Hydrogelated cells for regenerative medicine applications

Abstract

Synthetic polymer networks have been introduced into mammalian cells, rendering them incapable of dividing while retaining cellular activity. A variety of mammalian cell types, useful for example in tissue regeneration and reducing inflammation in mammals, have been generated by the inventors and found to produce a number of desirable growth factors, chemokines, cytokines and other factors, indicating uses in therapy for mammals in need of these factors.

Description

PATENT Attorney Docket No.: 081906-1513146-256110PC Client Reference No.: UC 2025-423-2 Hydrogelated Cells for Regenerative Medicine Applications CROSS-REFERENCES TO RELATED APPLICATIONS

[0001] The present application claims priority to US Provisional Appln. 63 / 694,581, filed September 13, 2024, which is incorporated herein by reference in its entirety. STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

[0002] This invention was made with Government support under Grant No. GM142788, awarded by the National Institutes of Health. The Government has certain rights in the invention. BACKGROUND OF THE INVENTION

[0003] There is a persistent need to develop new therapeutic platforms to serve as the basis for new treatments against advanced chronic or critical diseases. Current therapeutic approaches rely on the use of cells, synthetic materials, or small molecules. Each of these therapies have specific limitations, including an optimal range of functionality, and circumstances where the therapeutic effect is lost. BRIEF SUMMARY OF THE INVENTION

[0004] In some embodiments, the disclosure provides a metabolically-active cell comprising a cross-linked hydrogel within the cell in sufficient amount to prevent cell replication, wherein the cell is a mammalian cell. In some embodiments, the cell has a therapeutic effect when introduced into a mammal or that can be used for cell therapy applications. In some embodiments, the cell is a placental mesenchymal stromal cell (PMSC). In some embodiments, the cell is a stem cell or a progenitor cell. In some embodiments, the stem cell is an induced pluripotent stem cell (iPSC), fetal stem cell, mesenchymal stem cell (MSC) or a hematopoietic stem cell (HSC). In some embodiments, the cell is a primary cell. In some embodiments, the cell is a cancer cell. In some embodiments, the cancer cell is a glioblastoma cell. In some embodiments, the cell is an immune cell. In some embodiments, the immune cell is a macrophage or a T-cell. 1 KILPATRICK TOWNSEND 797856131

[0005] In some embodiments, the cell expresses a heterologous polynucleotide and / or polypeptide.

[0006] In some embodiments, the cell secretes one or more growth factors, cytokines, and chemokines including but not limited to EFG, FGF-2, HGF, IFN alpha, IFN gamma, IL-1 beta, IL1-RA, IL-2, IL-4, IL-6, IL-8, IL-9, IL-13, IL-21, IL-22, IL-23, IL-31, NGF-beta, Rantes, TNF beta and VEGF-D.

[0007] In some embodiments, the cell is a human, murine, or canine cell.

[0008] In some embodiments, the hydrogel comprises monosaccharide or polysaccharide monomer subunits, and wherein the hydrogel is a homopolymer or co-polymer. In some embodiments, the hydrogel comprises substituted or unsubstituted poly(ethylene glycol) monomer subunits. In some embodiments, the hydrogel comprises poly(dimethyl siloxane) (PDMS), poly(ethylene oxide) (PEO), poly(vinyl alcohol) (PVA), poly(propylene fumarate) (PPF), alginate, guanosine mono phosphate (GMP), cyclodextrin (CD), fibrin, collagen, polypeptides, decellularized extracellular matrix, or nucleic acids. In some embodiments, the hydrogel is substituted.

[0009] Also provided is a therapeutic biomaterial derived from metabolically-active cell as described above or elsewhere herein.

[0010] Also provided is a method of making the metabolically-active cell as described above or elsewhere herein. In some embodiments, the method comprises providing a plurality of mammalian cells, introducing a polymerization inducer and monomer units of a hydrogel into the mammalian cells; and causing the polymerization inducer to initiate formation in the cells of a hydrogel formed from the monomer units thereby forming living metabolically- active mammalian cells comprising the hydrogel. In some embodiments, the monomer units are at a concentration of 1-80% or 1-60% (e.g., 30-80%, 50-70%, e.g., 60%) w / w. In some embodiments, the introducing occurs at 20-25° C. In some embodiments, the polymerization inducer is lithium phenyl-2,4,6 trimethylbenzoylphosphinate. In some embodiments, at least 99% (e.g., at least 99.9% or 100%) of the cells comprise the hydrogel. In some embodiments, the monomer units are poly(ethylene glycol) diacrylate.

[0011] Also provided are methods of regenerating tissue in a mammal. In some embodiments, the method comprises administering the metabolically-active cell as described above or elsewhere herein to the mammal, wherein the cells stimulate regeneration of a tissue 2 KILPATRICK TOWNSEND 797856131in the mammal. In some embodiments, the mammal is a human. In some embodiments, the metabolically-active cell secretes FGF-2 and IL-22. In some embodiments, the metabolically- active cell promotes tissue repair, angiogenesis and / or wound healing in the mammal. In some embodiments, the metabolically-active cell secretes IL-RA. In some embodiments, the metabolically-active cell reduces inflammation in the mammal. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] FIG.1A-1D: Production of Cyborg Human Cells. FIG.1A) Cyborg Human Cells as a new class of enhanced therapeutic biomaterials. FIG.1B) Schematic illustrating the process of creating Cyborg Human Cells Through Controlled Intracellular Hydrogelation using PMSCs cells as a starting material (Methods M4). Representative fluorescence microscopy images (n= 3 independent experiments, Scale Bar = 50 µm and 20 µm). FIG. 1C) The untreated, fixed, and cyborg cell populations (PEG-DA; 20, 40 and 60% v / v) were differentiated using flow cytometry and Fluorescein Diacrylate labeling (Methods M21). Average Geometric Mean (n= 3 independent experiments). FIG.1D) Stability of untreated, fixed, and cyborg cells (PEG-DA; 40% v / v) over 6 weeks were quantified by high content confocal microscopy (Methods M6) (n= 3 independent experiments).

[0013] FIG.2A-2F: Cyborg Human Cells Preserve Metabolic Activity, Intracellular Architecture and Organelle Function. FIG.2A) Cyborg Human Cells preserve metabolic activity regardless of the percentage of PEG-DA in the intracellular hydrogel as measured by PrestoBlue HS staining (Methods M7).100 x Fluorescence Microscope Images (n= 3 independent experiments, Scale Bar = 20 µm). FIG.2B) The metabolic activity was quantified as the pixel intensity from fluorescence microscopy images of all cells in the FOV in the channel, averaged and reported as fluorescence (A.U). FIG.2C) Cytoskeleton morphology was preserved after Intracellular Hydrogelation (Methods M8). Cyborg and control cells were stained with AF647 Phalloidin.60 x Confocal Microscopy Images (n= 3 independent experiments, Scale Bar = 100 µm, Zoom = 20 µm). FIG.2D) Radial intensity distribution of AF 647 Phalloidin in Cyborg and Control cells, indicate the preservation of the actin organization when the cells were hydrogelated. Controls show a shift in organization consistent with cell attachment (n = 3 independent experiments). FIG.2E) Golgi dynamic function is preserved in Cyborg Human Cells. TR-Ceramide was used to stain the Golgi apparatus in Cyborg and control cells (Methods M9). Golgi activity was quantified from the resulting fluorescence detected in 60 x Confocal images in cells incubated with TR- 3 KILPATRICK TOWNSEND 797856131Ceramide. Preservation of dynamic function was confirmed by Brefeldin A treatment (BFA; 1 µM) (Methods M9) (n= 3 independent experiments, Scale Bar = 50 µm). FIG.2F) Lysosome dynamic function is preserved after intracellular hydrogelation. Cyborg and control cells were incubated with the pH sensitive fluorescent dye LysoTracker DeepRed to verify lysosomal integrity. Fluorescence intensity quantified from 60 x Confocal images indicate preservation of lysosomal function. Exposure of Cyborg and control cells stained with LysoTracker to H2O2 (10% v / v) resulted in reduced lysosomal activity indicating the preservation of dynamic function of the lysosomes after hydrogelation (n= 3 independent experiments, Scale Bar = 50 µm).

[0014] FIG.3A-3F: Cyborg Human Cells Preserve Protein Expression and Therapeutic Molecule Secretion Capabilities. Cyborg Human Cells derived from stably transfected cell lines are capable of expressing the fluorescent reporters FIG.3A) TdT and FIG.3B) GFP. Cyborg and control cells were cultured under standard conditions (Methods M2; 37°C, 5% CO2) for 7 days to allow the degradation of pre-expressed proteins. Following incubation, the expression of fluorescent reporters were analyzed using high content confocal microscopy. Confocal imaging confirms that Cyborg and untreated control cells express TdT and GFP at similar levels (Methods M5-M6, 20x air objective, n = 4 independent replicates, scale bar = 200 µm. (10x air objective, n = 4 independent replicates, scale bar 100 µm). FIG.3C) Flow cytometry analysis of Cyborg and control cells expressing EGFP 48 hours after transient transfection (n= 3 independent experiments). FIG.3D) Secreted Alkaline Phosphatase (SEAP) can be detected 21 days after transfection in Cyborg and control cells (Methods M11) (n= 3 independent experiments). FIG.3E) Cyborg Human Cells are capable FVIII expression and secretion. Control and Cyborg Cells hydrogelated using 20-60% v / v PEG-DA (Methods M4) were incubated for 7 days with a media change at day 4 and the supernatant was assayed for FVIII through ELISA (Methods M20). Results were normalized to show secretion in ng ml-1 per 1E6 Cells (n = 3 independent replicates). FIG.3F) Secretome analysis of Cyborg Human Cells derived from PMSCs. Control and Cyborg Cells were incubated in growth media under culture conditions (Methods M2; 37°C, 5% CO2), and the supernatant was collected and assayed after 7 days using a 45plex Luminex Panel (Methods M16). The results are normalized to show secretion in pg ml-1 per 1E6 Cells.

[0015] FIG.4A-4G: Cyborg Human Cells Are Resistant to Extracellular Stressors. FIG. 4A) Cyborg Human Cells are Resistant to hypotonic conditions. To assess resistance to osmotic stress, Cyborg Human Cells (Methods M4) and untreated controls, were incubated 4 KILPATRICK TOWNSEND 797856131in solutions with varying ionic strengths (150 mM, 7.5 mM, and 3 mM) for a duration of 12 hours (37°C, 5% CO2; Methods M12). Morphological changes were monitored using phase- contrast and fluorescence microscopy (Methods M5). FIG.4B) Key morphological changes were monitored and recorded to assess the extent of the damage caused by different degrees of osmotic stress. After 12 hours, the metabolic activity of the cells and the nuclear morphology was investigated using PrestoBlue HS and Hoechst 33342 according to the procedure described in Methods M7. All experiments were carried out using µ-Slide 15 Well angiogenesis slides (ibidi USA, Inc.) during incubation and imaging. (n= 3 independent experiments, Scale Bar = 20 µm). FIG.4C) Cyborg Mammalian Cells are resistant to high oxidative stress. Resistance to oxidative stress was evaluated by incubating untreated controls and Cyborg Cells derived from PMSCs, and HEK293 FT cells in an oxidizing environment with 10% hydrogen peroxide (H₂O₂) for up to 12 hours (37°C, 5% CO2; Methods M13). Morphological changes were monitored during the incubation period using phase-contrast and fluorescence microscopy (Methods M5). At every time point during the 12-hour experiment, cellular metabolic activity and nuclei morphology were measured using PrestoBlue HS (FIG.4D) and Hoechst 33342 (Methods M7). (n= 3 independent experiments, Scale Bar = 20 µm). FIG.4E) Cyborg Mammalian Cells can withstand a lethal concentration of staurosporine. To assess cell resistance to apoptotic inducers, untreated controls and Cyborg Cells derived from PMSCs, and HEK293 FT cells were incubated in D10 media supplemented with 10 µM of the apoptotic inducer Staurosporine for up to 12 hours (37°C, 5% CO2; Methods M14). At every time point during the experiment, cellular metabolic activity and nuclei morphology were measured using PrestoBlue HS and Hoechst 33342 (Methods M7). (n= 3 independent experiments, Scale Bar = 20 µm). FIG.4F) The number of cells was quantified and T= 0 and T= 17 hours. All cells in the FOV were quantified, and the cell counts were normalized by the initial cell counts of each condition. (n= 3 independent experiments, 100> cells per experiment). FIG.4G) Cyborg Mammalian Cells are resistant to in vitro hypoxia conditions. To assess cell resistance to a simulated hypoxic environment (low O2, limited nutrients), Cyborg Cells and controls derived from PMSCs, and HEK293 FT cells were incubated in DMEM for up to 72 hours (37°C, 1% O2, 5% CO2) Methods M15. At every timepoint cells were stained with Hoechst 33342 according to the procedure described in Methods M7, and the number of cells was quantified by high content confocal microscopy Methods M6. (n= 4 independent experiments). 5 KILPATRICK TOWNSEND 797856131

[0016] FIG.5A-5D. Cyborg Human Cells as Stable Therapeutic Agents. FIG.5A shows a schematic of the experimental setup for evaluating the stability of Control and Cyborg Cells and their ability to secret human FVIII. FIG.5B) Stability evaluation of control and Cyborg Human Cells in NSG mice. Untreated control or Cyborg Human Cells derived from PMSC cells stably transfected with nanoluc / GFP were injected subcutaneously into both hind limbs of NSG and C57BL / 6J mice (Methods M17). Bioluminescence imaging revealed sustained and localized signal from both control and Cyborg Cell implants throughout the duration of the experiment (n=7 biological replicates). FIG.5C) Stability evaluation of control and Cyborg Human Cells in immunocompetent C57BL / 6J mice. Untreated control or Cyborg Human Cells derived from PMSC cells stably transfected with nanoluc / GFP were injected subcutaneously into both hind limbs of NSG and C57BL / 6J mice (Methods M17). Only Cyborg Human Cells exhibited persistent, localized luminescence at the injection sites over a four-week period. FIG.5B-5C) Implanted cells were monitored weekly for up to 4 weeks by in vivo bioluminescence imaging using a Lago optical imaging system (Methods M18). Total bioluminescence intensity is reported. Baseline intensity was determined by using the same defined area where there is no positive signal in the same animal. n=7 biological replicates. FIG.4D) Evaluation of human FVIII secretion by implanted Control and Cyborg Human Cells in a murine model. Human FVIII concentration in the blood of NSG mice implanted with tdTomato / BDDFVIII nanoluc / GFP expressing cells was evaluated for 4 weeks (Methods M19-M20) (n=7 biological replicates). DEFINITIONS

[0017] Practicing this invention utilizes routine techniques in the field of molecular biology. Basic texts disclosing the general methods of use in this invention include Sambrook and Russell, Molecular Cloning, A Laboratory Manual (3rd ed.2001); Kriegler, Gene Transfer and Expression: A Laboratory Manual (1990); and Current Protocols in Molecular Biology (Ausubel et al., eds., 1994)).

[0018] For nucleic acids, sizes are given in either kilobases (kb), base pairs (bp), or nucleotides (nt). Sizes of single-stranded DNA and / or RNA can be given in nucleotides. These are estimates derived from agarose or acrylamide gel electrophoresis, from sequenced nucleic acids, or from published DNA sequences. For proteins, sizes are given in kilodaltons (kDa) or amino acid residue numbers. Protein sizes are estimated from gel electrophoresis, from sequenced proteins, from derived amino acid sequences, or from published protein sequences. 6 KILPATRICK TOWNSEND 797856131

[0019] Oligonucleotides that are not commercially available can be chemically synthesized, e.g., according to the solid phase phosphoramidite triester method first described by Beaucage and Caruthers, Tetrahedron Lett. 22:1859-1862 (1981), using an automated synthesizer, as described in Van Devanter et. al., Nucleic Acids Res. 12:6159-6168 (1984). Purification of oligonucleotides can be performed using any art-recognized strategy, e.g., native acrylamide gel electrophoresis or anion-exchange high performance liquid chromatography (HPLC), e.g., as described in Pearson and Reanier, J. Chrom.255: 137-149 (1983).

[0020] As used herein, the following terms have the meanings ascribed to them unless specified otherwise.

[0021] The terms “a,” “an,” or “the” as used herein not only include aspects with one member, but also include aspects with more than one member. For instance, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a cell” includes a plurality of such cells, and so forth.

[0022] The term “nucleic acid” or “polynucleotide” refers to deoxyribonucleic acids (DNA) or ribonucleic acids (RNA) and polymers thereof in either single- or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogs of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).

[0023] The term “gene” means the segment of DNA involved in producing a polypeptide chain. It may include regions preceding and following the coding region (leader and trailer) as well as intervening sequences (introns) between individual coding segments (exons).

[0024] A "promoter" is defined as an array of nucleic acid control sequences that direct transcription of a nucleic acid. As used herein, a promoter includes necessary nucleic acid sequences near the start site of transcription, such as, in the case of a polymerase II type promoter, a TATA element. A promoter also optionally includes distal enhancer or repressor 7 KILPATRICK TOWNSEND 797856131elements, which can be located as much as several thousand base pairs from the start site of transcription. The promoter can be a heterologous promoter. In some embodiments, the promoter is a prokaryotic promoter. Typical prokaryotic promoters include elements such as short sequences at the -10 and -35 positions upstream from the transcription start site, such as a Pribnow box at the -10 position typically consisting of the six nucleotides TATAAT, and a sequence at the -35 position, e.g., the six nucleotides TTGACA. In some embodiments, the promoter is a eukaryotic promoter.

[0025] An “expression cassette” is a nucleic acid construct, generated recombinantly or synthetically, with a series of specified nucleic acid elements that permit transcription of a particular polynucleotide sequence in a host cell. An expression cassette may be part of a plasmid, viral genome, or nucleic acid fragment. Typically, an expression cassette includes a polynucleotide to be transcribed, operably linked to a promoter. The promoter can be a heterologous promoter. In the context of promoters operably linked to a polynucleotide, a “heterologous promoter” refers to a promoter that would not be so operably linked to the same polynucleotide as found in a product of nature (e.g., in a wild-type organism).

[0026] As used herein, a first polynucleotide or polypeptide is "heterologous" to an organism or a second polynucleotide or polypeptide sequence if the first polynucleotide or polypeptide originates from a foreign species compared to the organism or second polynucleotide or polypeptide, or, if from the same species, is modified from its original form. For example, when a promoter is said to be operably linked to a heterologous coding sequence, it means that the coding sequence is derived from one species whereas the promoter sequence is derived from another, different species; or, if both are derived from the same species, the coding sequence is not naturally associated with the promoter (e.g., is a genetically engineered coding sequence).

[0027] “Polypeptide,” “peptide,” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues. All three terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers. As used herein, the terms encompass amino acid chains of any length, including full-length proteins, wherein the amino acid residues are linked by covalent peptide bonds.

[0028] The terms “expression” and “expressed” refer to the production of a transcriptional and / or translational product, e.g., of a RNA and / or a nucleic acid sequence encoding a protein. 8 KILPATRICK TOWNSEND 797856131In some embodiments, the term refers to the production of a transcriptional and / or translational product encoded by a gene (or a portion thereof. The level of expression of a DNA molecule in a cell may be assessed on the basis of either the amount of corresponding mRNA that is present within the cell or the amount of protein encoded by that DNA produced by the cell.

[0029] As used herein, the terms “hydrogelation” and “intracellular hydrogelation” are used interchangeably and refer to a process or method of forming a synthetic polymer within a cell. Typically, a synthetic monomer and a polymerization inducer are first added to a cell in a manner that allows them to enter the cell. Then, an initiation signal is applied to the cell such that the inducer is able to cause the formation of the synthetic polymer, i.e., a cross- linked hydrogel. Hydrogelated cells contain cross-linked hydrogel and are unable to replicate but can perform certain cellular processes, such as metabolic pathways, cellular respiration, protein synthesis, motility, etc.

[0030] As use herein, the term “therapeutically effective amount” is an amount of a compound, such as the cells or vaccine of the present disclosure that (i) treats the particular disease, condition, or disorder; (ii) attenuates, ameliorates, or eliminates one or more symptoms of the particular disease, condition, or disorder; (iii) prevents or delays the onset of one or more symptoms of the particular disease, condition, or disorder described herein; (iv) prevents or delays progression of the particular disease, condition or disorder; or (v) at least partially reverses damage caused by the condition prior to treatment.

[0031] Successful “treatment” of a condition according to this disclosure may have any effect that is beneficial to the subject being treated. This includes decreasing severity, duration, or progression of a condition, or of any adverse signs or symptoms resulting therefrom. Treatment may also be unsuccessful, resulting in no improvement in typical signs and symptoms of the condition. The subject has still been “treated” if the intention of the managing clinician has been at least in part the improvement or alteration of a condition referred to. A concurrent objective of therapy is to minimize adverse effects on the target tissue or elsewhere in the treated subject.

[0032] “Metabolically-active” as used herein means that the cells continue cellular activities aside from cellular replication. Examples of cellular activity includes transcription and translation and secretion of growth factors, chemokines, or cytokines as discussed herein. 9 KILPATRICK TOWNSEND 797856131DETAILED DESCRIPTION OF THE INVENTION

[0033] For over 25 years, bioengineers have been modifying cells that exist in two natural living states. The first state comprises cells that replicate continuously or conditionally, undergoing cell cycles while carrying out their functions. The second state encompasses fully differentiated, non-dividing cells with specialized functions, which can be engineered using genetic circuits or coupled to bioelectronic interfaces. However, both natural cell states present long-standing challenges in practical applications.

[0034] As disclosed herein, a distinct “Third” state in cells has been engineered by introducing synthetic polymer networks, referred to herein as “intracellular hydrogelation,” into mammalian cells. The engineered cells are incapable of dividing while retaining cellular activity, i.e., being metabolically active. These engineered cells maintain metabolic functions, protein synthesis, and biomolecule secretion, despite their arrested proliferation. Moreover, these engineered cells exhibit resistance to stressors typically lethal to human cells, including osmotic, apoptotic, and oxidative stress. Importantly, they persist within immune- incompatible hosts, continuing to synthesize biomolecules in vivo. This work opens a new frontier in human cell engineering, leveraging intracellular material-based strategies to modulate mammalian cell function and paves the way for durable and effective cell-based therapies across diverse biomedical applications. A variety of mammalian cell types, useful for example in tissue regeneration and reducing inflammation in mammals, have been generated by the inventors and found to produce a number of desirable growth factors, chemokines, cytokines and other factors, indicating uses in therapy for mammals in need of these factors.

[0035] A new class of cellular therapeutics is therefore provided using controlled intracellular hydrogelation of mammalian cells to achieve semi-living function that can be used as micro therapeutic factories in vitro or in vivo. The approach can be applied for example but not limited to, primary cells as well as stem cells, cancer cells, and immune cells. These new semi-living cells comprising non-lethal intracellular synthetic hydrogel to stabilize the cells, make them resistant to extracellular stressors and prevent their replication. In addition to being resistant to conditions lethal for untreated cells, the cells described herein are metabolically-active, i.e., they preserve their secretion capabilities, protein synthesis and in some cases the capability to produce angiogenesis and tissue regeneration promoting factors. These cells demonstrate remarkable resilience against extracellular stressors commonly encountered in therapeutic contexts, including oxidative environments, hypoxic 10 KILPATRICK TOWNSEND 797856131conditions, osmotic shock, and apoptotic inducers at lethal concentrations, resulting in increased stability both in vitro and in vivo. In some embodiments, in vivo cell clearance due to the degradation of the cells is avoided or reduced by use of the cells described herein in mammals compared to equivalent cells lacking the hydrogels. Applications of the cells described herein can include, but are not limited to, tissue regeneration, revascularization, neuroprotection, angiogenesis, wound healing, disease management, cell-based therapies, immunotherapies, and cancer therapies.

[0036] Synthetic polymer networks have been introduced into mammalian cells, rendering them incapable of dividing while retaining metabolic activity. A variety of mammalian cell types, useful for example in tissue regeneration and reducing inflammation in mammals, have been generated by the inventors and found to produce a number of desirable growth factors, chemokines, cytokines and other factors, indicating uses in therapy for mammals in need of these factors.

[0037] Synthetic polymer networks can be introduced into a variety of mammalian cells, for instance but not limited to as shown in the Examples. Specific mammalian cells that can be used include, but are not limited to, stem cells or progenitor cells, placental mesenchymal stromal cells (PMSCs), cancer cells or immune cells. Exemplary stem cells can include, for example, induced pluripotent stem cells (iPSCs), fetal stem cells, mesenchymal stem cells (MSCs) or hematopoietic stem cells (HSCs). Exemplary cancer cells can include, for example, glioblastoma cells, colon cancer cells, rectal cancer cells, pancreatic cancer cells, stomach cancer cells, esophageal cancer cells, cervical cancer cells, ovarian cancer cells, skin cancer cells, lung cancer cells, oral cancer cells, thyroid cancer cells, non-small cell lung cancer cells, osteosarcoma cells, brain tumor cells, pituitary tumor cells, gallbladder cancer cells, bile duct cancer cells, bladder cancer cells, leukemia cells, peritoneal cancer cells, adrenal cancer cells, spleen cancer cells, tongue cancer cells, small intestine cancer cells, kidney cancer cells, heart cancer cells, eye cancer cells, urethra cancer cells, liver cancer cells, or breast cancer cells. Exemplary immune cells can include, for example, macrophage cells, B-cells, T-cells or natural killer cells.

[0038] In some embodiments, the mammalian cells are primary cells. In some embodiments, primary cells are isolated from a mammal (e.g., a human or other non-human mammal), synthetic polymer networks are introduced in the cells (e.g., as described elsewhere here). In some embodiments, the cells are re-introduced into the mammal wherein 11 KILPATRICK TOWNSEND 797856131the cells are metabolically-active cells comprising a cross-linked hydrogel within the cell in sufficient amount to prevent cell replication.

[0039] In some embodiments, the mammalian cells are cultured cells, e.g., that have been immortalized, or that have been passed through more than 2, 3, 4 or more generations from primary cells.

[0040] The cells described herein (e.g., those having synthetic polymer networks within the cells) can be formulated into pharmaceutical compositions, e.g. further comprising a comprising a pharmaceutically acceptable carrier or excipient. The pharmaceutical composition can additionally contain other therapeutic agents that are suitable for treating or preventing a given disorder. Pharmaceutically carriers can enhance or stabilize the composition, or to facilitate preparation of the composition. Pharmaceutically acceptable carriers include solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible.

[0041] A pharmaceutical composition comprising the cells as described herein can be administered by a variety of methods known in the art. The route and / or mode of administration vary depending upon the desired results. Administration can be intravenous, intramuscular, intraperitoneal, or subcutaneous, or administered proximal to the site of the target. The pharmaceutically acceptable carrier should be suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion).

[0042] The cells can be formulated into pharmaceutically acceptable dosage forms as desired. Dosage regimens can be adjusted to provide the desired response (e.g., a therapeutic response). In determining a therapeutically or prophylactically effective dose, a low dose can be administered and then incrementally increased until a desired response is achieved with minimal or no undesired side effects. For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit contains a predetermined quantity of cells calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. 12 KILPATRICK TOWNSEND 797856131

[0043] Actual dosage levels of the cells in the pharmaceutical compositions can be varied so as to obtain an amount of the cells that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient. The selected dosage level depends upon a variety of pharmacokinetic factors including but not limited to the route of administration, the time of administration, the duration of the treatment, other drugs, compounds and / or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors.

[0044] Mammalian hydrogelated cells can be used for a variety of therapeutic scenarios. As shown here, a variety of mammalian hydrogelated cells have been shown to produce different amounts of various growth factors, chemokines, cytokines and other factors. Accordingly, by implanting the hydrogelated cells into a mammal, the cells will produce these factors and have a therapeutic effect. As a number of the factors shown to be produced by these cells can promoter angiogenesis and / or have tissue regenerative and / or anti-inflammatory effects, the cells can be implanted into mammals in need of angiogenesis and / or tissue regeneration and / or are experiencing inflammation or an inflammatory disease. Exemplary factors produced by the cells described herein can include, but are not limited to, EFG, FGF-2, HGF, IFN alpha, IFN gamma, IL-1 beta, IL1-RA, IL-2, IL-4, IL-6, IL-8, IL-9, IL-13, IL-21, IL-22, IL-23, IL-31, NGF-beta, Rantes, TNF beta and / or VEGF-D. Accordingly, in some embodiments, the hydrogelated cells described herein, producing one or more of these factors is implanted into a mammal that has a therapeutic benefit from the secreted factor(s). Diseases that can benefit from these factors include but not limited to aging (e.g., IL-2, IL-4, HGF, NGF-beta, FGF-2, IL-21, and IL-31 secreted from the cells described herein can be used to ameliorate aspects of aging). Exemplary inflammatory diseases that can be treated with the cells described herein include but are not limited to rheumatoid arthritis, Alzheimer’s disease, multiple sclerosis, and atherosclerosis. Exemplary diseases or conditions that benefit from increased angiogenesis or tissue regeneration can include but are not limited to, diabetic wound healing (FGF-2, HGF, VEGF-D, IL-1 beta secreted from the cells described herein can be used to improve diabetic wound healing) and hind limb ischemia (VEGF-D, FGF-2, HGF, IL-8, Rantes secreted from the cells described herein can be used to ameliorate aspects of hind limb ischemia). In some embodiments, the hydrogelated cells have neuroprotective effects. Exemplary diseases or conditions that benefit from increased neuroprotective effects can include but are not limited to, spinal cord injury and traumatic brain injury (NGF-beta, 13 KILPATRICK TOWNSEND 797856131IL6, HGF, IL4, IL22, IFN gamma secreted from the cells described herein can be used to ameliorate aspects of these injuries).

[0045] The mammalian hydrogelated cells implanted into a mammal can be autologous or allogeneic cells. In some embodiments, the allogenic cells are from the same species as the recipient mammal. In some embodiments, the cells are HLA or MHC-matched to the recipient mammal to reduce or avoid graft-host rejection. The location in the body of the mammal in which the hydrogelated cells are implanted will depend upon the specific disease to be treated or ameliorated. Hydrogelated cells can be implanted for treatment through various methods, each tailored to target specific diseases or tissues. Intravenous (IV) injection delivers stem cells systemically via the bloodstream, making it suitable for conditions affecting multiple organs or autoimmune disorders. Intrathecal injection can be employed to deliver stem cells into the cerebrospinal fluid to treat neurological disorders like spinal cord injuries or neurodegenerative diseases. In direct tissue injection, stem cells can be implanted directly into the damaged area, such as the heart or joints, to promote localized repair. Scaffold implantation involves seeding stem cells onto biodegradable scaffolds, which are then implanted to aid tissue regeneration, particularly in tissue engineering. Lastly, endoscopic delivery can allow for minimally invasive placement of stem cells in specific areas, such as the gastrointestinal or respiratory tract. Each method can be chosen to optimize therapeutic outcomes based on the disease and targeted tissue.

[0046] In some embodiments, the methods will comprise obtaining cells from a donor mammal (e.g., donor human), rendering the cells into hydrogelated cells, and introducing the cells into the recipient mammal. As noted above, the donor and recipient can be the same (autologous) or different (allogeneic).

[0047] Methods of making hydrogelated cells can vary to obtain optimal results. The cells comprising the introduced hydrogel polymer networks lose their ability to replicate (e.g., to divide). Nevertheless, the cells retain metabolic and other cellular activities for a time period (e.g., a period of days), and thus can be used for one or more of their cellular activities without expansion of the cell population. For example, essentially all cellular functions aside from replication will continue to function. Exemplary cellular activity can include but is not limited to, transcription and translation, enzymatic functions, motility, REDOX reactions, homeostasis, and active response to stimuli in the environment. In some embodiments, the cells comprising the hydrogel polymer network will gain additional abilities or functions. For 14 KILPATRICK TOWNSEND 797856131example as described in the Examples, the cells can gain the ability to survive exposure to oxidative stress (e.g., H2O2(10% w / w, 3M) for 3 hours at 37°C).

[0048] Moreover, in some embodiments, the cell can be engineered to express one or more heterologous polynucleotide. In some embodiments, the expressed polynucleotide is an RNA that encodes a polypeptide. Thus, in some embodiments, the cells comprise one or more heterologous polynucleotide (e.g., DNA) that is integrated into its genome or provided on a plasmid or other extrachromosomal vector. In some embodiments, the polynucleotide is operably linked to an endogenous or heterologous promoter that controls expression of the polynucleotide. The promoter can, in some embodiments, be constitutive or inducible. When under an inducible promoter, expression of the gene product can be controlled by exposure of the cells to an agent that induces expression from the inducible promoter. In some embodiments, the cells produce or express one or more anti-cancer molecules, including but not limited to those described in, e.g., Briolay et al., Mol. Cancer.20, 1–24 (2021). In some embodiments, the anti-cancer molecule is a RNA-guided nuclease, which can include but is not limited to Cas9, optionally with one or more guide RNAs.

[0049] The hydrogel polymeric network in the cells can be composed of different hydrogel components as desired. The hydrogel will be formed from monomer subunits that are polymerized once introduced into the cell as discussed in more detail below. The monomer subunits can comprise for example poly- and / or mono-saccharides and / or proteins. As exemplified in the Examples, the hydrogel can be composed of poly (ethylene glycol) monomers of any of a variety of lengths. In other embodiments, the hydrogel can comprise for example, poly(dimethyl siloxane) (PDMS), poly(ethylene oxide) (PEO), poly(vinyl alcohol) (PVA), or poly(propylene fumarate) (PPF). Polymeric networks in cells can also be formed from components such as, but not limited to, alginate, guanosine mono phosphate (GMP), cyclodextrins (CD), fibrin, collagen, polypeptides, decellularized extracellular matrix, and nucleic acids. A discussion of hydrogels can be found in, e.g., Ahmed, Journal of Advanced Research, Volume 6, Issue 2, March 2015, Pages 105-121; Drury JL, Mooney DJ. 2003 Biomaterials.24(24):4337-4351; and Tibbitt MW, Anseth KS.2009. Biotechnol. Bioeng.103(4):655-663.

[0050] The hydrogel monomers can be substituted at one or more side chains, e.g., with a side chain moiety or otherwise conjugated to other molecules, providing further functionality or reactivity of the polymer in the cell. For example, some or all of the monomers of the 15 KILPATRICK TOWNSEND 797856131polymers can be conjugated to, for example, metals, nucleic acids, nanoparticles, peptides, and drugs. Generally, conjugation to the monomers will occur before the monomers are introduced into the cells but in some embodiments, conjugation can occur after the monomer subunits are introduced into the cells. In some embodiments, the conjugation is carried out using activated monomers that can be conjugated to peptides, drugs, enzymes, etc. An activated monomer is a monomer that has been modified with a reactive (electrophilic) group. For example, PEG or other monomers can be modified with aryl chloride residues, reactive acyl groups, or modified with alkylating reagents or optionally can be purchased commercially.

[0051] Cells having an internal hydrogel polymer can be generated by providing cells, introducing hydrogel monomeric subunits and depending on the monomeric subunit, a polymerization inducer (an initiator of polymerization), into the cells and then polymerizing the monomeric subunits in the cells to form the polymer network in the cells. As noted above, any cell can be used as a starting material. Generally, the cells will be cultured such that the cells are actively dividing. As an example, for bacterial cells, they can be cultured so that the cells are in exponential growth phase. Other cells may not have an exponential growth phase, but can nevertheless be exposed to new culture and nutrients such that the cells are in a phase of cell division.

[0052] The monomer subunits and polymerization inducer can be introduced into the cells as desired. In some embodiments, for example, the cells can be exposed to a freeze / thaw cycle to increase the cells’ ability to uptake the monomeric subunits and polymerization inducer. The freezing will be selected to avoid excessive cell death while improving the entry of the monomer subunits. For example, the cells can be “flash’ frozen, e.g., immersed in a liquid below zero degrees C, e.g., at -80 C, for less than five minutes, e.g., 1-3 minutes. In some embodiments, one or more cryoprotectant is incubated with the cells during the freeze / thaw cycle to protect the cells from the process. Exemplary cryoprotectants include, but are not limited to, dimethyl sulfoxide (DMSO) or glycerol. In other embodiments, the monomer subunits can be introduced into the cell by chemical or electrical (e.g., electroporation)-based methods, or methods including but not limited to, e.g., osmotic shock, sonoporation, electroporation, laser, surfactant-based permeabilization, or shearing. Once the monomers are introduced into the cells, the cells can be washed to remove excess monomers on the exterior of the cells. 16 KILPATRICK TOWNSEND 797856131

[0053] The concentration of monomers introduced into the cells can be selected for optimum results and can vary depending on the monomer and resulting polymers used. In some embodiments, the concentration of monomers is 1-80% or 1-60% (e.g., 30-80%, 50- 70%, e.g., 60%) w / w. In some embodiments, the monomers are introduced to the cells at room temperature, e.g., 20-25° C.

[0054] In some embodiments, the monomers are polyethylene glycol (PEG) molecules. As one example, the PEG monomers can be of an average weight of 500-2000 daltons. In some embodiments, the PEG monomers comprise end moieties to assist in polymerization. For example, in some embodiments, the PEG is a diacrylate (e.g., PEG-DA), meaning both ends comprise acrylate moieties. The inventors have found that the concentration of monomer

[0055] In some embodiments, the monomers are sodium alginate, a non-toxic, biocompatible, and biodegradable polysaccharide. The formation of hydrogels from alginate can occur by interactions of the anionic alginates with multivalent inorganic cations through a typical ionotropic gelation method. Exemplary non-limiting divalent cations can include, for example, calcium or magnesium. The monomer and the crosslinker (divalent cation) can be introduced into the cell as desired. In some embodiments, they are introduced together or separately by freezing and thawing, osmotic shock, sonoporation, electroporation, laser, surfactant-based permeabilization, or shearing.

[0056] In some embodiments, the monomers are guanosine. In these embodiments, hydrogelation can occur as self-assembly, e.g., to form a guanosine mono phosphate (GMP) hydrogel. In some embodiments, the hydrogel can be stabilized using hydrazides, aldehydes, or cations such as but not limited to K+, which can also be introduced into the cells. The monomer and the stabilizer can be introduced for example by freezing and thawing, osmotic shock, sonoporation, electroporation, laser, surfactant-based permeabilization, or shearing.

[0057] In some embodiments, the monomers are cyclodextrin (CD), i.e., a cyclic oligosaccharide of glucopyranoside units linked through α-1,4 glycosidic bonds. Once introduced into cells, a CD polymer can be formed. In some embodiments, the polymer is formed via chemical cross-linking, for example by free-radical polymerization cross-linking- based methods; nucleophilic addition / substitution-based methods; cross-linking methods based on ‘click’ reactions and / or incorporation of CDs through post-gelation attachment. The CD monomer and any crosslinkers can be introduced, for example, by freezing and thawing, 17 KILPATRICK TOWNSEND 797856131osmotic shock, sonoporation, electroporation, laser, surfactant-based permeabilization, or shearing.

[0058] In some embodiments, the monomers are fibrinogen (e.g., a glycoprotein 45 nm in length). Once introduced into cells, fibrin polymerization can be initiated by the action of the proteolytic enzyme, thrombin, which can also be introduced into the cells. The monomer and polymerizing enzyme (e.g., thrombin) can be introduced, for example, by freezing and thawing, osmotic shock, sonoporation, electroporation, laser, surfactant-based permeabilization, or shearing.

[0059] In some embodiments, the monomers are collagen. For example, in some embodiments, the collagen is type I collagen subunits (triple-helical protein formed of 67-nm periodic polypeptide chains with a total molecular weight near 300 kDa). Polymerization can be achieved, for example with a mixture of temperature, pH, and ionic strength. In some embodiments, the monomers can be introduced by freezing and thawing, osmotic shock, sonoporation, electroporation, laser, surfactant-based permeabilization, or shearing. In some embodiments, crosslinking factors is controlled, for example, independently of permeation. For example, one can change temperature, ionic strength, and pH of the cell culture medium to induce cross-linking without needing to introduce external factors into the cell.

[0060] In some embodiments such as extracellular matrix (ECM), the monomers variate and their precise composition can depend on the tissue that it is derived from. For example, the composition is a mixture of collagen, glycosaminoglycans, proteoglycans, and ECM proteins. Hydrogel formation can be induced in cells by collagen-based self-assembly, which can also be regulated, for example, by a mixture of temperature, pH, and ionic strength. The ECM components can be introduced by freezing and thawing, osmotic shock, sonoporation, electroporation, laser, surfactant-based permeabilization, or shearing. In some embodiments, crosslinking factors are controlled independently of permeation. For example, one can change temperature, ionic strength, and pH to induce cross-linking without needing to introduce external factors into the cell.

[0061] In some embodiments, the monomers are nucleic acids or nucleotides. Nucleic acid hydrogels can be formed within the cells. In some embodiments, different classes of nucleic acids (e.g., DNA molecules) with different properties and shapes (e.g., X-shaped, Y-shaped or T-shaped DNA molecules). In some embodiments, the nucleic acids are chemically or enzymatically cross-linked to form a hydrogel in the cells. In some embodiments, the nucleic 18 KILPATRICK TOWNSEND 797856131acids and, if included, crosslinking enzymes (e.g., ligases used to polymerize different shapes of DNA), can be introduced, for example, by freezing and thawing, osmotic shock, sonoporation, electroporation, laser, surfactant-based permeabilization, or shearing.

[0062] In some embodiments, the monomers are polydimethylsiloxane-isocyanatomethyl- 3,5,5-trimethylcyclohexyl isocyanate-2-hydroxyethylmethacrylate (PDMS-IPDI-HEMA) and acrylamide. In some embodiments, these components can be polymerized in the cells, for example, by micellar copolymerization (compartmentalization of PDMS-IPDI-HEMA inside SDS or other detergent micelles). PDMS-IPDI-HEMA micelles and acrylamide can be introduced to cells, for example by freezing and thawing, osmotic shock, sonoporation, electroporation, laser, surfactant-based permeabilization, or shearing.

[0063] In some embodiments, the monomers are poly(ethylene oxide) (PEO). The PEO monomers can be any of a variety of weights as desired. In some embodiments, the lengths of the PEO monomers differ. In some embodiments, the PEO monomers can be of average length of 10000-10 million daltons weights (e.g., 35,000; 900,000; or 5,000,000 Da). In some embodiments, the PEO monomers are of different molecular weights (e.g., 35,000; 900,000; and / or 5,000,000 Da). Polymerization of the monomers, once introduced into the cells, can be achieved for example, by γ-irradiation or photo-crosslinking using UV light. PEO monomers can be introduced into cells, for example, by freezing and thawing, osmotic shock, sonoporation, electroporation, laser, surfactant-based permeabilization, or shearing.

[0064] In some embodiments, the monomers are poly(vinyl alcohol) (PVA). Following introduction into the cells, polymerization can be carried out, for example, by low- temperature crystallization. PVA can be introduced into cells, for example, by osmotic shock, sonoporation, electroporation, laser, surfactant-based permeabilization, or shearing.

[0065] In some embodiments, the monomers are poly(propylene fumarate) (PPF). In some embodiments, the monomers are a mixture of PPF and PEG. For example, in some embodiments, a 1:1 w / w mixture of PPF and PEG can be introduced into cells and then formed into a hydrogel with low cytotoxicity. In some embodiments, a polymerization inducer is also introduced into the cell. Exemplary polymerization inducer can include, for example, a benzoyl peroxide initiator mixed with a vinyl monomer, N-vinyl pyrrolidinone. The reaction can be further accelerated with, for example, N,N-dimethyl-p-toluidine. These components can be introduced into the cells to initiate polymerization. Monomers and other components described above can be introduced into the cells, optionally in gradual steps, by 19 KILPATRICK TOWNSEND 797856131freezing and thawing, osmotic shock, sonoporation, electroporation, laser, surfactant-based permeabilization, or shearing.

[0066] Depending on the monomer and polymerization inducer employed, the polymerization inducer can be introduced into the cells with the monomers or in a separate step. For efficiency, it can be helpful to introduce both at the same time. However, in circumstances in which mixture of the monomer and inducer causes significant polymerization before the components can be introduced into the cell together, it can be advantageous to introduce each component separately into the cells. The inducer used will depend on the cross-reaction chemistry involved in linking the monomers in the cell. In some embodiments, the inducer can be selected from, for example, 2-hydroxyl-4′-(2- hydroxyethoxy)-2-methylpropiophenone, 2-hydroxy-4′-(2-hydroxyethoxy)-2- methylpropiophenone (Irgacure 2959), Eosin-Y, and lithium phenyl-2,4,6-tri- methylbenzoylphosphinate.

[0067] Once the monomer subunits and polymerization inducer are introduced into the cells, polymerization can be induced. In embodiments in which the inducer is a photo- inducer, the appropriate wavelength and intensity of light can be exposed to the cells for sufficient time to polymerize the monomer subunits thereby forming a polymer network within the cell. In other embodiments, the inducer can be activated by temperature, or other environmental stimuli, or in some embodiments, simply by being in proximity to the monomer subunits.

[0068] In some embodiments, following polymerization, the cells are exposed to a replication-specific toxin and / or antibiotics that target the replication of cells. This will kill cells that remain capable of replication, thereby enriching cells that comprise the hydrogel polymer and that are no longer capable of replication. Exemplary toxins or antibiotics can include, but are not limited to, carbenicillin, which kills replicated bacterial cells.

[0069] In some embodiments, following polymerization and / or treatment with an antibiotic, the cells are sorted such that hydrogelated cells are isolated from non-hydrogelated cells. In some embodiments, a marker is introduced to the cells to distinguish hydrogelated cells from non-hydrogelated cells. The marker can be useful for assessing the permeation of the hydrogel components into the bacteria and confirm the success of intracellular hydrogelation. In some embodiments, the marker is introduced to the cells before, after, or in the step that hydrogel monomer units are introduced to the cells. In some embodiments, the 20 KILPATRICK TOWNSEND 797856131marker is a fluorescent dye, for example, without limitations, fluorescein O’O–diacrylate (also referred to as “fluorescein diacrylate). In some embodiments, the cells are sorted using fluorescence-activated cell sorting (FACS) using an appropriate channel. For example, as discussed in the Examples below, the incorporation of fluorescein O’O–diacrylate into intracellular hydrogel was detected using the fluorescein (FITC) channel, where the fluorescein is excited at about 498 nm and its emission is detected at about 517 nm. In some cases, the cells comprising fluorescent dye can be analyzed by fluorescence microscopy.

[0070] In some embodiments, in a population of treated cells, before or following antibiotic treatment and / or cell sorting, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% of cells comprise cross-linked hydrogel.

[0071] The resulting cell population will comprise cells having an internal polymeric network that prevents the cells from dividing but that nevertheless retain other biological activity. These cells can then be used in a large variety of biological assays, utilizing the native biological mechanisms of the cell or one or more heterologous activity in the cell resulting from, for example, expression of one or more heterologous polynucleotide in the cells.

[0072] In some embodiments, one can modulate one or more metabolic pathways in the cells. This can be achieved to maximize the funneling of resources towards metabolic routes where cellular resources are necessary therefore maximizing the function of the cells and increasing their life span. In some embodiments, for example, one can inhibit Dihydrofolate reductase (DHFR) and the folate pathway by contacting the cells, for example, by using Trimethoprim. In some embodiments, for example, one can inhibit fatty-acid and lipid synthesis, for example using ACHN-975, Cerulenin, or Platencin. In some embodiments, for example, one can inhibit RNA polymerase and RNA synthesis, for example, using Rifampicin and / or Norfloxacin. In some embodiments, for example, one can inhibit ribosomes and protein synthesis, for example, using Kanamycin. In some embodiments, for example, one can inhibit protein degradation, for example, using Z-LY-CMK and Bortezomib.

[0073] In some embodiments, a biological activity of the cells can be measured, e.g., in response to exposure to one or more agents, expressed gene product, or environmental 21 KILPATRICK TOWNSEND 797856131change. In some embodiments, the cells can be exposed to one or more cell-permeable probes to measure a biological activity. Exemplary cell-permeable probes include but are not limited to, resazurin-based PrestoBlueTM (ThermoFisher Scientific). In other embodiments, one can measure the ATP / ADP ratio of the cells using a protein biosensor. An exemplary protein biosensor can include but is not limited to, Perceval (see, e.g., Tantama, et al., Nat Commun. 2013; 4: 2550). In yet other embodiments, the cells can be measured for proteomic and metabolomic changes in response to exposure to one or more agents, expressed gene products, or environmental experience. EXAMPLES Example 1: Controlled Intracellular Hydrogelation Produces Metabolically Active and Enhanced Mammalian Cells 1.1 Overview

[0074] Mammalian cells were infused with a chemically stable and nondegradable synthetic hydrogel with low biological reactivity. The selected hydrogel chemistry consists of poly(ethylene glycol) diacrylate monomer (PEG-DA; Mn 700) and Lithium phenyl-2,4,6 trimethylbenzoylphosphinate (LAP; Sigma-Aldrich) (1 mg / mL, in DPBS) at 0.1% (w / v) as the photoinitiator. Fluorescein O'O – diacrylate was incorporated as a fluorescent dye to check the permeation of the hydrogelation components into the cells and the success of intracellular hydrogelation. The final protocol for the intracellular hydrogelation of mammalian cells was adapted from a protocol originally intended for bacterial hydrogelation (Contreras-Llano et al.2023, Adv Sci (Weinh) Mar;10(9):e2204175). However, 6 rounds of optimization with different cell lines and hydrogel chemistries were tested to identify an optimal protocol that would work to produce stable and active hydrogelated mamallian cells (Table 1). 1.2. Protocol Summary

[0075] Placental Mesenchymal Stem Cells (PMSC) were seeded in cell treated T-150 flasks at a density of 5000 cells per cm2 using growth media (DMEM supplemented with 5% FBS, 20 ng / mL fibroblast growth factor, 20 ng / mL epidermal growth factor, and 1% penicillin / streptomycin), flasks were incubated at 37 C°, 5% CO2 and media was replaced every 72 hours. When the cells reached 80-90% confluency, growth media was decanted out and the cells were washed with 1x DPBS for 5 min at RT. Immediately after, the washing 22 KILPATRICK TOWNSEND 797856131solution was decanted out and adherent cells were incubated with 1x TrypLE for 10 min at 37 C°, 5% CO2 followed by enzyme inactivation using growth media. Cells in suspension were recovered from the flask and centrifuged at 300g for 5 min to be collected at the bottom of a 50 ml conical tube. Cells were then washed twice using 1x DPBS (300g, 5 min), and the total number of cells was quantified, and their viability assessed using an automated cell counter and 0.4% Trypan Blue Solution.5 e6 cells were collected at the bottom of a 15 ml conical tube (300g, 5 min), discarded the supernatant and resuspended the cells using hydrogelation buffer (Lithium phenyl-2,4,6 trimethylbenzoylphosphinate (1 mg / mL, in DPBS) at 0.1% (w / v), 20-60% (v / v) poly(ethylene glycol) diacrylate (PEG-DA; Mn=700 Da), & 1 mg / mL of Fluorescein), and incubated for 10 min at RT (25C°) protected from light. After incubation, cells were collected at the bottom of the 15 ml conical tube (300g, 5 min), supernatant was discarded, and cells were washed once using 5 ml 1x DPBS (300g, 5 min). Following the washing step, cells were resuspended in 1ml 1x DPBS and transferred to a transparent 12- well plate. Cells in the 12-well plate were irradiated with UV light (wavelength of 365 λ) in an ultraviolet crosslinker for ~3 minutes (831 mJ / cm2). Cells were then recovered from the 12-well plate and washed twice with 1x DPBS in a 15 ml conical tube (300g, 5 min) before being resuspended in growth media for further use. Table 1. Protocol optimization summary for hydrogelation of mammalian cells. The condition highlighted in bold is the experimental variable changed for each optimization round. In total 6 rounds of optimization were necessary to achieve a protocol that would successfully produce intracellularly hydrogelated and active mammalian cells. This table does not include additional modifications unrelated to hydrogelation such as cell fitness, passage number, confluency, disassociation technique, and culture media. Sequence Experiment Cell Temperature PEG- UV Photoactivator Result ⇂ Line Tested DA% Dosage used and Observed t on t on s n / of ells23 KILPATRICK TOWNSEND 797856131RAW hydrogelation 293.9 of all media 5 Protocol modified HEK Heat Shock 4 20, 40 831 LAP 0.1% ( Some Cells n / of e e on ic1.3. Protein Expression and Therapeutical Molecule Secretion Is preserved In Hydrogelated Mammalian Cells

[0076] Intracellularly Hydrogelated Placental Mesenchymal Stem Cells preserve or have an enhanced cytokine, chemokine and growth factor secretion capabilities as quantified by Luminex analysis (Tables 2&3). Moreover, hydrogelated Placental Mesenchymal Stem Cells have a distinct secretion pattern with some of the secreted molecules being detected above the untreated control cells (Table 3). Among the molecules detected in this analysis, the therapeutically relevant FGF-2 is involved in tissue repair and angiogenesis, IL-RA is an anti- inflammatory protein, and IL-22 is involved in tissue repair and regeneration. It was determined that metabolic activity was inversely proportional to the concentration of PEG- DA monomers in the intracellular hydrogel, thereby allowing for a mechanism for regulating activity by using a particular concentration of hydrogel monomer. 1.4. Intracellular Hydrogelation Preserves the Functionality of Mammalian Cells In Challenging Extracellular Environments

[0077] The intracellular hydrogel core created by our optimized protocol confers the cells resistance to osmotic and oxidative stress levels that otherwise kill their untreated counterparts. Additionally, intracellularly hydrogelated cells can tolerate lethal concentrations of Staurosporine (1 µM) that otherwise kill untreated control PMSC cells. Lastly, intracellularly hydrogelated cells are resistant to hypoxic environments (1% O2). Altogether, the increased resistance of intracellularly hydrogelated cells makes them ideal for applications were necrotic, apoptotic or toxic factors are present due to disease or tissue damage, make them ideal options for tissue regeneration, wound healing, and ischemia recovery applications. 24 KILPATRICK TOWNSEND 797856131

[0078] Using essentially the same protocols, glioblastoma cells, murine macrophage-like cells (RAW 264.7) and HEK293FT cells were hydrogelated as described above, and the resulting cells were metabolically active as measured by cell staining, preserved cytoskeleton and Golgi apparatus and protein expression while being resistant to hydrogen peroxide and Staurosporine compared to non-hydrogelated cells.   Table 2: Normalized expression analysis of 45 chemokines, cytokines and growth factors. The analysis was carried out using a Cytokine / Chemokine / Growth Factor Convenience 45-Plex Human ProcartaPlex™ Panel 1 (ThermoFisher Scientific). The panel was analyzed using a Luminex 200 instrument. Secretome data was analyzed using ProcartaPlex Analysis App and Python.H IFN BD EG Eotaxin FG GM- GRO alpha G alph Results Normalized er 100000 cells NF F (CCL11) F-2 CSF (CXCL1) F a 09 07 12 12L- gamma alpha beta 1RA 2 4 IL-5 IL-6 IL-7 (CXCL8) 9 10 24 06 R R12p70 13 15 (CTLA-8) 18 21 22 23 27 31 LIF (CCL2) 74 0625 KILPATRICK TOWNSEND 797856131MIP-1 MIP-1 VE VE alpha beta NGF PDG PlG RANTES SC SDF-1 TNF TNF GF- GF- (CCL3) (CCL4) beta F-BB F-1 (CCL5) F alpha alpha beta A D 38 19 29 O <26 KILPATRICK TOWNSEND 797856131Table 3: Normalized expression analysis of selected chemokines, cytokines and growth factors where hydrogelated PMSCs show a comparable or higher expression when compared to the untreated controls. The analysis was carried out using a Cytokine / Chemokine / Growth Factor Convenience 45-Plex Human ProcartaPlex™ Panel 1 (ThermoFisher Scientific). The panel was analyzed using a Luminex 200 instrument. Secretome data was analyzed using ProcartaPlex Analysis App and Python. F IL- I I G IFN IL-1 1 IL- L L I I I I I I Results Normalized per F- alp alph bet 1R - - L L- L- L- L- L- 31 8. 94 5. 58 19 .9 7 31 .4 7(CCL3) NGF beta (CCL5) TNF beta 6 2 9 9

[0079] Hydrogelated mammalian cells accelerate the healing of “simulated wounds”. Experiments carried out by “scratching” S5Y5 cells in culture creating empty culture space that the cells need to re-populate show that in the presence of intracellularly hydrogelated PMSCs, these “simulated wounds” close faster compared to controls.

[0080] Hydrogelated PMSC cells protect neurons, astrocytes, and microglia from damage from apoptotic agents. Experiments carried out co-culturing neurons, astrocytes, and microglia treated with a lethal concentration of Staurosporine (apoptotic agent), show that the hydrogelated cells protect against damage with this apoptotic agent. Example 2: The Materials and Methods.

[0081] The materials and methods disclosed in this Example relate to Examples 3-7 below. 27 KILPATRICK TOWNSEND 797856131

[0082] M1. Cell Lines: Human Placenta-Derived Mesenchymal Stromal Cells (PMSCs) were isolated from chorionic villus tissue from deidentified, discarded second trimester human placentas and characterized as previously described (56). PMSCs expressing human B Domain Deleted Factor VIII (BDDFVIII) and the fluorescent protein TdTomato (TdT) were stably transfected using a lentiviral construct generated and characterized as previously described (67). PMSCs expressing human BDDFVIII, TdT, NanoLuc (68) (Nluc) and green fluorescent protein (GFP) were produced by stably transfecting BDDFVIII / TdT positive PMSC cells with lentiviral construct pLenti-PalmGRET (Methods M3). All PMSC cell lines were used between passages P3 and P7 for all experiments. Human Embryonic Kidney (HEK) 293 FT Cells were purchased from ThermoFisher® Scientific and were used between passages 5 to 25 in all experiments.

[0083] M2. Mammalian Cell Culture: PMSCs were expanded in T150 tissue culture- treated vented flasks (CorningTM) with D5 media containing Dulbecco’s Modified Eagle’s Medium (DMEM, GibcoTM) with high glucose, 5% fetal bovine serum (FBS, CorningTM), 20 ng / mL recombinant human basic fibroblast growth factor (bFGF, R&D Systems®), 20 ng / mL epithelial growth factor (EGF, R&D SystemsTM), 100 U / mL penicillin and 100 μg / mL streptomycin at 37°C, and 5% CO2 until they reached 90% confluence. For secretome experiments, media without recombinant human basic fibroblast growth factor (bFGF) or epithelial growth factor (EGF) was used. HEK 293FT Cells were expanded in T150 tissue culture-treated vented flasks (CorningTM) using a media composed of DMEM (High Glucose + Glutamine + Sodium Pyruvate, GibcoTM), 10% FBS, 0.1 mM NEAA (Non- Essential Amino acids), 2.0 mM glutamine, 1x pen strep, 3.7 g / L Sodium Bicarbonate, 5 mg / 100 mL Geneticin (G418 Sulfate) and incubated at 37°C, 5% CO2 until they reached 90% confluence. HUVECs were expanded T150 tissue culture-treated vented flasks (Corning®) using Ham’s F-12K (Kaighn) Medium (GibcoTM) supplemented with 10% fetal bovine serum (FBS, CorningTM), 0.1 mg mL-1heparin (Sigma), and 15 mg Endothelial Cell Growth Supplement (ECGS, CorningTM).

[0084] M3. Lentiviral Vector Production: Lentiviral vectors were produced using a second-generation lentiviral packaging system, consisting of the packaging plasmid psPAX2 and the envelope plasmid pMD2.G (Addgene plasmids # 12260 #12259; n2t.net / addgene:12260; RRID:Addgene_12260; n2t.net / addgene:12259;transfer plasmid pLenti-PalmGRET (69; Addgene plasmid # 28 KILPATRICK TOWNSEND 797856131158221; n2t.net / addgene:158221 ; RRID:Addgene_158221) was used to create the PMSCs labeled with GFP and nano luciferase used for in vivo studies.

[0085] Scientific) cells were used as the lentiviral vector production cell line due to their high transfection efficiency. HEK 293FT cells were seeded at a density of 1×105viable cells / cm2in tissue culture treated 24-well plates (Cellvis) using a media composed of DMEM (High Glucose + Glutamine + Sodium Pyruvate, GibcoTM), 10% FBS, 0.1 mM NEAA (Non-Essential Amino acids), 2.0 mM glutamine, 1x pen strep, 3.7 g / L Sodium Bicarbonate, 5 mg / 100 mL Geneticin (G418 Sulfate) and incubated at 37°C, 5% CO2 until the cells reached 60-70% confluence.

[0086] Plasmid DNA was introduced into HEK 293FT cells using Lipofectamine LTX with Plus Reagent (Thermo Fisher Scientific), following the manufacturer’s protocol. Briefly, the transfer plasmid, psPAX2, and pMD2.G were mixed using a 4:3:1 ratio, combined with Lipofectamine LTX and Plus reagent in Opti-MEM medium, and incubated for 20 minutes at room temperature before adding the transfection mixture to the cells. The medium was changed 12–16 hours post-transfection to remove excess transfection reagents and minimize toxicity.

[0087] Viral supernatants were collected at 48- and 96-hours post-transfection, centrifuged at 3000g for 10 minutes to pellet down cell debris and filtered through 0.45 µm syringe filter before aliquoting and storing at -80 °C for further use. Viral titers were estimated by performing a dilution series on HEK293 FT cells followed by flow cytometry analysis to quantify GFP-positive cells. Briefly, serial dilutions of the viral supernatant were applied to HEK293 FT cells in the presence of 8 µg / mL polybrene (Sigma-Aldrich) to enhance transduction efficiency. After 48 hours, cells were washed with PBS, trypsinized, and resuspended in FACS buffer (PBS with 2% FBS). Flow cytometry (CytoFLEX LX, Beckman Coulter) was used to determine the percentage of GFP-positive cells, and viral titers were calculated based on the number of transduced cells per milliliter of viral supernatant.

[0088] All lentiviral preparations were handled under Biosafety Level 2 (BSL-2) conditions in accordance with institutional biosafety guidelines.

[0089] M4. Production of Cyborg Human Cells: Human Embryonic Kidney (HEK 293FT) cells or Placental Mesenchymal Stem Cells (PMSC) were expanded to 90% confluency (Methods M2). Cells were harvested (300g, 5 minutes, 20°C), resuspended in 1× Dulbecco’s Phosphate-Buffered Saline (DPBS; Gibco) and counted to ensure a density of 29 KILPATRICK TOWNSEND 7978561315×106cells mL-1. Cells were then centrifuged (300g, 5 minutes, 20°C) and resuspended in hydrogelation buffer consisting of 1% (w / v) Lithium phenyl-2,4,6- trimethylbenzoylphosphinate (LAP; Sigma-Aldrich), 10–60% (v / v) poly(ethylene glycol) diacrylate (Mn: 700) (PEG-DA; Sigma-Aldrich), and 0.1% (w / v) fluorescein O,O′-diacrylate (fluorescein-DA; Sigma-Aldrich). Cells were resuspended by gently pipetting up and down to ensure homogeneity and the cell suspension was incubated at room temperature (20°C; room temperature (RT)) and protected from light for 10 minutes to allow for passive permeation of the hydrogel components.

[0090] Following incubation, the cells were pelleted down (300g, 5 minutes, 20 °C) in a 15 mL conical tube. The supernatant was then carefully aspirated to remove hydrogelation buffer without disturbing the cell pellet. Cells were resuspended in 1×DPBS and centrifuged again (300g, 5 minutes, 20°C). This washing step was performed twice by gently pelleting down the cells and resuspending them in 1 mL 1×DPBS. The cell suspension was transferred to a sterile 1.5 mL centrifuge tube and intracellular polymerization was triggered by irradiating the samples with UV light (UV-A, 831 mJ cm-2) using an UV crosslinker (Analytikjen, model no. CL-3000L). UV-A was used to trigger polymerization since it emits light outside of the absorbance range of nucleic acids, thus minimizing damage to cellular DNA (70). To verify the correct preparation and functionality of the hydrogelation buffer, 1 mL of hydrogel buffer was aliquoted in a clean 1.5 mL microcentrifuge tube and applied the same UV irradiation conditions as with the cell suspension (UV-A, 831 mJ cm-2).

[0091] Upon completion of UV irradiation, cells were washed once with 1× DPBS (300g, 5 minutes, 20 °C). Finally, the Cyborg Human Cells were resuspended in the appropriate culture medium for each cell type (Methods M2) and maintained under standard culture conditions (37 °C, 5% CO2) for short time storage (10 to 60 min) until further experimentation.

[0092] M5. Fluorescence Microscopy: For high magnification microscopy and cell line development a Nikon Eclipse Ti fluorescence microscope (Nikon Instruments Inc. Melville, NY, USA) with Perfect Focus System 3 was used. The microscope was equipped with an Andor Zyla sCMOS camera and NIS-Elements software. The fluorescence microscope was equipped with 10× / 0.25 Plan air, 40× / 0.95 Plan Apo Lambda air, and 100× / 1.4 Plan Apo Lambda oil objectives. The exposure settings for bright field and fluorescein used in this protocol were as indicated below: GFP / Fluorescein: exposure time of 300 ms. Microscope 30 KILPATRICK TOWNSEND 797856131filter settings: excitation, 450–490 nm; emission, 500–550 nm; dichroic mirror ≥ 495 nm. PrestoBlue / CTC / mCherry / : exposure time 200 ms. Microscope filter settings: excitation, 532–557 nm; emission, 570–640 nm; dichroic mirror ≥ 565 nm. Hoechst 33342 / DAPI: exposure time 100 ms. Microscope filter settings: excitation, 340–380 nm; emission, 435–485 nm; dichroic mirror ≥ 400 nm. Brightfield: exposure time of 56 ms. Emission: 699.5. Cellular metabolic activity was quantified as the pixel intensity from fluorescence microscopy images of all cells in the FOV in the channel, averaged and reported as fluorescence (A.U).

[0093] Image analysis and fluorescence intensity quantification were carried out using the open-source platform for biological imaging analysis Fiji (fiji.sc / cgi-bin / gitweb.cgi / ). Batch image analysis including object (cell) identification, mean object pixel intensity, maximum and minimum object pixel intensity, object area and standard deviation of these values was carried out using an automated macro.

[0094] M6. Confocal Microscopy: To acquire high-resolution images, an Olympus FV3000 laser scanning confocal microscope (Olympus Corporation, Tokyo, Japan) was used. The microscope was equipped with 10× air, 20× air, and 60× oil immersion objectives. The appropriate laser configuration was selected for each fluorophore in our samples from the available 405, 488, 514, 561, and 640 nm lasers. Images were captured using high-sensitivity spectral detectors. Image analysis and fluorescence intensity quantification were performed using Fiji (fiji.sc / cgi-bin / gitweb.cgi / ).

[0095] For high content confocal imaging, timelapse experiments, and continuous tracking of samples in culture conditions a Cytation C10 confocal microscope (Agilent, Santa Clara, CA, USA) was used. The microscope was equipped with a Hamamatsu sCMOS camera (Dynamic Range (dB): 90.37; Pixel Well Saturation Capacity: 30,000), temperature and environmental gas controllers for CO2(0-20%) and O2(1-19%), and GEN5 IPRIME software. The microscope was equipped with 4× and 20× Phase Contrast objectives, and a 60× Plan objective. The microscope was equipped with DAPI, GFP, TRITC, CY5 and CFP confocal filter cubes and with spinning disks with 40 µm and 60 µm pinhole sizes. Image analysis, pixel intensity quantification, cell quantification and tracking were carried out using the GEN5 IPRIME software (Version 3.16).

[0096] M7. Assessing metabolic activity of Cyborg Human Cells: To assess, visualize, and compare the metabolic activity of cyborg mammalian cells intracellularly hydrogelated with varying percentages of PEG-DA (Methods M4) against untreated controls, the non- 31 KILPATRICK TOWNSEND 797856131toxic resazurin-based reagent PrestoBlue HS (ThermoFisher Scientific) was used. Upon entering metabolically active cells, resazurin is reduced to resorufin, a highly fluorescent compound (Ex: 560 nm; Em: 590), in response to cellular redox reactions. The fluorescence intensity correlates directly with the metabolic activity of the cells, providing insight into their viability and physiological state post-hydrogelation. This well-established (71, 71), fluorescence and colorimetric assay enables real-time monitoring of cellular redox activity via fluorescence or confocal microscopy (Methods M5 & M6) or plate reader measurements (BioTek Synergy).

[0097] After hydrogelation (Methods M4), resuspended around 1x105Cyborg and untreated control cells were resuspended in 270 µl of media appropriate for mammalian cell culture (Methods M2). The cells were incubated with 30 µl of room temperature PrestoBlue HS (1:10 dilution). After a 10-minute incubation at 37°C, fluorescence intensity was measured using a plate reader (Ex: 560 nm; Em: 590; BioTek Synergy) in a 96-well plate (150 µl / well). In parallel, 150 µl of the PrestoBlue treated cells was spined down in a 1.5 ml microcentrifuge tube (300g, 5 min, RT), the resulting pellet was resuspended gently in 1x DPBS by gently pipetting up and down. This washing step was repeated twice before the cells were resuspended in 20 µM Hoechst 33342 (ThermoFisher Scientific; nuclear counterstain for cell detection and laser autofocus) diluted in 1x DPBS. After a 20-minute incubation at RT, cells were plated in a 96-well plate (150 µl / well) with a glass-like polymer coverslip bottom (Cellvis). Confocal or fluorescence microscopy was performed, and pixel intensity was analyzed for all detected objects in each image (Methods M4 & M5).

[0098] For the experiments detailed in Methods M11, M12, M13 & M14 the procedure was modified to allow for the assessment of the metabolic activity with minimal disruption of the experimental conditions. The hydrogelated cells and controls incubated under the conditions described below (Methods M11, M12, M13 & M14) using µ-Slide 15 Well angiogenesis slides (ibidi USA, Inc.) were treated with 5.5 µl of PrestoBlue HS (1:10 dilution) and Hoechst 33342 to a final concentration of 20 µM. After a 20 minutes incubation under experimental conditions, confocal or fluorescence microscopy was performed, and pixel intensity was analyzed for all detected objects in each image (Methods M4 & M5).

[0099] M8. Assessment of Cytoskeleton Morphology preservation: The preservation of the cytoskeleton in Cyborg Cells derived from PMSCs and HEK293 FT cells was investigated by using the filamentous actin (F-actin) binding fluorescent Alexa Fluor dye 32 KILPATRICK TOWNSEND 797856131conjugate AF647-Phalloidin (ThermoFisher Scientific; excitation, 650 nm; emission, 668 nm). This far-red F-actin stain binds to both large and small actin filaments with similar affinity in a 1:1 stoichiometry between phallotoxin and actin subunits. The reaction allows for the labeling, identification and quantification of F-actin filaments. Cyborg Cells (Methods M4) and untreated controls were incubated in D10 growth media supplemented with 10 µg mL-1of AF647-Phalloidin under culture conditions (37°C, 5% CO2) for 16 hours in µ-slide 4- well IbiTreat (ibidi USA, Inc). Cyborg Cells and controls were treated with Hoechst 33342 according to the procedure described before (Methods M7) 15 hours into the incubation. Cells were imaged using an Olympus FV3000 laser scanning confocal microscope (Olympus Corporation, Tokyo, Japan). Morphological changes were analyzed and the pixel intensity corresponding to the fluorescence of the AF647-Phalloidin stain was quantified and compared to the intensity of the untreated controls (Methods M6).

[0100] M9. Assessment of Golgi Apparatus Morphology preservation: The Golgi apparatus plays a critical role in protein and lipid trafficking, processing, and sorting within the cell. TR-Ceramide is a fluorescent lipid analog that integrates into the Golgi membrane, where its accumulation serves as a strong indicator of Golgi functionality and integrity in addition to providing knowledge on the overall efficiency of vesicular transport. TR- Ceramide stains the Golgi by exploiting the natural lipid processing pathways within the cell. Ceramides are synthesized in the endoplasmic reticulum (ER) and transported to the Golgi, where they are metabolized into complex sphingolipids. TR-Ceramide, being a fluorescent analog of natural ceramides, follows this same trafficking pathway and accumulates in the Golgi membrane, resulting in a perinuclear fluorescent signal. After generation of Cyborg Human Cells from (Methods M4), cells were stained with a 5 µM solution of TR-ceramide +10 µM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) at 4°C for 30 min, followed by one rinse with ice cold 1x Hanks' balanced salt solution (HBSS) and a final incubation HBSS at 37°C for 30 min. The intensity and distribution of fluorescence (Methods M5) can be used to assess Golgi activity, morphology, and trafficking efficiency. After visualization, cells were treated with 1 µM BFA for 2 hours to investigate Cyborg Mammalian Cell susceptibility to disruption. BFA disrupts vesicular trafficking by inhibiting ARF1 GTPase, leading to Golgi disassembly and a corresponding loss of TR-Ceramide fluorescence. This allows for a direct assessment of Golgi integrity and its ability to maintain structural organization under stress conditions. 33 KILPATRICK TOWNSEND 797856131

[0101] M10. Assessment of Lysosome Morphology preservation: Lysosomes are essential for cellular homeostasis, playing key roles in protein degradation, membrane turnover, and autophagy. LysoTracker Deep Red (ThermoFisher Scientific) is a fluorescent dye that selectively stains acidic organelles and lysosomes, due to its protonated amine group. The intensity and distribution of LysoTracker fluorescence provide a direct measure of lysosomal acidity and functional activity. After generation of Cyborg Mammalian Cells from (Methods M4) Cyborg Mammalian Cells are stained with. To compare the ability of Cyborg Cells to resist extracellular stressors, cells were rinsed once with 1X HBSS before being incubated in a 50 nM solution of LysoTracker in HBSS for 30 min at 37°C under standard cell culture conditions (5% CO₂) to maintain physiological pH and cellular activity. After rising once with HBSS, the cells were visualized using (Methods M5). Lastly, to evaluate the ability of Cyborg Human Cells to resist oxidative stress, the cells were treated with 500 µM hydrogen peroxide in HBSS. Hydrogen peroxide induces oxidative stress, which can damage lysosomes. The Cyborg Human Cells were incubated with H₂O₂ at 37°C for 1 hour under standard cell culture conditions, then rinsed with fresh HBSS and immediately re-stained with LysoTracker Deep Red following the same staining procedure described above.

[0102] M11. Transient protein production: Lipofectamine LTX with Plus Reagent (Thermo Fisher Scientific) was used to transiently transfect plasmid DNA into HEK 293FT cells according to the manufacturer’s protocol. Briefly, plasmids expressing either EGFP (pCDNA3-EGFP) or SEAP (pCMV-SEAP) (both under CMV promoters) were combined with Lipofectamine LTX and Plus reagent in Opti-MEM medium and incubated for 20 minutes at room temperature. Then, the transfection mixture was added to the cells. The medium was changed 12–16 hours post-transfection to remove excess transfection reagents and minimize toxicity. Plasmid pcDNA3-EGFP is as described in Addgene plasmid #13031; n2t.net / addgene:13031; RRID:Addgene_13031. Plasmid CMV-SEAP is as described inRRID:Addgene_24595. Cells were then cultured according to the methods described in (Methods M2). EGFP production was quantified using fluorescence microscopy imaging (Methods M5) and SEAP production was quantified using the Phospha-Light™ SEAP Reporter Gene Assay System (ThermoFisher) following the manufacturers protocol. Briefly, 200 µL of cell culture supernatant was extracted and proceeded to spin it down to precipitate any cell debris (500g, 5 min). Then, 50 µL of supernatant was mixed with 1X Dilution Buffer at the bottom of a 1.5 mL microcentrifuge tube and incubated the mixture for 30 min at 65°C in a thermoblock. 34 KILPATRICK TOWNSEND 797856131Immediately after, the tube was placed on ice and after 5 minutes 50 µL of the diluted sample was added into a well of a 96-well plate (Corning). To each well, 50 µL of Assay Buffer was added and mixed well by pipetting up and down and incubated for 5 min at RT. Then, 50 µL of Reaction Buffer was added per well and incubated for 20 min at RT. Immediately after, the luminescence intensity (450 nm) of each well was measured using a plate reader (Tecan Infinite m1000) using and integration time of 500 ms with no settle time and no attenuation. Cells were maintained in culture without passaging, tracking cell density through microscopy using the methods described in (Methods M5) for normalization. Untreated (WT) cells were not passaged during this process, and allowed to reach confluency, with regular media extractions and replacement every 3 days. Supernatant samples were stored at -80°C until measurement

[0103] M12. Assessing resistance of Cyborg Human Cells to osmotic stress: To assess the resistance of Cyborg Human Cells derived from PMSCs and HEK293 FT cells (Methods M4) and untreated controls to osmotic stress, cells were incubated in solutions with varying ionic strengths (150 mM, 7.5 mM, and 3 mM) for a duration of 12 hours (37°C, 5% CO2). These concentrations were selected to represent isotonic (1× DPBS; 150 mM; NaCl 137 mM, KCl 2.7 mM, Na₂HPO₄ 8.1 mM, and KH₂PO₄ 1.5 mM), hypotonic (1:20 dilution of 1× DPBS; 7.5 mM; NaCl 6.85 mM, KCl 0.135 mM, Na₂HPO₄ 0.405 mM, and KH₂PO₄ 0.075 mM), and extreme hypotonic (1:50 dilution of 1× DPBS; 3 mM; NaCl 2.74 mM, KCl 0.054 mM, Na₂HPO₄ 0.162 mM, and KH₂PO₄ 0.03 mM) environments, allowing for a comprehensive evaluation of cellular responses to osmotic pressure variations. Throughout the incubation period, morphological changes were monitored using phase-contrast and fluorescence microscopy (Methods M5). Key morphological parameters such as cell swelling, membrane blebbing, detachment, and lysis were systematically recorded to quantify the extent of the damage caused by different degrees of osmotic stress. After 12 hours, the metabolic activity of the cells and the nuclear morphology was assessed using PrestoBlue HS and Hoechst 33342 according to the procedure described before (Methods M7). All experiments were carried out using µ-Slide 15 Well angiogenesis slides (ibidi USA, Inc.) during incubation and imaging. This approach aimed to determine whether the cyborg modifications confer enhanced resilience to osmotic fluctuations compared to their untreated counterparts.

[0104] M13. Assessing resistance of Cyborg Human Cells to high oxidative stress: The resistance of Cyborg Human Cells to oxidative stress was assessed. by incubation in an 35 KILPATRICK TOWNSEND 797856131environment with 10% hydrogen peroxide (H₂O₂) for up to 12 hours (37°C, 5% CO2). This concentration was selected to induce significant oxidative stress, enabling the evaluation of cellular resilience and potential protective effects conferred by the intracellular hydrogel structure present in the Cyborg Mammalian Cells.

[0105] In this experiment, hydrogelated cells derived from PMSCs and HEK293 FT cells (Methods M4) and controls were prepared and resuspended in DMEM (with no phenol red) and 10% H₂O₂. Morphological changes were monitored during the incubation period using phase-contrast and fluorescence microscopy (Methods M5). Key indicators of oxidative damage, such as membrane blebbing, cytoplasmic shrinkage, and nuclear condensation, were systematically recorded. At every time point during the 12-hour experiment, cellular metabolic activity and nuclei morphology were measured using PrestoBlue HS and Hoechst 33342 following the procedure detailed in Methods M7. For every timepoint, experimental condition, and biological replicate, a different well in the µ-Slide 15 Well angiogenesis slide was stained and imaged.

[0106] M14. Assessing resistance of Cyborg Human Cells to Staurosporine: To assess resistance to apoptosis caused by extracellular apoptotic inducers, Cyborg Human Cells and untreated controls were exposed to 10 µM Staurosporine (Sigma-Aldrich), a broad-spectrum kinase inhibitor known to induce apoptosis in a wide range of mammalian cell types. Cells were incubated in phenol red–free DMEM containing staurosporine at 37°C in 5% CO₂ for 6 hours.

[0107] Hydrogelated cells were derived from PMSCs and HEK293FT cells (Methods M4), and all samples were processed in parallel. Morphological changes were monitored using fluorescence microscopy (Methods M5), with cell count and key apoptotic markers such as: membrane blebbing, nuclear fragmentation, and cytoplasmic condensation systematically recorded over time.

[0108] At defined time points, cell metabolic activity was assessed using PrestoBlue HS, while cell counts, nuclear morphology and integrity were evaluated via Hoechst 33342 staining, following the procedure outlined in Methods M7. For each condition, time point, and biological replicate, individual wells in a µ-Slide 15 Well angiogenesis slide were independently stained and imaged to ensure consistency and avoid cross-contamination.

[0109] M15. Assessing resistance of Cyborg Human Cells to hypoxic conditions: To evaluate the ability of Cyborg Human Cells to withstand prolonged hypoxic stress, 36 KILPATRICK TOWNSEND 797856131hydrogelated and non-hydrogelated control cells were cultured under 1% O₂ conditions for 72 hours and continuously monitored using a Cytation C10 high content confocal microscope (Agilent, Santa Clara, CA, USA) (Methods M6). Cells were seeded in cell culture treated 96- well plates (Nunc) using phenol red–free DMEM (Gibco) and maintained at 37 °C in 5% CO₂ and 1% O₂ to mimic chronic hypoxia. Conditions were maintained using the temperature and environmental gas controllers of the Cytation C10 confocal microscope (Methods M6).

[0110] Cyborg Cells were derived from PMSCs and HEK293 FT cells (Methods M4), and all samples were treated in parallel. Morphological changes were monitored every 24 hours using high content confocal microscopy (Methods M6) using automated cell count through Hoechst 33342 nuclear staining (Methods M7).

[0111] M16. Secretome analysis: Cyborg and control cells were resuspended in growth media, seeded in a T-150 cell culture treated vented flask at the same cellular density and incubated under culture conditions (37°C, 5% CO2) for 7 days with a complete growth media change at day 4. All collected supernatants were briefly centrifuged (5 min, 500g, RT) and filtered using a 0.45 µm syringe filter before aliquoting them into 1.5 mL microcentrifuge tubes and storing them at -80 °C until further analysis. Samples were thawed on ice right before analysis and all supernatants were processed and data was analyzed according to the protocol described by the manufacturer for supernatant samples. Secretome profile was analyzed using a 45plex Luminex Panel (ProcartaPlex Human Cytokine / Chemokine / Growth Factor Convenience Panel 1; ThermoFisher Scientific) and a Luminex 200 detection system.

[0112] Raw fluorescence intensity data were initially processed using the ProcartaPlex App (Software available through the Thermo Fisher Scientific cloud-based platform Connect), which facilitated standard curve generation, background correction, and preliminary concentration calculations based on manufacturer-supplied calibration standards. Further data analysis, including normalization, statistical evaluation, and visualization, was performed using Python (Python 3.9.16). Statistical analyses included normalization across samples, outlier detection, and comparative analysis between experimental groups to identify significant changes in secretome composition over time.

[0113] M17. In vivo stability analysis: All animal procedures were approved by The University of California, Davis (UCD) institutional animal care and use committee (IACUC). All facilities used during the study period were accredited by the Association for the 37 KILPATRICK TOWNSEND 797856131Assessment and Accreditation of Laboratory Animal Care International (AAALAC). NSG (NOD / SCID / IL2Rγ− / −, 4-week-old) immunodeficient mice, and (C57BL / 6 J, 8-week-old) were used in this study and were purchased from The Jackson Laboratory. Cyborg Human Cells (20% PEG-DA) were derived from nanoluc / GFP labeled PMSCs 812 using the procedure described in Methods M4. Cells were resuspended in D10 media and mixed with 30% Matrigel (BD Biosciences). Cells were injected subcutaneously to both limbs on each mouse.5 × 105nanoluc / GFP-labeled PMSCs were used in all experiments.

[0114] M18. Bioluminescence Imaging: Cells transplanted in NSG and C57BL / 6J mice were monitored by in vivo bioluminescence imaging using a Lago optical imaging system (Spectral Instruments Imaging). At designated timepoints animals were injected intraperitoneally with Nano-Glo Fluorofurimazine In vivo Substrate (FFz; Promega) (0.2875 µmoles per animal diluted in cold DPBS) and maintained under anesthesia with 3% inhaled isoflurane for 10 min before imaging. The NSG and C57BL / 6J mice were imaged at the day of transplantation and weekly after transplantation for 4 weeks. Images were analyzed by using Aura Imaging Software (Spectral instruments Imaging). Total intensity was measured within a defined area of the signal. Baseline intensity was determined by using the same defined area where there is no positive signal in the same animal.

[0115] M19. In vivo factor replacement model: All animal procedures were approved by The University of California, Davis (UCD) institutional animal care and use committee (IACUC). All facilities used during the study period were accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care International (AAALAC). NSG (NOD / SCID / IL2Rγ− / −, 4-week-old) immunodeficient mice were used in this study and were purchased from The Jackson Laboratory. Cyborg Human Cells (40% PEG-DA) were derived from PMSCs expressing tdTomato / BDDFVIII and nanoluc / GFP using the procedure described in Methods M4. Cells were resuspended in D10 media and mixed with 30% Matrigel (BD Biosciences). Cells were injected subcutaneously to both limbs on each mouse. 5 × 105tdTomato / BDDFVIII nanoluc / GFP expressing PMSCs were used in each injection site.

[0116] 90 µl of blood was collected per mice for up to 4 weeks using lateral tail vein blood collection. Syringes were preloaded with 10 µL of citrate anticoagulant buffer (4% Sodium Citrate) and the citrated blood mixture was transferred to a prechilled 1.5 mL microcentrifuge tube. Samples were stored at 4°C for 1 hour and then centrifuged for 15 minutes, 2,000g. 38 KILPATRICK TOWNSEND 797856131Supernatant (citrated plasma) was carefully collected and transferred to a prechilled 1.5 mL microcentrifuge tube and then stored at -80°C until further analysis.

[0117] M20. Enzyme-linked immunosorbent assay (ELISA): For in vitro experiments: Cyborg Cells derived from tdT / BDDFVIII PMSCs were seeded in T150 vented flasks using D10 growth media and incubated at 37°C, 5% CO2. After 48 h, the conditioned media was collected. Cell debris was removed by centrifugation at 1500 rpm for 10 min. Supernatant was filtered using a 45 µm syringe filter and analyzed immediately after collection. For in vivo experiments: Citrated plasma collected according to the procedure described in Methods M19 was thawed on ice and diluted 1 in 32 in the dilution buffer indicated by the human FVIII ELISA kit manufacturer (Affinity Biologicals).

[0118] Quantification of human FVIII protein secreted by cells present in undiluted conditioned media was assessed using human FVIII ELISA kit according to the protocol provided by the manufacturer. Citrated plasma from non-treated control mice and from huma FVIII deplete serum were used as negative controls. The standard was generated by human calibrator plasma (Affinity Biologicals) according to the manufacturer’s specifications.

[0119] M21. Flow Cytometry: A Beckman Coulter CytoFLEX LX flow cytometer was used for flow cytometry experiments. It is equipped with 4 activated lasers and 14 color parameters. The 488-nm laser was used for the quantification of cyborg stem cells. The detection of fluorescence was carried out using high-performance customized photomultiplier tube modules. Samples were run at a flow rate of 30 µl min−1. The instrument was calibrated by using CytoFLEX Daily QC fluorospheres. The Flowjo software was utilized to analyze flow cytometry results, and geometric means were calculated based on log-distributed data with n=5000 total events. Fluorescent Cyborg Mammalian Cells were gated based on negative / non-fluorescent WT controls using laser light scatter (forward vs. side angle scatter) to exclude cell debris and waste / aggregates.

[0120] M22: Statistical Analysis: Statistical tests were performed using standard two- tailed t-test, assuming unequal variances. Significant results were defined as those with p- values less than 0.05 and were indicated in each figure by adding an asterisk. The number of replicates contributing to the calculation is listed in the figure legends. 39 KILPATRICK TOWNSEND 797856131Example 3: Intracellular Assembly of Synthetic Hydrogel Networks to Create Hydrogelated Cells (“Cyborg Human Cells”)

[0121] In this Example, therapeutic semi-synthetic cellular systems were constructed by assembling synthetic polymer networks within cultured human cells. This process generates an artificial cytoskeleton that transforms cells into metabolically-active, non-dividing cellular devices that are stabilized and protected by the intracellular hydrogel matrix thus creating hydrogelated cells, referred to herein as “Cyborg Human Cells” (FIG.1A). Intracellular hydrogelation was seen as a cell fixation method to preserve membrane fluidity after cell death, essentially creating “taxidermized” cells (44, 52). As discussed previously, hydrogelation can convert bacteria into semi-living micromachines (53, 54). It was hypothesized that applying precise and rapid intracellular hydrogelation to human cells would yield a hybrid living material combining the capabilities of living cells and the stability of a synthetic material.

[0122] To test this, an intracellular hydrogelation protocol was implemented in Human Embryonic Kidney Cells (HEK293 FT). A chemically stable, non-degradable synthetic hydrogel with minimal biological reactivity was used (55). The hydrogel formulation consisted of poly(ethylene glycol) diacrylate monomer (PEG-DA; Mn 700) and Lithium phenyl-2,4,6 trimethylbenzoylphosphinate (LAP, Sigma-Aldrich; 1 mg mL-1, in DPBS) at 0.1% (w / v) as the photoinitiator. Fluorescein O'O–diacrylate was included as a fluorescent tracer to monitor intracellular diffusion and polymerization.

[0123] An intracellular hydrogelation protocol in HEK293 FT cells was optimized. The steps of the protocol were: 1) cell harvesting, 2) hydrogel infusion, and 3) photoinitiated hydrogel crosslinking (FIG.1B, Methods M4). In step 1), the cells were grown in cell culture flasks to a confluency of 80-90% and detached from the surface of the flask using a cell scraper, cells are then harvested and placed in a 15 ml conical centrifuge tube. In step 2), the cells were spun down and resuspended in hydrogel buffer with different percentages of PEG-DA (20, 40 and 60% v / v). After a 10 min incubation, the cells were spun down again, resuspended in DPBS and intracellular hydrogel was polymerized by UV-A exposure. In step 3), the resulting Cyborg Cells are incubated in fresh media in a new cell culture treated flask under culture conditions (37°C, 5% CO2). The infusion process leverages passive diffusion, allowing the hydrogel components to permeate the cells over a 10-minute incubation period. Following infusion, the cells were carefully washed to remove any extracellular hydrogel 40 KILPATRICK TOWNSEND 797856131components and cell debris, and ultraviolet light (UV-A) was applied to initiate crosslinking of the intracellular PEG-DA monomers. UV-A was selected to minimize genotoxicity during photopolymerization. Across PEG-DA concentrations from 20% to 60% (v / v), rapid and complete intracellular hydrogelation was observed (Methods M21). Time-course experiments confirmed that intracellularly hydrogelated cells stopped dividing and remained structurally stable for at least 6 weeks in culture, and in nutrient deficient conditions (Methods M6).

[0124] Next, the above protocol was applied to create Cyborg Human Cells derived from a therapeutically relevant cell line, placental mesenchymal stem cells (PMSCs), which possess well stablished immunomodulatory, neuroprotective and wound healing properties (56, 57). Using the same conditions established to create Cyborg HEK 293 FT cells (Methods M4), highly efficient intracellular hydrogelation was achieved in PMSCs (FIG.1B). Complete hydrogelation was observed with all the PEG-DA concentrations tested (20-60% v / v) (FIG. 1C, Methods M21). Hydrogelated / Cyborg PMSCs also showed complete cell cycle arrest and retained structural integrity for at least six weeks (FIG.1D, Methods M6). Collectively, these results demonstrate a robust and rapid intracellular hydrogelation method that can be implemented across different cultured human cell lines, enabling the creation of stabilized, non-replicative Cyborg Human Cells. Example 4: Cyborg Human Cells Preserve Metabolic Activity, Spatial Organization, and Organelle Functionality

[0125] Having established the feasibility and robustness of intracellular hydrogelation across different human cell types in the above Examples, next, the Cyborg Human Cells were assessed for the ability to retain essential cellular functions despite their structural transformation. Specifically, the extent to which the Cyborg Human Cells maintain metabolic activity, intracellular architecture, and organelle function was assessed.

[0126] The non-toxic resazurin-based assay PrestoBlue HS (ThermoFisher Scientific; Methods M7) to measure metabolic activity following intracellular hydrogelation with varying PEG-DA concentrations (20-60% v / v; Methods M4). In this assay, intracellular redox reactions reduce resazurin to resorufin, a fluorescent compound. The fluorescence generated inside the cell can be directly correlated with the metabolic activity of the cells, providing insight into their viability and physiological state post-hydrogelation. The results demonstrate preserved metabolic activity in both Cyborg HEK293FT and PMSC cells across 41 KILPATRICK TOWNSEND 797856131all hydrogelation conditions tested (FIG.2A-2B, Methods M6-M7). This is the first report showing metabolic function in intracellularly hydrogelated mammalian cells, suggesting that these semi-synthetic cellular systems can sustain their metabolic output even after hydrogel- induced cell cycle arrest.

[0127] To further investigate the preservation of the cell architecture, fluorescence and confocal microscopy was used to visualize key structural components in the cell. Cytoskeletal integrity was assessed with Alexa Fluor 647-conjugated phalloidin (AF647 Phalloidin, Methods M8) which binds to both large and small actin filaments with similar affinity in a 1:1 stoichiometry between phallotoxin and actin subunits, allowing the labeling, identification and quantification of F-actin filaments. Microscopy analysis shows that Cyborg Human Cells exhibit a distinct radial distribution pattern of actin filaments (FIG.2C-2D) indicating preservation of the cytoskeletal architecture without evidence of filament collapse or reorganization. Nuclear structure was examined using staining with Hoechst 33342 (Methods M7), and no significant changes in nuclear size, shape, or roundness were observed 72 hours post-hydrogelation (FIG.2A and 2C), confirming that intracellular hydrogelation does not lead to nuclear fragmentation or compromise chromatin integrity.

[0128] Preservation of dynamic organelle function was preserved in hydrogelated cells was also assessed. The Golgi apparatus, responsible for protein processing and trafficking, was labeled with TR-ceramide (ThermoFisher Scientific; Methods M9), which localizes to folds in the active Golgi membrane structures. According to the data, Cyborg HEK293FT Cells (40% v / v PEG-DA) showed significantly higher mean fluorescence intensity than untreated cells indicating preserved and elevated Golgi activity (FIG.2E). Treatment with the Golgi disruptor Brefeldin A (BFA; 1 µM) (Methods M9) was used to confirm functional responsiveness and cellular dynamic function in both Cyborg and untreated HEK293FT Cells (Fig.2E). Lysosomal integrity was evaluated using LysoTracker DeepRed (ThermoFisher Scientific; Methods M10), a pH-sensitive dye that accumulates in active lysosomes resulting in a fluorescent signal. Untreated and Cyborg HEK293FT (40% v / v PEG-DA) Cells displayed comparable median fluorescence intensity values indicating no difference in activity in addition to preservation in punctate lysosomal morphology (FIG.2F), which indicate sustained lysosomal activity. Exposure to oxidative stress (Methods M10) reduced MFI in both untreated and Cyborg HEK293FT Cells, confirming that lyssoosmal function remains sensitive to external perturbations after intracellular hydrogelation (FIG.2F). 42 KILPATRICK TOWNSEND 797856131

[0129] Together, these results demonstrate that intracellular hydrogelation preserves metabolic activity, intracellular organization, and dynamic functionality of membrane-bound organelles. Cyborg Human Cells, therefore, retain the defining features of viable, functional cells, while gaining enhanced stability and lacking proliferative capacity. These features enable their use as safe, bioactive, non-replicative devices for biomedical applications. Example 5: Protein Expression and Therapeutic Molecule Secretion Are Preserved in Cyborg Human Cells

[0130] Next, the preservation of protein expression and secretion capabilities of Cyborg Human Cells were assessed. If Cyborg Human Cells can maintain metabolic activities while not replicating, they could execute more complex activities involving protein synthesis and secretion. In particular, their ability to express exogenous reporters and secrete endogenous therapeutic proteins were assessed. Initially, Cyborg PMSCs (Methods M1 & M3) were assessed for maintained expression of fluorescent reporter proteins. PMSCs engineered to constitutively express TdTomato (TdT), and Green Fluorescent protein (GFP) were hydrogelated using our standardized protocol (Methods M4). Cyborg Cells and untreated controls were then cultured under standard conditions (Methods M2; 37°C, 5% CO2) for 7 days to allow the degradation fluorescent proteins expressed before intracellular hydrogelation, taking into consideration the known half-lives of GFP (t1 / 2 = 26h (58)) and TdT (t1 / 2 = 84h (59)). High content confocal and fluorescence microscopy revealed stable expression of TdT (FIG.3A) and GFP (FIG.3B) in Cyborg Human Cells, quantitatively comparable to untreated control cells, as indicated by the analysis of fluorescence intensity (Bottom Panels, FIG.3A-3B, Methods M5-M6). These results confirm that intracellular hydrogelation preserves sustained expression of exogenously integrated fluorescent proteins.

[0131] To further evaluate protein expression capabilities, HEK293FT cells were transiently transfected with constructs encoding cytoplasmic EGFP and secreted embryonic alkaline phosphatase (SEAP) (Methods M11). Post-transfection validation confirmed robust reporter expression. Following hydrogelation (Methods M4), EGFP expression was monitored over 7 days in Cyborg (40% v / v PEG-DA) and untreated HEK293FT cells seeded at a density of 50,000 cells per well in 96-well plates (37°C, 5% CO2). The percentage of cells expressing EGFP was higher in the Cyborg Cell group (72.6%) compared to untreated control cells (35.4%) (FIG.3C). The results in FIG.3A-3B demonstrate that Cyborg Human Cells retain fluorescent reporter expression capabilities when derived from stably or 43 KILPATRICK TOWNSEND 797856131transiently transfected cell lines. The data suggest that protein expression is comparable and under certain conditions higher in the Cyborg Human Cell population. SEAP secretion was similarly evaluated by collecting and analyzing cell culture supernatants at specific timepoints (Methods M11). Cyborg Human Cells exhibited stable SEAP secretion for up to 21 days (FIG.3D), in contrast to untreated controls that demonstrated declining secretion over time. Although Cyborg HEK293FT Cells showed an initially lower secretion rate, their stable long-term output highlights their potential for sustained exogenous therapeutic protein delivery.

[0132] Next, Cyborg Human Cells were assessed for the ability to produce and secrete proteins with clinical relevance, specifically human B Domain Deleted Factor VIII (BDDFVIII). Coagulation factor VIII deficiency underlies Hemophilia A (HA), a hereditary hemorrhagic disorder affecting over 800,000 individuals globally (60), characterized by severe bleeding episodes (61) that require sustained prophylactic treatment (62). Cyborg MCSCs Cells stably expressing BDDFVIII using varying PEG-DA concentrations (20, 40 and 60% v / v; Methods M4) derived from a PMSC cell line were used (Methods M1). Cyborg and untreated PMSCs were incubated under standard culture conditions for 7 days (37°C, 5% CO2) with a growth media change at day 4. Secreted BDDFVIII levels were measured via ELISA (Methods M20). Cyborg PMSCs secreted BDDFVIII throughout the duration of the experiment across all PEG-DA concentrations tested (FIG.3E), underscoring their therapeutic potential for prolonged protein secretion in clinical applications.

[0133] Finally, considering the critical therapeutic mechanism of stem cells via paracrine signaling (63), cytokine, chemokine, and growth factor secretion profiles of Cyborg PMSCs (40% v / v PEG-DA, Methods M1 and M4) were evaluated. Cyborg PMSCs and untreated PMSCs were cultured for 7 days with a media change at day 4, and supernatants were collected and analyzed using a 45plex Luminex Panel (ProcartaPlex Human Cytokine / Chemokine / Growth Factor Convenience Panel 1; ThermoFisher Scientific; Methods M16) and a Luminex 200 detection system. Cyborg PMSCs exhibited a distinct secretome compared to control cells, notably demonstrating enhanced secretion of therapeutically relevant molecules such as Fibroblast Grow Factor-2 (FGF-2), Interleukin -1 Receptor Agonist (IL-RA), and Interleukin-22 (IL-22) (FIG.3F). These cytokines are essential in mediating tissue repair and regeneration (64), angiogenesis (65), and immunomodulation (66). Collectively, these results establish that Cyborg Human Cells preserve and, in some cases, have an enhanced capacity for sustained therapeutic protein 44 KILPATRICK TOWNSEND 797856131expression and secretion, expanding their potential utility as programable synthetic cell therapy platforms for diverse clinical applications. Example 6: Intracellular Hydrogelation Enhances Resilience of Cyborg Human Cells in Challenging Extracellular Environments

[0134] The resilience of Cyborg Human cells against extracellular stressors was investigated. Examples of frequently encountered stressors during cell-based clinical interventions, including osmotic shock, oxidative stress, chemical apoptotic inducers, and prolonged hypoxia.

[0135] Under isotonic conditions (e.g., 150 mM), Cyborg PMSCs (40% v / v PEG-DA) and untreated PMSCs exhibited similar morphology, nuclear dimensions, and metabolic activity, as confirmed by phase-contrast and fluorescence microscopy. However, exposure to hypotonic (7.5 mM) and extreme hypotonic (3 mM) environments resulted in noticeable morphological changes to untreated PMSCs, including pronounced cellular and nuclear swelling, membrane blebbing, detachment, and eventual lysis (FIG.4A, Methods M12). In contrast, Cyborg PMSCs (40% v / v PEG-DA) maintained structural integrity and demonstrated minimal morphological changes. Quantitative metabolic assessments further confirmed higher viability in Cyborg PMSCs under hypotonic stress (FIG.4B; Methods M7 and M12), indicating effective preservation of cellular functionality and resilience to osmotic fluctuations.

[0136] To evaluate cellular response to oxidative stress, Cyborg PMSCs (40% v / v PEG- DA) and untreated PMSCs were exposed to 10% hydrogen peroxide (H₂O₂) for up to 12 hours (Methods M13). After incubation in this hyper-oxidative environment, untreated PMSCs exhibited extensive membrane blebbing, cytoplasmic shrinkage, and nuclear condensation, all hallmarks of oxidative damage. In contrast, Cyborg Human Cells retained stable morphology with reduced signs of damage due to oxidative stress (FIG.4C). These observations were supported by metabolic activity assays, which showed significantly higher viability in Cyborg Cells compared to controls (FIG.4D; Methods M7&M13), underscoring the protective role of intracellular hydrogelation against oxidative stress.

[0137] Additionally, the resistance of Cyborg PMSCs (40% v / v PEG-DA) to chemically induced apoptosis was tested.10 µM Staurosporine was used (Methods M14). Cell-tracking analysis via phase-contrast and fluorescence microscopy indicated no significant reduction on the number of Cyborg PMSCs (40% v / v PEG-DA) over the experimental timeframe (FIG. 45 KILPATRICK TOWNSEND 7978561314E-4F). In contrast, untreated PMSCs displayed significant reductions in cell number, apoptotic multinucleation, membrane blebbing, and cell detachment (FIG.4F-4F). These observations support the protective role of intracellular hydrogelation in mitigating chemically induced apoptosis.

[0138] Finally, cell resilience to prolonged hypoxia (1% O₂ for 72 hours; Methods M15) was assessed. High-content confocal microscopy revealed that Cyborg HEK293FT and PMSCs created using varying PEG-DA concentrations (20%, 40%, and 60% v / v) were markedly less susceptible to hypoxic stress compared to untreated controls, showing reduced signs of morphological deterioration and significantly lower cell loss (FIG.4G). Metabolic assays further confirmed sustained viability of Cyborg Cells under hypoxic conditions (FIG. 4H), indicating that intracellular hydrogelation imparts substantial protection against prolonged oxygen deprivation.

[0139] Taken together, the stress exposure results underscore the enhanced robustness of Cyborg Human Cells, demonstrating that intracellular hydrogelation significantly enhances resilience to clinically relevant extracellular stressors. Example 7: Cyborg Human Cells Are Stable add Capable of BDD Factor VIII Secretion In Vivo

[0140] The in vivo stability of Cyborg Human Cells was evaluated in immunodeficient and immunocompetent murine models (FIG.5A). A total of 5 × 105nanoluc / GFP-labeled Cyborg or untreated control cells were injected subcutaneously into both hind limbs of NSG and C57BL / 6J mice (Methods M17-M18). In NSG mice, bioluminescence imaging revealed sustained and localized signal from both Cyborg and untreated cell implants throughout the duration of the experiment (FIG.5B). In contrast, in immunocompetent C57BL / 6J mice, only Cyborg PMSCs exhibited persistent, localized luminescence at the injection sites over a four- week period, indicating prolonged cell survival and minimal dispersion (FIG.5C). Untreated PMSCs exhibited a rapid decline in signal intensity, consistent with immune-mediated clearance, as expected when transplanting cells into incompatible hosts. These findings suggest that intracellular hydrogelation confers enhanced in vivo stability and cell retention, even within immunocompetent hosts.

[0141] Building on the in vitro findings above demonstrating robust BDDFVIII secretion by Cyborg PMSCs (FIG.3E), their capacity for therapeutic protein secretion was evaluated in vivo.5 × 105nanoluc / TdT / -labeled BDDFVIII-secreting Cyborg PMSCs (40% v / v PEG- 46 KILPATRICK TOWNSEND 797856131DA) or untreated PMSCs cells were implanted subcutaneously into both hind limbs of NSG mice (Methods M17). Plasma from NSG mice transplanted with Cyborg or untreated PMSCs were obtained for up to 4 weeks (Methods M19). The concentration of human BDDFVIII in each sample was quantified using ELISA (Methods M20). The results show that even though Cyborg and control cells showed localized luminescence at the injection sites for the duration of the experiment (FIG.5A). However, the concentration of FVIII in plasma increased in mice implanted with Cyborg PMSCs compared to the concentration in plasma from mice injected with control PMSCs (FIG.5D). These results confirm that Cyborg Human Cells retain protein synthesis and section in vivo, reinforcing their potential as a therapeutic delivery platform.

[0142] Collectively, the results demonstrate that intracellular hydrogelation converts PMSCs into immune-resilient micro-bioreactors that sustain therapeutic outputs for weeks. The functional decoupling of biological activity from proliferation enables sustained, site- restricted therapeutic protein release and directly addresses clearance, dispersion, and safety constraints that limit current cell therapies. Cyborg Human Cells meet the key criteria for clinical use and support the application of this synthetic “Third” cellular state as a practical platform for long-acting biologic delivery without systemic immunosuppression. Discussion

[0143] Examples 2-7 above demonstrate the engineering of human cells into programmable, non-replicative therapeutic platforms by leveraging intracellular hydrogelation. Through the formation of synthetic polymer networks within the cytoplasm, a “Third” cellular state that produces dells displaying a unique convergence of biological functionality and synthetic stability. Unlike natural cell states, which are limited by short in vivo persistence, uncontrolled proliferation, or environmental fragility, Cyborg Human Cells maintain metabolic activity, organelle integrity, and protein secretion while demonstrating robust resilience to oxidative, osmotic, apoptotic, and hypoxic stressors.

[0144] Intracellular hydrogelation enables functional decoupling of cell viability and replication, a long-standing barrier in cell-based therapy design. This allows for the generation of therapeutic cells that are structurally stable and metabolically active without posing the risks of tumorigenicity or graft overgrowth. In mouse models, Cyborg Human Cells stably expressed and secreted human coagulation factor VIII, and persisted in vivo for weeks without dispersal or degradation, highlighting their translational promise. 47 KILPATRICK TOWNSEND 797856131

[0145] The implications of this approach extend far beyond hemophilia. Intracellular hydrogelation represents a broadly applicable, modular chassis for synthetic cell therapies. Cyborg Human Cells can be customized with diverse parental cell types and therapeutic cargos, offering a generalizable solution for regenerative medicine, oncology, autoimmune diseases, and beyond. By embedding programmable functions within a non-dividing, resilient cellular scaffold, this strategy redefines what is possible in the design of semi-living therapeutic systems.

[0146] In summary, Cyborg Human Cells mark a leap forward in the engineering of cell therapies. By combining the durability of synthetic materials with the functional versatility of living cells, our platform offers a robust, tunable, and safe solution for next-generation cell- based interventions. This intracellular hydrogelation strategy lays the groundwork for creating long-lived, responsive, and non-replicative therapeutic devices, advancing the vision of programmable, adaptive, and intelligent biologic systems in clinical medicine. References Related to the Examples:

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[0148] It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes. 56 KILPATRICK TOWNSEND 797856131

Claims

WHAT IS CLAIMED IS:

1. A metabolically-active cell comprising a cross-linked hydrogel within the cell in sufficient amount to prevent cell replication, wherein the cell is a mammalian cell.

2. The metabolically-active cell of claim 1, wherein the cell has a therapeutic effect when introduced into a mammal or that can be used for cell therapy applications.

3. The metabolically-active cell of claim 1, wherein the cell is a placental mesenchymal stromal cell (PMSC).

4. The metabolically-active cell of claim 1, wherein the cell is a stem cell or a progenitor cell.

5. The metabolically-active cell of claim 4, wherein the stem cell is an induced pluripotent stem cell (iPSC), fetal stem cell, mesenchymal stem cell (MSC) or a hematopoietic stem cell (HSC).

6. The metabolically-active cell of claim 1, wherein the cell is a primary cell.

7. The metabolically-active cell of claim 1, wherein the cell is a cancer cell.

8. The metabolically-active cell of claim 7, wherein the cancer cell is a glioblastoma cell.

9. The metabolically-active cell of claim 1, wherein the cell is an immune cell.

10. The metabolically-active cell of claim 9, wherein the immune cell is a macrophage or a T-cell.

11. The metabolically-active cell of any one of claims 1-10, wherein the cell expresses a heterologous polynucleotide and / or polypeptide.

12. The metabolically-active cell of any one of claims 1-10, wherein the cell secretes one or more growth factors, cytokines, and chemokines including but not limited 57 KILPATRICK TOWNSEND 797856131to EFG, FGF-2, HGF, IFN alpha, IFN gamma, IL-1 beta, IL1-RA, IL-2, IL-4, IL-6, IL-8, IL- 9, IL-13, IL-21, IL-22, IL-23, IL-31, NGF-beta, Rantes, TNF beta and VEGF-D.

13. The metabolically-active cell of any one of claims 1-12, wherein the cell is a human, murine, or canine cell.

14. The metabolically-active cell of any one of claims 1-13, wherein the hydrogel comprises monosaccharide or polysaccharide monomer subunits, and wherein the hydrogel is a homopolymer or co-polymer.

15. The metabolically-active cell of any one of claims 1-14, wherein the hydrogel comprises substituted or unsubstituted poly(ethylene glycol) monomer subunits.

16. The metabolically-active cell of any one of claims 1-15, wherein the hydrogel comprises poly(dimethyl siloxane) (PDMS), poly(ethylene oxide) (PEO), poly(vinyl alcohol) (PVA), poly(propylene fumarate) (PPF), alginate, guanosine mono phosphate (GMP), cyclodextrin (CD), fibrin, collagen, polypeptides, decellularized extracellular matrix, or nucleic acids.

17. The metabolically-active cell of any one of claims 1-16, wherein the hydrogel is substituted.

18. A therapeutic biomaterial derived from metabolically-active cell of any one of claims 1-17.

19. A method of making the metabolically-active cell of any one of claims 1-17, the method comprising, providing a plurality of mammalian cells, introducing a polymerization inducer and monomer units of a hydrogel into the mammalian cells; and causing the polymerization inducer to initiate formation in the cells of a hydrogel formed from the monomer units thereby forming living metabolically-active mammalian cells comprising the hydrogel.

20. The method of claim 19, wherein the monomer units are at a concentration of 1-80% or 1-60% (e.g., 30-80%, 50-70%, e.g., 60%) w / w. 58 KILPATRICK TOWNSEND 79785613121. The method of claim 19, wherein the introducing occurs at 20-25° C.

22. The method of claim 19, wherein the polymerization inducer is lithium phenyl-2,4,6 trimethylbenzoylphosphinate.

23. The method of claim 19, wherein at least 99% (e.g., at least 99.9% or 100%) of the cells comprise the hydrogel.

24. The method of any one of claims 19-23, wherein the monomer units are poly(ethylene glycol) diacrylate.

25. A method of regenerating tissue in a mammal, the method comprising, administering the metabolically-active cell of any one of claims 1-17 to the mammal, wherein the cells stimulate regeneration of a tissue in the mammal.

26. The method of claim 25, wherein the mammal is a human.

27. The method of claim 25 or 26, wherein the metabolically-active cell secretes fibroblast grow factor-2 (FGF-2) and interleukin-22 (IL-22).

28. The method of claim 27, wherein the metabolically-active cell promotes tissue repair, angiogenesis and / or wound healing in the mammal.

29. The method of claim 25 or 26, wherein the metabolically-active cell secretes interleukin-1 receptor agonist (IL-RA).

30. The method of claim 29, wherein the metabolically-active cell reduces inflammation in the mammal. 59 KILPATRICK TOWNSEND 797856131

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