WO2026015902A1 - Matrice synthétique pour encapsulation de cellules souches - Google Patents

Matrice synthétique pour encapsulation de cellules souches

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WO2026015902A1
WO2026015902A1 PCT/US2025/037598 US2025037598W WO2026015902A1 WO 2026015902 A1 WO2026015902 A1 WO 2026015902A1 US 2025037598 W US2025037598 W US 2025037598W WO 2026015902 A1 WO2026015902 A1 WO 2026015902A1
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seq
hydrogel matrix
matrix system
stem cells
cell
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Jacob SCHIMELMAN
Shaochen Chen
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University of California Berkeley
University of California San Diego UCSD
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University of California Berkeley
University of California San Diego UCSD
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/0012—Cell encapsulation
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
    • C08F2/00—Processes of polymerisation
    • C08F2/46—Polymerisation initiated by wave energy or particle radiation
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    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J3/00—Processes of treating or compounding macromolecular substances
    • C08J3/02—Making solutions, dispersions, lattices or gels by other methods than by solution, emulsion or suspension polymerisation techniques
    • C08J3/03—Making solutions, dispersions, lattices or gels by other methods than by solution, emulsion or suspension polymerisation techniques in aqueous media
    • C08J3/075—Macromolecular gels
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
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    • C08J3/00—Processes of treating or compounding macromolecular substances
    • C08J3/24—Crosslinking, e.g. vulcanising, of macromolecules
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B33—ADDITIVE MANUFACTURING TECHNOLOGY
    • B33Y—ADDITIVE MANUFACTURING, i.e. MANUFACTURING OF THREE-DIMENSIONAL [3D] OBJECTS BY ADDITIVE DEPOSITION, ADDITIVE AGGLOMERATION OR ADDITIVE LAYERING, e.g. BY 3D PRINTING, STEREOLITHOGRAPHY OR SELECTIVE LASER SINTERING
    • B33Y10/00—Processes of additive manufacturing
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    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/28—Phosphorus compounds with one or more P—C bonds
    • C07F9/30—Phosphinic acids [R2P(=O)(OH)]; Thiophosphinic acids ; [R2P(=X1)(X2H) (X1, X2 are each independently O, S or Se)]
    • C07F9/307—Acids containing the structure -C(=X)-P(=X)(R)(XH) or NC-P(=X)(R)(XH), (X = O, S, Se)
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
    • C08F32/00—Homopolymers and copolymers of cyclic compounds having no unsaturated aliphatic radicals in a side chain, and having one or more carbon-to-carbon double bonds in a carbocyclic ring system
    • C08F32/08—Homopolymers and copolymers of cyclic compounds having no unsaturated aliphatic radicals in a side chain, and having one or more carbon-to-carbon double bonds in a carbocyclic ring system having two condensed rings
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    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/45—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from artificially induced pluripotent stem cells
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    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/30—Synthetic polymers
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    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/30—Synthetic polymers
    • C12N2533/40—Polyhydroxyacids, e.g. polymers of glycolic or lactic acid (PGA, PLA, PLGA); Bioresorbable polymers
    • C—CHEMISTRY; METALLURGY
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    • C12N2537/00—Supports and/or coatings for cell culture characterised by physical or chemical treatment
    • C12N2537/10—Cross-linking
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/0068—General culture methods using substrates
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0657—Cardiomyocytes; Heart cells

Definitions

  • 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.
  • iPSCs induced pluripotent stem cells
  • 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.
  • Matrigel Coming
  • GelTrex ThermoFisher
  • 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.
  • these companies greatly reduce (but cannot eliminate) the concentration of the growth factors, which would otherwise induce differentiation.
  • 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.
  • 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.
  • 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.
  • 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.
  • ROCKi Rho-kinase inhibitor
  • ROCKi 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.
  • 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.
  • 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.
  • the current state-of-the-art in bioprinting involves spatially patterning primary or differentiated cell types within a 3D hydrogel microenvironment.
  • 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.
  • 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.
  • 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.
  • PEG polyethylene glycol
  • 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.
  • 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.
  • 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).
  • a functional group including norbomene, carboxylic acid, alkyne, diarylcyclooctyne, azide, acryloyl/acrylate, tetrazine, trans- cyclooctene (TCO), acrylamide, methacryloyl/me
  • 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.
  • MMP matrix metalloprotease
  • 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.
  • 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.
  • a functional amino acid group or a C- or N- terminus modification including norbornene, carboxylic acid, alkyne, diarylcyclooctyne, azide, acryloy
  • 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).
  • a method for encapsulating and proliferating stem cells inside of the hydrogel matrix system of claim 1. 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.
  • the method may also include enzymatically digesting the hydrogel matrix system prior to removing the stem cells.
  • the stem cells are human or mammalian induced pluripotent stem (iPS) cells or embryonic stem (ES) cells.
  • the crosslinking is spontaneous upon combining the dissociated stem cells with the components of the hydrogel matrix system.
  • 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.
  • 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.
  • the stem cells are differentiated into cardiomyocytes cells or neural stem cells using a standardized differentiation protocol.
  • the hydrogel matrix system has the stiffness varying from 0.1 kPa to 15 kPa.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • Day 0 is the day of encapsulation bioprinting.
  • the dotted line indicates cell population doubling threshold.
  • 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.
  • 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.
  • 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”.
  • 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.
  • 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.
  • 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. 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.
  • the iPSCs were collected at post-encapsulation
  • 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.
  • PSC pluripotent stem cell
  • somatic i.e., adult, differentiated markers
  • 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.
  • RHO GTPase Cycle pathway which is the signaling pathway that the ROCKi small molecule media supplement specifically inhibits.
  • 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.
  • PEG poly(ethylene glycol)
  • 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).
  • 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.
  • 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.
  • 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.
  • the crosslinker component may include an enzymatically degradable peptide, such as one that is cleavable by matrix metalloproteinases (MMPs).
  • MMPs matrix metalloproteinases
  • 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).
  • X may be any amino acid residue, allowing for further tuning of degradation behavior and crosslinking dynamics.
  • 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.
  • the hydrogel matrix system may include a photoinitiator to enable photoinduced polymerization and/or crosslinking.
  • a photoinitiator is lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP), which is commonly used for visible light-activated crosslinking of cell-laden PEG-based hydrogels.
  • LAP lithium phenyl-2,4,6-trimethylbenzoylphosphinate
  • 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.
  • MMPs matrix metalloproteases
  • CYIGSR Cys-Tyr-Ile-Gly- Ser-Arg
  • 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.
  • the method may also include in situ differentiation of the encapsulated iPS or ES cells within the 3D matrix.
  • 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.
  • the formulation is optimized for digital light processing (DLP) bioprinting or photopatterning to generate three-dimensional hydrogels for stem cell encapsulation.
  • DLP digital light processing
  • 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.
  • 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.
  • 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).
  • lithium phenyl-2,4,6-trimethylbenzoylphosphinate 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).
  • LAP lithium phenyl-2,4,6-trimethylbenzoylphosphinate
  • Hydrogel stiffness was modulated by adjusting the concentration of the 8-arm PEG-norbornene and the light exposure parameters:
  • YIGSR Tetyr-Ile-Gly-Ser-Arg
  • the YIGSR (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.
  • YIGSR SEQ ID NO: 12
  • laminin-511 E8 fragment which is widely used as a scaffold for routine 2D stem cell passaging and is commercially available.
  • 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).
  • 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.
  • a combination of enzymatically degradable and non-degradable dithiol peptide crosslinkers is employed.
  • human induced pluripotent stem cells iPSCs
  • iPSCs human induced pluripotent stem cells
  • 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.
  • the ROCKi small molecule Y-27632 can be supplemented in the culture medium after encapsulation to maintain viability.
  • 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.
  • RNA-seq Bulk RNA sequencing
  • iPSCs Human induced pluripotent stem cells
  • 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.
  • 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.
  • Example 1 Human iPSC Sternness and Proliferation Encapsulated in a 3D Synthetic Matrix
  • 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/cm 2 to 88 mW/cm 2 can photocrosslink the hydrogel and encapsulate stem cells at a density of 10 million cells/ml to 100 million cells/ml (FIG. 2).
  • FOG. 2 FOG. 2
  • 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
  • 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.
  • 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.
  • 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.
  • 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
  • iPSCs induced pluripotent stem cells
  • Solution A 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.
  • the cell-laden solution may be cast into a compatible mold prior to transfer for confined, geometrically defined 3D culture environments.
  • PEG polyethylene glycol
  • PEG-AC polyethylene glycol
  • PEG-NB 8-arm PEG norbornene
  • PEG-MAL 8-arm PEG maleimide
  • CYIGSR SEQ ID NO: 14
  • FAM-CYIGSR SEQ ID NO: 17
  • MMP matrix metalloproteinase-sensitive crosslinkers
  • KCVPMSMRGGCK SEQ ID NO: 1
  • KEECEVPMSMRGGECEEK SEQ ID NO: 15
  • MMP-degradable crosslinker KCGMMPVSRGCK (SEQ ID NO: 18), non-enzymatically degradable scrambled photo-crosslinker
  • Lithium phenyl(2,4,6- trimethylbenzoyl)phosphinate was purchased from TCI America (Oregon, USA).
  • iPSCs Human pluripotent stem cells
  • E8 Essential 8
  • Geltrex Geltrex
  • iPSCs were cultured to 90% confluence and were dissociated in Accutase for 5 minutes.
  • 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.
  • the hydrogel formulation was optimized for stem cell culture.
  • 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.
  • 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.
  • 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.
  • Hydrogel layer patterns were designed using Adobe Photoshop and exported as PNG files for compatibility with the bioprinter software.
  • PDMS polydimethylsiloxane
  • 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.
  • 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.
  • 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
  • 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.
  • 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.
  • Example 11 Immunofluorescence staining and imaging 3D passaging
  • hydrogels were degraded using collagenase, and cells were collected by centrifugation.
  • 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.
  • 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.
  • NSCs neural stem cells
  • NEM neural induction medium
  • NEM neural induction medium
  • NEM neural expansion medium
  • 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.
  • 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® UltraTM 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.
  • IGM UCSD Institute for Genomic Medicine
  • 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.
  • PC A Principal component analysis
  • DEGs significantly differentially expressed genes
  • FDR false discovery rate
  • 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.
  • ROCKi Rho- kinase inhibitor
  • iPSCs 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 iPSCs morphologically appear to have formed aggregates, which is expected since they were not exposed to ROCKi after encapsulation.
  • the 0.5 kPa and 5 kPa conditions resulted in similar very high positive staining for both the sternness and proliferation markers.
  • 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.
  • This 3D passaging process was repeated 6 times. After the 6 th passage, 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, 1 st passage of 3D culture, and 6 th passage of 3D culture conditions.
  • RNA-seq 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.
  • the normalized counts of the expression of well-established markers for both pluripotent stem cells and somatic cells i.e., differentiated
  • 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.
  • DMD digital micromirror device
  • encapsulated undifferentiated stem cells can be efficiently recovered by enzymatic digestion of the hydrogel (e.g., with collagenase) and subsequently passaged for continued culture.
  • 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.

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Abstract

Un système de matrice d'hydrogel synthétique combine un composant PEG, un agent de réticulation et un ligand adhérant aux cellules pour l'encapsulation et la culture de cellules souches. Est prévu un procédé d'encapsulation et de prolifération des cellules souches par combinaison des cellules souches à l'intérieur du système de matrice d'hydrogel à base de PEG. Les cellules souches peuvent en outre être passées pour la cryoconservation, la culture continue, l'expansion ou la différenciation dirigée.
PCT/US2025/037598 2024-07-12 2025-07-14 Matrice synthétique pour encapsulation de cellules souches Pending WO2026015902A1 (fr)

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Citations (5)

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US20110008444A1 (en) * 2008-02-29 2011-01-13 Ipr-Systems Sweden Ab Composition for the formation of gels
US20220089821A1 (en) * 2020-09-24 2022-03-24 University Of Wyoming Hydrogel-encapsulated beta cells, beta-cell encapsulation process, and uses thereof
US20220396792A1 (en) * 2019-10-30 2022-12-15 Nec Solution Innovators, Ltd. Method for producing nucleic acid molecule, biomaterial, and method for producing biomaterial
US20230365940A1 (en) * 2020-11-06 2023-11-16 The Board Of Trustees Of The Leland Stanford Junior University Hydrogel compositions and methods of use thereof
US20240033283A1 (en) * 2020-12-03 2024-02-01 Pmidg, Llc Functionalized and crosslinked polymers

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20110008444A1 (en) * 2008-02-29 2011-01-13 Ipr-Systems Sweden Ab Composition for the formation of gels
US20220396792A1 (en) * 2019-10-30 2022-12-15 Nec Solution Innovators, Ltd. Method for producing nucleic acid molecule, biomaterial, and method for producing biomaterial
US20220089821A1 (en) * 2020-09-24 2022-03-24 University Of Wyoming Hydrogel-encapsulated beta cells, beta-cell encapsulation process, and uses thereof
US20230365940A1 (en) * 2020-11-06 2023-11-16 The Board Of Trustees Of The Leland Stanford Junior University Hydrogel compositions and methods of use thereof
US20240033283A1 (en) * 2020-12-03 2024-02-01 Pmidg, Llc Functionalized and crosslinked polymers

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