Synthetic matrix for stem cell encapsulation

Abstract

A synthetic hydrogel matrix system combines a PEG component, a crosslinker and a cell-adhesive ligand for stem cell encapsulation and culture. A method is provided for encapsulating and proliferating the stem cells by combining the stem cells within the PEG-based hydrogel matrix system. The stem cells can be further passaged for cryopreservation, continued culture, expansion or directed differentiation.

Description

[0001] SYNTHETIC MATRIX FOR STEM CELL ENCAPSULATION

[0002] RELATED APPLICATIONS

[0003] This application claims the benefit of the priority of U.S. Provisional Application No. 63 / 670,603, filed July 12, 2024, which is incorporated herein by reference in its entirety.

[0004] INCORPORATION OF SEQUENCE LISTING

[0005] The Sequence Listing submitted concurrently herewith as an XML file named "UCSD-24430-2WO_SL.xml", created on 2025-07-14, and having a size of 17KB, is hereby incorporated by reference in its entirety.

[0006] FIELD OF THE INVENTION

[0007] The present invention relates to a synthetic hydrogel matrix system for stem cell encapsulation, and a method for culturing, proliferation and differentiation of the stem cells using the hydrogel matrix system.

[0008] BACKGROUND

[0009] The fields of stem cell biology, neuroscience, tissue engineering, and regenerative medicine, among many others, relies upon the use of human and mammalian induced pluripotent and embryonic stem cells. Both in answering fundamental biological questions as well as for clinically relevant cellular therapies. The method for generating induced pluripotent stem cells (iPSCs) from adult somatic cells was first described by Takahashi and Yamanaka in 2006. This discovery has enabled generating patient-specific stem cell lines to study human development and disease modeling from a highly diverse genetic background. Clinically, it has generated significant interest in developing stem cell therapies based on allogeneic or autologous stem cell sources to treat a variety of diseases, such as neurodegenerative diseases, spinal cord injuries, blindness related to injuries and diseases of the eye, volumetric muscle loss, among many others. One of the primary bottlenecks of translating iPSC technology for commercial and clinical applications are the technical challenges of large scale production.

[0010] Stem cells require a substrate for adherent 2D culture. Traditionally, this has been done using a feeder cell layer comprised of mouse embryonic fibroblasts. This method is still used in the generation and production of iPSCs, where newer techniques have transitioned away from the xenogeneic conditions to using primary human fibroblast lineages as the feeder cell layer. The most widely used acellular substrate for iPSC culture, propagation, and differentiation, such as in organoid model generation, is a commercially available naturally derived extracellular matrix obtained from a decellularized Englebreth- Holm-Swarm mouse sarcoma. The most commonly available commercial products of this material are Matrigel (Coming), GelTrex (ThermoFisher), and Cultrex (R&D Systems). This material will be hereafter referred to as Matrigel for simplicity. Matrigel is composed primarily of basement membrane proteins collagen Type IV, laminin, entactin, and heparan sulfate proteoglycans as well as a cocktail of growth factors. For stem cell culture, these companies greatly reduce (but cannot eliminate) the concentration of the growth factors, which would otherwise induce differentiation. For 2D culture, a thin layer of Matrigel is deposited in the culture dish and allowed to spread to evenly coat it. Matrigel is soluble below 10°C and irreversibly gels above that, a process which can take anywhere from 5-30 minutes depending on the environmental temperature. As such, it needs to be handled relatively quickly and with cooled pipette tips to prevent premature gelation. The plate is then washed with buffer solution and plated with cells. For 3D culture, the Matrigel solution is quickly yet gently mixed with a concentrate of spun down cells and transferred to the dish to typically form a hemispherical dome-shaped construct. For organoid generation, a dilute Matrigel solution is mixed with cells and deposited into specialized aggregate-forming well plates. Matrigel has proven itself to be highly versatile; however, its xenogeneic and biologically derived nature is an inherent limitation for many stem cell applications. There is great interest in a fully synthetic, xenogeneic-free, chemically defined matrix composition for 2D and 3D stem cell culture, expansion, and differentiation applications.

[0011] One such product that has proven successful, though is only applicable for 2D cell culture, is recombinant full-protein laminin and laminin fragments, specifically laminin-511 (commercially known as iMatrix-511). iMatrix-511 can be used in one of two ways: a direct coating for stem cell attachment, similar to Matrigel, or directly mixed in with a stem cell mixture before depositing the cells unto an uncoated dish. After stem cells are passaged, it is typically necessary to supplement their medium with a Rho-kinase inhibitor (ROCKi), as the Rho kinase pathway regulates cell proliferation and migration, which is related to cellcell interactions which are greatly reduced during passaging, triggering apoptosis. The most common ROCKi is a small molecule, Y-27632. It has been shown that when stem cells are mixed with, and thus coated with, iMatrix-511, they can be successfully passaged without the need for adding the ROCKi Y-27632. This was a surprising finding that demonstrated the importance of laminin, specifically its 511 fragment, in regulating stem cell maintenance.

[0012] Bioprinting has enabled the development of physiologically relevant 3D cell culture models to study development, diseases, and drug efficacy and toxicity in vitro. Additionally, bioprinted constructs have enabled novel and efficacious regenerative medicine therapeutic approaches. However, the ability to use iPSCs directly has been elusive as they are highly sensitive to outside stressors and their extracellular matrix environment, both of which typically result in either cell death or uncontrolled differentiation.

[0013] For 3D cell culture, there have been demonstrations of successfully encapsulating stem cells in a synthetic matrix, yet all were only shown to be effective with the inclusion of the ROCKi small molecule Y-27632. There has yet to be developed a synthetic, chemically defined hydrogel matrix for stem cell 3D culture that can be employed without ROCKi supplementation.

[0014] The current state-of-the-art in bioprinting involves spatially patterning primary or differentiated cell types within a 3D hydrogel microenvironment. There is a desire for bioprinting to be used to similarly pattern naive stem cells into precisely designed functional tissues or organoids. Accordingly, the need remains for a synthetic hydrogel design that can overcome the limitations of the current technologies such that stem cells can be encapsulated in a 3D matrix and maintain their viability and sternness until either harvesting or directed differentiation.

[0015] BRIEF SUMMARY

[0016] The inventive hydrogel matrix system provides a viable method to encapsulate human and mammalian stem cells within a polyethylene glycol)-based hydrogel matrix. The inventive hydrogel matrix system provides the culture of human stem cells with long term maintenance, and further allows the stem cells to differentiate into any cell types. The hydrogel system components are modular and are readily interchangeable for optimizing mechanical, physical, and biochemical properties for sternness maintenance, proliferation, spheroid formation, and differentiation into functional tissue.

[0017] The hydrogel matrix system comprises a polyethylene glycol) (PEG) component, a crosslinker, and one or more cell-adhesive ligands, for encapsulation and culture of the stem cells.

[0018] In one aspect, the PEG component is a multi-arm PEG derivative functionalized with norbomene, carboxylic acid, alkyne, diarylcyclooctyne, azide, acryloyl / acrylate, tetrazine, trans-cyclooctene (TCO), acrylamide, methacryloyl / methacrylate, maleimide, macrocyclic cyclodextrins (CDs), cucurbiturils (CBs), crown ethers, pillararenes, calixarenes, hydrazide, thiol, primary amine, N-hydroxysuccinimide (NHS) ester, or para-nitrophenyl carbonate (NPC). The multi-arm PEG derivative can be 4-arm or 8-arm PEG derivative, or other similar functionalized multi-arm polymeric backbone.

[0019] In another aspect, the crosslinker is selected from the group consisting of a linear PEG oligomer, an enzymatically degradable peptide, a non-enzymatically degradable peptide, a multi-arm PEG prepolymer and combinations thereof.

[0020] The crosslinker is difunctionalized with a functional group including norbomene, carboxylic acid, alkyne, diarylcyclooctyne, azide, acryloyl / acrylate, tetrazine, trans- cyclooctene (TCO), acrylamide, methacryloyl / methacrylate, maleimide, macrocyclic cyclodextrins (CDs), cucurbiturils (CBs), crown ethers, pillararenes, calixarenes, hydrazide, thiol, primary amine, N-hydroxysuccinimide (NHS) ester, or para-nitrophenyl carbonate (NPC).

[0021] The crosslinker can be an enzymatically degradable peptide, such as matrix metalloprotease (MMP) degradable peptide having a sequence of KCVPMSMRGGCK (SEQ ID NO: 1), KCGPQGIAGQCK (SEQ ID NO: 2), KCGPQGIWGQCK (SEQ ID NO: 3), KCIPVSLRSGCK (SEQ ID NO: 4), KCRPFSMIMGCK (SEQ ID NO: 5), KCVPLSLTMGCK (SEQ ID NO: 6), KCVPLSLYSGCK (SEQ ID NO: 7), KCIPESLRAGCK (SEQ ID NO: 8), KCSGESPAYYTACK (SEQ ID NO: 9), and combinations thereof. Other enzymatically degradable amino acid sequence relevant for stem cell or somatic cell matrix remodeling can be used as well. The reaction kinetics can be modified by substituting the amino acid C (Cys, cysteine) with a peptide having the sequence XCXX, such as ECEE (Glu-Cys-Glu-Glu) (SEQ ID NO: 10). X can be selected from any other amino acid residues.

[0022] In another aspect, cell-adhesive ligand comprises a synthetic linear or cyclic peptide having a sequence selected from the group consisting of RGD, IKVAV (SEQ ID NO: 11), YIGSR (SEQ ID NO: 12), RRETAWA (SEQ ID NO: 13), and combinations thereof. The cell-adhesive ligand may further include a functional amino acid group or a C- or N- terminus modification including norbornene, carboxylic acid, alkyne, diarylcyclooctyne, azide, acryloyl / acrylate, tetrazine, trans-cyclooctene (TCO), acrylamide, methacryloyl / methacrylate, maleimide, macrocyclic cyclodextrins (CDs), cucurbiturils (CBs), crown ethers, pillararenes, calixarenes, hydrazide, thiol, primary amine, N- hydroxysuccinimide (NHS) ester, or para-nitrophenyl carbonate (NPC) for attachment to the PEG component.

[0023] In yet another aspect, the hydrogel matrix system may also include a photo-initiator for photoinduced polymerization and / or crosslinking. The photo-initiator can be lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP).

[0024] In some embodiments, a method is provided for encapsulating and proliferating stem cells inside of the hydrogel matrix system of claim 1. The method includes the steps of: a) combining dissociated stem cells with the components of the hydrogel matrix systemin a liquid media; b) crosslinking the hydrogel matrix system, wherein the stem cells are encapsulated; c) incubating the stem cells for cell proliferation in a predetermined period of time; and d) removing the stem cells from the hydrogel matrix system.

[0025] The method may also include enzymatically digesting the hydrogel matrix system prior to removing the stem cells.

[0026] In one aspect of the invention, the stem cells are human or mammalian induced pluripotent stem (iPS) cells or embryonic stem (ES) cells.

[0027] In another aspect of the invention, the crosslinking is spontaneous upon combining the dissociated stem cells with the components of the hydrogel matrix system.

[0028] In another aspect of the invention, the crosslinking is by light stimulation (i.e., photoinduced crosslinking, photocrosslinking, photopolymerization), such as a light-based digital light processing system. The light-based digital light processing system provides a light exposure from a LED light source within a wavelength range of 365nm to 405nm.

[0029] In another aspect of the invention, the method further includes the steps of passaging the stem cells for cryopreservation, or continued cell culture and expansion. The method can also include the steps of differentiating the stem cells within the hydrogel matrix system.

[0030] In another aspect of the invention, the stem cells are differentiated into cardiomyocytes cells or neural stem cells using a standardized differentiation protocol.

[0031] In yet another aspect of the invention, the hydrogel matrix system has the stiffness varying from 0.1 kPa to 15 kPa. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] FIG. 1 is a diagram of an embodiment of the inventive hydrogel matrix system design. Figure discloses SEQ ID NOS 1 and 14, respectively, in order of appearance.

[0033] FIG. 2 provides bioprinting parameters and results from experiments characterizing mechanical, biochemical, and enzymatic degradation properties. Figure discloses “FAM- YIGSR” as SEQ ID NO: 19.

[0034] FIG. 3 provides photomicrographs comparing high cell density post-encapsulation human iPSC viability at Day 1 (upper panel) and Day 7 (lower panel). Scale bar is 200 pm. BF is brightfield.

[0035] FIG. 4 provides a set of photomicrographs of immunofluorescence staining showing maintenance of sternness and proliferation for Day 7 post-encapsulation for 0.5 kPa stiffness hydrogel condition with no ROCKi supplementation. Sternness markers: SOX2, NANOG, OCT4A. Proliferation marker: Ki-67.

[0036] FIG. 5 provides results from a DNA quantification assay experiment that was performed on the low cell density and high cell density encapsulation conditions to evaluate the quantified cell count over time, where Day 0 is the day of encapsulation bioprinting. The dotted line indicates cell population doubling threshold.

[0037] FIG. 6 is a diagram of an embodiment of the inventive hydrogel matrix system design for spontaneous crosslinking. Figure discloses SEQ ID NOS 15 and 14, respectively, in order of appearance.

[0038] FIG. 7 provides an exemplary scheme of repeated 3D culture, dissociation, and "3D passaging", whereby "3D passaging" indicates 3D encapsulating iPSCs that were previously cultured in a 3D encapsulated matrix environment. Below the scheme is a set of photomicrographs showing nuclear counterstain DAPI and sternness markers OCT4A and NANOG of human iPSCs that were cultured on a 2D GelTrex-coated well plate after undergoing six rounds of hydrogel encapsulation and “3D passaging”.

[0039] FIG. 8A-8B provide two different 3D in situ differentiation protocols using standardized commercial differentiation kits. FIG. 8A provides a diagram of the commercially standardized cardiomyocyte differentiation protocol, starting from bioprinting human iPSCs, and photomicrographs of immunofluorescence staining at Day 14 post-encapsulation of the cardiomyocyte-specific markers cTNT and NKX2.5 as well as F-actin staining for cell morphology representation. FIG. 8B provides a diagram of the commercially standardized neural stem cell differentiation protocol, starting from bioprinting human iPSCs, and photomicrographs of immunofluorescence staining at Day 8 post-encapsulation of the neural stem cell specific markers PAX6 and Nestin. Additionally, photomicrographs of immunofluorescence staining of sternness markers OCT4A and NANOG are included to confirm the virtually complete differentiation of the encapsulated cells.

[0040] FIG. 9A-9D provide a set of bulk RNA sequencing data performed on human iPSCs cultured conventionally via adherent 2D culture in a plate and 3D cultured within the disclosed matrix formulation, with and without the medium supplement of the ROCK inhibitor small molecule Y-27632. For the 3D culture condition, the iPSCs were collected at post-encapsulation day 1 and 4. FIG. 9A demonstrates the experimental design for the stem cell encapsulation inside of the hydrogel matrix system, propagation and collection process. FIG. 9B provides a heatmap of the top 500 upregulated genes of the 2D cultured iPSCs and compares the normalized counts for these genes among all groups. FIG. 9C provides the expression levels of characteristic pluripotent stem cell (PSC) and somatic (i.e., adult, differentiated) markers of the 2D and 3D encapsulation culture groups. The results clearly show that the 3D-cultured iPSCs, independent of ROCKi inclusion, have no statistical difference in sternness or somatic markers as the conventional 2D-cultured iPSCs. FIG. 9D provides a heatmap of the genes involved in the RHO GTPase Cycle pathway, which is the signaling pathway that the ROCKi small molecule media supplement specifically inhibits. As expected, it is clearly shown that only the Day 1 timepoint of the 3D-cultured iPSCs that were not supplemented with ROCKi had the RHO GTPase Cycle pathway gene expression activated.

[0041] DETAILED DESCRIPTION OF EMBODIMENTS

[0042] A hydrogel matrix system optimized for high-viability stem cell encapsulation and culture is described herein. This system comprises three primary components: (I) a poly(ethylene glycol) (PEG) component, (2) one or more crosslinkers, and (3) one or more cell-adhesive ligands.

[0043] The PEG component is a multi-arm PEG derivative that is functionalized — either uniformly or in a mixed fashion — with reactive groups such as norbornene, carboxylic acid, alkyne, diarylcyclooctyne (e.g., dibenzocyclooctyl (DBCO)), azide, acryloyl or acrylate, tetrazine, trans-cyclooctene (TCO), acrylamide, methacryloyl or methacrylate, maleimide, or host-guest chemistry moieties including macrocyclic compounds like cyclodextrins (CDs), cucurbiturils (CBs), crown ethers, pillararenes, and calixarenes. Additional functional groups may include hydrazide, thiol, primary amine, N-hydroxysuccinimide (NHS) ester, or para-nitrophenyl carbonate (NPC).

[0044] The crosslinker is difunctionalized to react compatibly with the functional groups on the PEG component to form a stable network. The specific chemical makeup and chain length of the crosslinker(s) are determined by the desired mechanical, physical, degradability and degradation rate properties. Suitable crosslinkers include linear PEG oligomers, enzymatically degradable peptides, non-enzymatically degradable peptides, and multi-arm PEG prepolymers (i.e., as a partially polymerized intermediate used to form the final hydrogel structure). Combinations of these crosslinker types may also be employed to tailor mechanical or degradative properties.

[0045] The cell-adhesive ligand is typically a synthetic peptide that promotes cell attachment and viability. Examples include RGD (Arg-Gly-Asp), IKVAV (Ile-Lys-Val-Ala- Val) (SEQ ID NO: 11), YIGSR (Tyr-Ile-Gly-Ser-Arg) (SEQ ID NO: 12), RRETAWA (Arg- Arg-Glu-Thr-Ala-Trp-Ala) (SEQ ID NO: 13), or combinations thereof. These peptides may include a terminal cysteine residue to enable covalent attachment to the PEG network through thiol -reactive chemistry.

[0046] The hydrogel matrix system described herein can be modified in various ways to suit specific applications. The multi-arm PEG derivative may, for example, be a 4-arm or 8- arm PEG structure, providing different network architectures and mechanical properties.

[0047] The crosslinker component may include an enzymatically degradable peptide, such as one that is cleavable by matrix metalloproteinases (MMPs). Representative MMP- sensitive peptide sequences, include:

[0048] • Lys-Cys-Val-Pro-Met-Ser-Met-Arg-Gly-Gly-Cys-Lys (KCVPMSMRGGCK) (SEQ ID NO: 1)

[0049] • Lys-Cys-Gly-Pro-Gln-Gly-Ile-Ala-Gly-Gln-Cys-Lys (KCGPQGIAGQCK) (SEQ ID NO: 2)

[0050] • Lys-Cys-Gly-Pro-Gln-Gly-Ile-Trp-Gly-Gln-Cys-Lys (KCGPQGIWGQCK) (SEQ ID NO: 3)

[0051] • Lys-Cys-Ile-Pro-Val-Ser-Leu-Arg-Ser-Gly-Cys-Lys (KCIPVSLRSGCK) (SEQ ID NO: 4)

[0052] • Lys-Cys-Arg-Pro-Phe-Ser-Met-Ile-Met-Gly-Cys-Lys (KCRPFSMIMGCK) (SEQ ID NO: 5) • Lys-Cys-Val-Pro-Leu-Ser-Leu-Thr-Met-Gly-Cys-Lys (KCVPLSLTMGCK) (SEQ ID NO: 6)

[0053] • Lys-Cys-Val-Pro-Leu-Ser-Leu-Tyr-Ser-Gly-Cys-Lys (KCVPLSLYSGCK) (SEQ ID NO: 7)

[0054] • Lys-Cys-Ile-Pro-Glu-Ser-Leu-Arg-Ala-Gly-Cys-Lys (KCIPESLRAGCK) (SEQ ID NO: 8)

[0055] • Lys-Cys-Ser-Gly-Glu-Ser-Pro-Ala-Tyr-Tyr-Thr-Ala-Cys-Lys

[0056] (KCSGESPAYYTACK) (SEQ ID NO: 9)

[0057] Other enzymatically degradable sequences relevant to stem cell or somatic cell matrix remodeling may also be used.

[0058] To adjust the reaction kinetics, the cysteine (Cys, C) residue can be replaced with a peptide containing the motif XCXX — for example, Glu-Cys-Glu-Glu (ECEE) (SEQ ID NO: 10). In this motif, "X" may be any amino acid residue, allowing for further tuning of degradation behavior and crosslinking dynamics.

[0059] These peptides allow for enzymatic degradation of the hydrogel for cellular matrix remodeling and to facilitate the gentle extraction of the stem cells proliferated in the hydrogel matrix system.

[0060] Additionally, the hydrogel matrix system may include a photoinitiator to enable photoinduced polymerization and / or crosslinking. An example of a suitable photoinitiator is lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP), which is commonly used for visible light-activated crosslinking of cell-laden PEG-based hydrogels.

[0061] An illustrative embodiment of the hydrogel matrix system is depicted in FIG. 1. In this example, the hydrogel is composed of an 8-arm PEG-norbomene macromer (average molecular weight: 40 kDa) serving as the structural backbone, a dithiol (via cysteine) synthetic peptide crosslinker (KCVPMSMRGGCK; Lys-Cys-Val-Pro-Met-Ser-Met-Arg- Gly-Gly-Cys-Lys) (SEQ ID NO: 1) that is enzymatically degradable by matrix metalloproteases (MMPs), and a cell-adhesive peptide ligand, CYIGSR (Cys-Tyr-Ile-Gly- Ser-Arg) (SEQ ID NO: 14), to support cell attachment and viability.

[0062] The method for culturing induced pluripotent stem (iPS) cells and embryonic stem (ES) cells in a xenogeneic-free, chemically-defined three-dimensional (3D) synthetic extracellular matrix involves combining dissociated iPS or ES cells with a liquid medium containing the hydrogel precursors shown in FIG. 1 to form a 3D matrix via crosslinking, thereby encapsulating the cells within the hydrogel network. Once encapsulated, the iPS or ES cells are incubated within the 3D matrix to promote cell proliferation and expansion. After a desired incubation period, the cells can be retrieved from the hydrogel by enzymatic digestion of the PEG-based matrix. The recovered cells may then be cryopreserved, further expanded, or subjected to directed differentiation using commercially available differentiation reagent kits.

[0063] The method may also include in situ differentiation of the encapsulated iPS or ES cells within the 3D matrix. Alternatively, the encapsulated cells may be maintained in an undifferentiated and proliferative state for at least seven days in vitro, supporting flexible downstream applications. This flexibility of stem cell culture and handling enables temporally controlled stem cell differentiation.

[0064] The formulation is optimized for digital light processing (DLP) bioprinting or photopatterning to generate three-dimensional hydrogels for stem cell encapsulation. Suitable DLP bioprinting systems and methodologies are described in U.S. Patents 10,351,819, 10,464,307, and 10,954,489, all assigned to The Regents of the University of California and incorporated herein by reference.

[0065] The hydrogel formulation can be designed for spontaneous crosslinking by combining the PEGmacromer, peptide crosslinker, peptide ligand, and stem cells — enabling in situ formation of a three-dimensional, cell-laden hydrogel matrix (as illustrated in FIG. 6). A variation of this system employs 8-arm PEG-maleimide instead of PEG-norbornene, allowing crosslinking through a thiol-maleimide Michael-type addition reaction. Since this reaction typically occurs within seconds at physiological pH — making it impractical for controlled handling — the reaction kinetics can be moderated by lowering the local pH around the cysteine thiol group. This is achieved by substituting cysteine with a short peptide sequence of the form XCXX, where X represents any amino acid. Based on findings in the scientific literature, glutamic acid (E, Glu) — which contains a carboxylic acid side chain — was selected to create the sequence ECEE (Glu-Cys-Glu-Glu) (SEQ ID NO: 10), effectively reducing the local pKa and slowing the crosslinking rate.

[0066] A non-degradable version of the hydrogel was also evaluated by replacing both the PEG-norbornene and the MMP-sensitive peptide crosslinker with 8-arm PEG-acrylate (40 kDa), forming a photoinduced, non-cleavable hydrogel network. One key advantage of this photo-crosslinking approach is the ability to study an ideal environment for stem cell proliferation and behavior in a 3D matrix environment for an extended period of time (e.g., >14 days). For photocrosslinking, lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) is used as the photoinitiator as it has been thoroughly characterized to be highly cytocompatible at or below 0.5% (w / v) and photocleavable at noncytotoxic light settings (i.e., wavelength and light intensity). For this stem cell bioprinting formulation, LAP was used at a concentration of 0.2% (w / v), activated with an LED light source in the 365-405 nm range at intensities of 10-20 mW / cm2. Hydrogel stiffness was modulated by adjusting the concentration of the 8-arm PEG-norbornene and the light exposure parameters:

[0067] • 0.5 kPa stiffness was achieved using 3% w / v PEG-norbornene,

[0068] • 5 kPa stiffness was achieved using 4% w / v PEG-norbornene.

[0069] These stiffness values are biologically relevant, as 0.5 kPa is optimal for maintaining stem cell viability and 5 kPa supports differentiation toward soft tissue lineages. The YIGSR (Tyr-Ile-Gly-Ser-Arg) (SEQ ID NO: 12) peptide was selected as the cell-adhesive ligand as it is one of the active integrin-binding domains of laminin, a key extracellular matrix protein critical for stem cell maintenance and is one of the primary constituents of Matrigel. Notably, YIGSR (SEQ ID NO: 12) is present in the laminin-511 E8 fragment, which is widely used as a scaffold for routine 2D stem cell passaging and is commercially available. In our system, CYIGSR (SEQ ID NO: 14) is incorporated at a concentration of 100 pg / mL, although this concentration can be readily adjusted for specific applications or optimization needs. This is the first documentation of maintaining mammalian (specifically human) stem cells in a 3D matrix with the only bioactive component being YIGSR (SEQ ID NO: 12).

[0070] The hydrogel matrix system is highly adaptable, allowing substitution or supplementation of YIGSR (SEQ ID NO: 12) with any other thiol-functionalized ligand, either in combination or as an alternative. This is a critical aspect of the invention for optimizing the hydrogel environment for specific differentiation lineages, e.g., for functional in vitro 3D models.

[0071] To finely control cell-mediated remodeling of the hydrogel, a combination of enzymatically degradable and non-degradable dithiol peptide crosslinkers is employed. For proof of concept, human induced pluripotent stem cells (iPSCs) were encapsulated at a density of 40 million cells / ml. This 3D culture method is compatible with a wide range of initial cell densities, provided minimal cell-cell interactions are maintained — something that can be optimized during cell dissociation to promote small embryoid body formation instead of single-cell dispersion. If low density, single-cell encapsulation is desired, the ROCKi small molecule Y-27632 can be supplemented in the culture medium after encapsulation to maintain viability.

[0072] We demonstrated that the 3D hydrogel system supports iPSC viability and maintenance of pluripotency across all tested conditions, including:

[0073] • Non-degradable variant using 8-arm PEG-acrylate,

[0074] • Enzymatically degradable variant,

[0075] • Soft (0.5 kPa) and stiffer (5 kPa) hydrogel formulations,

[0076] • With or without the ROCK inhibitor Y-27632,

[0077] • Low (10M cells / ml) and higher (40M cells / ml) cell densities,

[0078] • As well as combinations of the above parameters.

[0079] Across multiple experimental setups, encapsulated iPSCs remained viable, proliferative, and undifferentiated for at least seven days in vitro. Furthermore, differentiation assays confirmed that encapsulated iPSCs could be readily directed into both cardiomyocyte and neural cell lineages using unmodified commercial differentiation kits and standardized protocol. Importantly, iPSCs could be enzymatically retrieved from the hydrogel, re-plated, and passaged while retaining their stem cell properties and genetic stability.

[0080] Collected data from validation experiments include:

[0081] 1. Immunofluorescence staining of sternness markers OCT4, NANOG, and SOX2 and the proliferation marker Ki-67 in both 2D cultures and 3D encapsulated iPSCs (with and without ROCKi) at Days 1, 4, and 7.

[0082] 2. Bulk RNA sequencing (RNA-seq) data to compare gene expression profiles of 2D controls and 3D-encapsulated iPSCs (with and without ROCKi) across the same time points (Days 1, 4, and 7).

[0083] 3. Molecular karyotyping and immunofluorescence staining data confirming genetic integrity after multiple back-to-back encapsulation, enzymatic dissociation, recovery, and re-encapsulation rounds (described as “3D passaging”).

[0084] 4. Immunofluorescence staining images from in situ 3D cardiomyocyte differentiation collected on Days 21 and 27, showing expected cardiomyocyte-specific markers, and from in situ 3D neural stem cell differentiation collected on Day 8, showing expected neural stem cell markers. Both differentiation protocols were initiated 24 hours after encapsulation in the hydrogel formulation. The inventive approach offers a photocrosslinkable synthetic prepolymer system that is enzymatically degradable and functionally versatile. This system is highly compatible with 3D stem cell culture, which is advantageous over the current commercially available options as this synthetic system is non-xenogeneic, chemically defined, and modular. The stiffness of the resulting 3D-printed hydrogel can be precisely tuned between 0.5 kPa and 5 kPa, while maintaining its structural integrity. Human induced pluripotent stem cells (iPSCs) can be successfully encapsulated within the hydrogel for at least seven days, exhibiting high viability throughout the culture period. The hydrogel environment also supports the maintenance of sternness and proliferative capacity of iPSCs for at least seven days, even in the absence of ROCK inhibitor, although compatibility with ROCK inhibitor is retained if desired. Notably, the encapsulated iPSCs can be directed to undergo lineagespecific differentiation within the 3D matrix using unmodified commercial differentiation kits originally designed for 2D culture — highlighting the translational potential of the system for advanced stem cell applications.

[0085] EXAMPLES: The invention is further illustrated by the following non-limiting examples, which include applications and descriptions of methods used for preparation and testing of materials in accordance with embodiments of the inventive approach.

[0086] Example 1 : Human iPSC Sternness and Proliferation Encapsulated in a 3D Synthetic Matrix The primary formulation of the bioink solution (used to encapsulate human iPSCs cells for 3D printing) contained 2% (w / v) 8-arm poly(ethylene glycol) norbomene (MW=40 kDa), the dithiol MMP-degradable crosslinker KCVPMSMRGGCK (SEQ ID NO: 1), the cell-adhesive ligand peptide with sequence CYIGSR (SEQ ID NO: 14), and the photoinitiator lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) all in IX DPBS.

[0087] Exposure to light ranging from 365 nm to 405 nm can initiate the photocrosslinking of the hydrogel. A 25-second to 50-second exposure of 365-nm light at 30 mW / cm2to 88 mW / cm2can photocrosslink the hydrogel and encapsulate stem cells at a density of 10 million cells / ml to 100 million cells / ml (FIG. 2). In this example, we comprehensively studied a low cell density (10 million cells / ml) and high cell density (40 million cells / ml) condition. The bioink formulation was optimized for encapsulating stem cells that can consistently maintain their sternness by developing a resultant hydrogel with a tunable stiffness of 0.5 kPa to 5 kPa and that displays integrin-binding ligands preferred by stem cells. The human iPSCs were bioprinted to characterize their sternness and proliferation over the course of 7 days (the day of bioprinted encapsulation is considered Day 0). Example 2: In Situ 3D Differentiation of Human iPSCs Encapsulated in a 3D Synthetic

[0088] Matrix

[0089] The objective of this study was to evaluate if standard protocols and / or commercial kits for human iPSC differentiation could be successfully used to differentiate iPSCs encapsulated within the synthetic hydrogel formulation as described in Example 1. Commercial differentiation kits for iPSC differentiation to neural stem cells (NSCs) and cardiomyocytes were used for this evaluation.

[0090] Example 3: Synthetic Matrix Formulation for Spontaneous Encapsulation

[0091] To enable cell encapsulation without the use of a light source, a modified hydrogel formulation has been developed (see FIG. 6). This formulation utilizes a spontaneous thiol— Michael addition reaction. Because conventional thiol-Michael chemistry proceeds within seconds, the reaction is typically too rapid to allow sufficient time for combining cells and reagents, mixing, and transferring the mixture to a culture plate or mold. Such rapid gelation can also lead to hydrogels with heterogeneous mechanical and physical properties. To address this, the formulation incorporates a modified MMP-degradable peptide crosslinker in which the terminal cysteine residues are substituted with the tetrapeptide ECEE (Glu- Cys-Glu-Glu) (SEQ ID NO: 10). This modification increases the local pKa of the cysteine thiol group, thereby reducing the proportion of reactive thiolate species at physiological pH and slowing the reaction kinetics. The resulting gelation time is approximately 60 seconds — sufficient for practical handling and pipetting during bioink preparation and deposition.

[0092] Modified Hydrogel Formulation

[0093] • 2% (w / v) 8-arm poly(ethylene glycol)-maleimide (MW = 40 kDa)

[0094] • MMP-degradable crosslinker: KEECEVPMSMRGGECEEK (SEQ ID NO: 15)

[0095] • Cell-adhesive ligand peptide: CYIGSR (SEQ ID NO: 14) or EECEYIGSR (SEQ ID

[0096] NO: 16) (depending on experimental context)

[0097] • Solvent: l x DPBS

[0098] Preparation Protocol

[0099] 1. Solution A (2x concentration): 8-arm PEG-maleimide in l x DPBS

[0100] 2. Solution B (2x concentration): MMP-degradable crosslinker KEECEVPMSMRGGECEEK (SEQ ID NO: 15) and cell-adhesive ligand (CYIGSR (SEQ ID NO: 14) or EECEYIGSR (SEQ ID NO: 16)) in 1 x DPBS

[0101] For encapsulating induced pluripotent stem cells (iPSCs) or other cell types, gently mix the cells with Solution A, then add an equal volume of Solution B. Mix carefully and transfer the mixture by pipette to a culture plate or dish to form an unconfined, hemispherical 3D hydrogel. Alternatively, the cell-laden solution may be cast into a compatible mold prior to transfer for confined, geometrically defined 3D culture environments.

[0102] Example 4: Materials

[0103] 8-arm polyethylene glycol (PEG) acrylate (PEG-AC) 40 kDa, 8-arm PEG norbornene (PEG- NB) 40 kDa, and 8-arm PEG maleimide (PEG-MAL) 40 kDa were purchased from Creative PEGWorks (North Carolina, USA). CYIGSR (SEQ ID NO: 14), fluorescein-labeled CYIGSR (FAM-CYIGSR (SEQ ID NO: 17)), and matrix metalloproteinase (MMP)-sensitive crosslinkers (KCVPMSMRGGCK (SEQ ID NO: 1), MMP- degradable photocrosslinker; KEECEVPMSMRGGECEEK (SEQ ID NO: 15), MMP-degradable crosslinker; KCGMMPVSRGCK (SEQ ID NO: 18), non-enzymatically degradable scrambled photo-crosslinker) were custom-ordered from GenScript (New Jersey, USA). Lithium phenyl(2,4,6- trimethylbenzoyl)phosphinate (LAP) was purchased from TCI America (Oregon, USA). Sterile IX Dulbecco’s phosphate buffer solution (DPBS), no magnesium, no calcium, was purchased from Thermo Fisher Scientific (Massachusetts, USA).

[0104] Example 5: Cell Culture

[0105] Human pluripotent stem cells (iPSCs) were maintained in either Essential 8 (E8) or E8 Flex medium on Geltrex (Gibco)-coated culture plates, with media changes performed every 24 hours. For 2D passaging and bioprinting preparation, iPSCs were cultured to 90% confluence and were dissociated in Accutase for 5 minutes. For expansion culture passaging, a 1 :6 dilution ratio was used. Cell counting, unless described otherwise, was performed manually using a hemocytometer, where each discernable individual cell was counted as a single cell and each cell aggregate where it was not possible to discern the total number of cells was counted as a single cell for consistency.

[0106] Example 6: Hydrogel Matrix Formulation

[0107] The hydrogel formulation was optimized for stem cell culture. For a completely non- degradable matrix, we employed a 2% (w / v) 8-arm PEG- AC prepolymer solution with 100 pg / ml CYIGSR (SEQ ID NO: 14) and 0.2% (w / v) LAP. For an enzymatically degradable matrix, we employed a 2% to 4% (w / v) 8-arm PEG-NB prepolymer solution with 3 : 1 molar ratio of the MMP-degradable photocrosslinker relative to 8-arm PEG-NB for crosslinking 75% of the PEG-NB arms, 100 pg / ml CYIGSR (SEQ ID NO: 14), and 0.2% (w / v) LAP.

[0108] Example 7: Bioprinting Process

[0109] Glass coverslips were functionalized with methacrylate groups to enhance hydrogel adhesion. During photopolymerization, methacrylate groups on the coverslips reacted with acrylate groups in the hydrogel, ensuring stable attachment. Functionalization was performed by first preparing a 1 : 10 mixture of acetic acid (Cat. #320099-500ML, Sigma- Aldrich) and 100% ethanol (Cat. #459836-lL, Sigma-Aldrich). A solution containing 1.75% (v / v) 3 -(trimethoxy silyl)-propyl methacrylate (TMSPMA) (Cat. #M6514-50ML, Sigma- Aldrich), 10.5% (v / v) acetic acid-ethanol mixture, and 87.75% (v / v) ethanol was prepared in a 50 mL conical tube. Five-millimeter round coverslips were submerged in this solution at a 2: 1 solution-to-coverslip volume ratio and incubated overnight at room temperature on a rocker. The next day, coverslips were sequentially washed twice with ethanol, twice with Milli-Q water, and air-dried before storage under aluminum foil at room temperature.

[0110] Hydrogel layer patterns were designed using Adobe Photoshop and exported as PNG files for compatibility with the bioprinter software. To control the Z-thickness of each layer, polydimethylsiloxane (PDMS) spacers were placed on the methacrylated coverslips. APDMS-coated coverslip was positioned as the top layer of the setup, and the prepolymer solution was pipetted between the spacers. The setup was transferred to a motorized stage, and photocrosslinking was initiated by projecting a light pattern onto the prepolymer solution.

[0111] For cell-laden hydrogel fabrication, the supernatant from an iPSC pellet was diluted to a final concentration of 10 - 40 million cells / mLby gently mixing with an equal volume of 2x concentrated prepolymer solution (1 : 1 dilution). The mixture was immediately transferred to the bioprinting stage to maintain cell viability. Exposure times vary from 25 seconds to 50 seconds, depending on the formulation and desired gel stiffness.

[0112] Example 8: Degradability Testing

[0113] Fluorescently labeled hydrogels (FAM-CYIGSR (SEQ ID NO: 17)) were used for degradation testing. Bioprinted hydrogels were incubated with 4 mg / mL collagenase solution, and fluorescence images were captured every 15 minute using a Leica fluorescence microscope. Example 9: Mechanical Testing

[0114] Nanoindentation was performed to assess the local stiffness of cell monolayers using a nanoindenter (Piuma, Optics 11 Life). The system is integrated with an optical microscope to visualize indentation positions. A soft probe with a small tip was calibrated on glass according to the manufacturer’s protocol before sample measurement.

[0115] Example 10: Cell Viability

[0116] Live / dead staining was performed to assess cell viability. After removing the culture medium, samples were washed twice with DPBS and incubated with a staining solution containing 1 pM calcein AM (live cell stain, Cat. #C3099, Invitrogen) and 2 pM ethidium homodimer-1 (dead cell stain, Cat. #P3566, Invitrogen) at room temperature for 30 minutes. Stained samples were imaged immediately using a Leica DMI 6000B microscope.

[0117] Example 11 : Immunofluorescence staining and imaging 3D passaging

[0118] To evaluate chromosomal stability of stem cells encapsulated in 3D bioprinted hydrogels, hydrogels were degraded using collagenase, and cells were collected by centrifugation.

[0119] The dissociated iPSCs were reprinted using identical bioprinting parameters and cultured for an additional four days. This process was repeated for a total of six passages.

[0120] Karyotyping analysis (BeadChip) was performed to compare cells at passage 1 (Pl) and passage 6 (P6) under 3D culture conditions.

[0121] Example 12: Cardiomyocyte Differentiation

[0122] For cardiomyocyte differentiation, 12-well well plates were coated with Matrigel, and hiPSCs were seeded at a 1 :6 ratio and cultured until reaching 80% confluency. Differentiation was initiated using the iPSC Cardiomyocyte Differentiation Kit (Cat. #A2921201, Thermo Fisher Scientific). Cells were cultured with differentiation medium A for 2 days, followed by differentiation medium B for another 2 days, and then maintained in differentiation maintenance medium for 8 days. After differentiation, cells were purified using RPMI medium without glucose (Cat. #11879020, Thermo Fisher Scientific) supplemented with 4 mM lactate (Cat. #129-02666, Wako Chemicals) for 6 days.

[0123] Example 13: Neural Stem Cell Differentiation

[0124] Human PSCs were differentiated into neural stem cells (NSCs) using the PSC Neural Induction Medium (Thermo Fisher Scientific, A1647801). Cells were cultured in neural induction medium (NIM), composed of 98% Neurobasal Medium (Thermo Fisher Scientific, 21103-049), 2% PSC Neural Induction Supplement (Thermo Fisher Scientific, A1647801), and 0.1% penicillin-streptomycin for 7 days. On day 7, cells were transitioned to neural expansion medium (NEM) for further expansion. NEM consisted of 49% Neurobasal Plus Medium (Thermo Fisher Scientific, A3582901), 49% Advanced DMEM / F- 12 (Thermo Fisher Scientific, 12634010), and 2% PSC Neural Induction Supplement.

[0125] Example 14: Bulk RNA Sequencing

[0126] RNA from 2D and 3D iPSCs were extracted using the Quick RNA Microprep Kit (Zymo Research; #R1050) per manufacturer’s instructions. RNA quantity and quality were analyzed using a NanoDrop 2000 (Thermo Scientific) and 4200 TapeStation (Agilent Technologies), respectively. RNA-seq libraries were generated using poly-A enriched (NEBNext® Poly(A) mRNA Magnetic Isolation Module; NEB; E7490L) samples and the NEBNext® Ultra™ II Directional RNA Library Prep Kit (NEB; E7760L). Sequencing was performed on a NovaSeq X Plus (100 bp paired-end reads, Illumina) at the UCSD Institute for Genomic Medicine (IGM) Core to obtain 25 million reads per sample.

[0127] Example 15: Bulk RNA-seq Analysis

[0128] Adapters were trimmed from FASTQ files using TrimGalore! (Galaxy Version 0.6.3) and reads were mapped to the human reference genome GRCh38.pl 3 using HISAT2 (Galaxy Version 2.2.1). Gene expression levels were quantified using FeatureCounts (Galaxy Version 2.0.1+) to determine counts for each gene and differential expression analysis was carried out between conditions using DESeq2 (Galaxy Version 2.11.40.6) which determines counts and differential expression via the median of ratios method for normalization.

[0129] Principal component analysis (PC A) was performed using DESeq2 (Galaxy Version 2.11.40.6) while hierarchical cluster map analyses were performed using Seaborn (version 0.12.0) and Matplotlib (version 3.6.0) in Python (version 3.8.9). Volcano plots were generated using ggplot2 (Version 3.3.5) with RStudio (running R version 4.1.2). Venn diagrams were generated using VennPainter (Version 1.2.0). Scatter plots depicting DESeq2 normalized count values of genes in each treatment group across replicates were made in GraphPad Prism 10 (Version 10.4.1). Differential expression data generated by DESeq2 was then used to identify significantly differentially expressed genes (DEGs) with normalized counts (median of ratios) >10, a Log2 (FoldChange) > 1 and a false discovery rate (FDR) < 0.05. DEGs were then input into GSEApy (version 1.0.4) to identify pathways as defined by Uniprot, Gene Ontology, Kyoto Encyclopedia of Genes and Genomes (KEGG, Version 99.1), Interpro (Version 86.0) and Simple Modular Architecture Research Tool (Version 9.0) that are significantly differentially expressed. Differentially expressed pathways that were considered statistically significant based upon an FDR < 0.05 were then used to generate a bar chart via Seaborn and Matplotlib which depicts each pathway and its corresponding FDR. Heatmaps of individual pathways identified by GSEApy which are known to be involved in retinal development were then used to generate heatmaps via Seaborn / Matplotlib comparing gene expression with each individual replicate of each treatment group based upon the counts value determined by DESeq2.

[0130] Example 16: Experimental Results

[0131] The MMP-degradable, YIGSR (SEQ ID NO: 12)-presenting 8-arm PEG hydrogel formulation (chemical formulation schematic shown in FIG. 1) was designed and optimized for 3D encapsulation of human and mammalian stem cells. The molecular weight of the 8- arm PEG hydrogel and the molar ratio of 8-arm PEG monomer and crosslinker was optimized for stem-cell appropriate mechanical properties as well as the rate of remodeling. The 8-arm PEG monomer molecular weight was chosen such that each PEG arm had a number-average molecular weight of 5 kDa (i.e., 40 kDa total) in order to achieve the desired low stiffness of 0.5 kPa that has been shown in the literature to be preferable for maintaining sternness and proliferation of stem cells in 3D culture. The MMP-degradable peptide crosslinker was based on a sequence known to be enzymatically degradable by MMP-1, a MMP variant produced by iPSCs. The photo-induced thiol-ene reaction was chosen for the hydrogel crosslinking and conjugation as it is a click chemistry reaction, so the reaction is orthogonal, stoichiometric, and it goes to completion. This resulted in highly homogenous crosslinking and photoconjugation, as is demonstrated visually in FIG. 2, both in the fluorescence image of FAM- YIGSR (SEQ ID NO: 19) showing its homogeneous distribution and in the narrow standard deviation shown in nanoindentation stiffness data, where each n was the average of measurements taken across each region of the hydrogel surface. The C YIGSR (SEQ ID NO: 14) concentration was optimized experimentally first for its homogeneous incorporation throughout the hydrogel and subsequently for stem cell viability, sternness maintenance, and proliferation. Enzymatic degradation of the hydrogel matrix was demonstrated by immersing it in a collagenase solution (FIG. 2). The degradation rate appeared to be independent of the hydrogel stiffness. Based on the optimized prepolymer solution formulation of 500 pM 8-arm PEG (40 kDa), 2.81 mM MMP-1 degradable crosslinker, 100 pg / ml CYIGSR (SEQ ID NO: 14), and 0.2% (m / v) LAP in IX PBS, a stem-cell and soft-tissue physiologically relevant order of magnitude stiffness range of 0.5 kPa to 5 kPa was demonstrated (FIG. 2). The formulation was optimized such that on average, per PEG monomer, 6-7 arms were used for crosslinking and 1-2 arms were used for CYIGSR (SEQ ID NO: 14) conjugation.

[0132] A previously validated human iPSC line was used for stem cell bioprinting assessment. For all bioprinting experiments, unless explicitly stated otherwise, no Rho- kinase inhibitor (ROCKi) was added to the culture medium after encapsulation (i.e., at Day 0). Based on stem cell bioprinting optimization, we used a cell density of 10 million cells per milliliter (lOM / ml) as the low cell density condition and a cell density of 40 million cells per milliliter (40M / ml) as the high cell density condition. Using the high cell density condition, a cell viability staining assay was performed, where iPSC-laden hydrogel constructs were assessed at 1 and 7 days after being bioprinted (FIG. 3). 1 day after bioprinting, the vast majority of iPSCs were viable, and the non-viable (dead) iPSCs were predominantly found in the periphery of the hydrogel construct. After 7 days in vitro, the iPSCs remain viable and appear to have expanded, with approximately less than 10% of cells stained as non-viable. This result is considered excellent for a bioprinted 3D culture. The following experiment assessed the ability for the hydrogel to maintain iPSC sternness and proliferation. As shown in FIG. 4, the encapsulated iPSCs highly expressed key sternness (NANOG, OCT4A, SOX2) and proliferation (Ki-67) markers after 7 days in vitro. Further, the iPSCs morphologically appear to have formed aggregates, which is expected since they were not exposed to ROCKi after encapsulation. Notably, the 0.5 kPa and 5 kPa conditions resulted in similar very high positive staining for both the sternness and proliferation markers. Thus, the hydrogel formulation appears to be highly compatible for maintaining sternness of iPSCs in stiff environments without ROCKi supplementation upon encapsulation. This is notable as the field of stem cell biology has previously shown that stiffness above 1 kPa lead to differentiation (in naturally derived matrices) and that ROCKi is necessary to maintain viability and sternness whenever stem cells are dissociated and passaged, for both 2D and 3D conditions.

[0133] Similar to reported commercial stem cell 3D suspension culture protocols, we observed a doubling rate of approximately 1 day for the low cell density 3D encapsulation condition (FIG. 5). The high cell density condition still expanded in cell number across 7 days in vitro but not to the extent of doubling, which was expected due to the relatively high amount of cell-cell interactions, which inhibits expansion. Notably, we did not need to add ROCK inhibitor small molecule Y-27632 to the initial 3D culture medium to achieve these results. To further demonstrate the viability, genetic stability, and function of the 3D- cultured bioprinted human iPSCs, the cells were “3D passaged” by degrading the matrix with collagenase and bioprinting the collected cells into a new encapsulated 3D hydrogel matrix (FIG. 7). This 3D passaging process was repeated 6 times. After the 6thpassage, a portion of the collected cells were plated using a standard 2D culture in a Geltrex-coated well plate, and the remaining cells were used for molecular karyotyping. The results from both of the 2D-plated culture of the 3D-passaged iPSCs and the molecular karyotyping indicated that there was no deleterious effects from the 3D encapsulation culture environment (FIG. 7). After the 2D culture seeding, ideal iPSC colony formation, morphology, and proliferation were observed. The molecular karyotyping experiment was performed for the standard 2D culture, 1stpassage of 3D culture, and 6thpassage of 3D culture conditions. The results showed that there were no chromosomal abnormalities (i.e., mutations) for any of the three conditions and that the chromosomal molecular signatures were virtually identical for each of the three conditions. These results definitively indicate that the hydrogel formulation in combination with the light-based bioprinting process is highly compatible for the encapsulation and sustained 3D culture of human stem cells with consistent maintenance of expected sternness and proliferation characteristics.

[0134] To further validate the effect on iPSCs of encapsulation within this hydrogel formulation, two different 3D in situ differentiation protocols were performed. To emphasize the universality of this hydrogel environment, standardized commercial differentiation kits were used. The successful differentiation of both cardiomyocytes (FIG. 8A) and neural stem cells (FIG. 8B) were demonstrated.

[0135] A bulk RNA sequencing (RNA-seq) (FIG. 9A) was performed to comprehensively evaluate the similarities and differences of genetic expression levels between human iPSCs cultured under standard 2D, 3D encapsulation without ROCKi supplementation, and 3D encapsulation with ROCKi supplementation conditions. The top 500 upregulated genes in the 2D culture condition were largely conserved throughout the 3D conditions, with the Day 4 time point being most similar (FIG. 9B). This is likely due to allowing the stem cells sufficient time to recover after undergoing the encapsulation bioprinting process. To comprehensively validate their sternness phenotype, the normalized counts of the expression of well-established markers for both pluripotent stem cells and somatic cells (i.e., differentiated) were compared. There were no statistical differences between the expression levels of the 2D and 3D culture conditions (FIG. 9C). This result indicated that the overall sternness of the hydrogel encapsulation 3D culture condition, both with and without ROCKi supplementation, met the accepted standard of the pluripotent stem cell phenotype. To validate that indeed one 3D culture condition was exposed to ROCKi while the other condition was not, the RHO GTPase Cyle pathway heatmap is provided (FIG. 9D). It shows that the Rho kinase pathway was activated only in the Day 1 timepoint of the 3D condition where the ROCKi supplement was not included.

[0136] The inventive hydrogel matrix formulation enables the encapsulation of human or mammalian induced pluripotent stem (iPS) cells or embryonic stem (ES) cells, and while maintaining their undifferentiated state for minimum seven days. During encapsulation, the stem cells also remain proliferative, demonstrating the matrix’s suitability for sustained 3D culture. This matrix system is fully compatible with light-based bioprinting and photopatterning technologies, such as digital micromirror device (DMD)-based systems. Importantly, encapsulated undifferentiated stem cells can be efficiently recovered by enzymatic digestion of the hydrogel (e.g., with collagenase) and subsequently passaged for continued culture.

[0137] The inventive approach represents the first hydrogel system to integrate stem cell viability, proliferation, and recovery with photo-patternable culture capabilities, highlighting its strong commercial potential for applications in regenerative medicine, tissue engineering, and stem cell research.

[0138] Although the inventive platform and techniques have been described in considerable detail with reference to certain preferred embodiments and examples, other modifications and implementations may become apparent to those of skill in the art based on the concepts and teachings provided herein. Accordingly, the scope of the appended claims should not be limited by the foregoing disclosure and description of preferred embodiments but should be construed to include obvious variations.

Claims

CLAIMS:

1. A hydrogel matrix system, comprising a polyethylene glycol) (PEG) component, a crosslinker, and one or more cell-adhesive ligands.

2. The hydrogel matrix system of claim 1, wherein the PEG component is a multi-arm PEG derivative functionalized with norbomene, carboxylic acid, alkyne, diarylcyclooctyne, azide, acryloyl / acrylate, tetrazine, trans-cyclooctene (TCO), acrylamide, methacryloyl / methacrylate, maleimide, macrocyclic cyclodextrins (CDs), cucurbiturils (CBs), crown ethers, pillararenes, calixarenes, hydrazide, thiol, primary amine, N- hydroxysuccinimide (NHS) ester, or para-nitrophenyl carbonate (NPC).

3. The hydrogel matrix system of claim 2, wherein the multi-arm PEG derivative is a 4-arm PEG derivative or an 8-arm PEG derivative.

4. The hydrogel matrix system of claim 1, wherein the crosslinker is selected from the group consisting of a linear PEG oligomer, an enzymatically degradable peptide, a non- enzymatically degradable peptide, a multi-arm PEG prepolymer and combinations thereof.

5. The hydrogel matrix system of claim 1, wherein the crosslinker is difunctionalized with a functional group comprising norbomene, carboxylic acid, alkyne, diarylcyclooctyne, azide, acryloyl / acrylate, tetrazine, trans-cyclooctene (TCO), acrylamide, methacryloyl / methacrylate, maleimide, macrocyclic cyclodextrins (CDs), cucurbiturils (CBs), crown ethers, pillararenes, calixarenes, hydrazide, thiol, primary amine, N- hydroxysuccinimide (NHS) ester, or para-nitrophenyl carbonate (NPC).

6. The hydrogel matrix system of claim 4, wherein the enzymatically degradable peptide is a matrix metalloprotease (MMP) degradable peptide.

7. The hydrogel matrix system of claim 6, wherein the enzymatically degradable peptide having a sequence of KCVPMSMRGGCK (SEQ ID NO: 1), KCGPQGIAGQCK (SEQ ID NO: 2), KCGPQGIWGQCK (SEQ ID NO: 3), KCIPVSLRSGCK (SEQ ID NO: 4), KCRPFSMIMGCK (SEQ ID NO: 5), KCVPLSLTMGCK (SEQ ID NO: 6),KCVPLSLYSGCK (SEQ ID NO: 7), KCIPESLRAGCK (SEQ ID NO: 8),KCSGESPAYYTACK (SEQ ID NO: 9), and combinations thereof.

8. The hydrogel matrix system of claim 7, wherein C (cysteine) of the sequence is optionally substituted with a peptide having a sequence of XCXX, wherein X is another amino acid residue.

9. The hydrogel matrix system of claim 8, wherein the XCXX is ECEE (SEQ ID NO: 10).

10. The hydrogel matrix system of claim 1, wherein the cell-adhesive ligand comprises a synthetic peptide having a sequence selected from the group consisting of RGD, IKVAV (SEQ ID NO: 11), YIGSR (SEQ ID NO: 12), RRETAWA (SEQ ID NO: 13), and combinations thereof.

11. The hydrogel matrix system of claim 1, further comprises a photo-initiator for photoinduced polymerization and / or crosslinking.

12. The hydrogel matrix system of claim 11, wherein the photo-initiator is lithium phenyl- 2,4,6-trimethylbenzoylphosphinate (LAP).

13. A method for encapsulating and proliferating stem cells inside of the hydrogel matrix system of claim 1, the method comprising: a) combining dissociated stem cells with the hydrogel matrix system in a liquid media; b) crosslinking the hydrogel matrix system, wherein the stem cells are encapsulated; c) incubating the stem cells of step b) for cell proliferation in a predetermined period of time; and d) removing the stem cells from the hydrogel matrix system.

14. The method of claim 13, wherein the stem cells are human or mammalian induced pluripotent stem (iPS) cells or embryonic stem (ES) cells.

15. The method of claim 13, further comprising: enzymatically digesting the hydrogel matrix system prior to removing the stem cells.

16. The method of claim 13, wherein the crosslinking is spontaneous.

17. The method of claim 13, wherein the crosslinking is by light stimulation.

18. The method of claim 17, wherein the light stimulation is by a light-based digital light processing system.

19. The method of claim 18, wherein the light-based digital light processing system provides a light exposure from a LED light source within a wavelength range of 365nm to 405nm.

20. The method of claim 13, further comprising: passaging the stem cells for cry opreservation, or continued cell culture and expansion.

21. The method of claim 13, optionally comprising: differentiating the stem cells within the hydrogel matrix system.

22. The method of claim 21, wherein the stem cells are differentiated into cardiomyocytes cells or neural stem cells using a standardized differentiation kit and protocol.

23. The method of claim 13, wherein the predetermined period time is at least 7 days.

24. The method of claim 13, wherein the hydrogel matrix system has the stiffness varying from 0.1 kPa to 15 kPa.

Images