced labor requirements andQuality: High risk ofDevelopment of qualityComparabilityability to implement automatedchanges in MSCassays of productprocess steps.product efficacy whenefficacy.Stirred bioreactors are wellmoving from existingcharacterized, allowing for changesplanar technology toin scale to be assessed prior tomicrocarrier-basedimplementation [21].process.Reduction in required clean roomcapacity and equipment (incubatorsetc.) allowing for facility andoperational cost savings.Product YieldAbility to manufacture >109cells toCost of Goods:Development and(perprovide >4000 doses per batch comparedIncreased MSCoptimization of culturebioreactorwith <108cells to provide <400 doses pernumber per batchmedium and processvolume)batch in planar culture technology [22].reduces the cost perparametersdose.AttachmentRequired for anchorage-dependent cellsSustainability:Development of reducedto maintian cell phenotype andReducing the amountor serum-free processpluripotency in the case of stem cells.of serum is critical inincluding methods forincreasing large-scalemicrocarrier modificationsustainability.to improve MSCCost of Goods:attachment [23].Reduced attachmentreduces growth rateand number of MSCsper batch.HarvestReduction in batch pooling and productQuality: IncreasedDevelopment of MSCholding times, improving processagitation rate duringdetachment protocolsscalability and product quality attributesdetachment reducesprior to implementation[24].product quality.at scale [25, 26].Cost of Goods:Increased detachmentefficiency and MSCsper batch reduces thecost per dose.SeparationImproved integration of up- and down-Quality: Method ofDevelopment of scale-stream unit operations, reducing processseparation may affectdown models to testtime and improving scalability.MSC CQAsproduct CQAs [27] andScalability: Processdownstream processingshould allow for timelytimes early inseparation, even atdevelopment [23].large scale.ProcessHomogeneous environment allowsQuality: Impact onIntegration of effectiveOptimizationfor monitoring and control and keyMSC product CQAs.process control systems,process parameters such asScalability: Limit tomedia optimization anddissolved oxygen, pH, nutrients andprocess scale.consideration of directmetabolites[28].aeration methods [13] inBioreactor systems allow for flexibleearly development.modes of operation such as batch,fed-batch or perfusion, allowing forprocess development activities toimprove product quality and yield.PurificationClosed system manufacture, reducing theQuality: Increase inIntegration of particulaterisk associated with contamination andrate of failed lots dueand impurity levels as afailed product lots.to impurities.screening criterion fordevelopment ofdownstream separationand volume reductionprocesses.

In suspension cultures, fluid shear advantageously prevents carrier sedimentation and cell aggregation while ensuring cell culture homogeneity; however, shear stress affects cell viability and morphology and can have a modulating effect on cell metabolism and differentiation states [29]. Moreover, shear stress from agitation results in cell damage due to microcarrier-to-microcarrier or microcarrier-to-impeller (or probe/insert) collisions. This damage increases with increasing microcarrier size, concentration, and agitation intensity, incurring a limitation on scale-up [30]. Smaller microcarriers can reduce shear-induced cell death and increase growth rates; however, decreasing the size of the microcarriers likewise decreases the available surface area for cell attachment and expansion. A minimum agitation rate has been estimated from an empirical correlation derived by Zwietering [101], which suggests that microcarriers should not remain at the vessel bottom for more than 1-2 s. Taking in view the Kolmogorov model [102], turbulent eddies in stirred-tank bioreactor microcarrier cultures are intermediate in size between the cells and the microcarriers. The Kolmogorov eddy size decreases as the agitation speed increases. The high rate of local energy dissipation due to these eddies interacting with the surface of microcarriers can cause shear rates that are sufficiently large to damage or even remove cells from the microcarrier surface. Additionally, foam formation from aeration leads to hydrodynamic stress in large-scale cultures, which is detrimental to cells growing on the surface of microcarriers [13, 31]. At the cellular level, exposure to a rigid microcarrier bead and shear fluid flow patterns can have dramatic impact on cell viability and mechanotransduction ultimately impacting the quality of cultured cells.

Efforts are beginning to direct towards protection of cells from shear forces expanded using the microcarrier technology; porous carriers such as the Cultisphere® (Sigma Aldrich, UK) have been developed that shield cells from shear-induced damage and allow higher cell growth through colonization of cells in the pores of the carrier [32-34]. Though a plausible solution, another important issue, identified by Gupta et al. [35], is that agitation of Cytodex® 3 microcarrier cultures (GE Healthcare, Marlborough, Mass.) above 25 rpm results in carrier breakage. As such, use of soft carriers, such as Cytodex® 1/3 (GE Healthcare, Marlborough, Mass.), raises safety concerns for in vivo applications where cells need to be free of any residual microcarriers or sub-particles. Current approaches to cell harvesting involve the use of cell strainers to separate expanded cells from their carriers; however, the breakage products are difficult to remove. These carrier impurities continue to be of concern and are a major obstacle for the clinical application of cell therapeutics cultured on microcarriers.

Methods and systems of the present invention provide alternatives to currently-available microcarriers that can circumvent one or more of the above-noted deficiencies, such as, for example, by promoting increased viability and purity of the final cell product.

In one embodiment, the invention is directed to a capsule for growing or storing cells that includes a shell defining an interior compartment and a substrate for cell attachment located within the interior compartment. The substrate comprises a polymer and one or more adhesion molecules. The adhesion molecules, also referred to as adhesion conjugates, can be, for example, adhesion peptides, adhesion proteins, or small molecules capable of attaching cells to a substrate and/or activating a differentiation pattern of attachment. For example, adhesive conjugates can be proteins (e.g., partial or full-length proteins), such as collagen type I, fibronectin, laminin, collagen type IV, Matrigel, or any combinations thereof. Adhesive conjugates can also be antibodies, which engage with cell surface receptors, such as T cell receptors. For example, the antibody can be an antibody that binds at least one of CD3, CD28, and CD40. Adhesive conjugates can also be or include nonpeptide small molecules that activate a differentiation program in cells to enhance adhesion, such as stemregulin or reversine, or a non-steroidal anti-inflammatory molecule.

Examples of capsules are shown inFIGS. 2A-2E. The substrate for cell attachment can be, for example, an inner surface of the shell, as shown inFIGS. 2A-2B, such that cells are attached to the capsule along an inner circumference of the capsule. Alternatively, or in addition, the substrate for cell attachment can be a hydrogel disposed within the interior compartment of the capsule, as shown inFIGS. 2C-2E.

With regard to materials for cell encapsulation, several natural and synthetic polymers have been investigated in rodent models; with alginate and polyethylene glycol (PEG) reaching pre-clinical and clinical trials. Alginate provides favorable gelling conditions; however, challenges associated with batch to batch variability, wide pore size distribution, encapsulated product size (e.g., engraftment volume) and scalability exist [10, 11]. PEG has been used for conformal coats on islets because it can react with amine groups in collagen and membrane proteins on the islet surface [23-25], which has the advantage of shorter distance for diffusion for oxygen, nutrients, and insulin and a smaller implantation site. Moreover, PEG can be functionalized with peptides and growth factors to stimulate insulin gene transcription, prevent (3-cells apoptosis and promote islet vascularization [31-33].

A comparison of methods and lead materials for producing encapsulated cells is shown in Table 2. While alginate and PEG are provided as examples, other materials that can be polymerized could potentially be combined with the encapsulation methods described herein, or other known encapsulation methods, for polymer matrix components. For example, one method of producing small monodisperse capsules in a high-throughput manner includes the pulsation of a jet or vibration of a nozzle during extrusion of the lead material, often referred to as a laminar jet breakup technique. The laminar jet breakup technique involves axisymmetric disturbances to break the jet from the nozzle into equally sized droplets. This technique can achieve production rates as high as 104 particles per second [22]. The vibration frequency, diameter of the nozzle, viscosity, and flow rate of the polymer-cell suspension govern the size and production rate of the microcapsules. In another example, a rotating disk or jet cutting method can be used to produce small monodisperse capsules. Jet cutting can provide a higher production rate for generating particles with the encapsulated material at a frequency of 10,000 Hz, or 104 particles per second, which translates to a 500 μm bead throughput of 60 mL/min [22].

Other methods include emulsification techniques, which, compared to extrusion drip methods, are not limited by scale. With emulsification techniques, a production rate is governed by the vessel size in which cell encapsulation takes place. Emulsification techniques also provide for the ability to produce cell-laden microcarriers in a single step as compared to the two-step procedure required with commercially available microcarriers. Appropriate dispersion devices and operating conditions, such as mixing rates and surfactants, can allow for reduction in capsule size.

In further embodiments, in addition to a polymer shell, capsules can further include a hydrogel core, as shown, for example, inFIGS. 2D-2F. The hydrogel core can comprise, for example, cross-linked polyethylene glycol (PEG), cross-linked polyethylene glycol diacrylate (PEGDA), or a combination thereof.

The polymer shell and/or the hydrogel core can be functionalized with one or more adhesion peptides that allow for cell attachment and/or spreading within the capsule. The adhesion peptides can be, for example, RGDS (SEQ ID NO:1), YIGSR (SEQ ID NO:2), IKVAV (SEQ ID NO:3), REDV (SEQ ID NO:4), GKKQRFRHRNRKG (SEQ ID NO:5), RNIAEIIKDI (SEQ ID NO:6), KTRWYSMKKTTMKIIPFNR (SEQ ID NO:7), or any combination thereof. Adhesion peptides can be peptides that affect cell viability, proliferation, survival, growth and/or differentiation.

For example, PEG can be chemically modified to include one or more adhesion peptides by reacting acrylate-PEG-SVA with an adhesion peptide in a sodium bicarbonate solution at a predefined molar ratio (e.g., 50 mM, pH8.5) overnight, followed by dialyzation to remove any unreacted peptides. The modified polymer material can, optionally, be lyophilized and stored. Adhesion peptide conjugation efficiency can be assessed by detecting any unreacted free amines by a ninhydrin assay. Acceptable conjugation efficiency can be determined based upon the desired application. For example, a conjugation efficiency of about 85% or greater could be considered acceptable. Examples of adhesion peptides, protein derivatives, and resulting PEGylated products are shown in Table 3.

The conjugated adhesion peptides (e.g., PEG-conjugated adhesion peptides, or acryl-PEG-AP) can be combined with a cell suspension (e.g., an MSC suspension) prior to undergoing a photopolymerization process (e.g., by being added to photopolymerizable PEG diacrylate (PEGDA) and undergoing free-radical polymerization) to form cell-laden PEG capsules. Concentrations and combinations of acryl-PEG-AP can be varied in the precursor solution to optimize cell attachment. For example, hydrogels can be formed by combining 0.1 g/mL 10 kDa PEGDA and 10±5 mM acryl-PEG-AP in 10 mM HEPES buffered saline (pH 7.4) and photoinitiators 1.5% v/v triethanolamine (TEOA), 37 mM 1-Vinyl-2-pyrrolidinone (NVP), and 10 μM eosin Y disodium salt. The solution can then be sterilized by being filtered, such as through a 0.2 μm filter. By combining the cells (e.g., MSCs) in the precursor solution, the cells can be encapsulated during photopolymerization and formation of the capsules. In an example procedure, a hydrophobic photoinitiator solution containing 2,2-dimethoxy-2-phenyl acetophenone in 1-vinyl-2-pyrrolidinone (300 mg/mL) can be combined in mineral oil (3 μL/mL), to which the cell-prepolymer suspension is added. The combined solution can then be vortexed for 4 seconds in ambient light, followed by an additional 3 seconds under white light. The vortex may then be stopped and the emulsion exposed to white light for 20 seconds with a vortex pulse at 10 seconds. Crosslinked microspheres can then be isolated by centrifugation at 300 g for 5 minutes, resuspended in media, and placed in Transwell® (Corning, Tewksbury, Mass.) cell culture inserts.

Morphological evaluation of the encapsulated cells and polymer shells/hydrogel core can be conducted using fluorescent stains. In an example of an evaluation procedure, images can be taken to verify the interaction of cells and the surrounding hydrogel matrix. Actin organization of the encapsulated MSCs can be analyzed to verify bioactivity. Upon encapsulation, MSCs may be attached along the inner membrane of the PEG capsules, anchoring to the PEGylated adhesion moieties. Cell-laden capsules (alternatively referred to as microcapsules) can be fixed in a 4% formaldehyde solution, permeabilized using a 1% Triton X-100 solution, then incubated for 30 min in a 4 unit/ml phalloidin rhodamine solution and mounted in a mounting solution containing a fluorescent stain, such as DAPI. Cell adhesion and spreading can be monitored by, for example, light microscopy and fluorescence microscopy, such as on an Axio Observer Z1 (Zeiss, Jena, Germany) equipped with an ApoTome system (Zeiss, Jena, Germany) to achieve optical sectioning. Selection of an adhesion peptide can be made by semi-quantitative analysis of, for example, 100 pictures of random cell capsules for N=3 batches per peptide to quantify spindle formation as compared to no peptide controls. An acceptable attachment efficiency can be determined based upon the desired application. For example, an attachment efficiency of at least about 60% could be considered acceptable. Alternatively, a desired adhesion peptide can be selected based on other factors. For example, it may be desirable to include RGDS because RGDS is well characterized for directing cell association with biomaterials [76-78].

While an example of forming encapsulated cells by photopolymerizing a precursor solution containing cells is described, other methods of encapsulating cells are possible. For example, a capsule can include a porous shell and/or a porous hydrogel core. The porous capsule can then be exposed to a cell suspension such that cells translocate into the interior compartment of the shell and adhere to the substrate.

Capsules having a pore size of about 10 nm or greater, or of about 20 nm or greater, can also allow for the diffusion of nutrients and waste products into and out of the capsules. A pore size can be adjusted by modifying a weight of the polymer precursor material used to create the hydrogel mesh that forms the shell and/or hydrogel core of the capsule. For example, PEG-SVA having a weight of 5 kDA can be used to form a hydrogel mesh that has a larger pore size than that of a hydrogel mesh made from 10 kDa PEG-SVA.

In another embodiment, capsules can include one or more growth factors. The inclusion of a growth factor can further enhance cell growth and/or proliferation within the capsule. The one or more growth factors can be conjugated to the polymer (e.g., the polymer shell, the hydrogel core, or both). Alternatively, the growth factor(s) can be soluble within a liquid core or a hydrogel core of the capsule. Examples of suitable growth factors include FGF, TGF-β1, VEGF, PDGF-BB, PDGF, IGF1, and BMP superfamily members, or any combination thereof.

Growth factors (GFs) may be required for some in vitro cell cultures and are typically added to culture media as soluble GFs. Capsules of the present invention can advantageously provide for a concentrated, transient, controlled supply of bioactive GFs and reduce the need for serum components typically used as a growth agent for cultured cells. For example, PEGylated GFs can be included in the shell and/or core of a capsule. Examples of PEGylated GFs, including FGF, TGF-β1 [79] [80], IGF1[81] and BMP [82] [83], are shown in Table 4.

In an example procedure, recombinant GFs can be conjugated to PEG by reaction with acrylate-PEG-SVA in a 1:15 (peptide:PEG) molar ratio in 50 mM sodium bicarbonate (pH 8.5) and then stirred under argon overnight, lyophilized, and stored at −80° C. A Western blot can be used to analyze the resulting acryl-PEG-GF. Soluble and PEG-conjugated GF can be separated on a 4-15% SDS-PAGE gel and transferred to a nitrocellulose membrane. The membrane can be incubated overnight at 4° C. with 5% milk in buffer containing 0.1% (vol/vol) Tween 20 in TBS (TBST). The membrane can be incubated with rabbit anti-GF for 1 h at room temperature. After two washes with TB ST, peroxidase-labeled goat anti-rabbit IgG can be added and incubated for 1 h at room temperature and treated with pico-chemiluminescence reagent for detection. The bioactivity of the PEG-GF conjugates can be assessed to verify that bioactivity was not affected by the conjugation process or due to steric hindrance. The PEG-GF conjugates can be assessed in capsules at a range of concentrations of GF (e.g., 1 μg/L, 2.5 μg/L or 5 μg/L.) Cell growth can be measured in the presence of (i) unmodified or soluble GF, and (ii) PEG-conjugated GF to determine suitability of conjugated GF for an application. For example, a 50% growth advantage by PEG-GF vs PEG acceptable growth advantage by PEG-GF versus PEG may be desirable. The MSCs can be seeded in 12-well plates at an estimated density of 5000 cells/capsule and incubated for a 7 day growth promotion assay in a 37° C./5% CO2 environment. After 7 days, cell capsules can be quantified by imaging and MTS assays to assess cell growth. Additional supplementation of GF to a cell culture media may not be needed in capsules comprising PEG-GF.

As noted above, the purification of cells from prior art microcarriers can be challenging, often resulting in product having residual microcarriers and subparticles present. To address this shortcoming, PEG hydrogels can include proteolytically degradable peptide sequences to control hydrogel biodegradation. In one embodiment, capsules of the present invention include an enzyme-sensitive, such as a protease-sensitive peptide (e.g., GGGPQG↓IWGQGK (SEQ ID NO:8), GGL↓GPAGGK, GGG↓LGPAGGK (SEQ ID NO:9). The enzyme-sensitive peptide can be included in the shell and/or in a hydrogel core of the capsule. After a cell expansion process has completed, the capsule material can be caused to degrade, and standard cell concentration methods can then be used to purify the cell material. Some examples of enzymes used are trypsin, collagenase, DNase, RNase, and horseradish peroxidase. Other sensitive structures can be doped into capsules that infer temperature sensitivity, light sensitivity, or small molecule sensitivity, as well as protein-based sensitivity. For example, covalent bulk immobilization of PEGylated GF(s) in a degradable PEG hydrogel construct can provide a controlled transient local release of GF(s) to stimulate MSC growth and expansion while also providing for capsule degradation. Examples of degradable PEG are shown in Table 5.

Hydrogels can be rendered degradable through, for example, the covalent incorporation of a collagenase-sensitive peptide sequence, such as GGGPQG↓IWGQGK (SEQ ID NO:8), (PQ), where ↓ indicates a cleavage point by collagenase between the leucine and glycine residues. Single and multisite PQ domains can be incorporated between acrylate groups. Example procedures for forming degradable capsules follow. For single domain, the PQ peptide can be dissolved in 50 mM NaHCO3(pH 8.0) and reacted with PEG-SVA (MW=3400 Da) in a 1:2 (PQ: PEG-SVA) molar ratio at room temperature overnight to attach PEG on both ends of the peptide sequence (PEG-PQ-PEG). The resulting products, acrylate-PEG-peptide-PEG-acrylate can be dialyzed, lyophilized, and stored frozen at −20° C. under argon. For multidomain PQ conjugation, the peptide can be reacted with acryl-PEG-SVA in equal molar ratios, then dialyzed and lyophilized to remove undesired products. This acryl-PEG-peptide can be further reacted with SVA-PEG-SVA (MW 3400 Da), dialyzed and lyophilized. The reaction step can be repeated to add additional PEG-peptide. In the final step, the previous product can be reacted with acryl-PEG-SVA to form the multisite PQ degradable PEGDA macromer. The final product acrylate-(PEG-peptide)3-PEG-acrylate can be dialyzed, lyophilized and stored at −20° C.

The rate of MSC invasion within collagenase-sensitive PEG hydrogels can be evaluated to determine the conduciveness of the microcapsule to MSC expansion within the degradable substrate with local access to GF(s). Hydrogel degradation kinetics can be determined by, for example, allowing the microcapsules to swell for 24 hrs in PBS with 1 mM CaCl2at 37° C. Microcapsules can then be incubated at 37, 33 or 25° C. with collagenase fromClostridium hystolyticumin PBS with 1 mM CaCl2. The change in wet weight of the microcapsules can be measured over time. Degradation conditions in terms of enzyme concentration, time, temperature, and neutralization can be determined.

Non-enzymatic methods of polymer degradation can also be included in methods and systems of the present invention. For example, PEG segments can be incorporated into tyrosine-derived polycarbonates, as described by Kohn et al. [84-86], and/or thiol functionalized PEG macromers can be used to provide degradability in response to reducing microenvironments, such as in the presence of glutathione [87]. This degradation can occur via a thiol-disulfide exchange reaction. This reaction will fragment the polymer into soluble units [103]. As such the microcapsules will be prepared by employing 4-arm PEG or PEG tetra acrylate (PEGTA) in which each arm will be terminated with a thiol group [104] to form PEG-diester-dithiol cross-linker. The presence of disulphide bonds will allow hydrogel degradation in the presence of glutathione. Alternatively, water soluble PEG microcapsules will also be prepared by reacting PEGTA with dithiotriethol (DTT) in 1:1 molar ratio of acrylate to thiol in triethanolamine (TEA) and allowed to gel at 37° C. [105, 106]. Degradation of the capsules will occur overtime as water breaks down the hydrolytically labile ester bonds where the degradation kinetics will be dependent on reaction [105]. To attach peptides such as cell-adhesive ligands, 4-arm PEG-vinyl sulfone (PEG-VS) will be dissolved in TEA buffer and the cysteine-terminating peptides added at a large stoichiometric deficit to VS groups to covalently attach to VS [106] [107].

In addition to soluble factors, such as growth factors, and biochemical cues, including adhesion peptides, cells may also be impacted by physical and mechanical cues, such as a surface topography and rigidity/stiffness of the substrate. Capsules can be further optimized to have a modulus that retains multipotency during cell expansion.

The rationale for optimizing the spatial organization of cell adhesion peptides and mechanical stiffness of the substrate is that such characteristics can play significant roles in regulating multipotent cell function [88-90]. With regard to microcarriers, characteristics such as stiffness and curvature influence cellular activities while the adhered cells are being cultured in a bioreactor through changes in the shear stress on cells in a bioreactor [91]. Thus far, limited efforts have been made to define the role of microcarrier properties and geometry on controlling multipotent cell function [92, 93]. The regulation of MSC cell fate is dependent on substrate rigidity, which modulates the extracellular matrix (ECM) of the MSC, adhesion peptides (e.g., integrin), and cytoskeleton interactions. A failure to control for these properties can lead to unwanted differentiation of MSCs and a loss of immunomodulatory potency.

The modulus of the substrate of capsules of the present invention can be tunable. For example, polymer molecular weights and concentrations in precursor solutions can be adjusted to provide for softer/harder substrates. In one example, 3400 Da PEG-SVA and 10% 10 kDa PEGDA can be used for microcapsule formation. Cell attachment and spreading can be analyzed and the polymer weight can be decreased if the matrix is too ‘soft’ for cell attachment. Several molecular weight PEGDA (e.g., 3.4 kDa and 5 kDa) can be utilized, and the concentration can be varied (e.g., 10% or 15%) to tailor substrate modulus to cell culture and reactor conditions in future aims. To determine if the stiffness of a substrate is suitable, cell proliferation with microcapsules can be followed for 14 days and measured via MTS assay at predetermined time points. Alternatively, a desired weight/concentration of polymer precursor materials can be selected based on other factors. For example, it may be desirable to include 10% 10 kDa PEGDA because such a combination provides a balance between substrate rigidity, porosity and degradation kinetics.

In some embodiments capsules of the present invention include a cell adhered to the substrate, such as, for example, a stem cell. While the Mesenchymal Stem Cell (MSC) has been used as an example of an adherent cell that can be encapsulated within capsules of the present invention, it should be understood that other adherent cells can alternatively be included. In addition to an MSC, examples of suitable cells include Chinese Hamster Ovary (CHO) cells, Madin-Darby Canine Kidney Epithelial (MDCK) cells, Vero cells, pancreatic islet, pancreatic precursor cells, embryonic stem cells, and pluripotent stem cells. Cells contained in capsules can be maintained in a viable state, such as by being maintained in a suspension culture. Encapsulated cells can also be stored for later use, such as by undergoing a cryopreservation processes.

Cell Counting Method & Apparatus

An example of a cell counting method and apparatus is shown inFIG. 24. An amount of solution containing suspended capsules can be drawn with a pipette and mixed with phosphate buffered saline (PBS) to create a counting solution. For example, 10 μL of sample can be drawn and mixed with 10 μL of PBS using a 200 μL pipette, as shown. The drawn samples can be placed in a hemocytometer with a silicon cover. For example, 5-6 μL samples can be drawn and placed in the hemocytometer with the solution occupying the middle square of the hemocytometer. Either four outer squares can be used for counting, or the middle square can be used. The middle square is shown as being used in the figure to confine capsules in area of known dimensions.

The sample can then be placed under a microscope, and capsules within the middle square counted. A total number of capsules/mL is equal to the number of capsules counted multiplied by the dilution factor and multiplied by 10,000 capsules/mL. Use of a slip cover may cause capsules to break. Silicone can be used to keep the solution in place instead of use of a slip cover.

Continuous Flow Capsule Production Using Light-Based Polymerization

An example of a continuous capsule production system is shown inFIG. 25. A polymer solution containing a hydrophilic photoinitiator is mixed with a hydrophobic photoinitiator in mineral oil. The terms hydrophilic and hydrophobic are used to designate the solution in which the photo initiators are present. The mineral oil solution (e.g., as illustrated, 54, acetophenone/NVP solution and 1 mL mineral oil) is hydrophobic. The PEGDA solution (e.g., as illustrated, 0.1 g PEGDA, 15 uL TEOA, 10 uL Eosin Y disodium salt, 3.754, NVP, 493.025 uL HBS Solution, and 10 uL Pluronic acid) is hydrophilic.

In the system illustrated inFIG. 25, a PEGDA pump pumps PEGDA solution to a reflective white light box while a mineral oil pump pumps mineral oil with hydrophobic photoinitiator to the white light box. The pumps and the reflective white light box can be connected by tubing. The white light box is connected to an illuminator.

The tubing passes through the white light box to a receptacle for storing the processed capsules. For example, the capsules can be collected in a 75 cm2T flask as the system processes capsules on a continuous basis continuous system.

The amount of time the solutions are present in the white light box can be dependent upon a number of light sources used with the reflective box, type of light sources, and setup of the reflective box. A pump speed can be adjusted accordingly to ensure that the solutions are exposed to light for an appropriate amount of time. For example, with system shown inFIG. 25, having one light source, the solution can be present in the white light box for approximately three minutes. Other configurations are possible, examples of which follow:Mineral Oil 144 mL/min and PEDGA 1.3 mL/min with 3 minutes of light exposure.Mineral Oil 144 mL/min and PEDGA 1.3 mL/min without using the light box and point blank exposing the mixed solution tube for 15 seconds for each inch of tubing.Mineral Oil 96 mL/min and and PEDGA 1.3 mL/min without using the light box and point blank exposing the mixed solution tube for 15 seconds for each inch of the tubing.

Pump speed can also affect capsule size. Increasing the pump speed of the mineral oil solution can reduce the size of the PEDGA solution when both the solutions are mixed. This can allow for a smaller capsule size.

Conjugation of Lentivirus to PEG for In Situ Cell Engineering

An example of a method for creating capsules comprising conjugated lentivirus is shown inFIGS. 26-29. As shown inFIG. 26, a first part of the process includes conjugation of lentiviral particles to PEG. The conjugation can be accomplished with, for example, amine crosslinker (NETS) reactive chemistry by which Acrylate-PEG-SVA (molecular weight (MW): 3,400) reacts with surface-exposed primary amines on lysine residues, part of the viral capsid glycoprotein subunit gp120 of lentiviral particles, thereby forming Acryl-PEG-VP.

For example, the method can include concentrating viral particles using ultracentrifugation (e.g. at 25,000 rpm for 2 hours at 4° C.) and resuspending the particles in PBS (e.g., 25 mL). The suspension can then be combined with the polymer (e.g., 10 mg PEG-SVA dissolved in 25 mL PBS) providing for a combined solution (e.g., a combined solution of 50 mL volume). The mixture can then be shaken for a period of time at suitable temperature (e.g., overnight at 4° C.).

As further shown inFIG. 27, the Acryl-SVA can be rendered degradable. For example, a separate polymer mixture can be made to render PEG hydrogels degradable through covalent incorporation of the collagenase-sensitive peptide sequence, GGGPQG↓IWGQGK (SEQ ID NO:8), (PQ). PQ peptide reacts with Acryl-PEG-SVA to create Acryl-PEG-PQ-PEG-Acryl.

As further shown inFIG. 28, the crosslinking of polymers and encapsulation of cells can be accomplished using a dual photoinitiator emulsion-based technique, for example, as shown and described above with respect toFIG. 25. Exposure to white light and a photoinitiator can break Acryl group double bonds to form VP-PEG-Acryl-PEG-PQ-PEG-Acryl polymeric chains. Emulsion polymers begin forming when a free radical, acting as an initiator, breaks a double bond between two carbon atoms in an acrylic monomer, starting a reaction that can cause monomer units (e.g., as many as 10,000 monomer units) to bind together into a polymer chain.

A bulk view of the process shown inFIG. 28is shown inFIG. 29. As illustrated cell cultures (e.g., NK cells) can be mixed with PEG-PQ, PEG-VP, and polymerization components (e.g., eosin T, triethanolamine, and 1-vinyl-2 pyrrolidinone) and exposed to white light.

Example 1. Mechanical Properties of Polyethylene Glycol (PEG) Capsules

Current microcarriers are generally not mechanically durable and are known to sustain damage during bioreactor agitation processes, resulting in e.g. fragmented debris. Polyethylene glycol diacrylate (PEGDA) capsules were tested in preliminary studies under fast agitation conditions and were evaluated for visual integrity.FIGS. 3A-3Cillustrate the mechanical durability of PEGDA capsules. PEGDA capsules were synthesized through a water-oil emulsification method and cultured in static (FIG. 3A), conventional speed (FIG. 3B), and high speed (FIG. 3C) agitation conditions. The photomicrographs ofFIGS. 3A-3Cshow that the capsules maintain their integrity even at high rotations per minute (rpms) which are beyond the traditional speeds (˜75 rpm) used in cultures.FIGS. 3A-3Calso show a lack of capsule fragment debris.

Example 2. PEG Capsules Support Cell Growth

Preliminary studies with non-decorated PEGDA capsules were performed under static conditions to validate biomaterial properties. Polyethylene glycol (PEG) is a versatile polymer with tunable biochemical and mechanical properties and its safety profile is well-established. PEG offers a blank-state which, on its own, repels protein adsorption and subsequent cell-surface interactions, but also provides for the addition of bioactive ligands. As such, PEG provides an opportunity to create surfaces that promote cell interactions and adhesion while suppressing non-specific protein adsorption and cell adhesion.

MSCs were encapsulated in PEGDA via white light polymerization. Cells were encapsulated at two different input cell concentrations to the cell-material bulk mixture. Viable cells detected by Calcein Acetoxymethyl (AM) staining were observed as MSC spheroids at the end of a 12-day culture process (FIG. 4A). Specifically, LIVE/DEAD™ Viability/Cytotoxicity Kit was used to detect live cells (green) with Calcein AM, and dead cells (red) with Ethidium Homodomer 1.

Encapsulation efficiency and cell proliferation were quantified over a 12 day culture process via Cell Titer 96® AQueous One Solution Cell Proliferation Assay. Results show an approximate 3-fold expansion in capsules over a 12-day period without inclusion of growth factors or adhesion peptides. Efficiencies of encapsulation correlated to the cell density per given feed material stock as expected. Encapsulated cells showed an approximate 2-3 day doubling time (within acceptable range for industrial MSC culture) without the addition of any growth factors or adhesion peptides to the capsule material (FIG. 4B).

Example 3A. RGDS-Conjugated PEG Capsules Promotes Cell Attachment and Spreading

The peptide RGDS was successfully conjugated to PEG by reacting the peptide with acrylate-PEG-SVA. Adhesion peptide conjugation efficiency was assessed by detecting any unreacted free amines via a ninhydrin assay. Glycine was used as free-amine standard. PEG conjugated RGDS (PEG-RGDS) was assayed to detect any unreacted free amines. An 87% conjugation efficiency was achieved (FIG. 5). MSCs were encapsulated in a pilot study within adhesion peptide (RGDS) functionalized PEGDA capsules with a polymerized central core and cultured under dynamic conditions (125 rpm). It should be noted that the material of the shell and the core can be the same and the polymerization technique can either create a polymerized shell or a polymerized shell and core (which is referred to as a polymerized central core). The difference is in the degree of crosslinking. In liquid core capsules, the central core of the capsule is left uncrosslinked and only the shell is polymerized. In hydrogel or polymerized core capsules, the central core is also cross-linked. Cell-laden capsules sampled on day 3 were stained with rhodamine phalloidin and DAPI to visualize actin organization of the encapsulated cells (FIG. 6). Specifically, confocal images of rhodamine phalloidin (red) and DAPI (blue) stained encapsulated MSCs were sampled on day 3 from dynamic spinner flask cultures. The image ofFIG. 6demonstrates preliminary evidence of cell attachment and spreading.

Example 3B. Effective Cell Attachment with Adhesive Capsules

Further testing was performed to assess the bioactivity of adhesives, the results of which are shown inFIGS. 14A-14I.

MSCs were encapsulated within adhesion peptide (RGDS) functionalized PEG capsules and cultured under dynamic conditions (125 rpm). PEG-RGDS at 5 mM (low) and 10 mM (high) concentration was used and cell spreading and attachment was assessed via confocal microscopy imaging of rhodamine phalloidin stained cells. No PEG-RGDS capsules were used as control. High cell spreading was demonstrated with 10 mM (high) adhesive on day 14 at the end of the culture process.

Example 4A. Effect of Growth Factor in Cell Proliferation

Cell-laden liquid core (LC) and polymerized core (PC) capsules were assessed under static cultures to study the effect of growth factors (GFs) on cell proliferation, the results of which are shown inFIG. 7. In particular, capsules were created with PEGylated RGDS and contained one of PEGylated FGF, soluble GF, or no GF. MSC were encapsulated in LC or PC capsules. Samples were taken on days 1, 4, and 6 to assess cell proliferation. The results show capsules including PEG-FGF, whether the PEG-FGF was located in a hydrogel core (i.e., PC capsules) or in a shell of the capsule (i.e., LC capsule) had the greatest cell proliferation while soluble FGF in PC capsules maintained cell numbers at the encapsulated seeding density.

Example 4B. Addition of Growth Factor in Material Improves Post-Encapsulation Viability

Further testing was performed to assess MSC viability in capsules with and without FGF. Varying concentrations of PEGylated adhesive peptide RGDS and PEGylated FGF peptide were tested, the results of which are shown inFIGS. 15A-15F.

MSCs were encapsulated within adhesion peptide (RGDS) and FGF functionalized PEG capsules and cultured under dynamic conditions (125 rpm). PEGylated FGF was added at 2.5 μg/L for each 0 mM, 5 mM, and 10 mM PEG-RGDS samples. Comparison against no FGF controls demonstrates higher post-encapsulation viability in the presence of PEGylated FGF.

Capsules with FGF peptide showed higher day 1 post-encapsulation viability than capsules lacking FGF.

Example 5A. Effect of Agitation on Cell Viability, Proliferation, and Carrier Integrity

Cell-laden polymerized core capsules with PEGylated RGDS and FGF were exposed to high (125 rpm) and low (30 rpm) agitation speeds in spinner flasks. The results are shown inFIGS. 8A-8D(high agitation culture) and9A-9D (low agitation culture). Samples were taken on days 1, 3, 6 and 10. High agitation cultures resulted in an approximate ˜18 fold increase in encapsulated cell number while low agitation cultures resulted in only a 3 fold increase in cell number at the end of the culture period. This might be attributed to inefficient nutrient and waste exchange at low agitation while also highlighting that high agitation/shear is not detrimental cells encapsulated within PEG capsules.

Example 5B. MSC Viability after High-Speed Agitation Culture

Further testing was performed to assess MSC viability after high-speed agitation culture (125 rpm) in spinner flasks in the presence of bioactive factors PEG-RGDS and PEG-FGF in degradable PEG capsules.

The results are shown inFIG. 18. The presence of both bioactive components, PEG-RGDS and PEG-FGF provided improved cell viability and significantly improved viability over soluble FGF.

Example 6. Properties of Polymerized Core (PC) Capsules

PC capsules were analyzed on a Coulter Counter to study carrier size distribution, surface area, and volume. Empty PEGDA capsules were produced as a polymerized core via photo-polymerization without adhesion peptides or GFs. Capsules of heterogeneous size distribution were produced within acceptable and expected diameter limits of 70 um-250 um. Average volume and surface area of the thus produced capsules were 3×107um3and 1.5×106um2, respectively for PC and 7.2×106um3and 3.4×105um2, respectively for LC. The results are shown inFIGS. 10A-10C.

Example 7. Properties of Liquid Core (LC) Capsules

LC capsules were analyzed on a Coulter Counter to study carrier size distribution, surface area, and volume. The LC capsules were formed with a liquid core via photo-polymerization without adhesion peptides or GFs. Capsules of heterogeneous size distribution were produced within acceptable and expected diameter limits of 70 um-250 um. Average volume and surface area of the thus produced capsules were 3×107 um3 and 1.5×106 um2, respectively for PC and 7.2×106 um3 and 3.4×105 um2, respectively for LC. The results are shown inFIGS. 11A-11C.

Example 8. Capsule Degradation

Collagenase degradable peptide (GGGPQG↓IWGQGK) (SEQ ID NO:8) was conjugated to PEG to form degradable capsules. Collagenase degradable peptide was conjugated in a 1:2 (peptide:PEG) molar ratio. The conjugated PEG was then used to form non-cell-laden (FIGS. 12A-12D) and cell-laden (FIGS. 13A-13B) PC capsules via white light polymerization. No adhesion peptides or GFs were used. The degradable capsules were then exposed to collagenase and capsule integrity was analyzed at different time points under static conditions (FIGS. 12A-12D) as well as under mechanical agitation conditions (FIGS. 13A-13B). The results are shown inFIGS. 12A-12D and 13A-13B. As shown inFIG. 12A, capsules having the protease-sensitive peptide and that were not incubated with collagenase were not degraded. As shown inFIG. 12B-D, the capsules doped with the protease-sensitive peptide show signs of degradation over time upon exposure to collagenase. The degradation without mechanical agitation is a process that can take hours, in this example, >24 hours.

As shown inFIGS. 13A-13B, the degradation of the cell-laden PC capsules occurred almost instantaneously after mechanical agitation, which, in this example, was performed by repeated extrusion of the solution. As shown in bothFIGS. 12D and 13B, no remaining subparticles over a size of about 1 μm in approximation were found. In the example of cell-laden capsules, the arrow ofFIG. 13Bpoints to harvested cells that remain after capsule degradation.

Example 9. MSC Morphology Maintained after Cell Purification

Further testing was performed to assess MSC morphology following harvesting from capsules that included varying concentrations of adhesive peptides, the results of which are shown inFIGS. 16A-16C. These tests were performed to confirm that the capsule materials (PEG, PQ or RGDS) did not have any detrimental effect on TCP attachment, spreading and proliferation of MSCs post-harvest. No difference in cell morphology was observed between the samples. PEG-RGDS at 10 mM demonstrated a higher cell yield.

Example 10. Comparison to Industry-Standard Carriers

Microcarriers were equilibrated in cell culture media, and MSC cells were allowed to adhere to the microcarrier surface under static conditions (FIG. 17A-B). Cytodex 3 (Corning) microcarriers were used. Microcarrier-adhered MSCs were cultured at 70 rpm under standard cell culture media conditions.

After forty-eight hours in dynamic culture, MSCs detached from the microcarrier surface and aggregate cell debris was visible in the spinner flask, as shown inFIG. 17C.

Example 11. Comparative Viability of MSCs in PEGDA with 0 mM or 10 mM RGDS

Testing was performed to assess the viability of MSCs in PEGDA with and without 10 mM conjugated RGDS. Samples were taken at Days 1, 7, and 14, the results of which are shownFIGS. 19A-19F. The purpose of this experiment was to determine if adhesive peptides effect the viability and proliferation of encapsulated MSCs. Encapsulated MSCs were cultured in spinner flasks at high agitation (125 rpm) and maintained in an incubator at 37° C./5% CO2. In the presence of a bioactive component, PEG-RGDS provided improved cell viability by Day 1, over the 0 mM PEG-RGDS cohort.

Example 12. Comparative Viability of MSCs in PEGDA with 10 mM RGDS and Varying Concentrations of bFGF

Testing was performed to assess the viability of MSCs in PEGDA with varying concentrations of bFGF (0 μg/L, 0.25 μg/L, and 25 μg/L). Samples were taken at days 1, 7, and 14, the results of which are shownFIGS. 20A-20I. Encapsulated MSCs were cultured in spinner flasks at high agitation (125 rpm) and maintained in an incubator at 37° C./5% CO2. MSC proliferation was positively affected by the concentration of bFGF. With higher concentrations of bFGF conjugated to the microcapsules, we have found denser pockets along the microcapsule wall of proliferating MSCs. As seen inFIGS. 20A-20I, the presence of both bioactive components, PEG-RGDS and PEG-FGF, provided improved cell viability and significantly improved viability over soluble FGF.

Example 13. Comparative Viability of MSCs in Non-Degradable and Degradable Capsules

Testing was performed to assess the viability of MSCs in non-degradable and degradable capsules. Capsules including 10 mM RGDS and 25 FGF were created with PEGDA (non-degradable) and PEGPQ (degradable). Samples were taken at Days 1, 7, and 14, the results of which are shownFIGS. 21A-21F.

Encapsulated MSCs were cultured in spinner flasks at high agitation (125 rpm) and maintained in an incubator at 37° C./5% CO2. MSCs in PEGDA (non-degradable) capsules supplemented with bFGF (25 μg/L) showed dense viable pockets along the microcapsule wall, suggestive of MSC proliferation. MSCs in PEGPQ (degradable) capsules produced dense viable pockets, however, were fewer in number compared to MSCs encapsulated in PEGDA. PQ peptide may be producing an acidic microenvironment within the capsule, which may negatively affect MSC viability.

Example 14. Comparative Viability of Multiple Cell Types in PEGDA

Testing was performed with multiple cell lineages, including AtT-20, 3T3, and JURKAT cells. The cells were encapsulated in synthesized PEGDA capsules with 0 mM and sampled at days 1, 7, and 14, the results of which are shown inFIGS. 22A-22G.

Encapsulated MSCs were cultured in spinner flasks at high agitation (125 rpm) and maintained in an incubator at 37° C./5% CO2. The purpose of this experiment was to determine if PEGDA capsules are not only compatible with MSCs, but also with additional cell lines that are relevant to the development of cell therapy products. We have found PEGDA capsules are indeed compatible with adherent cell lines that include AtT-20's and 3T3's, as opposed to Jurkat cell lines grown in suspension. Photos for AtT-20 at Days 7 and 14 were not provided as these studies are ongoing.

Testing was performed to compare the efficiency of PBMC stimulation with soluble CD3/CD28 vs PEGylated CD3/CD28, the results of which are shown inFIGS. 23A-23C. CFSE stained cells were encapsulated in PEGPQ capsules containing soluble PHA/IL2 or soluble CD3/CD28 or PEGylated CD3/CD28. Cells were harvested on day 4 post-stimulation, and CFSE signal was assessed using flow cytometry analysis. No difference in stimulation efficiency was observed between soluble and PEGylated CD3/CD28. PBMC activation was also achieved with PHA/IL2 in the capsule suggesting that stimulation efficiency is not lost during the encapsulation process or in the capsules compare to suspended cells.

Example 16. Viral Particles

Testing was performed in which Lentivirus (LV) particles were conjugated and encapsulated with HEK 293T cells by the process described above with respect toFIGS. 25-29. Results are shown inFIG. 30. RFP (Red fluorescent protein) is the gene construct that is carried by the lentivirus particle (e.g., an expression marker to assess transduction efficiency based on integration of the RFP gene into the host cell HEK293T genome). RFP in this image corresponds to the fluorophore that fluoresces red when excited, such as when the gene is in the cell of interest, which can confirm the occurrence of lentiviral transduction of HEK293T cells with the gene of interest. GFP (green fluorescent protein) in the image corresponds to live cells. (GFP from Calcein AM staining) is a dye used to determine cell viability because it readily permeates intact, live cells. In particular, 2 μL of Calcein was added to 1 mL of media and encapsulated cells prior to imaging using a ZEISS microscope upon which the image ofFIG. 30was collected. In this particular experiment, we were testing the proof of concept that conjugated lentiviral particles could transduce target cells with a gene of interest (RFP) and that, after a period of time, capsules will still contain live cells (GFP stain). We concluded the ability to encapsulate cells and conjugate lentiviral particles successfully inside of PEG capsules, while maintaining the infectivity of the particles by showing successful transduction of encapsulated HEK293T cells with lentivirus using the RFP marker.

REFERENCES