Systems and methods for preparing biologically active molecules

Abstract

Disclosed herein are compositions comprising a hydrogel scaffold, methods of generating a hydrogel scaffold, and systems for and methods of using the hydrogel scaffold to produce biologically active molecules.

Description

SYSTEMS AND METHODS FOR PREPARING BIOLOGICALLY ACTIVE MOLECULESI. CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No. 63 / 635,987 filed 18 April 2024, which is incorporated herein in its entirety.IL STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] This invention was made with government support under R01NS094599 and R01 AI152568 awarded by the National Institutes of Health. The government has certain rights in the invention.III. REFERENCE TO THE SEQUENCE LISTING

[0003] The Sequence Listing submitted with this application is an XML file named ‘23-2074-WO Sequence Listing.xmT, created on 9 April 2025 and having a size of 60.4 KB is hereby incorporated by reference pursuant to 37 C.F.R. § 1.52(e)(5).IV. BACKGROUND

[0004] Viruses, DNA plasmids, and mRNA can be used to deliver transgenes encoding recombinant proteins or native bioactive signals, as well as gene regulatory systems for RNA interference or gene editing technologies. Viruses have been the primary method for FDA- approved gene therapies, but they are cost-prohibitive due to scale-up challenges and strict manufacturing guidelines, aside from risks of immunogenicity. However, viruses remain the most effective vector for therapeutic expression and are still highly relevant for the clinic. Adeno- associated virus (AAV) is one of the most common gene therapies on the market, with many rare diseases able to be treated. As clinical use shifts to more common disease, focus has been on better methods to improve yield for the growing patient population.

[0005] The current AAV production approach has been to either use many stackable layered flasks for static 2D cell culture, or to expand in suspension culture at different volumes from shaker flasks to bioreactors. Much of the focus in enhancing AAV production has been in either streamlining the production scheme or optimizing the cell transfection and culture conditions to improve titer output. Areas to improve virus production efficiency, beyond engineering new proteins for immune-evasion and tropism, would greatly impact their availability for larger patient populations.

[0006] To circumvent these limitations and make nonviral delivery applicable for long-term therapeutic gene expression or biologies production, delivery approaches that retain the nucleic acid cargo at the local cell or tissue interface are beneficial. Thus, there remains an unmet need toimprove methods concerning simultaneous culture and nonviral gene delivery for protein production or cell modification, particularly for long-term production runs.V. BRIEF SUMMARY

[0007] Disclosed herein is a method of producing a biologically active molecule, the method comprising, loading cells into a hydrogel scaffold, wherein the hydrogel scaffold comprises disclosed hydrogel particles and disclosed nanoparticles complexed with one or more nucleic acid, and maintaining the cells.

[0008] In an aspect, the method can further comprise adding a medium to a disclosed hydrogel scaffold. In an aspect, the method can further comprise recovering the biologically active molecule.

[0009] In some aspects of the method, cells can be human, primate, murine, leporine, canine, feline, bovine, equine, ovine, caprine, avian. In an aspect, cells can comprise a human embryonic kidney (HEK) cells, Chinese hamster ovary (CHO) cells, DXB-11, DG-44, baby hamster kidney (BHK) cells, African green monkey kidney cells, Hela cell, adenocarcinomic human alveolar basal epithelial cells, human lung cell, human hepatoma (Hep) cells, mouse mammary tumor (MMT) cells, TRI cells, MRC5 cells, FS4 cells, mammalian myeloma cells, E. coli cells, yeast cells, or any combination thereof.

[0010] In an aspect, loading the cells comprises topical seeding of the cells into a disclosed hydrogel scaffold. In an aspect, topical seeding of the cells comprises combining the cells with a disclosed hydrogel scaffold at about 5,000 cells / pL scaffold volume to 15,000 cells / pL scaffold volume. In an aspect, loading the cells comprises encapsulating the cells in a disclosed hydrogel scaffold. In an aspect, encapsulating the cells comprise encapsulating cells at about 100 cells / pL scaffold volume to 10,000 cells / pL scaffold volume.

[0011] In an aspect, a disclosed hydrogel scaffold comprises hyaluronic acid (HA). In an aspect, the HA further comprise one or more acrylamide functional groups (HA-AC), one or more RGD ligands (RGDSP - SEQ ID NO:36), one or more Q-peptides (NQEQVSPLGGERCG -SEQ ID NO:2), one or more K-peptides (FKGGERCG - SEQ ID NO:3), or any combination thereof.

[0012] In an aspect, the one or more nucleic acids complexed to disclosed nanoparticles can comprise at least two nucleic acids. In an aspect, the one or more nucleic acids complexed to disclosed nanoparticles can comprise at least three nucleic acids. In an aspect, the one or more nucleic acids complexed to disclosed nanoparticles can comprise more than three nucleic acids.

[0013] In an aspect, the disclosed nanoparticle complexed with a nucleic acid can be prepared by combining polyethyleneimine (PEI), the nucleic acid, and HA comprising one or more norbomene functional groups (HA-NB), at N / P of about 20 and an HA:PEI ratio of about 5.

[0014] In an aspect, the medium comprises Ham’s F10, Minimal Essential Medium (MEM), RPMI-1640, Dulbecco’s or Modified Eagle’s Medium (DMEM), Ex-cell GTM3, Freestyle 5FM293 Hyclone 5FM293, RPMI1640 medium, GIT medium, Ex-cell 302 medium, IMDM medium, Hybridoma-SFM or any combination thereof.

[0015] In an aspect, the nucleic acid can encode a biologically active molecule comprising an oligonucleotide, a DNA molecule, an RNA molecule, a virus, or any combination thereof. In an aspect, the biologically active molecule is an Adeno-associated virus (AAV). In an aspect, the nucleic acid can encode a biologically active molecule comprising a peptide, a polypeptide, a protein, an antibody, or any combination thereof. In an aspect, the biologically active molecule can be a therapeutic protein, therapeutic peptide, recombinant protein, monoclonal antibody, recombinant antibody, chimeric antibody, humanized antibody, non-human antibody, multispecific antibody, antibody fragments, Fc fusion protein, single chain variable fragments (scFv), chimeric antigen receptors (CAR), or a combination thereof. In an aspect, the biologically active molecule can be an enzyme, hormone, growth factor cytokine, chemokine, or any combination thereof. In an aspect, the biologically active molecule can be human, primate, murine, leporine, canine, feline, bovine, equine, ovine, caprine, avian, or any derivatives thereof. In an aspect, the biologically active molecule can comprise human vascular endothelial growth factor (VEGF), interleukin 4 (IL-4), or any combination thereof.

[0016] In an aspect, maintaining the cells can comprise removing spent medium and adding fresh medium. In an aspect, the biologically active molecule can be recovered at least about 1 x 1010genomic copies or at least about 1000 pg / mL. In an aspect, maintaining the cells can comprise maintaining for at least 14 days. In an aspect, maintaining the cells can comprise maintaining for at least 21 days. In an aspect, maintaining the cells can comprise maintaining for at least 28 days. In an aspect, the maintaining the cells can comprise maintaining for more than 28 days.

[0017] Further provided herein is a method of nucleic acid delivery, the disclosed method can comprise loading cells into a hydrogel scaffold, wherein a disclosed hydrogel scaffold comprises disclosed hydrogel particles and disclosed nanoparticles complexed with one or more nucleic acid.

[0018] In an aspect, the disclosed method can further comprise adding a medium. In an aspect, the disclosed method can further comprise maintaining the cells for at least 14 days. In an aspect, the disclosed method can further comprise maintaining the cells for at least 21 days. In an aspect, the disclosed method can further comprise maintaining the cells for at least 28 days. In an aspect, the disclosed method can further comprise maintaining the cells for more than 28 days. In an aspect, maintaining the cells comprises removing spent medium and adding fresh medium. In an aspect, the disclosed method maintains the expression of delivered nucleic acid in the cells for atleast 14 days. In an aspect, the disclosed method maintains the expression of delivered nucleic acid in the cells for at least 21 days. In an aspect, the disclosed method maintains the expression of delivered nucleic acid in the cells for at least 28 days. In an aspect, the disclosed method maintains the expression of delivered nucleic acid in the cells for more than 28 days.

[0019] In some aspects of the disclosed method, the nucleic acid can encode a biologically active molecule comprising an oligonucleotide, a DNA molecule, an RNA molecule, a virus, or any combination thereof. In an aspect, the biologically active molecule can be an Adeno-associated virus (AAV).

[0020] In an aspect, a disclosed nucleic acid can encode a biologically active molecule comprising a peptide, a polypeptide, a protein, an antibody, or any combination thereof. In an aspect, the biologically active molecule can be a therapeutic protein, therapeutic peptide, recombinant protein, monoclonal antibody, recombinant antibody, chimeric antibody, humanized antibody, non-human antibody, multispecific antibody, antibody fragments, Fc fusion protein, single chain variable fragments (scFv), chimeric antigen receptors (CAR), or any combination thereof. In an aspect, the biologically active molecule can be an enzyme, hormone, growth factor cytokine, chemokine, or any combination thereof. In an aspect, the biologically active molecule can be human, primate, murine, leporine, canine, feline, bovine, equine, ovine, caprine, avian, or any derivatives thereof. In an aspect, the biologically active molecule can comprise human vascular endothelial growth factor (VEGF), interleukin 4 (IL-4), or any combination thereof. In an aspect, the disclosed method can further comprise recovering the biologically active molecules.

[0021] In an aspect, the cell can be human, primate, murine, leporine, canine, feline, bovine, equine, ovine, caprine, avian. In an aspect, cells can comprise a human embryonic kidney (HEK) cell, Chinese hamster ovary (CHO) cell, DXB-11, DG-44, baby hamster kidney (BHK) cell, African green monkey kidney cell, Hela cell, adenocarcinomic human alveolar basal epithelial cell, human lung cell, human hepatoma (Hep) cell, mouse mammary tumor (MMT) cell, TRI cell, MRC5 cell, FS4 cell, mammalian myeloma cell, E. coli cell, yeast cell, or any combination thereof.

[0022] In an aspect, loading cells can comprise topical seeding of cells into a disclosed hydrogel scaffold. In an aspect, topical seeding of cells can comprise combining the cells with a disclosed hydrogel scaffold at about 5,000 cells / pL scaffold volume to 15,000 cells / pL scaffold volume. In an aspect, loading cells can comprise encapsulating the cells in the scaffold. In an aspect, encapsulating cells can comprise encapsulating cells at about 100 cells / pL scaffold volume to 10,000 cells / pL scaffold volume.

[0023] In an aspect, a disclosed hydrogel scaffold can comprise hyaluronic acid (HA). In an aspect, the HA further can comprise one or more acrylamide functional groups (HA-AC), one or more RGD ligands (RGDSP - SEQ ID NO:36), one or more Q-peptides (NQEQVSPLGGERCG - SEQ ID NO:2), one or more K-peptides (FKGGERCG - SEQ ID NO:3), or any combination thereof.

[0024] In an aspect, the one or more nucleic acid complexed to disclosed nanoparticles can comprise at least two nucleic acids. In an aspect, the one or more nucleic acid complexed to disclosed nanoparticles can comprise at least three nucleic acids. In an aspect, the one or more nucleic acid complexed to disclosed nanoparticles can comprise more than three nucleic acids. In an aspect, the disclosed nanoparticle complexed with a nucleic acid can be prepared by combining PEI, the nucleic acid, and HA comprising one or more norbomene functional groups (HA-NB), at N / P of about 20 and an HA:PEI ratio of about 5.

[0025] In an aspect, the medium can comprise Ham’s F10, Minimal Essential Medium (MEM), RPMI-1640, Dulbecco’s or Modified Eagle’s Medium (DMEM), Ex-cell GTM3, Freestyle 5FM293 Hyclone 5FM293, RPMH640 medium, GIT medium, Ex-cell 302 medium, IMDM medium, Hybridoma-SFM, or any combination thereof.

[0026] In an aspect, the biologically active molecule can be recovered at least about 1 x 1012genomic copies or at least about 1000 pg / mL.

[0027] In an aspect, the disclosure further encompasses a method of producing a biologically active molecule in a three-dimensional cell culture device, comprising, adding a hydrogel scaffold comprising disclosed nanoparticles complexed with one or more nucleic acids, into a culture device, loading cells by topical seeding of cells, into a disclosed hydrogel scaffold, and maintaining the cells in a disclosed hydrogel scaffold. In an aspect, topical seeding of cells comprises combining the cells with a disclosed hydrogel scaffold at about 5,000 cells / pL scaffold volume to 15,000 cells / pL scaffold volume

[0028] In an aspect, provided further is a method of producing a biologically active molecule in a three-dimensional cell culture device, comprising, loading cells by encapsulating cells, in a hydrogel scaffold comprising disclosed nanoparticles complexed with one or more nucleic acids, adding a disclosed hydrogel scaffold into a culture device, maintaining the cells. In an aspect, encapsulating cells comprise encapsulating cells at about 100 cells / pL scaffold volume to 10,000 cells / pL scaffold volume.

[0029] In an aspect, the three-dimensional cell culture device comprises a culture tube, culture bottle, flask, microfluidic device, perfusion device, a bioreactor, or any combination thereof.

[0030] In an aspect, a disclosed hydrogel scaffold comprises hyaluronic acid (HA). In an aspect, the HA can further comprise one or more acrylamide functional groups (HA-AC), one or more RGD ligands (RGDSP - SEQ ID NO: 36), one or more Q-peptides (NQEQVSPLGGERCG - SEQ ID NO: 2), one or more K-peptides (FKGGERCG - SEQ ID NO: 3), or any combination thereof. In an aspect, the one or more nucleic acid complexed to disclosed nanoparticles can comprise at least two nucleic acids. In an aspect, the one or more nucleic acid complexed to disclosed nanoparticles can comprise at least three nucleic acids. In an aspect, the one or more nucleic acid complexed to disclosed nanoparticles can comprise more than three nucleic acids. In an aspect, the nanoparticle complexed with a nucleic acid is prepared by combining polyethyleneimine (PEI), the nucleic acid, and HA comprising one or more norbomene functional groups (HA-NB), at N / P of about 20 and an HA:PEI ratio of about 5.

[0031] In an aspect, the method can further comprise adding medium to the culture device. In an aspect, the medium comprises Ham’s F10, Minimal Essential Medium (MEM), RPMI-1640, Dulbecco’s or Modified Eagle’s Medium (DMEM), Ex-cell GTM3, Freestyle 5FM293 Hyclone 5FM293, RPMH640 medium, GIT medium, Ex-cell 302 medium, IMDM medium, Hybridoma- SFM, or any combination thereof.

[0032] In an aspect, maintaining the cells can comprise removing spent medium and adding fresh medium.

[0033] In an aspect, the nucleic acid can encode a biologically active molecule comprising an oligonucleotide, a DNA molecule, an RNA molecule, a virus, or any combination thereof. In an aspect, the biologically active molecule is an Adeno-associated virus (AAV). In an aspect, the nucleic acid encodes a biologically active molecule comprising a peptide, a polypeptide, a protein, an antibody, or any combination thereof. In an aspect, the biologically active molecule is a therapeutic protein, therapeutic peptide, recombinant protein, monoclonal antibody, recombinant antibody, chimeric antibody, humanized antibody, non-human antibody, multispecific antibody, antibody fragments Fc fusion protein, single chain variable fragments (scFv), chimeric antigen receptors (CAR), or any combination thereof. In an aspect, the biologically active molecule can be an enzyme, hormone, growth factor cytokine, chemokine, or any combination thereof. In an aspect, the biologically active molecule can be human, primate, murine, leporine, canine, feline, bovine, equine, ovine, caprine, avian, or any derivatives thereof. In an aspect, the biologically active molecule comprises human vascular endothelial growth factor (VEGF), interleukin 4 (IL- 4), or any combination thereof.

[0034] In an aspect, the biologically active molecule can be recovered at least about 1 x 1012genomic copies or at least about 1000 pg / mL. In an aspect, maintaining the cells can comprisemaintaining for at least 14 days. In an aspect, maintaining the cells can comprise maintaining for at least 21 days. In an aspect, maintaining the cells can comprise maintaining for at least 28 days.

[0035] In an aspect, the cells can be maintained for more than 28 days. In an aspect, the method can maintain the expression of nucleic acid in the cells for at least 14 days. In an aspect, the method can maintain the expression of nucleic acid in the cells for at least 21 days. In an aspect, the method can maintain the expression of nucleic acid in the cells for at least 28 days. In an aspect, the method can maintain the expression of nucleic acid in the cells for more than 28 days.

[0036] The disclosure further encompasses a method of producing an AAV, the method can comprise loading cells into a disclosed hydrogel scaffold comprising disclosed nanoparticles complexed with one or more plasmids, and maintaining the cells.

[0037] In an aspect, the disclosed nanoparticles can be complexed with at least one plasmid. In an aspect, the disclosed nanoparticles can be complexed with at least two plasmids. In an aspect, the disclosed nanoparticles can be complexed with at least three plasmids. In an aspect, the disclosed nanoparticles can be complexed with more than three plasmids. In an aspect, the plasmid can be a Helper plasmid, a Replication / Capsid plasmid, transgene insert (ITR) plasmid, or any combination thereof.

[0038] In further aspects, the method can further comprise adding a medium to a disclosed hydrogel scaffold.

[0039] In an aspect, the method can further comprise recovering AAV. In an aspect, the AAV is AAV1, AAV2, AAV3 (including 3 A and 3B), AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV8 bp, AAV7M8, AAVAnc80, AAVrhlO, AAVPHP.B, AAV type rh32.33, AAV type rh8, AAV type rh74, AAV type hu.68, avian AAV, bovine AAV, canine AAV, equine AAV, ovine AAV, snake AAV, bearded dragon AAV, AAV2i8, AAV2g9, AAV-LK03, AAV7m8, AAV Anc80, AAV-PHP.eB, AAV-TT, AAVv66, rAAV2 / l, rAAV2 / 8, rAAV2 / 9, AAV-DJ, AAV-HAE1, AAV-HAE2, AAVM41, AAV- 1829, AAV2 Y / F, AAV2 T / V, AAV2i8, AAV2.5, AAV9.45, AAV9.61, AAV-B1, AAV-AS, AAV9.45A-String, AAV9.45Angiopep, AAV9.47-Angiopep, AAV9.47-AS, AAV-PHP.B, AAV-PHP.eB, AAV- PHP.S, AAV-F, AAVcc.47, or AAVcc.81.

[0040] In an aspect, the cell can be human, primate, murine, leporine, canine, feline, bovine, equine, ovine, caprine, avian. In an aspect, cells can comprise a human embryonic kidney (HEK) cell, Chinese hamster ovary (CHO) cell, DXB-11, DG-44, baby hamster kidney (BHK) cell, African green monkey kidney cell, Hela cell, adenocarcinomic human alveolar basal epithelial cell, human lung cell, human hepatoma (Hep) cell, mouse mammary tumor (MMT) cell, TRI cell,MRC5 cell, FS4 cell, mammalian myeloma cell, E. coli cell, yeast cell, or any combination thereof.

[0041] In an aspect, loading cells can comprise topical seeding of cells into a disclosed hydrogel scaffold. In an aspect, topical seeding of cells can comprise combining the cells with a disclosed hydrogel scaffold at about 5,000 cells / pL scaffold volume to 15,000 cells / pL scaffold volume. In an aspect, loading cells can comprise encapsulating the cells in the scaffold. In an aspect, encapsulating cells can comprise encapsulating cells at about 100 cells / pL scaffold volume to 10,000 cells / pL scaffold volume.

[0042] In an aspect, a disclosed hydrogel scaffold can comprise hyaluronic acid (HA). In an aspect, the HA can further comprise one or more acrylamide functional groups (HA-AC), one or more RGD ligands (RGDSP - SEQ ID NO: 36), one or more Q-peptides (NQEQVSPLGGERCG - SEQ ID NO: 2), one or more K-peptides (FKGGERCG - SEQ ID NO: 3), or any combination thereof. In an aspect, the nanoparticle complexed with plasmids can be prepared by combining polyethyleneimine (PEI), the plasmid nucleic acid, and HA comprising one or more norbomene functional groups (HA-NB), at N / P of about 20 and an HA:PEI ratio of about 5.

[0043] In an aspect, the medium can comprise Ham’s F10, Minimal Essential Medium (MEM), RPMI-1640, Dulbecco’s or Modified Eagle’s Medium (DMEM), Ex-cell GTM3, Freestyle 5FM293 Hyclone 5FM293, RPMH640 medium, GIT medium, Ex-cell 302 medium, IMDM medium, Hybridoma-SFM, or any combination thereof. In an aspect, maintaining the cells can comprise removing spent medium and adding fresh medium.

[0044] In an aspect, the AAV can be recovered at least about 1 x 1012genomic copies. In an aspect, maintaining a disclosed hydrogel scaffold can comprise maintaining for at least 14 days. In an aspect, maintaining a disclosed hydrogel scaffold can comprise maintaining for at least 21 days. In an aspect, maintaining a disclosed hydrogel scaffold can comprise maintaining for at least 28 days. In an aspect, the cells can be maintained for more than 28 days.

[0045] In further aspect, disclosed herein is a method of expressing a nucleic acid in one more cells, the method can comprise loading one or more cells into a hydrogel scaffold described herein.

[0046] In an aspect, expressing of one or more nucleic acid in one or more cells can be sustained for at least 14 days. In an aspect, expressing of one or more nucleic acid in one or more cells can be sustained for at least 21 days. In an aspect, expressing of one or more nucleic acid in one or more cells can be sustained for at least 28 days. In an aspect, expressing of one or more nucleic acid in one or more cells can be sustained for more than 28 days.

[0047] In an aspect, further provided is a perfusion device for production of a biologically active molecule, the device can comprise a chamber comprising hydrogel scaffold loaded with cells, amedia channel comprising an inlet and an outlet, a membrane separating the chamber from the media channel, a media reservoir, and a peristaltic pump.

[0048] In some aspects of the device, a disclosed hydrogel scaffold can comprise nanoparticle complexed with one or more nucleic acid.

[0049] In some aspects of the device, the cell can be human, primate, murine, leporine, canine, feline, bovine, equine, ovine, caprine, avian. In an aspect, cells can comprise a human embryonic kidney (HEK) cell, a Chinese hamster ovary (CHO) cell, a DXB-11 cell, a DG-44 cell, a baby hamster kidney (BHK) cell, an African green monkey kidney cell, a Hela cell, an adenocarcinomic human alveolar basal epithelial cell, a human lung cell, a human hepatoma (Hep) cell, a mouse mammary tumor (MMT) cell, a TRI cell, a MRC5 cell, a FS4 cell, a mammalian myeloma cell, an E. coli cell, a yeast cell, or any combination thereof.

[0050] In some aspects of device, a disclosed hydrogel scaffold comprises hyaluronic acid (HA). In an aspect, the HA further comprise one or more acrylamide functional groups (HA-AC), one or more RGD ligands (RGDSP - SEQ ID NO:36), one or more Q-peptides (NQEQVSPLGGERCG - SEQ ID NO:2), one or more K-peptides (FKGGERCG - SEQ ID NO:3), or any combination thereof.

[0051] In some aspects of the device, the disclosed nanoparticles can be complexed with two nucleic acids. In an aspect, the disclosed nanoparticles can be complexed with three nucleic acids. In an aspect, the disclosed nanoparticles can be complexed with more than three nucleic acids. In an aspect, the disclosed nanoparticles can be complexed with one or more nucleic acid plasmids.

[0052] In some aspects of the device, the disclosed nanoparticle complexed with one or more nucleic acid can be prepared by combining PEI, nucleic acid vector and HA comprising one or more norbomene functional groups (HA-NB) at N / P of about 20 and an HA:PEI ratio of about 5. In an aspect, the nucleic acid can encode a biologically active molecule comprising an oligonucleotide, a DNA molecule, an RNA molecule, a virus, or any combination thereof. In an aspect, the biologically active molecule is an Adeno-associated virus (AAV). In an aspect, the nucleic acid can encode a biologically active molecule comprising a peptide, a polypeptide, a protein, an antibody, or any combination thereof. In an aspect, the biologically active molecule can be therapeutic protein, therapeutic peptide, recombinant protein, monoclonal antibody, recombinant antibody, chimeric antibody, humanized antibody, non-human antibody, multispecific antibody, antibody fragments Fc fusion protein, single chain variable fragments (scFv), chimeric antigen receptors (CAR), or any combination thereof. In an aspect, the biologically active molecule can be an enzyme, hormone, growth factor cytokine, chemokine, or any combination thereof. In an aspect, the biologically active molecule can be human, primate,murine, leporine, canine, feline, bovine, equine, ovine, caprine, avian, or any derivatives thereof. In an aspect, the biologically active molecule can comprise human vascular endothelial growth factor (VEGF), interleukin 4 (IL-4), or any combination thereof.

[0053] In an aspect, the disclosed device can further comprise a medium, wherein the medium is Ham’s F10, Minimal Essential Medium (MEM), RPMI-1640, Dulbecco’s or Modified Eagle’s Medium (DMEM), Ex-cell GTM3, Freestyle 5FM293 Hyclone 5FM293, RPMI1640 medium, GIT medium, Ex-cell 302 medium, IMDM medium, Hybridoma-SFM, or any combination thereof.VI. BRIEF DESCRIPTION OF THE FIGURES

[0054] Figure la - Figure li show loading and transfection from two-plasmid loaded FLIP scaffolds. Figure la illustrates direct nanoparticle embedding in FLIP and transfection over time from cells grown within and degrading the scaffold. Figure lb depicts soluble transfection from nanoparticles in solution electrostatically associating to the FLIP surface. Figure 1c shows the nucleic acid nanoparticle delivery from loaded scaffolds seeded with HEK293 cells, using either scaffolds embedded with lyophilized nanoparticles or with surface-exposed particles in solution for more direct cell transfection. Compared over time for new expression at each time point, and final cumulative expression. N = 5, analyzed by two-way ANOVA with post hoc testing, or student's t-test for cumulative comparison (***p < 0.005). Likewise, Figure Id shows the longterm expression from loaded scaffolds seeded with CHO-K1 cells, with both expression at each time point and cumulative endpoint expression. N = 5, analyzed by two-way ANOVA with post- hoc testing, or student's t-test for cumulative comparison (n.s., p > 0.05). Figure le illustrates long-term transfection from loaded FLIP scaffolds for DI MSCs, with repeated measure data across a 27-day period. Data is reported as both per-day rate and cumulative expression for protein and cytokine expression. Figure If shows plasmid loading method comparing directly encoded transgenes on a single plasmid. Figure 1g depicts time study on VEGF production from embedded and surface loaded FLIP scaffolds. Figure Ih illustrates Dual-plasmid delivery encoding a dCas9- VP64 and sgRNA for endogenous gene activation from FLIP. Figure li shows similar time study as in Figure 1g for both embedded FLIP and soluble delivery of nanoparticles for endogenous VEGF expression. Figure Ij shows plasmid loading method comparing directly encoded transgenes on a single plasmid for monoclonal antibody expression. Figure Ik shows anti-GFP antibody production from CHO cultured in embedded and soluble nanoparticle scaffolds. Inverse ELISA was run for each timepoint collected sample, and the cumulative yield was reported as before for VEGF. N = 3, analyzed by two-way ANOVA with post hoc testing, or student’s t-test for cumulative comparison.

[0055] Figure 2a - Figure 2i show embedded loading of HA-coated lyophilized DNA / PEI nanoparticles (NP) in FLIP and effect on material properties. Figure 2a shows confocal imaging of loaded and non -loaded FLIP scaffolds. Red = FLIP, green = YOYO-1 stained pDNA. Scaffolds were imaged for 500 pm z-stacks at 2 pm step-size, and rendered in IMARIS. FLIP was prepared at 1 pg / pL gel, at 30 pL scaffolds. Figure 2b depicts mechanical properties of loaded and nonloaded bulk hydrogels and FXIII-annealed FLIP scaffolds, based on storage modulus (G’) using shear rheology. Figure 2c illustrates nucleic acid nanoparticle delivery from loaded FLIP, using either FLIP embedded with lyophilized nanoparticles or FLIP surface-exposed to particles in solution for more direct cell transfection. Compared across “difficult-to-transfecf ’ primary mouse bone marrow derived macrophage (BMDM) and astrocytes. Transfection is reported for Gaussia luciferase expression in terms of relative luminescence units (RLU), measured after 48 hours of cell culture. Figure 2d shows comparison of direct single plasmid and dCas9 / sgRNA dualplasmid endogenous VEGF expression from FLIP in different cell types, including difficult-to- transfect HUVEC cells, compared to a control single plasmid containing just a Gaussia reporter construct. Figure 2e illustrates IL-4 production from both direct single plasmid transgene expression and dual plasmid dCas9 endogenous gene activation. Figure 2f depicts time study on IL-4 production from embedded and soluble FLIP scaffolds. Figure 2g shows a time study in HEK293 for endogenous IL-4 production using the dual-plasmid dCas9 / sgRNA system. Figure 2h shows the metabolic activity from long-term HEK293 culture in loaded and non-loaded scaffolds, quantified over time by absorbance from the PrestoBlue viability assay. N=3, two-way ANOVA with post-hoc. By the endpoint, each condition was significantly different, with plain scaffolds the most viable (p<0.001) followed by embedded scaffolds (p<0.001). Figure 2i shows the viability of HEK293 from dead cell staining (ZombieViolet) and flow cytometry, collected as individual endpoints at each time point. N=3, two-way ANOVA with post-hoc. As with PrestoBlue, condition was significantly different at the endpoint, with plain scaffolds the most viable (p<0.005) followed by embedded scaffolds (p<0.001).

[0056] Figure 3a - Figure 3g show AAV production from FLIP scaffolds and improved longterm expression in static culture. Figure 3a illustrates static cell culture in FLIP scaffolds embedded with the triple plasmid cocktail for AAV. Figure 3b is a schematic of AAV production from static FLIP scaffolds assessed over time. Figure 3c shows long-term AAV production from transfected cultures, with samples collected at Days 3, 5, and 9. Titers were again determined by qPCR. N = 3 for all conditions, run in triplicate. Figure 3d depicts cumulative virus titer after the month-long period for both 2D plate and FLIP culture. N = 3, one-way ANOVA with Tukey's post hoc (n.s., p > 0.05). Figure 3e illustrates the infectivity of the AAV produced from staticscaffold culture in DI mouse MSCs using a fixed MOI, quantified as reporter luciferase expression, and assessed by two-way ANOVA with post-hoc (N = 4-5). Figure 3f shows endpoint brightfield images of HEK cells in a 2D plate and scaffold culture. Figure 3g shows the viability from Zombie staining at each time point and quantified for a number of live cells. N = 3, two-way ANOVA with post hoc. Figure 3h shows the cell division (proliferation) tracking of fluorescent dye-labelled cells after 5-7 days of culture, where cells were collected at each time point to quantify the percent of cells at each number of divisions (N = 3). Figure 3i depicts the genome copy number tracking via BrdU labelling for cell proliferation. Samples were collected at each time point, acid-treated, fixed, and antibody labelled for confocal imaging. Positive labeled cells quantified based on co-localization to DAPI and nuclei counting in FIJI. N=3, two-way ANOVA with post-hoc. Similarly, Figure 3j depicts the proliferation tracking via Ki67 labelling, assessing each time point and quantifying from co-localization. N=3, two-way ANOVA with post-hoc.

[0057] Figure 4a - Figure 4e characterize the scaffold produced AAV. Figure 4a shows 48-hour reporter Gaussia luciferase expression from triple-plasmid transfected HEK293T in either 2D tissue culture or 3D embedded FLIP scaffolds. Figure 4b depicts 48-hour GFP expression from transfected cells based on flow cytometry. Figure 4c illustrates 48-hour viability from transfected cells based on PI staining and flow. Figure 4d shows AAV titers from the two methods based on qPCR. Figure 4d illustrates infectivity studies in DI mouse MSCs using produced AAV at fixed MOI for AAV produced at Day 7 from static FLIP culture (N = 3). Figure 4e shows infectivity of AAV from 2D and 3D static FLIP culture in HEK293 cells. Figure 4f depicts the CFSE-labelled cell division tracking for 2D plate cultured cells, cells within embedded nanoparticle FLIP scaffolds, and cells within non-loaded FLIP scaffolds with soluble nanoparticles. N=3 for each condition.

[0058] Figure 5a - Figure 5h show AAV production from FLIP scaffolds and improved longterm expression in suspension culture. Figure 5a shows comparison across non-FLIP suspension cells in bolus transfection, cells encapsulated in the nanoparticle embedded FLIP scaffolds, and cells encapsulated in non-loaded FLIP with soluble nanoparticle delivery. Figure 5b - Figure 5d illustrate time study on suspension FLIP AAV9 production. Samples were processed for Gaussia luciferase expression (Figure 5b), metabolic activity via PrestoBlue assay (Figure 5c), and AAV titer via qPCR, following media purification and concentration (Figure 5d). All samples were N = 3 and assessed with one-way ANOVA within groups, along with Tukey's post hoc. Figure 5e shows resulting cumulative AAV titers, based on the data from Figure 5d. N = 5 for all conditions, run in duplicate and assessed via one-way ANOVA with Tukey's post-hoc (n.s.,p > 0.05). Figure 5f shows infectivity studies in DI mesenchymal stem cells (MSCs) using the suspension-producedAAV, comparing cells alone in suspension (no FLIP) to the two FLIP scaffold conditions, and again quantified as reporter luciferase expression. N = 3, two-way ANOVA with post hoc. Figure 5g illustrates AAV titers from gradient centrifuge purified samples for adherent cells in 2D tissue culture, compared to both static and suspension FLIP scaffolds, either embedded or surface transfected. Titers again determined by qPCR. Figure 5h shows capsid loading based on relative AAV titer determined from qPCR compared to titer based on total whole-capsid protein ELISA. N = 5, one-way ANOVA, Tukey's post hoc (*p < 0.05).

[0059] Figure 6a - Figure 61 show suspension FLIP improve viability and gene delivery. Figure 6a - Figure 6b show considerations for FLIP culture, from cell type, volume of gel relative to suspension volume, and sub-dividing the gel volume for increased surface area Figure 6c - Figure 6d show comparison on cell seeding density for cells in (Figure 6b) at a fixed gel volume and number (1 x 30 pL gel in 30 pL media). Samples were run in triplicate and processed at Day 5, based on the PrestoBlue assay for metabolic activity or processed with flow cytometry for number of cell divisions, as determined by CFSE staining and fluorescence distribution (Figure 6c). Figure 6d illustrates using cells at a fixed 300 or 600 cells / pL scaffold and gel-to-media ratio of 1 : 10 (v / v), assessed the best surface area ratio by sub-dividing the FLIP solution volume into “clusters”, cultured in the same well. Samples were processed for metabolic activity as before at Day 5. Figure 6e shows the viability for the samples in Figure 6d from Zombie staining and flow cytometry. Figure 6f is a schematic of overview of the different loading schemes for FLIP scaffolds, now applied to suspension culture. Figure 6g - Figure 6h show suspension conditions with and without scaffolds were compared for Gaussia luciferase expression from pDNA / PEI nanoparticle transfection. FLIP loaded at 0.5 pg / pL with the lyophilized nanoparticle formulation were compared to the same amount in freely suspended cells (“3D dose”) and the standard concentration of 500 ng / pL used in 2D plate cell culture (“2D dose”). Nanoparticle-embedded and plain FLIP were prepared as before, but with the additional step of encapsulating cells within the scaffold prior to annealing. Cells were processed at Day 5 for luciferase expression (Figure 6g) and metabolic activity (Figure 6h). Figure 6i - Figure 6j illustrate suspension production of therapeutic proteins IL-4 (Figure 6i) and VEGF (Figure 6j) over time, comparing HEK293F suspension cells without FLIP to HEK293T adherent cells encapsulated in FLIP either embedded with nanoparticles or non-loaded with soluble delivery. N = 3 for each condition, production determined by protein ELISA. Figure 6k shows HEK cells seeded in non-loaded suspension FLIP scaffold “microcarriers,” stained to show cell spreading after 1 week of culture. Maximum intensity projection was generated from confocal microscopy and 3D rendered.

[0060] Figure 7a - Figure 7d characterize the scaffold produced AAV. Figure 7a depicts capsid loading assessment of a select few batches of AAV, produced either in standard 150 mm plates, using 3D static device culture, or from the suspension schemes. Purified AAV samples were processed using a sandwich ELISA against AAV9 and compared titer from standard dilutions to the titers determined from qPCR. Capsid loading is represented as the ratio of the qPCR for loaded to total ELISA capsid result. The accepted literature value for capsid loading at -60-70% is shown with the dashed line. High batch variability was present, but likely due to the sensitivity of the assay as well. Figure 7b - Figure 7c represent scale-up attempt using HA-Alginate scaffolds at 3 mL scaffold in 30 mL media. Media was processed for Gaussia reporter expression (Figure 7b) and AAV titer (Figure 7c) following purification and qPCR. Figure 7d shows infectivity study at fixed MOI for AAV produced over time from static and suspension culture (N = 3).

[0061] Figure 8a - Figure 8f show prototype testing for in-line static FLIP scaffolds for AAV production. Figure 8a is a schematic showing possible perfusion culture through an annealed FLIP scaffold loaded with DNA nanoparticles and seeded with cells. Figure 8b (Left to Right) illustrates reverse mold for perfusion device channel, with inlet and outlet sides. Photos of a PDMS perfusion device, with a 40 pm mesh separating the scaffold side (with injection port) and the media flow channel. For perfusion culture through an annealed FLIP scaffold loaded with DNA nanoparticles and seeded with cells. Luer-lock barb adapters join the device to the peristaltic pump with the media source connected in-line. The samples were then removed after 30 days and stained for DAPI and actin for imaging via confocal microscopy. Figure 8c shows the process used for perfusion FLIP culture using the prototype device, with FLIP and cells on one side of the mesh, and media flowing in parallel on the other side, connected to the pump. Figure 8d shows Gaussia luciferase expression per day of 3D static FLIP culture compared to the perfusion device over a one-month period. Figure 8e depicts metabolic activity of cells from static and perfusion culture based on PrestoBlue assay. Figure 8f illustrates AAV titer determined by qPCR from sampled media at each day of culture. Media was fully exchanged, collected, and purified prior to concentration and titering. Figure 8g shows cumulative AAV titer after 30 days of perfusion or static FLIP culture. Figure 8h shows infectivity of AAV produced from either static or perfusion culture in embedded FLIP scaffolds. AAV was collected across various time points, purified, and then transduced in DI MSCs. N = 3, two-way ANOVA with post hoc Figure 8i shows Capsid loading efficiency of AAV collected over time, following cumulative AAV pooling and purification, and determined by the ratio of titer from qPCR to titer from whole-capsid ELISA. N = 3, student’s t-test.

[0062] Figure 9a - Figure 9d show design process for FLIP bioreactors. Figure 9a is a schematic showing possible perfusion culture through an annealed FLIP scaffold loaded with DNA nanoparticles and seeded with cells. Figure 9b shows photograph of the perfusion device setup connected to a circulating media line and peristaltic pump. Figure 9c shows possible designs for in-line direct scaffold perfusion devices. For a fixed chamber volume of 200 pL, several designs (left to right: disc, tube, and box) are shown with the CAD render and the SLA 3D printed part. Figure 9d shows challenge with direct scaffold perfusion and compression of softer gels resulting in pressure buildup and scaffold ejection or line snapping.

[0063] Figure lOa-Figure 10g show FLIP scaffolds prepared with polyethylene glycol. Figure 10a shows PEG hydrogel chemistry with PEG-maleimide polymer and an L-MMP di-thiol crosslinker. Figure 10b shows the preparation of shredded PEG hydrogel microparticles, where the hydrogel is passed through a 70 pm filter during centrifugation at 18,000G for 5 minutes. Figure 10c shows a process of annealing shredded PEG hydrogel particles into a FLIP scaffold. Figure lOd shows images and size distribution of shredded PEG hydrogel particles within a FLIP scaffold. Figure lOe shows cell viability of NIH3T3 fibroblasts following exposure to DNA polyplexes at 2 pg / pL or 1 pg / pL in solution or embedded within a FLIP scaffold. Figure lOf shows gene expression between exposure to DNA polyplexes in solution or embedded within a FLIP scaffold. Figure 10g shows images of NIH3T3 cells cultured in DNA loaded PEG-FLIP scaffolds resulting in transfection of the NIH3T3 cells and GFP expression.

[0064] Figure 11 is a schematic of different methods using nucleic acid nanoparticle loaded granular FLIP hydrogel scaffolds for sustained perfusion cell culture and production of biologies.

[0065] Figure 12a - Figure 12k show plasmid maps of the plasmids used in the studies described.

[0066] Figure 13 shows normalized cross-analysis of the various biomanufacturing schemes from granular hydrogel scaffolds. AAV titers are reported as the total yield for the given volume of FLIP scaffolds relative to the number of cells seeded into the scaffolds. The infectivity from previous figures is shown, to compare samples collected over time following transducing cells at a fixed titer (fixed MOI). An arbitrary metric of “active” AAV particles was derived by multiplying the titer-per-cell (top row) by the infectivity (center row), which helps visualize the trends among the sample conditions and the three production schemes: (left) Cell-normalized AAV from long-term static scaffold culture, comparing embedded and soluble nanoparticle loading methods to the standard 2D plate control. Scaffolds again are prepared at 10 000 cells pL-1 scaffold and 12 pL media per pL scaffold, (center) Normalized AAV from suspension scaffold culture, prepared at 6000 cells pL1scaffold, and 10 pL media per pL scaffold. Again, FLIP scaffolds were clustered in 30 pL increments, (right) Continuous flow reactor AAVproduction from perfusion scaffold culture seeded similar to static scaffold culture at 10,000 cells pL1scaffold. Current device limitations are for 60 pL scaffold per device, with flowing 40 pL media per pL scaffold. All statistical analyses are the same as in the previous respective figures.VII. DETAILED DESCRIPTION

[0067] The present disclosure describes hydrogel scaffolds and use of a disclosed hydrogel scaffold for protein and AAV production. The inventors have identified and optimized a granular hydrogel scaffold compatible with a variety of nucleic acids, including vectors, plasmids and minicircle DNA, for cell transfection, with sustained cell viability, and transgene expression over long period of time. It is to be understood that the inventive concepts are not limited to specific methods unless otherwise specified, or to particular reagents unless otherwise specified, as such can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only and is not intended to be limiting. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, example methods and materials are now described.

[0068] All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention.A. Definitions

[0069] Before the present compounds, compositions, articles, systems, devices, and / or methods are disclosed and described, it is to be understood that they are not limited to specific synthetic methods unless otherwise specified, or to particular reagents unless otherwise specified, as such can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only and is not intended to be limiting. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, example methods and materials are now described.

[0070] This disclosure describes inventive concepts with reference to specific examples. However, the intent is to cover all modifications, equivalents, and alternatives of the inventive concepts that are consistent with this disclosure.

[0071] As used in the specification and the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise.

[0072] The phrase ‘consisting essentially of limits the scope of a claim to the recited components in a composition or the recited steps in a method as well as those that do not materially affect thebasic and novel characteristic or characteristics of the claimed composition or claimed method. The phrase ‘consisting of excludes any component, step, or element that is not recited in the claim. The phrase ‘comprising’ is synonymous with ‘including’, ‘containing’, or ‘characterized by’, and is inclusive or open-ended. ‘Comprising’ does not exclude additional, unrecited components or steps.

[0073] As used herein, when referring to any numerical value, the term ‘about’ means a value falling within a range that is ± 10% of the stated value.

[0074] Ranges can be expressed herein as from ‘about’ one particular value, and / or to ‘about’ another particular value. When such a range is expressed, a further aspect includes from the one particular value and / or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent ‘about,’ it will be understood that the particular value forms a further aspect. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as ‘about’ that particular value in addition to the value itself. For example, if the value ‘ 10’ is disclosed, then ‘about 10’ is also disclosed. It is also understood that each unit between two particular units are also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

[0075] References in the specification and concluding claims to parts by weight of a particular element or component in a composition denotes the weight relationship between the element or component and any other elements or components in the composition or article for which a part by weight is expressed. Thus, in a compound containing 2 parts by weight component X and 5 parts by weight component Y, X and Y are present at a weight ratio of 2:5, and are present in such ratio regardless of whether additional components are contained in the compound.

[0076] As used herein, the terms ‘optional’ or ‘optionally’ means that the subsequently described event or circumstance can or cannot occur, and that the description includes instances where said event or circumstance occurs and instances where it does not. In an aspect, a disclosed method can optionally comprise one or more additional steps, such as, for example, repeating an administering step or altering an administering step.

[0077] The present disclosure also contemplates that In an aspect, any feature or combination of features set forth herein can be excluded or omitted. To illustrate, if the specification states that a complex comprises components A, B and C, it is specifically intended that any of A, B or C, or a combination thereof, can be omitted and disclaimed singularly or in any combination.

[0078] As used herein, the term “nucleic acid” refers to isolated, purified, natural, recombinant, synthetic deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form, composed of monomers (nucleotides) containing a sugar, phosphate and a base that is either a purine or pyrimidine. 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) and complementary sequences as well as the reference sequence explicitly indicated.

[0079] The term “oligonucleotide” as used herein is defined as a molecule comprised of two or more deoxyribonucleotides or ribonucleotides, preferably more than three, and usually more than ten. There is no precise upper limit on the size of an oligonucleotide. However, in general, an oligonucleotide is shorter than about 250 nucleotides, preferably shorter than about 200 nucleotides and more preferably shorter than about 100 nucleotides. The exact size will depend on many factors, which in turn depends on the ultimate function or use of the oligonucleotide. The oligonucleotide can be generated in any manner, including chemical synthesis, DNA replication, reverse transcription, or a combination thereof.

[0080] As used herein, the term “FLIP” scaffold refers to scaffolds comprising Flowable Linked Irregular Particles.

[0081] As used herein, the term “hydrogel particles” refers to the particles created by passing a crosslinked hydrogel through a sieve or mesh (e.g., a cell strainer) with an average pore size smaller than the hydrogel. The crosslinked hydrogel is fractionated into pieces sized according to the average pore size of the sieve or mesh. Exemplary average pore sizes can be about 1000 pm, about 750 pm, about 500 pm, about 400 pm, about 300 pm, about 200 pm, about 150 pm, about 100 pm, about 85 pm, about 70 pm, about 60 pm, about 50 pm, about 40 pm, about 30 pm, about 20 pm, about 10 pm, or about 1 pm.

[0082] As used herein, the term “annealing” refers to heating a material below its peak melting point in order to strengthen and relieve stress on the polymer, particularly those attributable to creating the physical bonding sites. Annealing time can be at least 1 minute to several weeks long. In one embodiment, the annealing time is about 4 hours to about 48 hours. In another embodiment, the annealing time is about 24 hours to about 48 hours. In another embodiment, the annealing time is about 24 hours.

[0083] As used herein, the term “nucleic acid complexing agent” refers to a molecule, compound or chemical moiety that interacts with one or more nucleic acids to enable packaging in disclosedhydrogel particles with reduced, minimized or prevented aggregation or other characteristics detrimental to packaging and delivery of such nucleic acid to a cell.

[0084] As used herein, the term “N / P ratio” or “N / P” refers to the ratio of positively-chargeable polymer amine (N = nitrogen) groups to negatively-charged nucleic acid phosphate (P) groups, and is a way to define mixture ratios for polymer-based nucleic acid delivery vehicles.

[0085] As used herein, “expression” refers to the process by which nucleic acid, e.g., DNA, is transcribed into mRNA and translated into peptides, polypeptides, or proteins. If the nucleic acid is derived from genomic DNA, expression can, if an appropriate eukaryotic host cell or organism is selected, include splicing of the mRNA.

[0086] As used herein, “transformation” or “transfection” or “nucleic acid delivery” or “gene delivery” refers to the process by which nucleic acids are introduced into cells. Transfection refers to the taking up of exogenous nucleic acid, by a host cell whether or not any coding sequences are in fact expressed. Methods and compositions of the disclosure are effective for transformation or transfection. Successful transfection is generally recognized by detection of the presence of the heterologous nucleic acid within the transfected cell, such as, for example, any visualization of the heterologous nucleic acid or any indication of the operation of a such nucleic acid within the host cell.

[0087] “Endogenous” with reference to a gene, protein, and / or nucleic acid refers to the natural presence of that gene, protein, and / or nucleic acid in a cell, such as an immune cell.

[0088] “Exogenous” refers to an introduced agent, such as a nucleic acid, gene, or protein, into a cell, for example from an outside source. A nucleic acid introduced into a cell is exogenous even if it encodes a protein which is naturally found in the cell. Such exogenous introduction of a nucleic acid encoding a protein can be used to increase the expression of the protein over the level that would naturally be found in the cell under similar conditions, e.g., without introduction of the exogenous nucleic acid.

[0089] As used herein, the term “humanized antibody” refers to forms of non-human (e.g., murine) antibodies that are specific immunoglobulin chains, chimeric immunoglobulins, or fragments thereof that contain minimal non-human sequences. Typically, humanized antibodies are human immunoglobulins in which residues from the complementary determining region (CDR) are replaced by residues from the CDR of a non-human species (e.g., mouse, rat, rabbit, hamster, etc.) that have the desired specificity, affinity, and capability. In some instances, the Fv framework region (FR) residues of a human immunoglobulin are replaced with the corresponding residues in an antibody from a non-human species that has the desired specificity, affinity, and capability. The humanized antibody can be further modified by the substitution of additionalresidue either in the Fv framework region and / or within the replaced non-human residues to refine and optimize antibody specificity, affinity, and / or capability. In general, the humanized antibody will comprise substantially all of at least one, and typically two or three, variable domains containing all or substantially all of the CDR regions that correspond to the non-human immunoglobulin whereas all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. The humanized antibody can also comprise at least a portion of an immunoglobulin constant region or domain (Fc), typically that of a human immunoglobulin.

[0090] As used herein, “lipid nanoparticles” or “LNPs” can deliver nucleic acid (e.g., DNA or RNA), protein (e.g., RNA-guided DNA binding agent), or nucleic acid together with protein. LNPs can comprise biodegradable, ionizable lipids. For example, LNPs can comprise (9Z,12Z)- 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl octadeca-9,12-di enoate, also called 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl (9Z, 12Z)-octadeca-9, 12-di enoate) or another ionizable lipid. In an aspect, the term cationic and ionizable in the context of LNP lipids can be used interchangeably, e.g., wherein ionizable lipids are cationic depending on the pH. As used herein, LNPs can include extracellular vesicles.

[0091] As used herein, “extracellular vesicles” (EVs) is a generic term that can refer to all membrane vesicles secreted in the extracellular space. As such, EVs include a broad and extremely heterogeneous population of vesicles, which possess different functions, biophysical properties, and have different biogenesis routes. Given the lack of a clear consensus on the nomenclature of EVs, the field has coined a multitude of terms to address the different types of vesicles, resulting in sub-categories that are often redundant and / or overlapping. Accordingly, the terms “ectosomes,” “shedding vesicles,” “microvesicles,” and “microparticles” usually refer to 150— 1000 nm vesicles that bud directly from the plasma membrane, while the term “exosomes” refers to smaller vesicles (30-100 nm), which are generated intracellularly by the inward budding of multivesicular bodies (MVB) and released in the extracellular space upon fusion of the MVBs with the plasma membrane. EVs can package different macromolecules including proteins, nucleic acids, and viruses, thereby making them an attractive therapeutic platform. (Pegtel DM, et al. 2019 Exosomes. Annu Rev Biochem. 88:487-514; Colombo M, et al. 2014 Annu Rev Cell Dev Biol. 30:255-289). Relevant to the disclosed compositions and methods, recombinant AAV capsids associated with exosomes can enable efficient gene transfer to the retina, the nervous system, the inner ear (Hudry E, et al. 2016 Gene Ther. 23(4):380-392; Gybrgy B, et al. 2017 Mol Ther. 25(2):379-391; Meliani A, et al. 2017 Blood Adv. l(23):2019-2031; Volak A, et al. 2018 JNeurooncol. 139(2):293-305) and appear shielded from anti-AAV neutralizing antibodies. (Meliani A, et al. 2017 Blood Adv. l(23):2019-2031).

[0092] As used herein “biologically active molecule” is a molecule that is capable of exerting a biological effect upon administration to an individual. As used herein a biologically active molecule is one which can exert its biological activity i.e., exhibits an effect at any level (biochemical, cellular and / or morphological) within the cell in which it is expressed, on the cell surface, effect the cell’s interactions with other cells or biologically active molecules or can be released or secreted from the cell in which it is made and exert its effect on a separate target cell (e.g., hormone, growth factor, soluble receptor, antibody, antibody fragment, anti -angiogenic factor, or cytokine). A biologically active molecule is any agent, such as a virus, protein, peptide, amino acid, lipid, carbohydrate, nucleic acid, nucleotide, drug, pro-drug or other substance that can have an effect on cells whether such effect is harmful, beneficial, or otherwise. In an aspect, nucleic acids can include DNA, plasmid DNA, ssDNA, dsDNA, cDNA, vector DNA, minicircle DNA, genomic DNA, DNA gene fragments, cDNA, viral DNA, bacterial DNA, microbial DNA, mRNA, pre-mRNA, siRNA, shRNA, ssRNA, antisense RNA oligonucleotide, antisense miRNA, trans-splicing RNA, guide RNA, single-guide RNA, crRNA, tracrRNA, trans-splicing RNA, or RNA vector antisense molecules, plasmids, vectors, etc.

[0093] As used herein “media”, “medium,” “cell culture medium,” and “culture medium” used herein refer to a solution containing nutrients that nourish growing cells, e.g., mammalian cells, and can also refer to medium in combination with cells.

[0094] As used herein, “effective amount” and “amount effective” can refer to an amount that is sufficient to achieve the desired result such as, for example, the treatment and / or prevention of a disease and / or disorder. As used herein, the terms “effective amount” and “amount effective” can refer to an amount that is sufficient to achieve the desired an effect on an undesired condition (a disease and / or disorder). For example, a “therapeutically effective amount” refers to an amount that is sufficient to achieve the desired therapeutic result or to have an effect on undesired symptoms, but is generally insufficient to cause adverse side effects.

[0095] In an aspect, “therapeutically effective amount” means an amount of a disclosed biologically active molecule 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, and / or (iii) delays the onset of one or more symptoms of the particular disease, condition, or disorder described herein. The specific therapeutically effective dose level for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the disclosed biologically active molecule, or any combination thereofemployed; the disclosed methods employed; the age, body weight, general health, sex and diet of the patient; the time of administration; the route of administration; the rate of excretion of the disclosed biologically active molecule, or any combination thereof employed; the duration of the treatment; drugs used in combination or coincidental with the disclosed biologically active molecule employed, and other like factors well known in the medical arts.

[0096] Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.B. Hydrogel Scaffold

[0097] Disclosed herein is a hydrogel scaffold. In further aspects, a hydrogel scaffold described herein and used in the methods described infra can comprise disclosed hydrogel particles and disclosed nanoparticles complexed with one or more nucleic acids. In an aspect, a disclosed hydrogel scaffold is both injectable and porous and provides a biocompatible structure to support in vitro cell culture growth, expansion and long-term survival. In an aspect, a disclosed hydrogel scaffold can be configured to be efficiently loaded with genetic material that can encode a biologically active molecule (e.g., a peptide, a polypeptide, a protein, an antibody, or combination thereof) or viruses for improved production of the biologically active molecule or virus. In an aspect, a disclosed hydrogel scaffold can allow for media and nutrients to flow through voids between the fragmented particles so secreted proteins produced by cells within a disclosed hydrogel scaffold can be easily collected in the supernatant without degrading the scaffold and / or separating the cells from the scaffold. In an aspect, a disclosed hydrogel scaffold facilitates transfection from both soluble and embedded transfection methods for cells within the scaffold.

[0098] In an aspect, a hydrogel scaffold disclosed herein and used in the methods described infra can comprise a polymer. Suitable polymers, non-limiting examples of which include hyaluronic acid (HA), chitosan, heparin, alginate, gelatin, fibrin, collagen, MATRIGEL®, polyvinyl alcohol, polyethylene glycol, sodium polyacrylate, acrylate polymers, copolymers, dithiol polymers (e.g., acrylamide), click-based composite hydrogels polyethylene glycol)-diacrylate, poly(ethylene glycol)-vinyl sulfone, or any combination thereof. In an aspect, hydrogel can comprise hyaluronic acid (HA). In an aspect, polymers can be functionalized polymers. Functionalization of the polymer can be confirmed with1H nuclear magnetic resonance spectroscopy, mass spectroscopy, Elman’s reagent, UV-Vis spectroscopy, infrared spectroscopy, and other methods known to those skilled in the art.

[0099] In an aspect, bulk hydrogel can be prepared by crosslinking hydrogel polymers with a polymer crosslinker. Polymer crosslinkers for use in the hydrogel preparation are known by those skilled in the art. In an aspect, crosslinker can be a multifunctional crosslinker for example, abifunctional polymer crosslinker. Non-limiting examples of bi-functional polymer crosslinkers and multifunctional polymer crosslinkers include polyethylene glycol dithiol (PEG-DT), protease- degradable crosslinkers and multi-arm polyethylene glycol) terminated with thiol (e.g., 4-arm PEG terminated with thiol). In an aspect, the crosslinker can comprise a protease-degradable crosslinker for example, matrix metalloproteinase (MMP)-degradable crosslinkers (MMP-1- degradable crosslinkers, MMP-2-degradable crosslinkers, MMP-9-degradable crosslinkers), Omi degradable sequences, or Heat-Shock Protein degradable sequences. In another aspect, the crosslinker sequences are hydrolytically degradable natural and synthetic polymers e.g., heparin, alginate, poly(ethylene glycol), polyacrylamides, polymethacrylates, polyesters, polyamides, and polyurethanes. In an aspect, the crosslinker can comprise a MMP-degradable crosslinker. In an aspect, bulk hydrogel can be prepared by crosslinking hydrogel polymer with a MMP dithiol crosslinker (AC-GCRDGPQGIWGQDRCG-NH2 - SEQ ID NO: 1). In an aspect, bulk hydrogel can prepared using crosslinking HA with MMP dithiol crosslinkers.

[0100] In an aspect, the hydrogel polymer in the scaffold can be modified with one or more functional groups. In an aspect, hydrogel polymer can be modified with a functional group norbornene, acrylamide, tetrazine, sulfate, cyclodextrin, adamantane, vinyl sulfone, acrylate, allyl, azide, alkyne, thiol, PEG, or any combination thereof. In an aspect, a disclosed hydrogel scaffold can comprise HA modified with acrylamide functional groups (HA-AC). In an aspect, a disclosed hydrogel scaffold can comprise HA modified with norbomene functional group (HA-NB).

[0101] In further aspects, hydrogel polymer can be further modified with a cell adhesion peptide. In an aspect, the cell adhesion peptide can be RGD ligand (RGDSP - SEQ ID NO:36). In other aspects, the hydrogel polymer can be modified with other ligands, Q peptide, K peptide, or any combination thereof. In an aspect, a disclosed hydrogel scaffold can comprise HA modified with one or more RGD ligands (RGDSP - SEQ ID NO:36), one or more Q-peptides (Ac- NQEQVSPLGGERCG-NH2 - SEQ ID NO:2), one or more K-peptides (Ac-FKGGERCG-NH2- SEQ ID NO:3), or any combination thereof.

[0102] In an aspect, one or more hydrogel particles of the disclosed scaffold can originate from a bulk hydrogel. For example, bulk hydrogel initially can comprise a size larger than a desired size for disclosed hydrogel particles. Such bulk hydrogels can be fractionated into two or more pieces until a desired hydrogel particle size can be achieved. In an aspect, one or more disclosed hydrogel particles can also be polymerized or annealed to from a disclosed hydrogel scaffold. In an aspect, one or more particles can originate from a bulk hydrogel and one or more particles can be annealed.

[0103] In an aspect, the bulk hydrogel can be shredded to prepare disclosed hydrogel particles. In an aspect, shredding can be performed by sieving the hydrogel through a sieve, mesh, or strainer. In an aspect, bulk hydrogel can be passed through a sieve or mesh with an average pore size, between about 1 pm and 1000 pm, or between about 5 pm and 750 pm, or between about 10 pm and 500 pm, or between about 15 pm and 400 pm, or between about 20 pm and 300 pm, or between about 30 pm and 200 pm, or between about 40 pm and 150 pm, or between about 40 pm and 100 pm, or about 10 pm, or about 20 pm, or about 30 pm, or about 40 pm, or about 50 pm, or about 60 pm, or about 70 pm, or about 85 pm, or about 100 pm, as well as other suitable pore sizes. In an aspect, the bulk hydrogel can be shredded using a 70 pm cell sieve.

[0104] In an aspect, the scaffold comprises disclosed hydrogel particles of two or more types. In an aspect, the scaffold comprises disclosed hydrogel particles comprising an irregular shape with an average surface area that can range from about 100 pm2and 1000000 pm2, as well as other size ranges. In an aspect, the irregularly shaped particles comprise shredded disclosed hydrogel particles. In an aspect, two or more particles are annealed together, wherein annealed together comprises covalent, electrostatic, hydrophobic and mechanical annealing.

[0105] In an aspect, the one or more disclosed hydrogel particles that comprise an irregular shape comprise an average surface area that can range from between about 100 pm2and 1000000 pm2, or between about 500 pm2and 500000 pm2, or between about 1000 pm2and 250000 pm2, or between about 2500 pm2and 100000 pm2, or between about 5000 pm2and 50000 pm2, or between about 7500 pm2and 40000 pm2, or between about 10000 pm2and 25000 pm2.

[0106] In some embodiments, two or more disclosed hydrogel particles, of any composition as described above, are annealed together, wherein annealing comprises covalent, electrostatic, hydrophobic and mechanical annealing. In an aspect, non-annealed scaffolds also can possess physical properties like stiffness in the range of some annealed scaffolds. Both annealed and nonannealed scaffolds are contemplated and useful for aspects of the disclosure.

[0107] In an aspect, the shredded hydrogels are further combined and annealed to generate a disclosed hydrogel scaffold. In an aspect, an annealing agent can comprise Factor XIII, Eosin Y, a free radical transfer agent, thrombin or any combination thereof. Functionalities that allow for annealing can be incorporated in the disclosed hydrogel particles. In an aspect, functionalities such as a,P-unsaturated carbonyl groups that can be activated for annealing through either radical initiated reaction with a,[3-unsaturated carbonyl groups on adjacent particles or Michael and pseudo-Michael addition reactions with nucleophilic functionalities, can be added to the disclosed hydrogel particles. In another aspect, functionalities that participate in Click chemistry which allow for attachment of adjacent disclosed hydrogel particles can be added. In further aspects, theannealing agent can comprise thrombin and Factor XIII. In an aspect, a disclosed hydrogel scaffold comprising the disclosed hydrogel particles can be granular.

[0108] In an aspect, a disclosed hydrogel scaffold can comprise HA- AC hydrogel particles, functionalized with RGD ligand, Q peptide, and is K peptide, annealed using thrombin and Factor XIII.

[0109] In an aspect, a disclosed hydrogel scaffold used in the methods described infra can comprise disclosed nanoparticles. In an aspect, disclosed nanoparticles can be polymer and / or lipid-based nanoparticles. In an aspect, disclosed nanoparticles can be generated using a cationic polymer. In an aspect, the cationic polymer can comprise linear poly(ethyleneimine) (PEI), branched PEI, poly(beta-amino esters), Poly(2-(dimethylamino)ethyl methacrylate) (pDMEAMA), poly(amido amine) (PAMAM), chitosan, imine-containing polyamines, polyurethanes, cyclodextrin, disulfide-containing poly(amido amine), or any combination thereof. In an aspect, disclosed nanoparticles can comprise a cationic lipid lipofectamine, DOSPA, DOPE, DSPE, DSTAP, DOTAP, DOTMA, DORIE, DMRIE, DOTIM, GAP-DLRIE, DDAB, DC-6-14, DODAP, DOTC, DOGS, or any combination thereof. In an aspect, disclosed nanoparticles are generated using linear PEI. In an aspect, PEI can be combined with HA at a HA:PEI ratio of about 20: 1 to about 1 :1, or about 20:1, about 19: 1, about 18: 1, about 17:1, about 16:1, about 15: 1, about 14: 1, about 13:1, about 12: 1, about 11 :1, about 10: 1, about 9:1, about 8: 1, about 7: 1, about 6:1, about 5:1, about 4: 1, about 3: 1, about 2: 1, or about 1 : 1. In some aspects the HA:PEI ratio can be about 5:1 or 5: 1.

[0110] In an aspect, disclosed nanoparticles can be complexed with one or more nucleic acid. In an aspect, the nanoparticle can be complexed with one or more nucleic acids, by combining linear PEI with one or more nucleic acids. In an aspect, disclosed nanoparticles and nucleic acids are combined at N / P ratio between about 1 and 50, or between about 10 and 30, or between about 15 and 25. In an aspect, disclosed nanoparticles and nucleic acids are combined at N / P ratio of about 50, about 45, about 40, about 35, about 30, about 25, about 20, about 15, about 10, about 5, or about 1. In an aspect, the disclosed nanoparticles and nucleic acids are combined at N / P ratio of about 20.

[0111] In further aspects, disclosed nanoparticles can be complexed with one or more nucleic acids, where nucleic acids can be encased within the disclosed nanoparticles, conjugated, either covalently or ionically, to the membrane of the disclosed nanoparticles, and / or attached to a carrier molecule that directs the nucleic acids to within the disclosed nanoparticle or to a surface (e.g., interior or exterior surface) of the disclosed nanoparticle. In an aspect, the nanoparticle can bind to or otherwise engage with the cell membrane (e.g., transfect) of cells within a disclosed hydrogelscaffold and the disclosed nanoparticle-complexed nucleic acids can be released into the cells to drive expression of the biologically active molecules.

[0112] In further aspects, the disclosed nanoparticles are further coated with a hydrogel. In an aspect, the hydrogel used for coating can be modified with one or more functional groups. In an aspect, the hydrogel used for coating can be hyaluronic acid (HA), chitosan, heparin, alginate, gelatin, fibrin, collagen, MATRIGEL®, polyvinyl alcohol, polyethylene glycol, sodium polyacrylate, acrylate polymers, copolymers, dithiol polymers (e.g., acrylamide), click-based composite hydrogels polyethylene glycol)-diacrylate, poly(ethylene glycol)-vinyl sulfone, or any combination thereof. In an aspect, the hydrogel used for coating can be modified with functional group norbornene, acrylamide, tetrazine, sulfate, cyclodextrin, adamantane, vinyl sulfone, acrylate, allyl, azide, alkyne, thiol, PEG, or any combination thereof. In an aspect, the hydrogel can comprise HA modified with norbomene functional group (HA-NB). In an aspect, a disclosed hydrogel scaffold can comprise disclosed nanoparticles coated with HA-NB.

[0113] In an aspect, the one or more disclosed nanoparticles and one or more coating hydrogel are mixed at a coating / complexing ratio. The coating / complexing ratio represents a weight / weight ratio. In some embodiments, the coating / complexing ratio can comprise between about 0.1 and 100, or between about 0.5 and 75, or between about 0.5 and 50, or between about 1 and 40, or between about 1 and 30, or between about 1 and 25, or between about 1 and 20. In an aspect, the coating / complexing ratio can be about 5.

[0114] In an aspect, the disclosed nanoparticles are further freeze-dried or lyophilized. In an aspect, the disclosed nanoparticles are lyophilized in the presence of a cryoprotectant. In an aspect, the cryoprotectant can comprise sucrose, trehalose, proline, lysine, lactose, lactosucrose, low melting point agarose, taurine, or any combination thereof. In an aspect, the cryoprotectant can comprise sucrose.

[0115] In an aspect, the disclosed nanoparticles can comprise one or more nucleic acids. In an aspect, some of the disclosed nanoparticles can comprise one of one or more nucleic acids. In an aspect, some of the disclosed nanoparticles can comprise two of one or more nucleic acids. In an aspect, some of the disclosed nanoparticles can comprise three of one or more nucleic acids.

[0116] In an aspect, the disclosed nanoparticles can comprise at least two nucleic acids. In an aspect, some of the disclosed nanoparticles can comprise one of the two nucleic acids. In an aspect, some of the disclosed nanoparticles can comprise two of the two nucleic acids.

[0117] In an aspect, some of the disclosed nanoparticles can comprise at least three nucleic acids. In an aspect, some of the disclosed nanoparticles can comprise one of the three nucleic acids. Inan aspect, some of the disclosed nanoparticles can comprise two of the three nucleic acids. In an aspect, some of the disclosed nanoparticles can comprise three of the three nucleic acids.

[0118] In an aspect, a disclosed nanoparticle can comprise the same nucleic acids (e.g., A, or A and A, or A, A, and A; B, or B and B, or B, B, and B; or C, C and C, or C, C, and C). In an aspect a disclosed nanoparticle can comprise different nucleic acids (e.g., A and B, or A and C, or B and C, or A, B, and C).

[0119] In an aspect, the concentration of the nucleic acid in a disclosed hydrogel scaffold comprises between about 0.1 mg / mL nucleic acid and 20 mg / mL nucleic acid, or between about 0.2 mg / mL nucleic acid and 18 mg / mL nucleic acid, or between about 0.25 mg / mL nucleic acid and 16 mg / mL nucleic acid, or between about 0.3 mg / mL nucleic acid and 14 mg / mL nucleic acid, or between about 0.4 mg / mL nucleic acid and 12 mg / mL nucleic acid, or between about 0.5 mg / mL nucleic acid and 10 mg / mL nucleic acid, or between about 0.5 mg / mL nucleic acid and 8 mg / mL nucleic acid, or between about 0.5 mg / mL nucleic acid and 7 mg / mL nucleic acid, or between about 0.5 mg / mL nucleic acid and 6 mg / mL nucleic acid, or between about 0.5 mg / mL nucleic acid and 5 mg / mL nucleic acid, or between about 0.6 mg / mL nucleic acid and 6 mg / mL nucleic acid, or between about 0.7 mg / mL nucleic acid and 7 mg / mL nucleic acid, or between about 0.8 mg / mL nucleic acid and 8 mg / mL nucleic acid, or between about 0.9 mg / mL nucleic acid and 9 mg / mL nucleic acid, or between about 1 mg / mL nucleic acid and 10 mg / mL nucleic acid, or between about 1 mg / mL nucleic acid and 7.5 mg / mL nucleic acid, or between about 1 mg / mL nucleic acid and 5 mg / mL nucleic acid.

[0120] In an aspect, the disclosed nanoparticles can be loaded into the scaffold by embedding. In an aspect, the disclosed nanoparticles can be embedded by combining the disclosed lyophilized or freshly prepared disclosed nanoparticles in hydrogel precursor solution, followed by crosslinking of the precursor solution, to generated disclosed nanoparticle embedded hydrogel. Such hydrogel can be further processed using the described methods to generate a disclosed hydrogel scaffold comprising embedded disclosed nanoparticles.

[0121] In an aspect, the disclosed nanoparticles can be loaded by mixing lyophilized or freshly prepared disclosed nanoparticles with the disclosed hydrogel particles prior to annealing, incubating the disclosed nanoparticles in the hydrogel and annealing the disclosed hydrogel particles to form a disclosed hydrogel scaffold.

[0122] In other aspects, the disclosed nanoparticles can be loaded by adding a solution comprising the disclosed nanoparticles, to a disclosed hydrogel scaffold.

[0123] In an aspect, the disclosed nanoparticles can be loaded with the disclosed hydrogel particles in a disclosed hydrogel scaffold at a volumetric ratio of about 1 :100 to about 1 :1. In anaspect, disclosed nanoparticles can be loaded at a volumetric ratio of about 1 : 100, about 1:50, about 1 :30, about 1 :20, about 1 : 10, about 1 :5, about 1 :2 or about 1 : 1. In an aspect, the disclosed nanoparticles can be loaded into hydrogel before annealing step, at a volumetric ratio of about 1 : 10.

[0124] In an aspect, the disclosed nanoparticles complexed with one or more nucleic acids can comprise DNA or RNA. In an aspect, DNA can comprise one or more of all forms of natural, synthetic, purified, isolated, recombinant, or any combination thereof, of deoxyribonucleic acid, e.g., oligonucleotide, DNA, plasmid DNA, ssDNA, dsDNA, cDNA, vector DNA, mini circle DNA, genomic DNA, DNA gene fragments, cDNA, viral DNA, bacterial DNA, microbial DNA. In an aspect, the RNA can comprise one or more of all forms of natural, synthetic, purified, isolated, recombinant, or any combination thereof, of ribonucleic acid, e.g., oligonucleotide, mRNA, pre-mRNA, siRNA, shRNA, ssRNA, antisense RNA oligonucleotide, antisense miRNA, trans-splicing RNA, guide RNA, single-guide RNA, crRNA, tracrRNA, trans-splicing RNA, or RNA vector.

[0125] In an aspect, the disclosed nucleic acid can be a plasmid. The disclosed plasmid can be any plasmid known to one skilled in the art.

[0126] In an aspect, the nucleic acid encodes a biologically active molecule. In an aspect, the nucleic acid can encode a peptide, a polypeptide, a protein, an antibody, or any combination thereof. In an aspect, the nucleic acid encodes a therapeutic protein, therapeutic peptide, recombinant protein, monoclonal antibody, recombinant antibody, chimeric antibody, humanized antibody, non-human antibody, Fc fusion protein, single chain variable fragments (scFv), chimeric antigen receptors (CAR) or any combination thereof. In an aspect, the biologically active molecule can be an enzyme, hormone, growth factor cytokine, chemokine, or any combination thereof. In an aspect, the biologically active molecule can be human, primate, murine, leporine, canine, feline, bovine, equine, ovine, caprine, avian, or any derivatives thereof.

[0127] In some aspects the nucleic acid encodes a biologically active molecule, wherein the biologically active molecule can be one or more of glucagon, Hepatitis B vaccine, polyketides, aromatic amino-acid-derived molecule, epidermal growth factor, blood coagulation factor Xllla, proinsulin, insulinotropin, cytochrome P450 enzyme, oestrogen receptor a, vascular endothelial growth factor (VEGF) such as e.g., short-form VEGF-D3; a vascular growth inhibitor (e.g., endostatin, angiostatin), thymidine kinase, an interferon (IFN, e.g., IFN-a, IFN-P, IFN-y) , an ATP -binding cassette (ABC) transport protein such as ABC1 member 4 (a / k / a ABCA4) or an ALD protein, e.g., the ABCD-1 protein, myosin VIIA; cyclooxygenase-2, a PGF2-alpha receptor; dopamine; the human hemoglobin subunit beta protein, tumor necrosis factor (TNF, e.g., TNF-a,TNF-P) , growth hormone (e.g., human growth hormone, bovine growth hormone), growth hormone releasing factor, parathyroid hormone, thyroid stimulating hormone, lipoproteins; alpha- 1 -antitrypsin, insulin A-chain, insulin B-chain, proinsulin, follicle stimulating hormone, calcitonin, luteinizing hormone, follicle stimulating hormone (FSH), chorionic gonadotropin (CG) factor VIIIC, factor IX, tissue factor, von Willebrands factor; Protein C; atrial natriuretic factor, lung surfactant, urokinase or human urine or tissue-type plasminogen activator (t-PA), bombesin, thrombin, hemopoietic growth factor, enkephalinase, RANTES (regulated on activation normally T-cell expressed and secreted), human macrophage inflammatory protein (MIP-1 -alpha), a serum albumin, human serum albumin, mullerian-inhibiting substance, relaxin A-chain, relaxin B-chain, prorelaxin, mouse gonadotropin-associated peptide; beta-lactamase, DNase, inhibin, activin, integrin, protein A or D, rheumatoid factor, a bone-derived neurotrophic factor (BDNF), neurotrophin-3, -4, -5, or -6 (NT-3, NT-4, NT-5, or NT-6), NGF-P, platelet-derived growth factor (PDGF), fibroblast growth factor, aFGF, bFGF, epidermal growth factor (EGF), transforming growth factor (TGF, e.g., TGF-a, TGF-P, including TGF-pi, TGF-P2. TGF-P3, TGF-P4, or TGF- P5), insulin-like growth factor-I and -II (IGF-I and IGF-II), des(l-3) -IGF-I (brain IGF-I), CD-3, CD-4, CD-8, CD- 19, erythropoietin, osteoinductive factors, immunotoxins, ; colony stimulating factors (CSFs, e.g., M-CSF, GM-CSF, or G-CSF), interleukins (e.g., IL-la, IL-lb, IL-RA, IL-2, IL-3, -4, IL-5, IL-7, IL-9, IL-10, IL-13, IL-14, IL-15, IL-16, IL-17), cytokines (e.g., CD154, 4- 1BBL, APRIL, TRANCE, TWEAK, TRANCE, TALL-1, LIGHT, OX40L, Flt-3L, stem cell factor, receptor activator of nuclear factor (RANKL)), C-C chemokines (e.g., CCL1, CCL2 (MCP- 1), CCL3 (MIP-la) CCL4 (MIP-ip), CCL5 (RANTES), CCL7 (MCP-3), CCL8 (MCP-2), CCL11 (eotaxin), CCL14 (MCP-4), CCL15 (HCC-1), CCL16 (HCC-2), CCL17 (TARC), CCL18 (MIP- 4), CCL19 (MIP-3P), CCL20 (MIP-3a), CCL21, CCL22, CCL23, CCL24, CCL25, CCL26 CCL27), C-X-C chemokines (e g., CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13), superoxide dismutase, or T- cell receptor.

[0128] In an aspect, the nucleic acid encodes a biologically active molecule, wherein the biologically active molecule can be an antibody, or antibody fragments. In an aspect, the antibody can be a monoclonal antibody, a recombinant antibody, a human chimeric antibody, a humanized antibody, a human antibody, non-human antibody, multispecific antibodies (e.g., bispecific antibodies), antibody fragments that can bind antigen (e.g., Fab, Fab', F(ab')2, Fv, single chain antibodies, diabodies), comprising complementarity determining regions (CDRs) of the foregoing as long as they exhibit the desired biologically activity, multimers or aggregates of intact molecules and / or fragments, including chemically derivatized antibodies. Antibodies of anyisotype class or subclass, including IgG, IgM, IgD, IgA, and IgE, IgGl, IgG2, IgG3, IgG4, IgAl and IgA2, or any allotype, are contemplated. Exemplary antibodies include a Rituximab, Trastuzumab, Alemtuzumab, Omalizumab, Bevacizumab, Panitumumab, Tocilizumab, blinatumomab, Pertuzumab, Avelumab, Atezolizumab, Brentuximab, Cetuximab, Daratumumab, Denosumab, Durvalumab, Ipilimumab, nivolumab, Nivolumab, Pembrolizumab, Polatuzumab, or Rituximab. Exemplary Fc fusion proteins include Etanercept, Alefacept, Abatacept, Rilonacept, or Belatacept

[0129] In an aspect, the nucleic acid encodes a virus. In an aspect, the nucleic encodes an Adeno- associated virus (AAV), a herpes simplex virus, a retrovirus, a lentivirus, and alphavirus, a flavivirus, a rhabdovirus, a measles virus, a Newcastle disease virus, a poxvirus, or a picomavirus. In an aspect, a disclosed virus can be AAV. In an aspect, a disclosed AAV can be AAV1, AAV2, AAV3 (including 3 A and 3B), AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV8 bp, AAV7M8, AAVAnc80, AAVrhlO, AAVPHP.B, AAV type rh32.33, AAV type rh8, AAV type rh74, AAV type hu.68, avian AAV, bovine AAV, canine AAV, equine AAV, ovine AAV, snake AAV, bearded dragon AAV, AAV2i8, AAV2g9, AAV-LK03, AAV7m8, AAV Anc80, AAV-PHP.eB, AAV-TT, AAVv66, rAAV2 / l, rAAV2 / 8, rAAV2 / 9. In an aspect, AAV can be a naturally isolated AAV variants including, but not limited to, AAV-DJ, AAV-HAE1, AAV-HAE2, AAVM41, AAV-1829, AAV2 Y / F, AAV2 T / V, AAV2i8, AAV2.5, AAV9.45, AAV9.61, AAV-B1, AAV-AS, AAV9.45A-String (e.g., AAV9.45-AS), AAV9.45Angiopep, AAV9.47-Angiopep, and AAV9.47-AS, AAV-PHP.B, AAV-PHP.eB, AAV-PHP.S, AAV-F, AAVcc.47, and AAVcc.81.C. Methods of Producing Biologically Active Molecules Using Hydrogel Scaffolds

[0130] Disclosed herein is a method of producing a biologically active molecule. The method can comprise loading cells into a hydrogel scaffold described herein, and maintaining the cells. In an aspect, a disclosed hydrogel scaffold can comprise disclosed hydrogel particles. In further aspects, a disclosed hydrogel scaffold can also comprise disclosed nanoparticles complexed with one or more nucleic acids.

[0131] In an aspect, the disclosed nucleic acids complexed to the disclosed nanoparticles encode a biologically active molecule. In an aspect, the biologically active molecule is an oligonucleotide, a DNA molecule, an RNA molecule, a virus, or any combination thereof. In an aspect, the biologically active molecule can be a AAV. In an aspect, the nucleic encodes an Adeno-associated virus (AAV), a herpes simplex virus, a retrovirus, a lentivirus, and alphavirus, a flavivirus, a rhabdovirus, a measles virus, a Newcastle disease virus, a poxvirus, or a picomavirus. In an aspect, a disclosed vims can be AAV. In an aspect, a disclosed AAV can be AAV1, AAV2, AAV3(including 3A and 3B), AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV8 bp, AAV7M8, AAVAnc8O, AAVrhlO, AAVPHP.B, AAV type rh32.33, AAV type rh8, AAV type rh74, AAV type hu.68, avian AAV, bovine AAV, canine AAV, equine AAV, ovine AAV, snake AAV, bearded dragon AAV, AAV2i8, AAV2g9, AAV-LK03, AAV7m8, AAV Anc80, AAV-PHP.eB, AAV-TT, AAVv66, rAAV2 / l, rAAV2 / 8, rAAV2 / 9. In an aspect, AAV can be a naturally isolated AAV variants including, but not limited to, AAV-DJ, AAV-HAE1, AAV-HAE2, AAVM41, AAV-1829, AAV2 Y / F, AAV2 T / V, AAV2i8, AAV2.5, AAV9.45, AAV9.61, AAV-B1, AAV-AS, AAV9.45A-String (e.g., AAV9.45-AS), AAV9.45Angiopep, AAV9.47-Angiopep, and AAV9.47-AS, AAV-PHP.B, AAV-PHP.eB, AAV-PHP.S, AAV-F, AAVcc.47, and AAVcc.81.

[0132] In an aspect, a biologically active molecule can be a peptide, a polypeptide, a protein, an antibody, or any combination thereof. In an aspect, biologically active molecule can be a therapeutic protein, therapeutic peptide, recombinant protein, monoclonal antibody, recombinant antibody, chimeric antibody, humanized antibody, non-human antibody, multispecific antibody, antibody fragments, Fc fusion protein, single chain variable fragments (scFv), chimeric antigen receptors (CAR), or any combination thereof. In an aspect, the biologically active molecule can be an enzyme, hormone, growth factor cytokine, chemokine, or any combination thereof. In an aspect, the biologically active molecule can be human, primate, murine, leporine, canine, feline, bovine, equine, ovine, caprine, avian, or any derivatives thereof. In an aspect, the biologically active molecule can be human vascular endothelial growth factor (VEGF), interleukin 4 (IL-4), or any combination thereof.

[0133] In an aspect, cells used in the method can be human, primate, murine, leporine, canine, feline, bovine, equine, ovine, caprine, avian. In an aspect, cells can comprise any cells suitable for production of the biologically active molecule. In an aspect, cells can be adherent cells or nonadherent cells. In an aspect, the cells can be prokaryotic or eukaryotic cells. Non-limiting examples of prokaryotic cell includes bacterial species, e.g., Escherichia coli. In an aspect, eukaryotic cells can be yeast and fungal cells (e.g., yeast, Aspergillus, Penicillium, Pichia pastoris, Yarrowia lipolytica), plant cell, or insect cells. In further aspects, eukaryotic cells can be mammalian or a non-mammalian cell. In an aspect, the cell can be a primary cell or a cell from an established or temporary cell line. The cell can be a naive (e.g., unmodified) cell, normal (e.g., healthy) cells, abnormal (e.g., sick or unhealthy cells), pharmacologically modified cell, a genetically modified cell (e.g., transfected cell, transformed cell, knockout cells, conditional knockout cells, knockin cells, conditional knockin cells, chimeric cells, polyploid cells, a fresh cell (e.g., a cell removed from an living organism and loaded into a disclosed hydrogel scaffold with no or minimalmanipulation), a cryopreserved (e.g., frozen) cell, thawed cells, stored cells, or a combination thereof. In an aspect, the cell is a human embryonic kidney (HEK) cell, Chinese hamster ovary (CHO) cell, DXB-11, DG-44, baby hamster kidney (BHK) cell, African green monkey kidney cell, Hela cell, adenocarcinomic human alveolar basal epithelial cell, human lung cell, human hepatoma (Hep) cell, mouse mammary tumor (MMT) cell, TRI cell, MRC5 cell, FS4 cell, mammalian myeloma cell, E. coli cell, yeast cell, or any combination thereof. In an aspect, the cell can be a HEK cell. In an aspect, the cell can be a HEK293 cell.

[0134] In an aspect, the disclosed e nanoparticles are complexed with one or more nucleic acids. The nucleic acid encodes one or more of any of the biologically active molecule, described herein. In an aspect, the disclosed nanoparticles are complexed at least two nucleic acids. In an aspect, the disclosed nanoparticles are complexed at least three nucleic acids. In an aspect, the disclosed nanoparticles are complexed with more than three nucleic acids. In an aspect, the disclosed nanoparticles deliver the complexed one or more nucleic acids to the cells within a disclosed hydrogel scaffold to drive production of a biologically active molecule.

[0135] In an aspect, a disclosed hydrogel scaffold can be loaded with one or more cells. In an aspect, loading cells can comprise topical seeding of the cells into a disclosed hydrogel scaffold. In an aspect, topical seeding of the cells can comprise combining the cells with the disclosed hydrogel scaffold. In an aspect, the cells are combined at about 5,000 cells / pL scaffold volume to about 15,000 cells / pL scaffold volume. In an aspect, the cells are combined with a disclosed hydrogel scaffolds at about 5,000 cells / pL scaffold volume, about 6,000 cells / pL scaffold volume, about 7,000 cells / pL scaffold volume, about 8,000 cells / pL scaffold volume, about 9,000 cells / pL scaffold volume, about 10,000 cells / pL scaffold volume, about 11,000 cells / pL scaffold volume, about 12,000 cells / pL scaffold volume, about 13,000 cells / pL scaffold volume, about 14,000 cells / pL scaffold volume, or about 15,000 cells / pL scaffold volume. In an aspect, the cells are seeded topically at about 10,000 cells / pL scaffold volume.

[0136] In an aspect, a disclosed hydrogel scaffold can be loaded with one or more cells by encapsulating the cells in the disclosed hydrogel scaffold. In an aspect, for encapsulation, cells are initially seeded into hydrogel before annealing step, based on volumetric ratio of 1 : 100 to 1 : 1. In an aspect, cells are seeded at a volumetric ratio of 1 : 100, 1 :50, 1 :30, 1 :20, 1: 10, 1 :5, 1 :2 or 1 : 1. In an aspect, the cells are seeded into hydrogel before annealing step, at a volumetric ratio of 1 : 10 and the cells are encapsulated within the scaffold during annealing. In an aspect, the cells are seeded at a cell density of about 100 cells / pL to about 10,000 cells / pL of scaffold volume. In an aspect, the cells are seeded at a density of about 100 cell / pL of scaffold volume, about 300 cell / pL of scaffold volume, about 500 cell / pL of scaffold volume, about 600 cell / pL of scaffold volumeabout 1000 cell / .L of scaffold volume, about 2000 cell / .L of scaffold volume, about 3000 cell / .L of scaffold volume, about 4000 cell / .L of scaffold volume, about 5000 cell / .L of scaffold volume, about 6000 cell / .L of scaffold volume, about 7000 cell / .L of scaffold volume, about 8000 cell / .L of scaffold volume, about 9000 cell / .L of scaffold volume, or about 10,000 cell / pL of scaffold volume. In an aspect, cells are seeded at about 300 cell / pL of scaffold volume. In an aspect, cells are seeded at about 600 cell / pL of scaffold volume. In an aspect, the cells are seeded at about 1000 cell / pL of scaffold volume. In an aspect, the cells are seeded at about 6000 cell / pL of scaffold volume.

[0137] In further aspects, the method can comprise adding a medium to a disclosed hydrogel scaffold. In an aspect, the medium can be any medium that supports the growth and expansion of the disclosed cells. Suitable medium for cell growth and expansion will also be apparent to the skilled person, based on the type of cells used. In an aspect, the medium used for growing cells provides at least one component from one or more an energy source, usually in the form of a carbohydrate such as glucose, all essential amino acids, and usually the basic set of twenty amino acids plus cysteine, vitamins and / or other organic compounds required at low concentrations, free fatty acids, trace elements, where trace elements are defined as inorganic compounds or naturally occurring elements that are typically required at very low concentrations, usually in the micromolar range, or any combination thereof. The medium can optionally be supplemented with one or more components, non-limiting examples of which include hormones and other growth factors (e.g., insulin, transferrin, and epidermal growth factor), salts and buffers (e.g., calcium, magnesium, and phosphate), nucleosides and bases (e.g., adenosine, thymidine, and hypoxanthine), and protein and tissue hydrolysates. In an aspect, medium can comprise Ham’s F10, Minimal Essential Medium (MEM), RPMI-1640, Dulbecco’s, Modified Eagle’s Medium (DMEM), Ex-cell GTM3, Freestyle 5FM293, Hyclone 5FM293, RPMI1640 medium, GIT medium , Ex-cell 302 medium, IMDM medium, Hybridoma-SFM or any combination thereof. In an aspect, the medium can comprise DMEM.

[0138] In an aspect, the medium can be added to a disclosed hydrogel scaffold at gel-to-media ratio (v / v) of about 1 : 100 to about 1 : 1. In an aspect, the medium can be added to a disclosed hydrogel scaffold at about 1 : 100, about 1 :50, about 1 :30, about 1 :20, about 1 : 10, about 1 :5, about 1 :2 or about 1 : 1. In an aspect, the medium can be added to a disclosed hydrogel scaffold at about 1 : 10.

[0139] In some aspects of the method, the cells are further maintained. In an aspect, maintaining the cells can comprise removing spent medium and adding fresh medium. In an aspect, removing spent medium and adding fresh medium can be done continuously (e.g., perfusion) or periodically.In an aspect, the spent medium can be removed and replaced with fresh medium at desired intervals for e.g., twice daily, daily, every 2-3 days, or every 5-7 days. In further aspects, the spent medium can be filtered to remove cells, using appropriate filtration means well known in the art. The spent medium can also be further processed to recover the biologically active molecule produced by the cells. In an aspect, maintaining the cells further can comprise maintaining the cells at conditions suitable for production of biologically active molecule, including, maintaining the volume of medium, maintaining the cell density, maintaining the medium nutrient components, maintaining culture conditions (e.g., pH, dissolved oxygen (dCh), temperature, air saturation). Such conditions used with the particular cells will be apparent to the ordinarily skilled the art. For, e.g, the pH can be adjusted to a level between about 6.5 and 7.5 using either an acid (e.g., HC1 or H2CO3) or abase (e.g., ISfeCCh orNaOH). A suitable temperature range for culturing mammalian cells such as CHO cells can be between about 30 to 38° C. and preferably about 37° C. and a suitable dCh can be between 5-90% of air saturation.

[0140] In an aspect, the cells are maintained for a period of time from about 7 days to about 60 days. In an aspect, the cells are maintained for about 7 days, about 10 days, about 20 days, about 30 days, about 35 days, about 40 days, about 45 days, about 50 days, about 55 days, or about 60 days. In an aspect, the cells are maintained at least 14 days. In an aspect, the cells are maintained for at least 21 days. In an aspect, the cells are maintained for at least 27 days. In an aspect, the cells are maintained for at least 30 days. In an aspect, the cells are maintained for more than 30 days.

[0141] Recovering the biologically active molecule can be done continuously, periodically, or at the end of the culture, from the spent medium (e.g., biologically active molecule released in the medium) or by recovering cells from the medium (e.g., biologically active molecule not released in the medium). In an aspect, recovering the biologically active molecule can be performed e.g., twice daily, daily, every 2-3 days, or every 5-7 days for a period of about 7 days to about 30 days, or more. The biologically active molecule can be recovered using any known methods in the art.

[0142] In an aspect, the disclosed method produces long-term, stable expression of the biologically active molecule. In an aspect, the expression of the biologically active molecules from the loaded cells in a disclosed hydrogel scaffold can be sustained for a period time from about 7 days to about 60 days. In an aspect, the expression of the biologically active molecules can be sustained for about 7 days, about 10 days, about 20 days, about 30 days, about 35 days, about 40 days, about 45 days, about 50 days, about 55 days, or about 60 days. In an aspect, the expression of the biologically active molecules can be sustained at least 14 days. In an aspect, the expression of the biologically active molecules can be sustained for at least 21 days. In an aspect, theexpression of the biologically active molecules can be sustained for at least 27 days. In an aspect, the expression of the biologically active molecules can be sustained for at least 30 days. In an aspect, the expression of the biologically active molecules can be sustained for more than 30 days. In an aspect, expression of the biologically active molecules can be measured, assessed, assayed, quantified, or otherwise evaluated at one or more timepoints during the period of time. The biologically active molecules can be isolated from a disclosed hydrogel scaffold, the cells within the scaffold, and / or the media provided to a disclosed hydrogel scaffold. In an aspect, the media from a disclosed hydrogel scaffold or perfusion device including a disclosed hydrogel scaffold can be harvested for immediate analysis or stored (e.g., refrigerated at about 4 °C, frozen at about 0 °C, and / or cryopreserved at less than 0 °C, including but not limited to about -20°C, about -40°C, or about -80°C) prior to analysis. In an aspect, the biologically active molecules can be measured, assessed, assayed, quantified, or otherwise evaluated by polymerase chain reaction (PCR), reverse transcriptase polymerase chain reaction (RT-PCR), Southern Blot analysis, Northern Blot analysis, spectrophotometry, fluorometry, gel electrophoresis, SDS-PAGE, Western Blot analysis, enzyme-linked immunosorbent assay (ELISA), enzyme immunoassays (EIA), radioimmunoassay (RIA), flow cytometry, or the like. In an aspect, expression of the biologically active molecule can be considered “sustained” if the amount of the biologically active molecule measured by one of the above assays or other assay is within a parameter identified by the artisan. In an aspect, sustained expression can include an amount of the biologically active molecule within the sample to be ± about 90%, ± about 85%, ± about 80%, ± about 75%, ± about 70%, ± about 65%, ± about 60%, ± about 55%, ± about 50%, ± about 45%, ± about 40%, ± about 35%, ± about 30%, ± about 25%, ± about 20%, ± about 15%, ± about 10%, and / or ± about 5% of a reference sample (e.g., the amount of biologically active molecule produced within 1 to 3 days of loading cells onto a disclosed hydrogel scaffold). In an aspect, expression of a biologically active molecule can be considered “sustained” if the amount of the biologically active molecule within a sample is above a threshold value.

[0143] In an aspect, the biologically active molecule can be recovered at least about 1 x 1010genomic copies (GC). In an aspect, the biologically active molecule can be recovered at about 1 x 1010GC, about 1.5 x 1010GC, about 2 x 1010GC, about 2.5 x 1010GC, about 3 x 1010GC, about 3.5 x 1010GC, about 4 x 1010GC, about 1 x 1011GC, about 1.5 x 1011GC, about 2 x 1011GC, about 2.5 x 1011GC, about 3 x 1011GC, about 3.5 x 1011GC, about 4 x 1011GC, about 1 x 1012GC, about 1.5 x 1012GC, about 2 x 1012GC, about 2.5 x 1012GC, about 3 x 1012GC, about 3.5 x1012GC, about 4 x 1012, about 1 x 1013GC, about 1.5 x 1013GC, about 2 x 1013GC, about 2.5 x1013GC, about 3 x 1013GC, about 3.5 x 1013GC, about 4 x 1013, about 1 x 1014GC, about 1.5 x1014GC, about 2 x 1014GC, about 2.5 x 1014GC, about 3 x 1014GC, about 3.5 x 1014GC, about 4 x 1014, about 1 x 1015GC, about 1.5 x 1015GC, about 2 x 1015GC, about 2.5 x 1015GC, about 3 x 1015GC, about 3.5 x 1015GC, or about 4 x 1015. In an aspect, the biologically active molecule can be recovered at least about 1 x 1012GC. In an aspect, the biologically active molecule can be recovered at least about 1 x 1013GC.

[0144] In an aspect, the biologically active molecule can be recovered at least about 1000 pg / mL. In an aspect, the biologically active molecule can be recovered at least about 1000 pg / mL, about 1500 pg / mL, about 2000 pg / mL, about 2500 pg / mL, about 3000 pg / mL, about 3500 pg / mL, about 4000 pg / mL, about 5000 pg / mL, about 6000 pg / mL, about 7000 pg / mL, about 8000 pg / mL, about 9000 pg / mL, about 10,000 pg / mL, about 12,000 pg / mL, about 13,000 pg / mL, about 14,000 pg / mL, about 15,000 pg / mL, or about 20,000 pg / mL.

[0145] In an aspect, the disclosed method provides a continued production of a biologically active molecule, which can be recovered at sufficient levels, for a period of time. In an aspect, the biologically active molecule can be recovered at least about 1 x 1012genomic copies for a period time from about 7 days to about 60 days. In an aspect, the biologically active molecule can be recovered at least about 1 x 1012genomic copies for about 7 days, about 10 days, about 20 days, about 30 days, about 35 days, about 40 days, about 45 days, about 50 days, about 55 days, or about 60 days. In an aspect, the biologically active molecule can be recovered at least about 1 x 1012genomic copies for about 14 days. In an aspect, the biologically active molecule can be recovered at least about 1 x 1012genomic copies for about 21 days. In an aspect, the biologically active molecule can be recovered at least about 1 x 1012genomic copies for about 27 days. In an aspect, the biologically active molecule can be recovered at least about 1 x 1012genomic copies for about 30 days. In an aspect, the biologically active molecule can be recovered at least about 1 x 1012genomic copies for more than 30 days.

[0146] In an aspect, the disclosed method provides a continued production of a biologically active molecule, which can be recovered at sufficient levels, for a period of time. In an aspect, the biologically active molecule can be recovered at least about 1000 pg / mL for a period time from about 7 days to about 60 days. In an aspect, the biologically active molecule can be recovered at least about 1000 pg / mL for about 7 days, about 10 days, about 20 days, about 30 days, about 35 days, about 40 days, about 45 days, about 50 days, about 55 days, or about 60 days. In an aspect, the biologically active molecule can be recovered at least about 1000 pg / mL for about 14 days. In an aspect, the biologically active molecule can be recovered at least about 1000 pg / mL for about 21 days. In an aspect, the biologically active molecule can be recovered at least about 1000 pg / mL for about 27 days. In an aspect, the biologically active molecule can be recovered at least about1000 pg / mL for about 30 days. In an aspect, the biologically active molecule can be recovered at least about 1000 pg / mL for more than 30 days.

[0147] In an aspect, a disclosed hydrogel scaffold of disclosed method provides improved viability of loaded cells. Viability of cells can be assessed using any known methods in the art, non-limiting examples of which include, staining and imaging, flow cytometry, reporter transgene expression and metabolic activity assays (e.g., using PrestoBlue). In an aspect, the viability of cells can be increased by about 10% to about 100%, compared to cells not loaded in a disclosed hydrogel scaffold. In an aspect, the viability can be increased by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, or more compared to cells not loaded in a disclosed hydrogel scaffold.

[0148] In an aspect, the methods disclosed herein can be further modified. As used herein, “modifying the method” can comprise modifying or changing one or more features or aspects of one or more steps of a disclosed method. In an aspect, a method can be altered for e.g., by changing the amount of cells loaded in a disclosed hydrogel scaffold, used in a disclosed method, or by changing the frequency of medium replenishment or removal of spent medium in a disclosed method, by changing the duration of time of maintaining cell in the hydrogel, or by substituting for one or more of the disclosed components used in a disclosed hydrogel scaffold with a similar or equivalent component and / or reagent.D. Method of Nucleic Acid Delivery

[0149] Disclosed herein is a method of nucleic acid delivery. The disclosed method can comprise loading cells into a disclosed hydrogel scaffold. In further aspects, a disclosed hydrogel scaffold can comprise disclosed hydrogel particles and disclosed nanoparticles complexed with one or more nucleic acid. In an aspect, the nucleic acid encodes a biologically active molecule. The method disclosed herein provides direct cell transfection with the nucleic acids complexed on the disclosed nanoparticles.

[0150] In an aspect, a method of expressing a nucleic acid in one more cells, the method comprising loading one or more cells into the disclosed hydrogel scaffold is further provided.

[0151] In some aspects of the method, the disclosed nanoparticles are complexed with one or more nucleic acids, and one or more of the loaded cells are transfected with one or more nucleic acids. In an aspect, the disclosed nanoparticles are complexed with at least two nucleic acids, and one or more of the loaded cells are transfected with one or more nucleic acids. In an aspect, the disclosed nanoparticles are complexed with at least three nucleic acids, and one or more of the loaded cells are transfected with one or more nucleic acids. In an aspect, the disclosed nanoparticles are complexed with more than three nucleic acids, and one or more of the loadedcells are transfected with one or more nucleic acids. In an aspect, a disclosed hydrogel scaffold brings the loaded cells and disclosed nanoparticles complexed with one or more nucleic acids into proximity sufficient to deliver the nucleic acids to the cell and drive expression of the biologically active molecules. In an aspect, one or more of the loaded cells are transfected with at least two of the nucleic acids. In an aspect, one or more of the loaded cells are transfected with three of the nucleic acids.

[0152] In further aspects, the method can comprise loading cells using any of the disclosed methods, in a disclosed hydrogel scaffold, adding a medium and maintaining the cells. In an aspect, the cells are maintained as described herein, for a period time from about 7 days to about 60 days. In an aspect, the cells are maintained at least 14 days. In an aspect, the cells are maintained for at least 21 days. In an aspect, the cells are maintained for at least 27 days. In an aspect, the cells are maintained for at least 30 days. In an aspect, the cells are maintained for more than 30 days. In an aspect, the expression of one or more nucleic acids can be sustained for a period time from about 7 days to about 60 days. In an aspect, the expression can be sustained at least 14 days. In an aspect, the expression can be sustained for at least 21 days. In an aspect, the expression can be sustained for at least 27 days. In an aspect, the expression can be sustained for at least 30 days. In an aspect, the expression can be sustained for more than 30 days.

[0153] In an aspect, a disclosed hydrogel scaffold of disclosed method provides improved transfection of nucleic acids into the loaded cells. Transfection of cells can be assessed using any known methods in the art, non-limiting examples of which include staining and / or imaging reporter gene expression, using plate reader or flow cytometry. In an aspect, the transfection of cells are increased by about 10% to about 100%, compared to cells not loaded in a disclosed hydrogel scaffold. In an aspect, the transfection can be increased by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, or more compared to cells not loaded in a disclosed hydrogel scaffold.

[0154] In an aspect, a disclosed hydrogel scaffold of disclosed method provides long-term improved transfection of loaded cells. In an aspect, the transfection of cells is increased by about 10% to about 100%, for a period time from about 7 days to about 60 days, compared to cells not loaded in a disclosed hydrogel scaffold. In an aspect, the transfection can be increased by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, or more, for a period time from about 7 days to about 60 days, compared to cells not loaded in a disclosed hydrogel scaffold.

[0155] In an aspect, a disclosed hydrogel scaffold of disclosed method provides long-term improved viability of loaded cells. In an aspect, the viability of cells are increased by about 10%to about 100%, for a period time from about 7 days to about 60 days, compared to cells not loaded in a disclosed hydrogel scaffold. In an aspect, the viability can be increased by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, or more, for a period time from about 7 days to about 60 days, compared to cells not loaded in a disclosed hydrogel scaffold.

[0156] In further aspects, the method further can comprise recovering the expressed biologically active molecules, using method disclosed herein. In an aspect, the biologically active molecule can be an oligonucleotide, a DNA molecule, an RNA molecule, a virus, or any combination thereof. In an aspect, the biologically active molecule can be a AAV. In an aspect, biologically active molecule can be a peptide, a polypeptide, a protein, an antibody, or any combination thereof. In an aspect, biologically active molecule can be a therapeutic protein, therapeutic peptide, recombinant protein, monoclonal antibody, recombinant antibody, chimeric antibody, humanized antibody, non-human antibody, multispecific antibody, antibody fragments, Fc fusion protein, single chain variable fragments (scFv), chimeric antigen receptors (CAR), or any combination thereof. In an aspect, the biologically active molecule can be an enzyme, hormone, growth factor cytokine, chemokine, or any combination thereof. In an aspect, the biologically active molecule can be human, primate, murine, leporine, canine, feline, bovine, equine, ovine, caprine, avian, or any derivatives thereof. In an aspect, the biologically active molecule can comprise human vascular endothelial growth factor (VEGF), interleukin 4 (IL-4), or any combination thereof.

[0157] In an aspect, the method of nucleic acid delivery provides recovery of biologically active molecule at least about 1 x 1010genomic copies (GC). In an aspect, the biologically active molecule is recovered at about 1 x 1010GC, about 1.5 x 1010GC, about 2 x 1010GC, about 2.5 x 1010GC, about 3 x 1010GC, about 3.5 x 1010GC, about 4 x 1010GC, about 1 x 1011GC, about 1.5 x 1011GC, about 2 x 1011GC, about 2.5 x 1011GC, about 3 x 1011GC, about 3.5 x 1011GC, about 4 x 1011GC, about 1 x 1012GC, about 1.5 x 1012GC, about 2 x 1012GC, about 2.5 x 1012GC, about 3 x 1012GC, about 3.5 x 1012GC, about 4 x 1012, about 1 x 1013GC, about 1.5 x 1013GC, about 2 x 1013GC, about 2.5 x 1013GC, about 3 x 1013GC, about 3.5 x 1013GC, about 4 x 1013, about 1 x 1014GC, about 1.5 x 1014GC, about 2 x 1014GC, about 2.5 x 1014GC, about 3 x1014GC, about 3.5 x 1014GC, about 4 x 1014, about 1 x 1015GC, about 1.5 x 1015GC, about 2 x1015GC, about 2.5 x 1015GC, about 3 x 1015GC, about 3.5 x 1015GC, or about 4 x 1015. In an aspect, the biologically active molecule is recovered at least about 1 x 1012GC. In an aspect, the biologically active molecule can be recovered at least about 1 x 1013GC.

[0158] In an aspect, the method of nucleic acid delivery provides biologically active molecule recovery at least about 1000 pg / mL. In an aspect, the biologically active molecule can be recovered at least about 1000 pg / mL, about 1500 pg / mL, about 2000 pg / mL, about 2500 pg / mL, about 3000 pg / mL, about 3500 pg / mL, about 4000 pg / mL, about 5000 pg / mL, about 6000 pg / mL, about 7000 pg / mL, about 8000 pg / mL, about 9000 pg / mL, about 10,000 pg / mL, about 12,000 pg / mL, about 13,000 pg / mL, about 14,000 pg / mL, about 15,000 pg / mL, or about 20,000 pg / mL.E. Production of Biologically Active Molecules Using Different Culture Methods

[0159] Disclosed herein is a method of producing biologically active molecules using a disclosed hydrogel scaffold and one or more culture methods. In an aspect, a hydrogel scaffold disclosed herein can be adapted to any of the cell culture methods of producing biologically active molecules. Such methods can include static cell culture, or suspension cell culture. In an aspect, the method can comprise introducing disclosed hydrogel scaffolds to a three-dimensional cell culture device, loading cells, and maintaining the cells.

[0160] In an aspect, the method can comprise a static cell culture. In such aspects a method of producing a biologically active molecule in a three-dimensional cell culture device can comprise, adding a disclosed hydrogel scaffold comprising disclosed nanoparticles complexed with one or more nucleic acids into a culture device, loading cells into a disclosed hydrogel scaffold by topical seeding, and maintaining the cells in a disclosed hydrogel scaffold. In an aspect, the method can comprise loading the disclosed nanoparticles in a disclosed hydrogel scaffold by embedding. In an aspect, the disclosed nanoparticles are embedded by combining disclosed nanoparticles in hydrogel precursor solution, followed by crosslinking of the precursor solution.

[0161] In further aspects, the three-dimensional cell culture device for use in static culture, can be any cell culture device that supports the growth and expansion of cells. In an aspect, the three- dimensional cell culture device can comprise a culture tube, culture bottle, flask, microfluidic device, perfusion device, a bioreactor, or any combination thereof. In an aspect, the disclosed hydrogel particles produced by the methods described herein, can be added to the culture device, and the annealing process for generating a disclosed hydrogel scaffold can be performed in the device. In an aspect, the disclosed hydrogel particles produced by the methods described herein, can be annealed and added as a disclosed hydrogel scaffold to the culture device.

[0162] In further aspects of the static culture using a disclosed hydrogel scaffold, disclosed nanoparticles can be complexed with one or more nucleic acids. In an aspect, the disclosed nanoparticles can be complexed at least two nucleic acids. In an aspect, the disclosed nanoparticles can be complexed at least three nucleic acids. In an aspect, the disclosed nanoparticles can be complexed with more than three nucleic acids.

[0163] In further aspects, the method can comprise adding a medium to the cell culture device. In further aspects, maintaining the cells can comprise removing spent medium and adding fresh medium, as described herein.

[0164] In an aspect, the method further can comprise recovering the expressed biologically active molecules, from the static culture, using method disclosed herein. In an aspect, the biologically active molecule can be an oligonucleotide, a DNA molecule, an RNA molecule, a virus, or any combination thereof. In an aspect, the biologically active molecule can be a AAV. In an aspect, biologically active molecule can be a peptide, a polypeptide, a protein, an antibody, or any combination thereof. In an aspect, biologically active molecule can be a therapeutic protein, therapeutic peptide, recombinant protein, monoclonal antibody, recombinant antibody, chimeric antibody, humanized antibody, non-human antibody, multispecific antibody, antibody fragments, Fc fusion protein, single chain variable fragments (scFv), chimeric antigen receptors (CAR), or any combination thereof. In an aspect, the biologically active molecule can be an enzyme, hormone, growth factor cytokine, chemokine, or any combination thereof. In an aspect, the biologically active molecule can be human, primate, murine, leporine, canine, feline, bovine, equine, ovine, caprine, avian, or any derivatives thereof. In an aspect, the biologically active molecule can be human vascular endothelial growth factor (VEGF), interleukin 4 (IL-4), or any combination thereof.

[0165] In an aspect, the disclosed static cell culture using disclosed hydrogel scaffolds provide long-term, stable expression of the biologically active molecule. In an aspect, the expression of the biologically active molecules from the loaded cells in a disclosed hydrogel scaffold in the static culture can be sustained for a period time from about 7 days to about 60 days. In an aspect, the expression of the biologically active molecules can be sustained for about 7 days, about 10 days, about 20 days, about 30 days, about 35 days, about 40 days, about 45 days, about 50 days, about 55 days, or about 60 days. In an aspect, the expression of the biologically active molecules can be sustained at least 14 days. In an aspect, the expression of the biologically active molecules can be sustained for at least 21 days. In an aspect, the expression of the biologically active molecules can be sustained for at least 27 days. In an aspect, the expression of the biologically active molecules can be sustained for at least 30 days. In an aspect, the expression of the biologically active molecules can be sustained for more than 30 days.

[0166] In an aspect, the disclosed static cell culture using disclosed hydrogel scaffolds provide recovery of a biologically active molecule at least about 1 x 1010genomic copies (GC). In an aspect, the biologically active molecule is recovered at about 1 x 1010GC, about 1.5 x 1010GC, about 2 x 1010GC, about 2.5 x 1010GC, about 3 x 1010GC, about 3.5 x 1010GC, about 4 x 1010GC, about 1 x 1011GC, about 1.5 x 1011GC, about 2 x 1011GC, about 2.5 x 1011GC, about 3 x 1011GC, about 3.5 x 1011GC, about 4 x 1011GC, about 1 x 1012GC, about 1.5 x 1012GC, about 2 x 1012GC, about 2.5 x 1012GC, about 3 x 1012GC, about 3.5 x 1012GC, about 4 x 1012, about 1 x 1013GC, about 1.5 x 1013GC, about 2 x 1013GC, about 2.5 x 1013GC, about 3 x 1013GC, about 3.5 x 1013GC, about 4 x 1013, about 1 x 1014GC, about 1.5 x 1014GC, about 2 x 1014GC, about 2.5 x 1014GC, about 3 x 1014GC, about 3.5 x 1014GC, about 4 x 1014, about 1 x 1015GC, about 1.5 x 1015GC, about 2 x 1015GC, about 2.5 x 1015GC, about 3 x 1015GC, about 3.5 x 1015GC, or about 4 x 1015. In an aspect, the biologically active molecule can be recovered at least about 1 x 1012GC. In an aspect, the biologically active molecule can be recovered at least about 1 x 1013GC.

[0167] In an aspect, the disclosed static cell culture using disclosed hydrogel scaffolds provide a recovery of the biologically active molecule at least about 1000 pg / mL. In an aspect, the biologically active molecule can be recovered at least about 1000 pg / mL, about 1500 pg / mL, about 2000 pg / mL, about 2500 pg / mL, about 3000 pg / mL, about 3500 pg / mL, about 4000 pg / mL, about 5000 pg / mL, about 6000 pg / mL, about 7000 pg / mL, about 8000 pg / mL, about 9000 pg / mL, about 10,000 pg / mL, about 12,000 pg / mL, about 13,000 pg / mL, about 14,000 pg / mL, about 15,000 pg / mL, or about 20,000 pg / mL.

[0168] In an aspect, the method can comprise a suspension cell culture. In such aspects, a method of producing a biologically active molecule in a three-dimensional cell culture device can comprise loading cells by encapsulating, in a disclosed hydrogel scaffold comprising disclosed nanoparticles complexed with one or more nucleic acids, adding a disclosed hydrogel scaffold into a culture device, and maintaining the cells. In an aspect, the method can comprise loading the disclosed nanoparticles in the hydrogel by embedding. In an aspect, the nanoparticles are embedded by combining disclosed nanoparticles in hydrogel precursor solution, followed by crosslinking of the precursor solution.

[0169] In further aspects, the three-dimensional cell culture device for use in suspension culture, can be any cell culture device that supports the growth and expansion of cells. In an aspect, the three-dimensional cell culture device can comprise a culture tube, culture bottle, flask, microfluidic device, perfusion device, a bioreactor, or any combination thereof. In an aspect, the disclosed hydrogel particles produced by the methods described herein, can be added to the culture device, and the annealing process for generating a disclosed hydrogel scaffold can be performed in the device. In an aspect, the disclosed hydrogel particles produced by the methods described herein, can be annealed and added as a hydrogel to the culture device.

[0170] In further aspects of the suspension culture using a disclosed hydrogel scaffold, disclosed nanoparticles are complexed with one or more nucleic acids. In an aspect, the disclosed nanoparticles are complexed at least two nucleic acids. In an aspect, the disclosed nanoparticles are complexed at least three nucleic acids. In an aspect, the disclosed nanoparticles are complexed with more than three nucleic acids.

[0171] In further aspects, the method can comprise adding a medium to the cell culture device. In further aspects, maintaining the cells can comprise removing spent medium and adding fresh medium, as described herein.

[0172] In an aspect, the method further can comprise recovering the expressed biologically active molecules, from the suspension culture, using method disclosed herein. In an aspect, the biologically active molecule can be an oligonucleotide, a DNA molecule, an RNA molecule, a virus, or any combination thereof. In an aspect, the biologically active molecule can be a AAV. In an aspect, biologically active molecule can be a peptide, a polypeptide, a protein, an antibody, or any combination thereof. In an aspect, biologically active molecule can be a therapeutic protein, therapeutic peptide, recombinant protein, monoclonal antibody, recombinant antibody, chimeric antibody, humanized antibody, non-human antibody, multispecific antibody, antibody fragments, Fc fusion protein, single chain variable fragments (scFv), chimeric antigen receptors (CAR), or any combination thereof. In an aspect, the biologically active molecule can be an enzyme, hormone, growth factor cytokine, chemokine, or any combination thereof. In an aspect, the biologically active molecule can be human, primate, murine, leporine, canine, feline, bovine, equine, ovine, caprine, avian, or any derivatives thereof. In an aspect, the biologically active molecule can be human vascular endothelial growth factor (VEGF), interleukin 4 (IL-4), or any combination thereof.

[0173] In an aspect, the disclosed suspension cell culture using disclosed hydrogel scaffolds provide long-term, stable expression of the biologically active molecule. In an aspect, the expression of the biologically active molecules from the loaded cells in a disclosed hydrogel scaffold in the suspension culture can be sustained for a period time from about 7 days to about 60 days. In an aspect, the expression of the biologically active molecules can be sustained for about 7 days, about 10 days, about 20 days, about 30 days, about 35 days, about 40 days, about 45 days, about 50 days, about 55 days, or about 60 days. In an aspect, the expression of the biologically active molecules can be sustained at least 14 days. In an aspect, the expression of the biologically active molecules can be sustained for at least 21 days. In an aspect, the expression of the biologically active molecules can be sustained for at least 27 days. In an aspect, the expressionof the biologically active molecules can be sustained for at least 30 days. In an aspect, the expression of the biologically active molecules can be sustained for more than 30 days.

[0174] In an aspect, the disclosed suspension cell culture using disclosed hydrogel scaffolds provide recovery of a biologically active molecule at least about 1 x IO10genomic copies (GC). In an aspect, the biologically active molecule can be recovered at about 1 x IO10GC, about 1.5 x IO10GC, about 2 x IO10GC, about 2.5 x IO10GC, about 3 x IO10GC, about 3.5 x IO10GC, about 4 x IO10GC, about 1 x 1011GC, about 1.5 x 1011GC, about 2 x 1011GC, about 2.5 x 1011GC, about 3 x 1011GC, about 3.5 x 1011GC, about 4 x 1011GC, about 1 x 1012GC, about 1.5 x 1012GC, about 2 x 1012GC, about 2.5 x 1012GC, about 3 x 1012GC, about 3.5 x 1012GC, about 4 x 1012, about 1 x 1013GC, about 1.5 x 1013GC, about 2 x 1013GC, about 2.5 x 1013GC, about 3 x1013GC, about 3.5 x 1013GC, about 4 x 1013, about 1 x 1014GC, about 1.5 x 1014GC, about 2 x1014GC, about 2.5 x 1014GC, about 3 x 1014GC, about 3.5 x 1014GC, about 4 x 1014, about 1 x1015GC, about 1.5 x 1015GC, about 2 x 1015GC, about 2.5 x 1015GC, about 3 x 1015GC, about 3.5 x 1015GC, or about 4 x 1015. In an aspect, the biologically active molecule can be recovered at least about 1 x 1012GC. In an aspect, the biologically active molecule can be recovered at least about 1 x 1013GC.

[0175] In an aspect, the disclosed suspension cell culture using disclosed hydrogel scaffolds provide a recovery of the biologically active molecule at least about 1000 pg / mL. In an aspect, the biologically active molecule can be recovered at least about 1000 pg / mL, about 1500 pg / mL, about 2000 pg / mL, about 2500 pg / mL, about 3000 pg / mL, about 3500 pg / mL, about 4000 pg / mL, about 5000 pg / mL, about 6000 pg / mL, about 7000 pg / mL, about 8000 pg / mL, about 9000 pg / mL, about 10,000 pg / mL, about 12,000 pg / mL, about 13,000 pg / mL, about 14,000 pg / mL, about 15,000 pg / mL, or about 20,000 pg / mL.F. System For Production of Biologically Active Molecule

[0176] Disclosed herein is a system for producing one or more biologically active molecules comprising using a disclosed hydrogel scaffold. The disclosure further encompasses a system for production of biologically active molecules. In an aspect, the system can comprise hydrogel scaffold disclosed herein. The system can comprise disclosed hydrogel particles and disclosed nanoparticles complexed with one or more nucleic acids in a disclosed hydrogel scaffold. In an aspect, the system can further comprise one or more cells.

[0177] In an aspect, the system can comprise a disclosed hydrogel scaffold comprising disclosed hydrogel particles and disclosed nanoparticles complexed with one or more nucleic acids. In an aspect, the disclosed nanoparticles can be complexed with one or more nucleic acids which encodes one or more of any of the biologically active molecule, described herein. In an aspect, thedisclosed nanoparticles can be complexed at least two nucleic acids. In an aspect, the disclosed nanoparticles can be complexed at least three nucleic acids. In an aspect, the disclosed nanoparticles can be complexed with more than three nucleic acids. In an aspect, the biologically active molecule can be an oligonucleotide, a DNA molecule, an RNA molecule, a virus, or any combination thereof. In an aspect, the biologically active molecule can be an AAV. In an aspect, biologically active molecule can be a peptide, a polypeptide, a protein, an antibody, or any combination thereof. In an aspect, biologically active molecule can be a therapeutic protein, therapeutic peptide, recombinant protein, monoclonal antibody, recombinant antibody, chimeric antibody, humanized antibody, non-human antibody, multispecific antibody, antibody fragments, Fc fusion protein, single chain variable fragments (scFv), chimeric antigen receptors (CAR), or any combination thereof. In an aspect, the biologically active molecule can be an enzyme, hormone, growth factor cytokine, chemokine, or any combination thereof. In an aspect, the biologically active molecule can be human, primate, murine, leporine, canine, feline, bovine, equine, ovine, caprine, avian, or any derivatives thereof. In an aspect, the biologically active molecule can be human vascular endothelial growth factor (VEGF), interleukin 4 (IL-4), or any combination thereof.

[0178] In an aspect, the disclosed nanoparticles are loaded into the scaffold by embedding. In an aspect, the scaffold can comprise the disclosed nanoparticles loaded by mixing disclosed nanoparticles complexed with one or more nucleic acids with the disclosed hydrogel particles prior to annealing, incubating the disclosed nanoparticles in the hydrogel and annealing the disclosed hydrogel particles to from a disclosed hydrogel scaffold. In other aspects, a disclosed hydrogel scaffold can comprise disclosed nanoparticles complexed with one or more nucleic acids loaded by adding a solution comprising the disclosed nanoparticles to a disclosed hydrogel scaffold.

[0179] In further aspects, the system can comprise a disclosed hydrogel scaffold loaded with cells. Cells can be chosen by one skilled in the art, to obtain optimum expression of the biologically active molecule. In an aspect, the system can comprise a disclosed hydrogel scaffold loaded by topical seeding of cells. In an aspect, the system can comprise a disclosed hydrogel scaffold loaded by encapsulating the cells in a disclosed hydrogel scaffold. In an aspect, cells can be adherent cells or non-adherent cells. In an aspect, the cells can be prokaryotic or eukaryotic cells. Non-limiting examples of prokaryotic cell includes bacterial species, e.g., Escherichia coli. In an aspect, eukaryotic cells can be yeast and fungal cells (e.g., yeast, Aspergillus, Penicillium, Pichia pastoris, Yarrowia lipolytica), plant cell, or insect cells. In further aspects, eukaryotic cells can be mammalian or a non-mammalian cell. In an aspect, the cell can be a primary cell or a cell from anestablished or temporary cell line. The cell can be a naive (e.g., unmodified) cell, pharmacologically modified cell, a genetically modified cell, a fresh cell a cryopreserved (e.g., frozen) cell, thawed cells, stored cells, or a combination thereof. In an aspect, the cell can be human, primate, murine, leporine, canine, feline, bovine, equine, ovine, caprine, avian. In an aspect, cells can be a human embryonic kidney (HEK) cell, Chinese hamster ovary (CHO) cell, DXB-11, DG-44, baby hamster kidney (BHK) cell, African green monkey kidney cell, Hela cell, adenocarcinomic human alveolar basal epithelial cell, human lung cell, human hepatoma (Hep) cell, mouse mammary tumor (MMT) cell, TRI cell, MRC5 cell, FS4 cell, mammalian myeloma cell, E. coli cell, yeast cell, or any combination thereof. In an aspect, the cell can be a HEK cell. In an aspect, the cell can be a HEK293 cell.

[0180] In an aspect, the system further can comprise a medium. The medium provided in the system can be any medium that supports the growth and expansion of the cells in the system. Suitable medium for cell growth and expansion will also be apparent to the skilled person, based on the type of cells used. In an aspect, medium of the system can comprise Ham’s F10, Minimal Essential Medium (MEM), RPMI-1640, Dulbecco’s, Modified Eagle’s Medium (DMEM), Excell GTM3, Freestyle 5FM293, Hyclone 5FM293, RPMH640 medium, GIT medium, Ex-cell 302 medium, IMDM medium, Hybridoma-SFM or any combination thereof.

[0181] In further aspects, the system can comprise a three-dimensional cell culture device. In an aspect, the three-dimensional cell culture device can comprise a culture tube, culture bottle, flask, microfluidic device, perfusion device, a bioreactor, or any combination thereof. In some aspects of the system, the disclosed hydrogel particles produced by the methods described herein, can be added to the culture device, and the annealing process for generating a disclosed hydrogel scaffold can be performed in the device. In an aspect, the disclosed hydrogel particles produced by the methods described herein, can be annealed and added as disclosed hydrogel scaffolds to the culture device.G. Tunable Method of Production of Biologically Active Molecules

[0182] In an aspect, the disclosure further provided a tunable method of production of biologically active molecules. In such aspects, the method can comprise using a hydrogel scaffold described herein. In an aspect, a disclosed hydrogel scaffold can comprise disclosed hydrogel particles and disclosed nanoparticles complexed with one or more nucleic acids in a disclosed hydrogel scaffold. In an aspect, a disclosed hydrogel scaffold can further comprise one or more cells.

[0183] In an aspect, a disclosed hydrogel scaffold can be generated with disclosed hydrogel particles and disclosed nanoparticles complexed with one or more nucleic acids, at a desired density. In an aspect, a disclosed hydrogel scaffold can be generated to have a desired cell densityof the loaded cells. In an aspect, the disclosed hydrogel scaffold can be adaptable to production of several different biologically active molecules. In an aspect, the disclosed hydrogel scaffold can be adaptable to loading any desired cells, for production of biologically active molecules. In an aspect, a hydrogel scaffold disclosed herein can be optimized to produce biologically active molecules using lower number of cells, than a method not using a disclosed hydrogel scaffold. In an aspect, a hydrogel scaffold disclosed herein can be optimized to produce biologically active molecules using lower volume of medium, than a method not using a disclosed hydrogel scaffold.

[0184] In an aspect, a disclosed hydrogel scaffold can be used with existing systems or methods for production of biologically active molecule. In an aspect, such systems can include for e.g., a bioreactor system. In an aspect, the methods can include for e.g., a fed batch culture. In such aspects, a disclosed hydrogel scaffold can be employed in the culturing vessel with a medium, and additional culture nutrients are fed, continuously or in discrete increments, to the culture during culturing, with or without periodic cell and / or recovery of the biologically active molecule before termination of culture. The fed batch culture can include, for example, a semi-continuous fed batch culture, wherein periodically whole cells and medium are removed and replaced by fresh medium. In further aspects, a disclosed hydrogel scaffold can be used in perfusion culturing wherein the culture medium can be continuously or intermittently introduced and removed from the culturing vessel.H. Production of Therapeutic Proteins

[0185] In an aspect, further provided herein can be a method for producing therapeutic proteins. In an aspect, a disclosed hydrogel scaffold can comprise disclosed hydrogel particles. In further aspects, a disclosed hydrogel scaffold can further comprise disclosed nanoparticles complexed with one or more nucleic acid encoding a therapeutic protein.

[0186] In an aspect, therapeutic protein, can be a recombinant peptide, recombinant protein, monoclonal antibody, recombinant antibody, chimeric antibody, humanized antibody, non-human antibody, multispecific antibody, antibody fragments, Fc fusion protein, single chain variable fragments (scFv), chimeric antigen receptors (CAR), or any combination thereof. In an aspect, the biologically active molecule can be an enzyme, hormone, growth factor cytokine, chemokine, or any combination thereof. In an aspect, the biologically active molecule can be human, primate, murine, leporine, canine, feline, bovine, equine, ovine, caprine, avian, or any derivatives thereof. In an aspect, the biologically active molecule can be human vascular endothelial growth factor (VEGF), interleukin 4 (IL-4), or any combination thereof. Nucleic acid sequences of the gene encoding several therapeutic proteins are well known in the art and can be obtained from publicly available databases. For e.g., a nucleotide sequences for human VEGF is available at NCBIdatabase under accession number NC 000006.12 and for human IL-4 is available at NCBI database under accession number NC_000005.10.

[0187] In an aspect, disclosed nanoparticles complexed with one or more nucleic acid can comprise one or more vector or plasmid constructs comprising the nucleotide sequence encoding the therapeutic protein. In an aspect, disclosed nanoparticles are complexed with at least one vector or plasmid. In an aspect, disclosed nanoparticles are complexed with at least two vectors or plasmids. In an aspect, disclosed nanoparticles are complexed with at least three vectors or plasmids. In an aspect, disclosed nanoparticles are complexed with more than three vectors or plasmids.

[0188] In further aspects, the method can comprise loading cells into a disclosed hydrogel scaffold. Cells can be chosen by one skilled in the art, to obtain optimum expression of the therapeutic protein. In an aspect, a disclosed hydrogel scaffold loaded by topical seeding of cells. In an aspect, a disclosed hydrogel scaffold loaded by encapsulating the cells in a disclosed hydrogel scaffold. In an aspect, cells can be adherent cells or non-adherent cells. In an aspect, the cells can be prokaryotic or eukaryotic cells. Non-limiting examples of prokaryotic cell includes bacterial species, e.g., Escherichia coli. In an aspect, eukaryotic cells can be yeast and fungal cells (e.g., yeast, Aspergillus, Penicillium, Pichia pastoris, Yarrowia lipolytica), plant cell, or insect cells. In further aspects, eukaryotic cells can be mammalian or a non-mammalian cell. In an aspect, the cell can be a primary cell or a cell from an established or temporary cell line. The cell can be a naive (e.g., unmodified) cell, pharmacologically modified cell, a genetically modified cell, a fresh cell, a cryopreserved (e.g., frozen) cell, thawed cells, stored cells, or a combination thereof. In an aspect, the cell can be human, primate, murine, leporine, canine, feline, bovine, equine, ovine, caprine, avian. In an aspect, cells can comprise a human embryonic kidney (HEK) cell, Chinese hamster ovary (CHO) cell, DXB-11, DG-44, baby hamster kidney (BHK) cell, African green monkey kidney cell, Hela cell, adenocarcinomic human alveolar basal epithelial cell, human lung cell, human hepatoma (Hep) cell, mouse mammary tumor (MMT) cell, TRI cell, MRC5 cell, FS4 cell, mammalian myeloma cell, E. coli cell, yeast cell, or any combination thereof.

[0189] In an aspect, the method further can comprise adding a medium. In further aspects, the method can comprise maintaining the cells. In an aspect, maintaining cells can comprise removing spent medium and adding fresh medium, as described herein.

[0190] In an aspect, the method further can comprise recovering the expressed therapeutic protein. In an aspect, therapeutic protein can comprise glucagon, Hepatitis B vaccine, polyketides, aromatic amino-acid-derived molecule, epidermal growth factor, blood coagulation factor Xllla, proinsulin, insulinotropin, cytochrome P450 enzyme, oestrogen receptor a, vascular endothelialgrowth factor (VEGF) such as e.g., short-form VEGF-D3; a vascular growth inhibitor (e.g., endostatin, angiostatin), thymidine kinase, an interferon (IFN, e.g., IFN-a, IFN-P, IFN-y), , an ATP -binding cassette (ABC) transport protein such as ABC1 member 4 (a / k / a ABCA4) or an ALD protein, e.g., the ABCD-1 protein, myosin VIIA; cyclooxygenase-2, a PGF2-alpha receptor; dopamine; the human hemoglobin subunit beta protein, tumor necrosis factor (TNF, e.g., TNF-a, TNF-P) , growth hormone (e.g., growth hormone, bovine growth hormone), growth hormone releasing factor, parathyroid hormone, thyroid stimulating hormone, lipoproteins; alpha- 1- antitrypsin, insulin A-chain, insulin B-chain, proinsulin, follicle stimulating hormone, calcitonin, luteinizing hormone, chorionic gonadotropin (CG), factor VIIIC, factor IX, tissue factor, von Willebrands factor; Protein C; atrial natriuretic factor, lung surfactant, urokinase or human urine or tissue-type plasminogen activator (t-PA), bombesin, thrombin, hemopoietic growth factor, enkephalinase, RANTES (regulated on activation normally T-cell expressed and secreted), human macrophage inflammatory protein (MIP-1 -alpha), a serum albumin, human serum albumin, mullerian-inhibiting substance, relaxin A-chain, relaxin B-chain, prorelaxin, mouse gonadotropin- associated peptide; beta-lactamase, DNase, inhibin, activin, integrin, protein A or D, rheumatoid factor, a bone-derived neurotrophic factor (BDNF), neurotrophin-3, -4, -5, or -6 (NT-3, NT -4, NT-5, or NT-6), NGF-P, platelet-derived growth factor (PDGF), fibroblast growth factor, aFGF, bFGF, epidermal growth factor (EGF), transforming growth factor (TGF), TGF-alpha, TGF-beta, including TGF-pi, TGF-P2. TGF-P3, TGF-P4, or TGF-P5, insulin-like growth factor-I and -II (IGF-I and IGF-II), des(l-3) -IGF-I (brain IGF-I), CD-3, CD-4, CD-8, CD-19, erythropoietin, osteoinductive factors, immunotoxins, colony stimulating factors (CSFs, e.g., M-CSF, GM-CSF, or G-CSF), interleukins (e g., IL-la, IL-lb, IL-RA, IL-2, IL-3, -4, IL-5, IL-7, IL-9, IL-10, IL-13, IL-14, IL-15, IL-16, IL-17), cytokines (e.g, CD154, 4-1BBL, APRIL, TRANCE, TWEAK, TRANCE, TALL-1, LIGHT, OX40L, Flt-3L, stem cell factor, receptor activator of nuclear factor (RANKL)), C-C chemokines (e g., CCL1, CCL2 (MCP-1), CCL3 (MIP-la) CCL4 (MIP-ip), CCL5 (RANTES), CCL7 (MCP-3), CCL8 (MCP-2), CCL11 (eotaxin), CCL14 (MCP-4), CCL15 (HCC-1), CCL16 (HCC-2), CCL17 (TARC), CCL18 (MIP-4), CCL19 (MIP-3P), CCL20 (MIP- 3a), CCL21, CCL22, CCL23, CCL24, CCL25, CCL26 CCL27), C-X-C chemokines (e.g., CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, superoxide dismutase, T-cell receptor, Rituximab, Trastuzumab, Alemtuzumab, Omalizumab, Bevacizumab, Panitumumab, Tocilizumab, blinatumomab, Pertuzumab, Avelumab, Atezolizumab, Brentuximab, Cetuximab, Daratumumab, Denosumab, Durvalumab, Ipilimumab, nivolumab, Nivolumab, Pembrolizumab, Polatuzumab, Etanercept, Alefacept, Abatacept, Rilonacept, or Belatacept. In an aspect, the therapeutic protein can comprisehuman vascular endothelial growth factor (VEGF), interleukin 4 (IL-4), or any combination thereof.

[0191] In an aspect, the disclosed method using disclosed hydrogel scaffolds provide long-term, stable expression of the therapeutic protein. In an aspect, the expression of the therapeutic proteins from the loaded cells in a disclosed hydrogel scaffold can be sustained for a period time from about 7 days to about 60 days. In an aspect, the expression of the therapeutic protein can be sustained for about 7 days, about 10 days, about 20 days, about 30 days, about 35 days, about 40 days, about 45 days, about 50 days, about 55 days, or about 60 days. In an aspect, the expression of the therapeutic protein can be sustained at least 14 days. In an aspect, the expression of the therapeutic protein can be sustained for at least 21 days. In an aspect, the expression of the therapeutic protein can be sustained for at least 27 days. In an aspect, the expression of the therapeutic protein can be sustained for at least 30 days. In an aspect, the expression of the therapeutic protein can be sustained for more than 30 days.

[0192] In an aspect, the disclosed method using disclosed hydrogel scaffolds provide a recovery of the therapeutic proteins at least about 1000 pg / mL. In an aspect, the therapeutic proteins is recovered at least about 1000 pg / mL, about 1500 pg / mL, about 2000 pg / mL, about 2500 pg / mL, about 3000 pg / mL, about 3500 pg / mL, about 4000 pg / mL, about 5000 pg / mL, about 6000 pg / mL, about 7000 pg / mL, about 8000 pg / mL, about 9000 pg / mL, about 10,000 pg / mL, about 12,000 pg / mL, about 13,000 pg / mL, about 14,000 pg / mL, about 15,000 pg / mL, or about 20,000 pg / mL.I. Production of AAV

[0193] Disclosed herein is a method of producing AAV. In an aspect, a disclosed hydrogel scaffold can comprise disclosed hydrogel particles. In further aspects, a disclosed hydrogel scaffold can further comprise disclosed nanoparticles complexed with one or more nucleic acids encoding an AAV.

[0194] In an aspect, a disclosed AAV can comprise AAV1, AAV2, AAV3 (including 3 A and 3B), AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV8 bp, AAV7M8, AAVAnc80, AAVrhlO, AAVPHP.B, AAV type rh32.33, AAV type rh8, AAV type rh74, AAV type hu.68, avian AAV, bovine AAV, canine AAV, equine AAV, ovine AAV, snake AAV, bearded dragon AAV, AAV2i8, AAV2g9, AAV-LK03, AAV7m8, AAV Anc80, AAV- PHP.eB, AAV-TT, AAVv66, rAAV2 / l, rAAV2 / 8, rAAV2 / 9. In an aspect, AAV can comprise a naturally isolated AAV variants including, but not limited to, AAV-DJ, AAV-HAE1, AAV- HAE2, AAVM41, AAV-1829, AAV2 Y / F, AAV2 T / V, AAV2i8, AAV2.5, AAV9.45, AAV9.61, AAV-B1, AAV-AS, AAV9.45A-String (e.g., AAV9.45-AS), AAV9.45Angiopep, AAV9.47- Angiopep, and AAV9.47-AS, AAV-PHP.B, AAV-PHP.eB, AAV-PHP.S, AAV-F, AAVcc.47,and AAVcc.81. Plasmids encoding AAVs are well known in the art and can be obtained from commercially available databases. For e.g., helper plasmid pAdDeltaF6 is available at Addgene under product number 112867.

[0195] In an aspect, disclosed nanoparticles complexed with one or more nucleic acid can comprise one or more plasmid constructs comprising the nucleotide sequence encoding the AAV. In an aspect, disclosed nanoparticles can be complexed with at least one plasmid. In an aspect, disclosed can be complexed with at least two plasmids. In an aspect, disclosed nanoparticles are complexed with at least three plasmids. In an aspect, disclosed nanoparticles can be complexed with more than three plasmids. In an aspect, the plasmid can be a Helper plasmid, a Replication / Capsid plasmid, transgene insert (ITR) plasmid, or any combination thereof. In an aspect, the disclosed nanoparticles can be complexed with Helper plasmids, Replication / Capsid plasmids, and transgene insert (ITR) plasmids.

[0196] In further aspects, the method can comprise loading cells in a disclosed hydrogel scaffold. Cells can be chosen by one skilled in the art, to obtain optimum expression of the AAV. In an aspect, a disclosed hydrogel scaffold loaded by topical seeding of cells. In an aspect, a disclosed hydrogel scaffold loaded by encapsulating the cells in a disclosed hydrogel scaffold. In an aspect, the cells loaded into the perfusion device can be prokaryotic or eukaryotic cells. Non-limiting examples of prokaryotic cell includes bacterial species, e.g., Escherichia coli. In an aspect, eukaryotic cells can be yeast and fungal cells (e.g., yeast, Aspergillus, Penicillium, Pichia pastoris, Yarrowia lipolytica), plant cell, or insect cells. In further aspects, eukaryotic cells can be mammalian or a non-mammalian cell. In an aspect, the cell can be a primary cell or a cell from an established or temporary cell line. The cell can be a naive (e.g., unmodified) cell, pharmacologically modified cell, a genetically modified cell, a fresh cell, a cryopreserved (e.g., frozen) cell, thawed cells, stored cells, or a combination thereof. In an aspect, the cell can be human, primate, murine, leporine, canine, feline, bovine, equine, ovine, caprine, avian. In an aspect, cells can comprise a human embryonic kidney (HEK) cell, Chinese hamster ovary (CHO) cell, DXB-11, DG-44, baby hamster kidney (BHK) cell, African green monkey kidney cell, Hela cell, adenocarcinomic human alveolar basal epithelial cell, human lung cell, human hepatoma (Hep) cell, mouse mammary tumor (MMT) cell, TRI cell, MRC5 cell, FS4 cell, mammalian myeloma cell, E. coli cell, yeast cell, or any combination thereof.

[0197] In an aspect, the method further can comprise adding a medium. In further aspects, the method can comprise maintaining the cells. In an aspect, maintaining cells can comprise removing spent medium and adding fresh medium, as described herein. In an aspect, the method further can comprise recovering the AAV. AAV can be recovered using any known methods in the art.

[0198] In an aspect, the disclosed method using disclosed hydrogel scaffolds provide long-term, stable expression of AAV. In an aspect, the expression of the AAV from the loaded cells in a disclosed hydrogel scaffold can be sustained for a period time from about 7 days to about 60 days. In an aspect, the expression of the AAV can be sustained for about 7 days, about 10 days, about 20 days, about 30 days, about 35 days, about 40 days, about 45 days, about 50 days, about 55 days, or about 60 days. In an aspect, the expression of the AAV can be sustained at least 14 days. In an aspect, the expression of the AAV can be sustained for at least 21 days. In an aspect, the expression of the AAV can be sustained for at least 27 days. In an aspect, the expression of the AAV can be sustained for at least 30 days. In an aspect, the expression of the AAV can be sustained for more than 30 days.

[0199] In an aspect, the disclosed method using disclosed hydrogel scaffolds provide recovery of a AAV at least about 1 x 1010genomic copies (GC). In an aspect, the AAV is recovered at about 1 x 1010GC, about 1.5 x 1010GC, about 2 x 1010GC, about 2.5 x 1010GC, about 3 x 1010GC, about 3.5 x 1010GC, about 4 x 1010GC, about 1 x 1011GC, about 1.5 x 1011GC, about 2 x 1011GC, about 2.5 x 1011GC, about 3 x 1011GC, about 3.5 x 1011GC, about 4 x 1011GC, about 1 x 1012GC, about 1.5 x 1012GC, about 2 x 1012GC, about 2.5 x 1012GC, about 3 x 1012GC, about3.5 x 1012GC, about 4 x 1012, about 1 x 1013GC, about 1.5 x 1013GC, about 2 x 1013GC, about2.5 x 1013GC, about 3 x 1013GC, about 3.5 x 1013GC, about 4 x 1013, about 1 x 1014GC, about1.5 x 1014GC, about 2 x 1014GC, about 2.5 x 1014GC, about 3 x 1014GC, about 3.5 x 1014GC, about 4 x 1014, about 1 x 1015GC, about 1.5 x 1015GC, about 2 x 1015GC, about 2.5 x 1015GC, about 3 x 1015GC, about 3.5 x 1015GC, or about 4 x 1015. In an aspect, the AAV can be recovered at least about 1 x 1012GC. In an aspect, the AAV can be recovered at least about 1 x 1013GC.J. Perfusion Device

[0200] Disclosed herein is a perfusion device. In an aspect, further provided is a perfusion device comprising the disclosed hydrogel scaffold. Such a device can be used for production of biologically active molecules.

[0201] In an aspect, the perfusion device for production of a biologically active molecule can comprise a chamber comprising a disclosed hydrogel scaffold loaded with cells, a media channel comprising an inlet and an outlet, a membrane separating the chamber from the media channel, a media reservoir, and a peristaltic pump. The perfusion device can be assembled using any known methods in the art. In an aspect, the perfusion device can be constructed in Fusion360 (Autodesk) and printed on an Elegoo Mars 2 SLA 3D printer using a resin or a polymer, for e.g., isopropanolsoluble resin. In an aspect, the perfusion device can be constructed by reverse molding and casting channels using a resin or a polymer, for e.g., Polydimethylsiloxane (PDMS).

[0202] In an aspect, the chamber comprising a disclosed hydrogel scaffold loaded with cells provides the location in which cells are maintained within the perfusion device. In an aspect, the chamber can be configured to receive a disclosed hydrogel scaffold, preloaded with cells or an empty hydrogel scaffold for later addition of cells to it. In an aspect, the chamber can be configured to receive and hold media in addition to a disclosed hydrogel scaffold. If some aspects, the chamber can be configured to include an inlet and an outlet, where media (e.g., fresh or new media) can be provided to the chamber by the inlet and media (e.g., spent or old media) can be removed from the chamber. In an aspect, the perfusion device can include a membrane configured to separate the chamber from the media channel. In an aspect, the membrane can be configured to affect or direct flow of media into or out of the chamber. In an aspect, the perfusion device can include one or more media reservoir to store fresh media for input into the chamber via a membrane channel or spent media received as output from the chamber via a membrane channel. In an aspect, the perfusion device can include a pump for directing media into and out of the chamber via the media channel. In an aspect, the pump can be a peristaltic pump, a diaphragm pump, a rotary pump, a syringe pump, or a pressure driven flow control.

[0203] In an aspect, a disclosed hydrogel scaffold of the perfusion device can comprise disclosed hydrogel particles and disclosed nanoparticles complexed with one or more nucleic acids. In an aspect, the disclosed nucleic acid complexed to the disclosed nanoparticles can encode a biologically active molecule. In an aspect, the biologically active molecule can be an oligonucleotide, a DNA molecule, an RNA molecule, a virus, or any combination thereof. In an aspect, the biologically active molecule can be an AAV. In an aspect, biologically active molecule can be a peptide, a polypeptide, a protein, an antibody, or any combination thereof. In an aspect, biologically active molecule can be a therapeutic protein, therapeutic peptide, recombinant protein, monoclonal antibody, recombinant antibody, chimeric antibody, humanized antibody, non-human antibody, multispecific antibody, antibody fragments, fragments Fc fusion protein, single chain variable fragments (scFv), chimeric antigen receptors (CAR), or any combination thereof. In an aspect, the biologically active molecule can be an enzyme, hormone, growth factor cytokine, chemokine, or any combination thereof. In an aspect, the biologically active molecule can be human, primate, murine, leporine, canine, feline, bovine, equine, ovine, caprine, avian, or any derivatives thereof. In an aspect, the biologically active molecule can be, human vascular endothelial growth factor (VEGF), interleukin 4 (IL-4), or any combination thereof.

[0204] In an aspect, cells used in the perfusion device can comprise any cells suitable for production of the biologically active molecule. In an aspect, the cells loaded into the perfusion device can be prokaryotic or eukaryotic cells. Non-limiting examples of prokaryotic cell includesbacterial species, e.g., Escherichia coli. In an aspect, eukaryotic cells can be yeast and fungal cells (e.g., yeast, Aspergillus, Penicillium, Pichia pastoris, Yarrowia lipolytica), plant cell, or insect cells. In further aspects, eukaryotic cells can be mammalian or a non-mammalian cell. In an aspect, the cell can be a primary cell or a cell from an established or temporary cell line. The cell can be a naive (e.g., unmodified) cell, pharmacologically modified cell, a genetically modified cell, a fresh cell, a cryopreserved (e.g., frozen) cell, thawed cells, stored cells, or a combination thereof. In an aspect, the cell can be human, primate, murine, leporine, canine, feline, bovine, equine, ovine, caprine, avian. In an aspect, cells can be a human embryonic kidney (HEK) cell, Chinese hamster ovary (CHO) cell, DXB-11, DG-44, baby hamster kidney (BHK) cell, African green monkey kidney cell, Hela cell, adenocarcinomic human alveolar basal epithelial cell, human lung cell, human hepatoma (Hep) cell, mouse mammary tumor (MMT) cell, TRI cell, MRC5 cell, FS4 cell, mammalian myeloma cell, E. coli cell, yeast cell, or any combination thereof. In an aspect, the cell can be a HEK cell. In an aspect, the cell can be a HEK293 cell.

[0205] In some aspects of the device, the disclosed nanoparticles are complexed with one or more nucleic acids. In an aspect, the disclosed nanoparticles are complexed at least two nucleic acids. In an aspect, the disclosed nanoparticles are complexed at least three nucleic acids. In an aspect, the disclosed nanoparticles are complexed with more than three nucleic acids.

[0206] In an aspect, a disclosed hydrogel scaffold within the chamber can include a polymer. Suitable polymers include, but are not limited to HA, chitosan, heparin, alginate, gelatin, fibrin, collagen, MATRIGEL®, polyvinyl alcohol, polyethylene glycol, sodium polyacrylate, acrylate polymers, copolymers, dithiol polymers (e.g., acrylamide), click-based composite hydrogels polyethylene glycol)-diacrylate, poly(ethylene glycol)-vinyl sulfone, or any combination thereof. In an aspect, hydrogel can comprise hyaluronic acid (HA).

[0207] In an aspect, the hydrogel polymer in the scaffold within the chamber can be modified with one or more functional groups. In an aspect, hydrogel polymer can be modified with a functional group norbornene, acrylamide, tetrazine, sulfate, cyclodextrin, adamantane, vinyl sulfone, acrylate, allyl, azide, alkyne, thiol, PEG, or any combination thereof. In an aspect, a disclosed hydrogel scaffold can comprise HA modified with acrylamide functional groups (HA- AC). In an aspect, a disclosed hydrogel scaffold can comprise HA modified with norbornene functional group (HA-NB). In further aspects, a disclosed hydrogel polymer can be further modified with a cell adhesion peptide. In some aspects the cell adhesion peptide can be RGD ligand (RGDSP - SEQ ID NO:36). In other aspects, the hydrogel polymer can be modified with other ligands, Q peptide, K peptide, or any combination thereof. In an aspect, a disclosed hydrogel scaffold can comprise HA modified with one or more RGD ligands (RGDSP - SEQ ID NO:36),one or more Q-peptides (Ac-NQEQVSPLGGERCG-NEE - SEQ ID NO:2), one or more K- peptides (Ac-FKGGERCG-NEE - SEQ ID NO:3), or any combination thereof.

[0208] In an aspect, a disclosed hydrogel scaffold used in the perfusion device described infra can comprise disclosed nanoparticles. In an aspect, disclosed nanoparticles can be polymer and / or lipid based nanoparticles. In an aspect, disclosed nanoparticles can be generated using a cationic polymer. In an aspect, the cationic polymer can comprise linear poly(ethyleneimine) (PEI), branched PEI, poly(beta-amino esters), Poly(2-(dimethylamino)ethyl methacrylate) (pDMEAMA), poly(amido amine) (PAMAM), chitosan, imine-containing polyamines, polyurethanes, cyclodextrin, disulfide-containing poly(amido amine), or any combination thereof. In an aspect, disclosed nanoparticles can comprise a cationic lipid lipofectamine, DOSPA, DOPE, DSPE, DSTAP, DOTAP, DOTMA, DORIE, DMRIE, DOTIM, GAP-DLRIE, DDAB, DC-6-14, DODAP, DOTC, DOGS, or any combination thereof. In an aspect, disclosed nanoparticles are generated using linear PEI.

[0209] In an aspect, disclosed nanoparticles within the perfusion device can be complexed with one or more nucleic acid. In an aspect, the nanoparticle can be complexed with one or more nucleic acids, by combining linear PEI with one or more nucleic acids. In an aspect, PEI can be combined with HA at a HA:PEI ratio of about 20 : 1 to about 1 : 1 , or about 20:1, about 19:1, about 18: 1, about 17: 1, about 16:1, about 15: 1, about 14:1, about 13: 1, about 12:1, about 11 : 1, about 10: 1, about 9: 1, about 8: 1, about 7: 1, about 6: 1, about 5: 1, about 4: 1, about 3: 1, about 2: 1, or about 1 : 1. In some aspects the HA:PEI ratio can be about 5: 1 or 5 : 1. In an aspect, nanoparticle and nucleic acids are combined at N / P ratio between about 1 and 50, or between about 10 and 30, or between about 15 and 25. In an aspect, the disclosed nanoparticles and nucleic acids are combined at N / P ratio of about 50, about 45, about 40, about 35, about 30, about 25, about 20, about 15, about 10, about 5, or about 1. In an aspect, the disclosed nanoparticles and nucleic acids are combined atN / P ratio of about 20.

[0210] In an aspect, a disclosed hydrogel scaffold within disclosed perfusion device can include one or more nucleic acid that encodes a biologically active molecule, including, but not limited to, an oligonucleotide, a DNA molecule, an RNA molecule, a virus, or any combination thereof. In an aspect, biologically active molecule can be a therapeutic protein, therapeutic peptide, recombinant protein, monoclonal antibody, recombinant antibody, chimeric antibody, humanized antibody, non-human antibody, multispecific antibody, antibody fragments, Fc fusion protein, single chain variable fragments (scFv), chimeric antigen receptors (CAR), or any combination thereof. In an aspect, the biologically active molecule can be an enzyme, hormone, growth factor cytokine, chemokine, or any combination thereof. In an aspect, the biologically active moleculecan be human, primate, murine, leporine, canine, feline, bovine, equine, ovine, caprine, avian, or any derivatives thereof. In an aspect, the biologically active molecule can be human vascular endothelial growth factor (VEGF), interleukin 4 (IL-4), or any combination thereof. In an aspect, the biologically active molecule can comprise an Adeno-associated virus (AAV). In an aspect, the AAV can comprise AAV1, AAV2, AAV3 (including 3A and 3B), AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV8 bp, AAV7M8, AAVAnc80, AAVrhlO, AAVPHP.B, AAV type rh32.33, AAV type rh8, AAV type rh74, AAV type hu.68, avian AAV, bovine AAV, canine AAV, equine AAV, ovine AAV, snake AAV, bearded dragon AAV, AAV2i8, AAV2g9, AAV-LK03, AAV7m8, AAV Anc80, AAV-PHP.eB, AAV-TT, AAVv66, rAAV2 / l, rAAV2 / 8, rAAV2 / 9, AAV-DJ, AAV-HAE1, AAV-HAE2, AAVM41, AAV- 1829, AAV2 Y / F, AAV2 T / V, AAV2i8, AAV2.5, AAV9.45, AAV9.61, AAV-B1, AAV-AS, AAV9.45A-String, AAV9.45Angiopep, AAV9.47-Angiopep, AAV9.47-AS, AAV-PHP.B, AAV-PHP.eB, AAV-PHP.S, AAV-F, AAVcc.47, or AAVcc.81.

[0211] In an aspect, the disclosed perfusion device provides long-term, stable expression of the biologically active molecule. In an aspect, the expression of the biologically active molecules from the loaded cells in a disclosed hydrogel scaffold in the perfusion device can be sustained for a period time from about 7 days to about 60 days. In an aspect, the expression of the biologically active molecules can be sustained for about 7 days, about 10 days, about 20 days, about 30 days, about 35 days, about 40 days, about 45 days, about 50 days, about 55 days, or about 60 days. In an aspect, the expression of the biologically active molecules can be sustained at least 14 days. In an aspect, the expression of the biologically active molecules can be sustained for at least 21 days. In an aspect, the expression of the biologically active molecules can be sustained for at least 27 days. In an aspect, the expression of the biologically active molecules can be sustained for at least 30 days. In an aspect, the expression of the biologically active molecules can be sustained for more than 30 days.

[0212] In an aspect, the disclosed perfusion device provides recovery of a biologically active molecule at least about 1 x 1010genomic copies (GC). In an aspect, the biologically active molecule can be recovered at about 1 x 1010GC, about 1.5 x 1010GC, about 2 x 1010GC, about 2.5 x 1010GC, about 3 x 1010GC, about 3.5 x 1010GC, about 4 x 1010GC, about 1 x 1011GC, about 1.5 x 1011GC, about 2 x 1011GC, about 2.5 x 1011GC, about 3 x 1011GC, about 3.5 x 1011GC, about 4 x 1011GC, about 1 x 1012GC, about 1.5 x 1012GC, about 2 x 1012GC, about 2.5 x1012GC, about 3 x 1012GC, about 3.5 x 1012GC, about 4 x 1012, about 1 x 1013GC, about 1.5 x1013GC, about 2 x 1013GC, about 2.5 x 1013GC, about 3 x 1013GC, about 3.5 x 1013GC, about 4 x 1013, about 1 x 1014GC, about 1.5 x 1014GC, about 2 x 1014GC, about 2.5 x 1014GC, about3 x 1014GC, about 3.5 x 1014GC, about 4 x 1014, about 1 x 1015GC, about 1.5 x 1015GC, about 2 x 1015GC, about 2.5 x 1015GC, about 3 x 1015GC, about 3.5 x 1015GC, or about 4 x 1015. In an aspect, the biologically active molecule can be recovered at least about 1 x 1012GC. In an aspect, the biologically active molecule can be recovered at least about 1 x 1013GC.

[0213] In an aspect, the disclosed perfusion device provides a recovery of the biologically active molecule at least about 1000 pg / mL. In an aspect, the biologically active molecule can be recovered at least about 1000 pg / mL, about 1500 pg / mL, about 2000 pg / mL, about 2500 pg / mL, about 3000 pg / mL, about 3500 pg / mL, about 4000 pg / mL, about 5000 pg / mL, about 6000 pg / mL, about 7000 pg / mL, about 8000 pg / mL, about 9000 pg / mL, about 10,000 pg / mL, about 12,000 pg / mL, about 13,000 pg / mL, about 14,000 pg / mL, about 15,000 pg / mL, or about 20,000 pg / mL.

[0214] In another aspect, disclosed is a method of producing a biologically active molecule with a disclosed hydrogel scaffold or perfusion device described supra, and can further comprise administering the disclosed biologically active molecule to a subject in need thereof. In an aspect, a disclosed biologically active molecule can be used in a method of treating a disease or disorder, which can include (but are not limited to) genetic diseases, neurological diseases, cancers, or combination thereof. In an aspect, biologically active molecules can be isolated from a disclosed hydrogel scaffold, perfusion device, and / or media perfusing a disclosed hydrogel scaffold or passed through the perfusion device. The biologically active molecules can be isolated through conventional means appropriate for the identity and chemistry of the molecule. The biologically active molecule can be one or more sufficient or effective to treat the diseases and disorders described below.

[0215] As used herein, “lipid nanoparticle uptake,” “LNP uptake,” “extracellular vesicle uptake,” or “EV uptake” refers to the interaction of one or more LNPs or EVs with a target cell. In an aspect, LNPs or EVs can bind to the cell surface via antigen-antibody interaction or ligandreceptor interactions and can potentially trigger signaling via surface receptors, even without LNP or EV entry into the target cell. As known to the art, the most common mode of EV uptake into target cells involves internalization via endocytotic processes, such as clathrin, caveolin, or lipid raft-mediated endocytosis, micropinocytosis, or phagocytosis. In an aspect, LNPs or EVs can also directly fuse with the plasma membrane of the target cell and release the encapsulated cargo directly into the cytoplasm.

[0216] In an aspect, LNPs or EVs can comprise one or more biologically active molecules, including an oligonucleotide, a protein, or a combination thereof. In some aspects, the biologically active molecules within the LNPs or EVs can be a DNA molecule, an RNA molecule. In an aspect, LNPs or EVs can comprise AAV particles. In an aspect, LNPs or EVs can comprise AAV1,AAV2, AAV3 (including 3 A and 3B), AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV1 1, AAV12, AAV13, AAV8 bp, AAV7M8, AAVAnc8O, AAVrhlO, AAVPHP.B, AAV type rh32.33, AAV type rh8, AAV type rh74, AAV type hu.68, avian AAV, bovine AAV, canine AAV, equine AAV, ovine AAV, snake AAV, bearded dragon AAV, AAV2i8, AAV2g9, AAV-LK03, AAV7m8, AAV Anc80, AAV-PHP.eB, AAV-TT, AAVv66, rAAV2 / l, rAAV2 / 8, rAAV2 / 9, AAV-DJ, AAV-HAE1, AAV-HAE2, AAVM41, AAV-1829, AAV2 Y / F, AAV2 T / V, AAV2i8, AAV2.5, AAV9.45, AAV9.61, AAV-B1, AAV-AS, AAV9.45A-String, AAV9.45Angiopep, AAV9.47-Angiopep, AAV9.47-AS, AAV-PHP.B, AAV-PHP.eB, AAV-PHP.S, AAV-F, AAVcc.47, or AAVcc.81. In an aspect, LNPs orEVs can comprise one or more biologically active molecules, including a peptide, a polypeptide, a protein, an antibody, or any combination thereof. In an aspect, LNPs or EVs can comprise a therapeutic protein, therapeutic peptide, recombinant protein, monoclonal antibody, recombinant antibody, chimeric antibody, humanized antibody, non-human antibody, multispecific antibody, antibody fragments, or any combination thereof.

[0217] In an aspect, biologically active molecules can be provided to a subject to treat a disease, disorder or condition described infra within a lipid nanoparticle (LNP), such as within an EV. In an aspect, the LNP can have a size of 10 nm to 900 nm, or any individual value or range of values within, inclusive of the upper and lower values, whether specifically recited or not. The LNPs can, for example, have a size of 50-500 nm, of 100-200 nm, or 150-200 nm. The average size may be, for example, about 100 nm, about 125 nm, about 150 nm, about 175 nm, or about 200 nm.

[0218] In an aspect, the LNP can have a zeta potential of about 2.25 mV.

[0219] In embodiments where the first nucleic acid sequence and the one or more additional nucleic acid sequences are mRNA, the weight ratio of the first nucleic acid sequence to the additional nucleic acid sequences within the NPs may be between 1 : 1 and 1 :2, inclusive, including about 1 : 1.5.

[0220] In an aspect, LNPs can be prepared by any suitable mechanism which can be readily ascertained by one of skill in the art. In an aspect, LNPs can be prepared by self- assembled nanoprecipitation. Biologically active molecules can be immobilized on the LNP surface by way of, e.g., carbodiimide coupling chemistry between the -COOH group of carboxy modified PEG and the amino group at the end of a biologically active molecule.

[0221] In an aspect, the LNPs can be formulated with selective organ targeting (SORT) technology, as disclosed in Cheng et al. (Nat. Nanotechnol.15: 313-320 (2020)), the contents of which are incorporated herein by reference. While LNPs typically comprise ionizable cationic lipids, amphipathic phospholipids, cholesterol and PEG lipids, the addition of charge-modifying agents can result in targeted delivery of the LNPs to particular organs. Formulating the LNPsinvention with SORT technology allows targeted therapeutic delivery to specific organs in need thereof, with minimal off-target effects. For example, LNPs of the invention for the treatment of disorders, diseases, and / or conditions described infra may be delivered to the relevant organs or tissue.

[0222] In an aspect, a disclosed LNP carrier can be a LNP that comprises a charge-modifying agent or it can be a modification thereof within the purview of one of skill in the art. In certain embodiments of the invention, the LNP carrier can be formulated with 1,2-di oleoyl- 3- trimethylammonium-propane (DOTAP), dimethyldioctadecylammonium (DDAB), 1,2- dimyristoyl-sn-glycero-3-ethylphosphocholine (EPC), 1,2-di oleoyl-3 -dimethylammoniumpropane (DODAP) or 5A2-SC81, 2-dioleoyl-sn-glycero-3- phosphate (18PA), 1,2-dimyristoyl- snglycero-3 -phosphate (14PA), or sn-(3 -oleoyl -2-hydroxy)- glycerol-l-phospho-sn-3'-(l',2'- dioleoyl)-glycerol (18BMP). In an aspect, the LNP carrier may be formulated and / or configured to be organ or tissue selective. In some embodiments, LNPs can further include D-Lin- MC3- DMA, DSPC, cholesterol, DMG-PEG2000 and DOTAP at, for example, a 25 / 5 / 19.3 / 0.5 / 50 ratio

[0223] In an aspect, the LNPs can be administered by any suitable technique, including, but not limited to oral, aerosol, intranasal, injection, systemic, parenteral, subcutaneous, intravenous, intramuscular, intrathecal, intraperitoneal and rectal. In some embodiments, the LNPs can be administered at a frequency established based on LNP half-life, which can readily be assessed by one of skill in the art. In an aspect, the LNPs can be administered to a subject, either alone or in combination with a pharmaceutically acceptable excipient and / or carrier, in an amount sufficient to induce an appropriate biological response (e.g., knock down of the desired target).

[0224] Genetic diseases and disorders can include, but are not limited to, diseases and disorders due to a defect in the following gene: dystrophin including mini- and micro-dystrophins (DMD); titin (TTN); titin cap (TCAP) a-sarcoglycan (SGCA), [3-sarcoglycan (SGCB), y-sarcoglycan (SGCG) or 5-sarcoglycan (SGCD); alpha- 1 -antitrypsin (Al-AT); myosin heavy chain 6 (MYH6); myosin heavy chain 7 (MYH7); myosin heavy chain 11 (MYH11); myosin light chain 2 (ML2); myosin light chain 3 (ML3); myosin light chain kinase 2 (MYLK2); myosin binding protein C (MYBPC3); desmin (DES); dynamin 2 (DNM2); laminin a2 (LAMA2); lamin A / C (LMNA); lamin B (LMNB); lamin B receptor (LBR); dysferlin (DYSF); emerin (EMD); insulin; blood clotting factors, including but not limited to, factor VIII and factor IX; erythropoietin (EPO); lipoprotein lipase (LPL); sarcoplasmic reticulum Ca2+-ATPase (SERCA2A), SI 00 calcium binding protein Al (S100A1); myotubularin (MTM); DM1 protein kinase (DMPK); glycogen phosphorylase L (PYGL); glycogen phosphorylase, muscle associated (PYGM); glycogen synthase 1 (GYSI); glycogen synthase 2 (GYS2); a-galactosidase A (GLA); a-N-acetylgalactosaminidase (NAGA); acid a-glucosidase (GAA), sphingomyelinase phosphodiesterase 1 (SMPD1); lysosomal acid lipase (LIPA); collagen type I al chain (COL1 Al); collagen type I a2 chain (COL1A2); collagen type III al chain (COL3A1); collagen type V al chain (COL5A1); collagen type V a2 chain (COL5A2); collagen type VI al chain (COL6A1); collagen type VI a2 chain (COL6A2); collagen type VI a3 chain (COL6A3); procollagen-lysine 2-oxoglutarate 5-dioxygenase (PLOD1); lysosomal acid lipase (LIPA); frataxin (FXN); myostatin (MSTN); P-N-acetyl hexosaminidase A (HEXA); P-N-acetylhexosaminidase B (HEXB); P- glucocerebrosidase (GBA); adenosine monophosphate deaminase 1 (AMPD1); P-globin (HBB); iduronidase (IDUA); iduronate 2-sulfate (IDS); troponin 1 (TNNI3); troponin T2 (TNNT2); troponin C (TNNC1); tropomyosin 1 (TPM1); tropomyosin 3 (TPM3); N-acetyl-a- glucosaminidase (NAGLU); N-sulfoglucosamine sulfohydrolase (SGSH); heparan-a- glucosaminide N-acetyltransferase (HGSNAT); integrin a 7 (IGTA7); integrin a 9 (IGTA9); glucosamine(N-acetyl)-6-sulfatase (GNS); galactosamine(N-acetyl)-6-sulfatase (GALNS); P- galactosidase (GLB1); P-glucuronidase (GUSB); hyaluronoglucosaminidase 1 (HYAL1); acid ceramidase (ASAHI); galactosylcermidase (GALC); cathepsin A (CTSA); cathepsin D (CTSA); cathepsin K (CTSK); GM2 ganglioside activator (GM2A); arylsulfatase A (ARSA); arylsulfatase B (ARSB); formylglycine-generating enzyme (SUMFI); neuraminidase 1 (NEU1); N- acetylglucosamine-1 -phosphate transferase a (GNPTA); N-acetylglucosamine-1 -phosphate transferase P (GNPTB); N-acetylglucosamine-1 -phosphate transferase y (GNPTG); mucolipin-1 (MCOLN1); NPC intracellular transporter 1 (NPC1); NPC intracellular transporter 2 (NPC2); ceroid lipofuscinosis 5 (CLN5); ceroid lipofuscinosis 6 (CLN6); ceroid lipofuscinosis 8 (CLN8); palmitoyl protein thioesterase 1 (PPT1); tripeptidyl peptidase 1 (TPP1); battenin (CLN3); DNAJ heat shock protein family 40 member C5 (DNAJC5); major facilitator superfamily domain containing 8 (MFSD8); mannosidase a class 2B member 1 (MAN2B1); mannosidase R (MANBA); aspartylglucosaminidase (AGA); a-L-fucosidase (FUCA1); cystinosin, lysosomal cysteine transporter (CTNS); sialin; solute carrier family 2 member 10 (SLC2A10); solute carrier family 17 member 5 (SLC17A5); solute carrier family 6 member 19 (SLC6A19); solute carrier family 22 member 5 (SLC22A5); solute carrier family 37 member 4 (SLC37A4); lysosomal associated membrane protein 2 (LAMP2); sodium voltage-gated channel a subunit 4 (SCN4A); sodium voltage-gated channel P subunit 4 (SCN4B); sodium voltage-gated channel a subunit 5 (SCN5A); sodium voltage-gated channel a subunit 4 (SCN4A); calcium voltage-gated channel subunit ale (CACNA1C); calcium voltage-gated channel subunit als (CACNA1S); phosphoglycerate kinase 1 (PGK1); phosphoglycerate mutase 2 (PGAM2); amylo-a-1,6- glucosidase, 4-a-glucanotransferase (AGL); potassium voltage-gated channel ISK-relatedsubfamily member 1 (KCNE1); potassium voltage-gated channel ISK-related subfamily member 2 (KCNE2); potassium voltage-gated channel subfamily J member 2 (KCNJ2); potassium voltagegated channel subfamily J member 5 (KCNJ5); potassium voltage-gated channel subfamily H member 2 (KCNH2); potassium voltage-gated channel KQT-like subfamily member 1 (KCNQ1); hyperpolarization-activated cyclic nucleotide-gated potassium channel 4 (HCN4); chloride voltage-gated channel 1 (CLCN1); carnitine palmitoyltransferase 1A (CPT1A); ryanodine receptor 1 (RYR1); ryanodine receptor 2 (RYR2); bridging integrator 1 (BINI); LARGE xylosyl- and glucuronyltransferase 1 (LARGE1); docking protein 7 (D0K7); fukutin (FKTN); fukutin related protein (FKRP); selenoprotein N (SELENON); protein O-mannosyltransferase 1 (P0MT1); protein O-mannosyltransferase 2 (P0MT2); protein O-linked mannose N- acetylglucosaminyltransf erase 1 (P0MGNT1); protein O-linked mannose N- acetylglucosaminyltransferase 2 (P0MGNT2); protein-O-mannose kinase (POMK); isoprenoid synthase domain containing (ISPD); plectin (PLEC); cholinergic receptor nicotinic epsilon subunit (CHRNE); choline O-acetyltransferase (CHAT); choline kinase P (CHKB); collagen like tail subunit of asymmetric acetylcholinesterase (COLQ); receptor associated protein of the synapse (RAPSN); four and a half LIM domains 1 (FHL1); P-l,4-glucuronyltransf erase 1 (B4GAT1); P-l,3-N-acetylgalactosaminyltransferase 2 (B3GALNT2); dystroglycan 1 (DAGI); transmembrane protein 5 (TMEM5); transmembrane protein 43 (TMEM43); SECIS binding protein 2 (SECISBP2); glucosamine (LTDP-N-acetyl)-2-epimerase / N-acetylmannosamine kinase (GNE); anoctamin 5 (AN05); structural maintenance of chromosomes flexible hinge domain containing 1 (SMCHD1); lactate dehydrogenase A (LDHA); lactate dehydrogenase B (LHDB); calpain 3 (CAPN3); caveolin 3 (CAV3); tripartite motif containing 32 (TRIM32); CCHC-type zinc finger nucleic acid binding protein (CNBP); nebulin (NEB); actin, al, skeletal muscle (ACTA1); actin, al, cardiac muscle (ACTC1); actinin a2 (ACTN2); poly(A)-binding protein nuclear 1 (PABPN1); LEM domain-containing protein 3 (LEMD3); zinc metalloproteinase STE24 (ZMPSTE24); microsomal triglyceride transfer protein (MTTP); cholinergic receptor nicotinic al subunit (CHRNA1); cholinergic receptor nicotinic a2 subunit (CHRNA2); cholinergic receptor nicotinic a3 subunit (CHRNA3); cholinergic receptor nicotinic a4 subunit (CHRNA4); cholinergic receptor nicotinic a5 subunit (CHRNA5); cholinergic receptor nicotinic a6 subunit (CHRNA6); cholinergic receptor nicotinic a7 subunit (CHRNA7); cholinergic receptor nicotinic a8 subunit (CHRNA8); cholinergic receptor nicotinic a9 subunit (CHRNA9); cholinergic receptor nicotinic alO subunit (CHRNA10); cholinergic receptor nicotinic pi subunit (CHRNB1); cholinergic receptor nicotinic P2 subunit (CHRNB2); cholinergic receptor nicotinic P3 subunit (CHRNB3); cholinergic receptor nicotinic P4 subunit (CHRNB4); cholinergic receptornicotinic y subunit (CHRNG1); cholinergic receptor nicotinic a subunit (CHRND); cholinergic receptor nicotinic E subunit (CHRNE1); ATP binding cassette subfamily A member 1 (ABCA1); ATP binding cassette subfamily C member 6 (ABCC6); ATP binding cassette subfamily C member 9 (ABCC9); ATP binding cassette subfamily D member 1 (ABCD1); ATPase sarcoplasmic / endoplasmic reticulum Ca2+transporting 1 (ATP2A1); ATM serine / threonine kinase (ATM); a tocopherol transferase protein (TTP A); kinesin family member 21 A (KIF21 A); paired-like homeobox 2a (PH0X2A); heparan sulfate proteoglycan 2 (HSPG2); stromal interaction molecule 1 (STIM1); notch 1 (NOTCHI); notch 3 (N0TCH3); dystrobrevin a (DTNA); protein kinase AMP -activated, noncatalytic y2 (PRKAG2); cysteine- and glycine-rich protein 3 (CSRP3); viniculin (VCL); myozenin 2 (MyoZ2); myopalladin (MYPN); junctophilin 2 (JPH2); phospholamban (PLN); calreticulin 3 (CALR3); nexilin F-actin-binding protein (NEXN); LIM domain binding 3 (LDB3); eyes absent 4 (EYA4); huntingtin (HTT); androgen receptor (AR); protein tyrosine phosphate non-receptor type 11 (PTPN11); junction plakoglobin (JUP); desmoplakin (DSP); plakophilin 2 (PKP2); desmoglein 2 (DSG2); desmocollin 2 (DSC2); catenin a3 (CTNNA3); NK2 homeobox 5 (NKX2-5); A-kinase anchor protein 9 (AKAP9); A-kinase anchor protein 10 (AKAP10); guanine nucleotide-binding protein a-inhibiting activity polypeptide 2 (GNAI2); ankyrin 2 (ANK2); syntrophin a-1 (SNTAT); calmodulin 1 (CALM1); calmodulin 2 (CALM2); HTRA serine peptidase 1 (HTRA1); fibrillin 1 (FBN1); fibrillin 2 (FBN2); xylosyltransf erase 1 (XYLT1); xylosyltransferase 2 (XYLT2); tafazzin (TAZ); homogentisate 1,2-di oxygenase (HGD); glucose-6-phosphatase catalytic subunit (G6PC); 1,4- alpha-glucan enzyme 1 (GBE1); phosphofructokinase, muscle (PFKM); phosphorylase kinase regulatory subunit alpha 1 (PHKA1); phosphorylase kinase regulatory subunit alpha 2 (PHKA2); phosphorylase kinase regulatory subunit beta (PHKB); phosphorylase kinase catalytic subunit gamma 2 (PHKG2); phosphoglycerate mutase 2 (PGAM2); cystathionine-beta-synthase (CBS); methylenetetrahydrofolate reductase (MTHFR); 5-methyltetrahydrofolate-homocysteine methyltransferase (MTR); 5-methyltetrahydrofolate-homocysteine methyltransferase reductase (MTRR); methylmalonic aciduria and homocystinuria, cblD type (MMADHC); mitochondrial DNA, including, but not limited to mitochondrially encoded NADEkubiquinone oxidoreductase core subunit 1 (MT-ND1); mitochondrially encoded NADH: ubiquinone oxidoreductase core subunit 5 (MT-ND5); mitochondrially encoded tRNA glutamic acid (MT-TE); mitochondrially encoded tRNA histidine (MT-TH); mitochondrially encoded tRNA leucine 1 (MT-TL1); mitochondrially encoded tRNA lysine (MT-TK); mitochondrially encoded tRNA serine 1 (MT- TS1); mitochondrially encoded tRNA valine (MT-TV); mitogen-activated protein kinase 1 (MAP2K1); B-Raf proto-oncogene, serine / threonine kinase (BRAF); raf-1 proto-oncogene,serine / threonine kinase (RAFI); growth factors, including, but not limited to insulin growth factor 1 (IGF-1); transforming growth factor P3 (TGFP3); transforming growth factor P receptor, type I (TGFpRl); transforming growth factor P receptor, type II (TGFPR2), fibroblast growth factor 2 (FGF2), fibroblast growth factor 4 (FGF4), vascular endothelial growth factor A (VEGF-A), vascular endothelial growth factor B (VEGF-B); vascular endothelial growth factor C (VEGF-C), vascular endothelial growth factor D (VEGF-D), vascular endothelial growth factor receptor 1 (VEGFR1), and vascular endothelial growth factor receptor 2 (VEGFR2); interleukins; immunoadhesins; cytokines; and antibodies.

[0225] Genetic diseases and disorders include, but are not limited to, diseases and disorders due to a defect in the following genes: ABCA1, ABCA12, ABCA13, ABCA2, ABCA3, ABCA4, ABCA5, ABCC1, ABCC2, ABCC6, ABCC8, ABCC9, ACAN, ADAMTS13, ADCY10, ADGRV1, AGL, AGRN, AHDC1, ALK, ALMS1, ALPK3, ALS2, ANAPC1, ANK1, ANK2, ANK3, ANKRD11, ANKRD26, APC, APC2, APOB, ARFGEF2, ARHGAP31, ARHGEF10, ARHGEF18, ARID1A, ARID1B, ARID2, ASH1L, ASPM, ASXL1, ASXL2, ASXL3, ATM, ATP7A, ATP7B, ATR, ATRX, BAZ1A, BAZ2B, BCOR, BCORL1, BDP1, BLM, BPTF, BRCA1, BRCA2, BRIM, BRWD3, C2CD3, C3, C5, CACNA1A, CACNA1B, CACNA1C, CACNA1D, CACNA1E, CACNA1F, CACNA1G, CACNA1H, CACNA1S, CAD, CAMTAI, CARMIL2, CC2D2A, CCDC88A, CCDC88C, CCNB3, CDH23, CDK13, CDK5RAP2, CELSR1, CEMIP2, CENPE, CENPF, CENPJ, CEP152, CEP164, CEP250, CEP290, CFAP43, CFAP44, CFAP65, CFTR / ABCC7, CHD1, CHD2, CHD3, CHD4, CHD7, CHD8, CIC, CIT, CLIP1, CLTC, CNOT1, CNTNAP1, COL11A1, COL11A2, COL12A1, COL17A1, COL18A1, COL1A1, COL1A2, COL27A1, COL2A1, COL3A1, COL4A1, COL4A2, COL4A3, COL4A4, COL4A5, COL4A6, COL5A1, COL5A2, COL6A3, COL7A1, CPAMD8, CPLANE1, CPS1, CPSF1, CRB1, CREBBP, CUBN, CUL7, CUX1, DCC, DCHS1, DEPDC5, DICER1, DIP2B, DLC1, DMD, DMXL2, DNAH1, DNAH11, DNAH17, DNAH2, DNAH5, DNAH7, DNAH8, DNAH9, DNMBP, DNMT1, DOCK2, DOCK3, DOCK6, DOCK7, DOCK8, DSCAM, DSP, DST, DU0X2, DYNC1H1, DYNC2H1, DYSF, EIF2AK4, EP300, EPG5, ERCC6, ERCC6L2, EXPH5, EYS, F5, F8, FANCA, FANCD2, FANCM, FAT1, FAT4, FBN1, FBN2, FLG, FLG2, FLNA, FLNB, FLNC, FLT4, FMN2, FN1, FRAS1, FREM1, FREM2, FSIP2, FYCO1, GLI2, GLI3, GPR179, GREB1L, GRIN2A, GRIN2B, GRIN2D, HCFC1, HECW2, HERC1, HERC2, HFM1, HIVEP1, HIVEP2, HMCN1, HSPG2, HTT, HUWE1, HYDIN, IFT140, IFT172, IGF1R, IGF2R, IGSF1, INSR, INTS1, IQSEC2, ITGB4, ITPR1, ITPR2, JMJD1C, KALRN, KANK1, KAT6A, KAT6B, KDM3B, KDM5B, KDM5C, KDM6A, KDM6B, KDR, KIAA0586, KIAA1109, KIAA1549, KIDINS220, KIF14, KIF1 A, KIF1B, KIF21 A, KIF26B, KIF7, KMT2A,KMT2B, KMT2C, KMT2D, KMT2E, KNL1, LAMA1, LAMA2, LAMA3, LAMA4, LAMA5, LAMB1, LAMB2, LAMC3, LCT, L0XHD1, LPA, LRBA, LRP1, LRP2, LRP4, LRP5, LRP6, LRPPRC, LRRK1, LRRK2, LTBP2, LTBP4, LYST, MACF1, MADD, MAGI2, MAP1B, MAP3K1, MAPK8IP3, MAPKBP1, MAST1, MBD5, MCM3AP, MED12, MED12L, MED13, MED13L, MED23, MEGF8, MET, MLH3, MPDZ, MSH6, MTOR, MYH10, MYH11, MYH14, MYH2, MYH3, MYH6, MYH7, MYH7B, MYH8, MYH9, MYLK, MYO15A, MYO18B, MYO3 A, MYO5A, MYO5B, MYO7A, MYO9A, NALCN, NBAS, NBEA, NBEAL2, NCAPD2, NCAPD3, NEB, NEXMIF, NEXMIF, NF1, NFASC, NHS, NIN, NIPBL, NLRP1, NOTCH1, NOTCH2, NOTCH3, NPHP4, NRXN1, NRXN3, NSD1, NSD2, NUP155, NUP188, NUP205, OBSCN, OBSL1, OTOF, OTOG, OTOGL, PARD3, PBRM1, PCDH15, PCLO, PCNT, PHIP, PI4KA, PIEZO1, PIEZO2, PIK3C2A, PIKFYVE, PKD1, PKD1L1, PKHD1, PLCE1, PLEC, PLEKHG2, PNPLA6, POGZ, POLA1, POLE, POLR1A, POLR2A, POLR3A, PRG4, PRKDC, PRPF8, PRR12, PRX, PTCHI, PTPN23, PTPRF, PTPRJ, PTPRQ, PXDN, QRICH2, RAB3GAP2, RAH, RALGAPA1, RANBP2, RB1CC1, RELN, RERE, REV3L, RIC1, RIMS1, RIMS2, RNF213, ROBO1, ROBO2, ROBO3, ROS1, RP1, RP1L1, RTTN, RUSC2, RYR1, RYR2, SACS, SAMD9, SAMD9L, SBF2, SCAPER, SCN10A, SCN11A, SCN1A, SCN2A, SCN3A, SCN4A, SCN5A, SCN8A, SCN9A, SETBP1, SETD1A, SETD1B, SETD2, SETD5, SETX, SHANK2, SHANK3, SHROOM4, SI, SIPA1L3, SLIT2, SLX4, SMARCA2, SMARCA4, SMCHD1, SNRNP200, SON, SPEF2, SPEG, SPG11, SPTA1, SPTAN1, SPTB, SPTBN2, SPTBN4, SRCAP, STRC, SVIL, SYNE1, SYNGAP1, SYNJ1, SZT2, TAF1, TANC2, TCF20, TCOF1, TDRD9, TECPR2, TECTA, TENM3, TENM4, TET3, TEX14, TEX15, TG, THOC2, TMEM94, TNC, TNIK, TNR, TNRC6B, TNXB, TOGARAMI, TONSL, TRIO, TRIOBP, TRIP11, TRIP12, TRPM1, TRPM6, TRPM7, TRRAP, TSC2, TTC37, TTN, TUBGCP6, UBR1, UNC80, USH2A, USP9X, VCAN, VPS13A, VPS13B, VPS13C, VPS13D, VWF, WDFY3, WDR19, WDR62, WDR81, WNK1, WRN, ZFHX2, ZFYVE26, ZNF142, ZNF292, ZNF335, ZNF407, ZNF462, ZNF469, or a portion thereof.

[0226] Neurological diseases or neurological disorders refer to a host of undesirable conditions affecting neurons in the brain of a subject. These diseases include but are not limited to the following: Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, Pick’s disease, Kuf s disease, Lewy body disease, neurofibrillary tangles, Rosenthal fibers, Mallory’s hyaline, senile dementia, myasthenia gravis, Gilles de la Tourette’s syndrome, multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS), progressive supranuclear palsy (PSP), epilepsy, Creutzfeldt- Jakob disease, deafness-dystonia syndrome, Leigh syndrome, Leber hereditary optic neuropathy (LHON), parkinsonism, dystonia, motor neuron disease, neuropathy-ataxia and retinitispigmentosa (NARP), maternal inherited Leigh syndrome (MILS), Friedreich ataxia, hereditary spastic paraplegia, Mohr-Tranebjaerg syndrome, Wilson disease, sporadic Alzheimer’s disease, sporadic amyotrophic lateral sclerosis, sporadic Parkinson’s disease, autonomic function disorders, hypertension, sleep disorders, neuropsychiatric disorders, depression, schizophrenia, schizoaffective disorder, Korsakoff s psychosis, mania, anxiety disorders, phobic disorder, learning or memory disorders, amnesia or age-related memory loss, attention deficit disorder, dysthymic disorder, major depressive disorder, obsessive-compulsive disorder, psychoactive substance use disorders, panic disorder, bipolar affective disorder, severe bipolar affective (mood) disorder (BP-1), migraines, hyperactivity and movement disorders.

[0227] As disclosed herein, a disclosed cancer can be one or more neoplasm or cancer. The neoplasm can be malignant or benign, the cancer can be primary or metastatic; the neoplasm or cancer can be early stage or late stage. Non-limiting examples of neoplasms or cancers include acute lymphoblastic leukemia, acute myeloid leukemia, adrenocortical carcinoma, AIDS-related cancers, AIDS-related lymphoma, anal cancer, appendix cancer, astrocytomas (childhood cerebellar or cerebral), basal cell carcinoma, bile duct cancer, bladder cancer, bone cancer, brainstem glioma, brain tumors (cerebellar astrocytoma, cerebral astrocytoma / malignant glioma, ependymoma, medulloblastoma, supratentorial primitive neuroectodermal tumors, visual pathway and hypothalamic gliomas), breast cancer, bronchial adenomas / carcinoids, Burkitt lymphoma, carcinoid tumors (childhood, gastrointestinal), carcinoma of unknown primary, central nervous system lymphoma (primary), cerebellar astrocytoma, cerebral astrocytoma / malignant glioma, cervical cancer, childhood cancers, chronic lymphocytic leukemia, chronic myelogenous leukemia, chronic myeloproliferative disorders, colon cancer, cutaneous T-cell lymphoma, desmoplastic small round cell tumor, endometrial cancer, ependymoma, esophageal cancer, Ewing's sarcoma in the Ewing family of tumors, extracranial germ cell tumor (childhood), extragonadal germ cell tumor, extrahepatic bile duct cancer, eye cancers (intraocular melanoma, retinoblastoma), gallbladder cancer, gastric (stomach) cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor, germ cell tumors (childhood extracranial, extragonadal, ovarian), gestational trophoblastic tumor, gliomas (adult, childhood brain stem, childhood cerebral astrocytoma, childhood visual pathway and hypothalamic), gastric carcinoid, hairy cell leukemia, head and neck cancer, hepatocellular (liver) cancer, Hodgkin lymphoma, hypopharyngeal cancer, hypothalamic and visual pathway glioma (childhood), intraocular melanoma, islet cell carcinoma, Kaposi sarcoma, kidney cancer (renal cell cancer), laryngeal cancer, leukemias (acute lymphoblastic, acute myeloid, chronic lymphocytic, chronic myelogenous, hairy cell), lip and oral cavity cancer, liver cancer (primary), lung cancers (non-small cell, small cell), lymphomas (AIDS-related, Burkitt, cutaneous T-cell, Hodgkin, non-Hodgkin, primary central nervous system), macroglobulinemia (Waldenstrom), malignant fibrous histiocytoma of bone / osteosarcoma, medulloblastoma (childhood), melanoma, intraocular melanoma, Merkel cell carcinoma, mesotheliomas (adult malignant, childhood), metastatic squamous neck cancer with occult primary, mouth cancer, multiple endocrine neoplasia syndrome (childhood), multiple myeloma / plasma cell neoplasm, mycosis fungoides, myelodysplastic syndromes, myelodysplastic / myeloproliferative diseases, myelogenous leukemia (chronic), myeloid leukemias (adult acute, childhood acute), multiple myeloma, myeloproliferative disorders (chronic), nasal cavity and paranasal sinus cancer, nasopharyngeal carcinoma, neuroblastoma, non-Hodgkin lymphoma, non-small cell lung cancer, oral cancer, oropharyngeal cancer, osteosarcoma / malignant fibrous histiocytoma of bone, ovarian cancer, ovarian epithelial cancer (surface epithelial-stromal tumor), ovarian germ cell tumor, ovarian low malignant potential tumor, pancreatic cancer, pancreatic cancer (islet cell), paranasal sinus and nasal cavity cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pineal astrocytoma, pineal germinoma, pineoblastoma and supratentorial primitive neuroectodermal tumors (childhood), pituitary adenoma, plasma cell neoplasia, pleuropulmonary blastoma, primary central nervous system lymphoma, prostate cancer, rectal cancer, renal cell carcinoma (kidney cancer), renal pelvis and ureter transitional cell cancer, retinoblastoma, rhabdomyosarcoma (childhood), salivary gland cancer, sarcoma (Ewing family of tumors, Kaposi, soft tissue, uterine), Sezary syndrome, skin cancers (nonmelanoma, melanoma), skin carcinoma (Merkel cell), small cell lung cancer, small intestine cancer, soft tissue sarcoma, squamous cell carcinoma, squamous neck cancer with occult primary (metastatic), stomach cancer, supratentorial primitive neuroectodermal tumor (childhood), T-Cell lymphoma (cutaneous), testicular cancer, throat cancer, thymoma (childhood), thymoma and thymic carcinoma, thyroid cancer, thyroid cancer (childhood), transitional cell cancer of the renal pelvis and ureter, trophoblastic tumor (gestational), unknown primary site (adult, childhood), ureter and renal pelvis transitional cell cancer, urethral cancer, uterine cancer (endometrial), uterine sarcoma, vaginal cancer, visual pathway and hypothalamic glioma (childhood), vulvar cancer, Waldenstrom macroglobulinemia, and Wilms tumor (childhood).

[0228] In an aspect, the biologically active molecule produced by methods described herein can be a therapeutic protein, therapeutic peptide, recombinant protein, monoclonal antibody, recombinant antibody, chimeric antibody, humanized antibody, non-human antibody, multispecific antibody, antibody fragments Fc fusion protein, single chain variable fragments (scFv), chimeric antigen receptors (CAR), or any combination thereof. In an aspect, thebiologically active molecule can be an enzyme, hormone, growth factor cytokine, chemokine, or any combination thereof that is sufficient and / or capable to treat the diseases, disorders, and / or conditions described supra.K. Kits

[0229] Disclosed herein is a kit comprising a hydrogel scaffold described herein. In an aspect, the kit can comprise a disclosed hydrogel scaffold, with disclosed hydrogel particles and one or more disclosed nanoparticles complexed with one or more nucleic acids. In further aspects, the kit can comprise cells and medium. In some aspects the kit can comprise a culture device. In an aspect, the culture device can have a disclosed hydrogel scaffold, with disclosed hydrogel particles and one or more disclosed nanoparticles complexed with one or more nucleic acids, cell, and a medium. In an aspect, the kits can further comprise instructions for use. In an aspect, a disclosed kit can comprise at least a disclosed hydrogel scaffold and cells constituting the kit. Together, a disclosed hydrogel scaffold and cells can be used for production of a biologically active molecule. A disclosed hydrogel scaffold and cells can be physically packaged together or separately. For example, a kit comprising an instruction for using the kit can or cannot physically include the instruction with other individual components. Instead, the instruction can be supplied as a separate component, either in a paper form or an electronic form which can be supplied on computer readable memory device or downloaded from an internet website, or as recorded presentation. In an aspect, a kit for use in a disclosed method can comprise one or more containers holding a disclosed hydrogel scaffold, disclosed cells, disclosed media, disclosed perfusion device, disclosed culture devices or package insert with instructions for use. In an aspect, suitable containers include, for example, bottles, vials, bags, etc. The containers can be formed from a variety of materials such as glass or plastic. The container can hold a disclosed hydrogel scaffold, disclosed cells, disclosed medium, disclosed perfusion device, disclosed culture devices, or a combination thereof, and can have a sterile access port. In an aspect, a disclosed kit can comprise a “package insert”. In an aspect, a package insert can refer to instructions, that contain information about the methods and usage. The label or package insert can describe how a disclosed component can be used. A kit can comprise additional components necessary for production of biologically active molecules such as, for example, other buffers, diluents, and cell nutrients.

[0230] In an aspect, a disclosed kit can be used to (i) produce biologically active molecules; (ii) deliver one or more nucleic acids; (iii) improve viability of cells; (iv) improve transfection in cells; (v) produce therapeutic proteins; (vi) produce of AAV, or (vii) any combination thereof.

[0231] In an aspect, a disclosed kit can be used in a disclosed method of producing biologically active molecules. In an aspect, a disclosed kit can be used in a disclosed method of nucleic aciddelivery. In an aspect, a disclosed kit can be used in a disclosed method of producing therapeutic proteins. In an aspect, a disclosed kit can be used in a disclosed method of producing AAV.L. Miscellaneous

[0232] Disclosed herein is a method of producing a biologically active molecule, the method comprising: loading cells into a disclosed hydrogel scaffold, wherein a disclosed hydrogel scaffold comprises disclosed hydrogel particles and disclosed nanoparticles complexed with one or more nucleic acids; and maintaining the cells.

[0233] In an aspect, a disclosed method can include one or more nucleic acids that encode a biologically active molecule comprising an oligonucleotide, a DNA molecule, an RNA molecule, a virus, or any combination thereof.

[0234] In an aspect, a disclosed biologically active molecule can comprise an adeno-associated virus (AAV), such as any one of AAV2, AAV3 (including 3A and 3B), AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV8 bp, AAV7M8, AAVAnc80, AAVrhlO, AAVPHP.B, AAV type rh32.33, AAV type rh8, AAV type rh74, AAV type hu.68, avian AAV, bovine AAV, canine AAV, equine AAV, ovine AAV, snake AAV, bearded dragon AAV, AAV2i8, AAV2g9, AAV-LK03, AAV7m8, AAV Anc80, AAV-PHP.eB, AAV-TT, AAVv66, rAAV2 / l, rAAV2 / 8, rAAV2 / 9, AAV-DJ, AAV-HAE1, AAV-HAE2, AAVM41, AAV- 1829, AAV2 Y / F, AAV2 T / V, AAV2i8, AAV2.5, AAV9.45, AAV9.61, AAV-B1, AAV-AS, AAV9.45A-String, AAV9.45Angiopep, AAV9.47-Angiopep, AAV9.47-AS, AAV-PHP.B, AAV-PHP.eB, AAV-PHP.S, AAV-F, AAVcc.47, or AAVcc.81.

[0235] In an aspect, a disclosed nucleic acid can encode a biologically active molecule comprising a peptide, a polypeptide, a protein, an antibody, or any combination thereof, such as a therapeutic protein, therapeutic peptide, recombinant protein, monoclonal antibody, recombinant antibody, chimeric antibody, humanized antibody, non-human antibody, multispecific antibody, antibody fragments, fragments Fc fusion protein, single chain variable fragments (scFv), chimeric antigen receptors (CAR), or any combination thereof. In an aspect, the biologically active molecule can be an enzyme, hormone, growth factor cytokine, chemokine, or any combination thereof. In an aspect, the biologically active molecule can be human, primate, murine, leporine, canine, feline, bovine, equine, ovine, caprine, avian, or any derivatives thereof. In an aspect, a disclosed biologically active molecule can be human vascular endothelial growth factor (VEGF), interleukin 4 (IL-4), or any combination thereof.

[0236] In an aspect, a disclosed method can further comprise adding a medium to a disclosed hydrogel scaffold, such as Ham’s F10, Minimal Essential Medium (MEM), RPMI-1640, Dulbecco’s or Modified Eagle’s Medium (DMEM), Ex-cell GTM3, Freestyle 5FM293 Hyclone5FM293, RPMH640 medium, GIT medium, Ex-cell 302 medium, IMDM medium, Hybridoma- SFM, or any combination thereof.

[0237] In an aspect, the disclosed cells in or embedded within a disclosed hydrogel scaffold can be human, primate, murine, leporine, canine, feline, bovine, equine, ovine, caprine, avian. In an aspect, cells can include a human embryonic kidney (HEK) cells, Chinese hamster ovary (CHO) cells, DXB-11 cells, DG-44 cells, baby hamster kidney (BHK) cells, African green monkey kidney cells, Hela cells, adenocarcinomic human alveolar basal epithelial cells, human lung cells, human hepatoma (Hep) cells, mouse mammary tumor (MMT) cells, TRI cells, MRC5 cells, FS4 cells, mammalian myeloma cells, E. coli cells, yeast cells, or any combination thereof.

[0238] In an aspect, a disclosed method can include loading the cells by topically seeding the cells into a disclosed hydrogel scaffold, including topically seeding the cells at about 5,000 cells / pL scaffold volume to 15,000 cells / pL scaffold volume. In an aspect, disclosed the loaded cells can be encapsulated in a disclosed hydrogel scaffold at about 100 cells / pL scaffold volume to 10,000 cells / pL scaffold volume.

[0239] In an aspect, a disclosed method can utilize a disclosed hydrogel scaffold comprising hyaluronic acid (HA), which can further comprise one or more acrylamide functional groups (HA- AC), one or more RGD ligands (RGDSP - SEQ ID NO: 36), one or more Q-peptides (NQEQVSPLGGERCG - SEQ ID NO: 2), one or more K-peptides (FKGGERCG - SEQ ID NO: 3), or any combination thereof.

[0240] In an aspect, disclosed nanoparticles within a disclosed hydrogel scaffold can be complexed with at least two nucleic acids, at least three nucleic acids, more than three nucleic acids, at least two plasmids, at least three plasmids, or more than three plasmids. In an aspect, a disclosed plasmid can comprise a Helper plasmid, a Replication / Capsid plasmid, transgene insert (ITR) plasmid, or any combination thereof. In an aspect, a disclosed nanoparticle within the hydrogel complexed with one or more nucleic acids can be prepared by combining polyethyleneimine (PEI), the nucleic acids, and HA comprising one or more norbornene functional groups (HA-NB), at N / P of about 20, and an HA:PEI ratio of about 5.

[0241] In an aspect, a disclosed method can further comprise recovering the biologically active molecule. In an aspect, a disclosed method can recover at least about 1 x 1010genomic copies or at least about 1000 pg / mL of the biologically active molecule.

[0242] In an aspect, a disclosed method can further include maintaining the cells by removing spent medium and adding fresh medium, for example wherein cells can be maintained for at least 14 days, at least 21 days, at least 28 days, or more than 28 days. In an aspect, a disclosed expressionof a biologically active molecule can be sustained for at least 14 days, at least 21 days, at least 28 days, or more than 28 days.

[0243] Disclosed herein is a perfusion device for production of a biologically active molecule can comprise: a chamber comprising a disclosed hydrogel scaffold loaded with cells; a media channel comprising an inlet and an outlet; a membrane separating the chamber from the media channel; a media reservoir; and a peristaltic pump. In an aspect, a disclosed hydrogel scaffold within the device can comprise disclosed hydrogel particles and disclosed nanoparticles complexed with one or more nucleic acids. The cells within the device can be human, primate, murine, leporine, canine, feline, bovine, equine, ovine, caprine, avian. In an aspect, cells can include embryonic kidney (HEK) cells, Chinese hamster ovary (CHO) cells, DXB-11 cells, DG-44 cells, baby hamster kidney (BHK) cells, African green monkey kidney cells, Hela cells, adenocarcinomic human alveolar basal epithelial cells, human lung cells, human hepatoma (Hep) cells, mouse mammary tumor (MMT) cells, TRI cells, MRC5 cells, FS4 cells, mammalian myeloma cells, E. coli cells, yeast cells, or any combination thereof.

[0244] In an aspect, a disclosed device can include a disclosed hydrogel scaffold comprising hyaluronic acid (HA), and can further comprise one or more acrylamide functional groups (HA- AC), one or more RGD ligands (RGDSP - SEQ ID NO: 36), one or more Q-peptides (NQEQVSPLGGERCG - SEQ ID NO:2)), one or more K-peptides (FKGGERCG - SEQ ID NO: 3), or any combination thereof.

[0245] In an aspect, disclosed nanoparticles within the device can be complexed with two nucleic acid, three nucleic acids, more than three nucleic acids, two plasmids, three plasmids, or more than three plasmids. In an aspect, a disclosed nanoparticle within the device can be complexed with one or more nucleic acids can be prepared by combining polyethyleneimine (PEI), the nucleic acids, and HA comprising one or more norbomene functional groups (HA-NB), at N / P of about 20, and an HA:PEI ratio of about 5.

[0246] In an aspect, a disclosed nucleic acid within the device can encode a biologically active molecule comprising an oligonucleotide, a DNA molecule, an RNA molecule, a virus, or any combination thereof. In an aspect, a disclosed biologically active molecule can comprise an Adeno-associated virus (AAV), such as any one of AAV1, AAV2, AAV3 (including 3 A and 3B), AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV8 bp, AAV7M8, AAVAnc80, AAVrhlO, AAVPHP.B, AAV type rh32.33, AAV type rh8, AAV type rh74, AAV type hu.68, avian AAV, bovine AAV, canine AAV, equine AAV, ovine AAV, snake AAV, bearded dragon AAV, AAV2i8, AAV2g9, AAV-LK03, AAV7m8, AAV Anc80, AAV- PHP.eB, AAV-TT, AAVv66, rAAV2 / l, rAAV2 / 8, rAAV2 / 9, AAV-DJ, AAV-HAE1, AAV-HAE2, AAVM41, AAV-1829, AAV2 Y / F, AAV2 T / V, AAV2i8, AAV2.5, AAV9.45, AAV9.61, AAV-B1, AAV-AS, AAV9.45A-String, AAV9.45Angiopep, AAV9.47-Angiopep, AAV9.47- AS, AAV-PHP.B, AAV-PHP.eB, AAV-PHP.S, AAV-F, AAVcc.47, or AAVcc.81.

[0247] In an aspect, a disclosed nucleic acid within the device can encode a biologically active molecule comprising a peptide, a polypeptide, a protein, an antibody, or any combination thereof, such as a therapeutic protein, therapeutic peptide, recombinant protein, monoclonal antibody, recombinant antibody, chimeric antibody, humanized antibody, non-human antibody, multispecific antibody, antibody fragments, Fc fusion protein, single chain variable fragments (scFv), chimeric antigen receptors (CAR), or a combination thereof. In an aspect, the biologically active molecule can be an enzyme, hormone, growth factor cytokine, chemokine, or any combination thereof. In an aspect, the biologically active molecule can be human, primate, murine, leporine, canine, feline, bovine, equine, ovine, caprine, avian, or any derivatives thereof. In an aspect, a disclosed biologically active molecule can comprise human vascular endothelial growth factor (VEGF), interleukin 4 (IL-4), or a combination thereof.

[0248] In an aspect, a disclosed device can further include a medium, wherein the medium comprises Ham’s F10, Minimal Essential Medium (MEM), RPMI-1640, Dulbecco’s or Modified Eagle’s Medium (DMEM), Ex-cell GTM3, Freestyle 5FM293 Hyclone 5FM293, RPMI1640 medium, GIT medium, Ex-cell 302 medium, IMDM medium, Hybridoma-SFM, or any combination thereof.VIII. EXAMPLES

[0249] The Examples that follow are illustrative of specific aspects of the invention, and various uses thereof. They set forth for explanatory purposes only and are not to be taken as limiting the invention.Methods

[0250] Preparation of hyaluronic acid-acrylamide (HA-AC): To modify hyaluronic acid (HA) to contain acrylamide functional groups, 1 g of 70 kDa sodium hyaluronan (Contipro, 50-90 kDa) was dissolved in 200 mL DI water (1 g / 200 mL). Adipic dihydrazide (ADH, Fisher Scientific) was added for a 1 :40 molar ratio with HA, at 18.35 g, to add amines to the carboxylic acid side chains, and pH adjusted with 1 M HC1 to 4.75 while stirring to dissolve. 2.02 g N-(3- Dimethylaminopropyl)-N’ -ethylcarbodiimide hydrochloride (ED AC HC1, VWR) was added for a molar ratio of 1 :4 for HA to EDC to activate the carboxylic acids. pH was maintained for 4 hours before allowing the reaction to proceed overnight at 25 °C with constant stirring. The reaction solution was then transferred to dialysis tubing (Fisherbrand, 6000-8000 Da), and dialyzed over 3 days in NaCl solutions of decreasing concentration, starting at 100 mM NaCl and ending with 24hours of DI water. The final product was then filtered, flash-frozen, and lyophilized. The extent of ADH modification was confirmed via 1H-NMR spectrometry. The integrations of the peaks were normalized to the peak corresponding to the methyl group on the HA monomer at 5 = 2.0 ppm to determine percent of HA monomers modified to contain ADH groups. After this, the HA- ADH was modified with 2.23 g N-Succinimidyl Acrylate (NHS-AC, TCI Chemicals) for a molar ratio of 1 :5 for HA to NHS-AC. The HA-ADH was resuspended in 200 mL 10 mM HEPES (1 g / 200 mL) with 150 mM NaCl, 10 mM EDTA, at pH 7.4. NHS-AC was dissolved in DMSO (100 mg / mL) and added to the HA solution, lowering the pH to 6.0 and maintain for 4 hours while stirring before reacting overnight at 25 °C. As before, the reaction solution was transferred to dialysis tubing and dialyzed over 3 days before the product was filtered, flash-frozen, and lyophilized. The extent of Ac modification was also confirmed via 1H-NMR spectrometry, normalizing to the HA peak.

[0251] Preparation of hyaluronic acid-norbornene (HA-NB):Lo modify hyaluronic acid (HA) to contain norbomene functional groups, 1 g of 70 kDa sodium hyaluronan (Contipro, 50-90 kDa) and 3.111 g 4-(4,6-dimethoxy-l,3,5-triazin-2-yl)-4-methyl-morpholinium chloride (DMTMM) (Thermo Fisher Scientific, Waltham, MA) were each dissolved in 40 mL 200 mM MES buffer pH 5.5 (molar ratio of ~1 :633 for HA to DMTMM). The two solutions were combined and stirred for 10 minutes to allow for activation of the carboxylic acid. 0.677 mL 5-norbomene-2 -methylamine (TCI America, Portland, OR) was added dropwise to the reaction mixture (molar ratio of -1 :343 for activated HA to NMA), which was then allowed to react overnight at 25 °C with constant stirring. The reaction product was then precipitated in ethanol, filtered to collect the solid, dissolved in 2 M NaCl in water, and dialyzed under running deionized water for 24 hours. The final product was then filtered, flash-frozen, and lyophilized. The extent of modification was confirmed via 1H-NMR spectrometry. 1H-NMR shifts of attached norbomene groups in the product in D2O are 5 = 6.33 and 6.02 (vinyl protons, endo), and 6.26 and 6.23 ppm (vinyl protons, exo). The integrations of these peaks were normalized to the peak corresponding to the methyl group on the HA monomer at 5 = 2.0 ppm to determine percent of HA monomers modified to contain norbomene groups.

[0252] Hydrogel formation and FLIP scaffolds: Briefly, bulk HA-AC hydrogels were formed using a dithiol crosslinker for Michael addition chemistry, and RGD-SP adhesion peptide. From past optimization, the coated, stabilized LNP were loaded into the HA hydrogel precursor, which was then combined with the crosslinker using microfluidic mixing to generate the spherical HMP. To prepare bulk gels with and without lyophilized particles, 3.5 wt% HA-AC gels were made with a matrix-metalloproteinase (MMP) dithiol crosslinker (AC-GCRDGPQGIWGQDRCG-NH2 (SEQID NO: 1), Genscript). HA-AC was dissolved in 0.3 M triethanolamine (TeOA), pH 8.8. Crosslinker was prepared for a SH / HA monomer ratio of 19.00, dissolving in DI water. In the case of particle-loaded bulk hydrogels, the lyophilized DNA / PEI particles were resuspended in the volume of nuclease-free water for the crosslinker solution and subsequently used directly to resuspend the crosslinker peptide. Both solutions were then combined and used to make 35 pL bulk gels by sandwiching with Sigmacoted glass slides using a 1 mm Teflon spacer, and then incubating at 37 °C for 60 mins. Following incubation, gels were transferred to IX PBS solution containing Al exaFlour 647-NHS (Thermo Fisher Scientific, 1 : 1000 dilution) to stain the HA using the residual ADH groups and YOYO-1 (Thermo Fisher Scientific, 1 : 10,000 dilution) to stain the DNA, swelling at 4 °C overnight. Following swelling, gels were imaged using a confocal microscope (Nikon C2 scanning confocal) at 20X and 40X magnification across z-stacks (300- 500 pm, at 25-50 slices) to visualize the DNA / PEI particle distribution. Z-stacks were then assessed in IMARIS (Bitplane) to generate 3D renders of the distribution and assess aggregation. Maximum intensity projections (MIPs) were generated to view the general particle size, and imported to Imaged (FIJI) for particle size analysis. Briefly, images were converted to binary and assessed using the built-in Watershed analysis tool to identify particles, and then measured with the built-in Particle analyzer tool to quantify the cross-section area. Assuming spherical particles, the diameter was calculated for the particle size distribution.

[0253] For annealed FLIP, HA-AC nanoporous gels were prepared similar to before, but the HA solution was modified with an RGD ligand (RGDSP (SEQ ID NO: 36), 1 mM) to improve cell adhesion, in addition to modification with Q-peptide (AC-NQEQVSPLGGERCG-NH2 (SEQ ID NO: 2), 0.75 mM) and K-peptide (Ac-FKGGERCG-NH2(SEQ ID NO: 3), 0.75 mM) for Factor XIII (FXIII) / Thrombin transamination to anneal the microgels into FLIP scaffolds. Peptide modification was clustered by initially reacting with 20% of the HA precursor for 15 mins at 25 °C before pooling back with the original solution. From the bulk gels prepared with and without DNA / PEI particles, shredded hydrogel microparticles (sHMP) with prepared by sieving the swelled gels stacked on a 70 pm cell sieve (pluriSelect-USA, Mini-Strainer, PET sieve) and washing with IX PBS. The resulting flow-through was centrifuged at maximum speed (~18,000g) to collect the particles. sHMP were then combined with Factor XIII (Fibrogammin 1250, CSL Behring) and Thrombin (with calcium, from bovine plasma, Sigma Life Science) at 0.01 U / pL gel and 0.002 U / pL gel respectively, mixed thoroughly by pipetting, and residual buffer aspirated following centrifugation. The scaffolds were allowed to anneal for 1 hour at 37 °C under humidified conditions to avoid drying out. The scaffolds were imaged using Nikon Ti Eclipseequipped with C2 laser LED excitation to obtain z-stacks, imported into IMARIS to generate surface renders.

[0254] Stiffness of both nonporous HA-AC hydrogels and annealed FLIP scaffolds was measured as the storage modulus (G’) using a plate-to-plate rheometer (Physica MCR, Anton Paar, Ashland, VA). A frequency sweep was performed on the hydrogels using a strain of 0.2% with an angular frequency range of 0.1 to 10 rad / s. To measure the storage modulus of an annealed FLIP scaffold, 50 pL microgels with FXIII / Thrombin were pipetted directly onto the rheometer stage. The measuring position was set to 1 mm and the gel was allowed to incubate with humidity at 37 °C for 1 hour to allow for annealing. Once the gel was annealed, a frequency sweep was performed on the hydrogels using a strain of 1% with an angular frequency range of 0.1 to 10 rad / s.

[0255] Nanoparticle formation: All reporter plasmids were either ordered from Addgene or cloned in lab. The method for nanoparticle formation was performed. Briefly, DNA / PEI particles were prepared by complexing plasmid DNA encoding for Gaussia or Firefly luciferase and eGFP with linear polyethylenimine (L-PEI, 25 kDa, Polyscience') at an N / P ratio of 20. Briefly, 1 pg DNA was diluted in 10 pL of 150 mM NaCl and the corresponding amount of L-PEI was diluted in a separate tube in 10 pL of 150 mM NaCl. The L-PEI solution was then added to the DNA solution, immediately vortexed, and allowed to incubate for 15 min at 25 °C to allow for complexation. For HA coating, HA-NB was added to the DNA / PEI solution following at a w / w ratios (HA to PEI) of 5 and then incubated another 15 min. Solutions were then flash frozen in liquid nitrogen and allowed to freeze-dry for at least overnight, with storage anhydrous at -20 °C until reconstituted in buffer, gel precursor prior to crosslinking, or granular microgels prior to annealing.

[0256] Cell culture and transfection: All cells were obtained and validated from ATCC or the Duke Cell Culture Facility (CCF). Primary cells were screened using established protocols in literature or the lab, based on morphology and / or marker analysis. Human cells (primary and cell lines) and mouse cells were used to perform transfection experiments to elucidate better methods of transfection (nonviral gene delivery). For cell culture, the HMP was annealed together into a porous FLIP scaffold using tethered peptide crosslinking, with conjugated Q-peptide (Ac- NQEQVSPLGGERCG-NH2) (SEQ ID NO: 2) and K-peptide (Ac-FKGGERCG-NH2) (SEQ ID NO: 3) for Factor XIII (FXIII) / Thrombin transamination. To determine the transfection-ability of loaded scaffolds, a range of nanoparticle concentrations were tested in 3D cell culture. Luciferase expression and toxicity were assessed, in comparison to embedded approach and soluble administration. Using a 3D cell culture device, the lyophilized or freshly prepared nanoparticles (pDNA / PEI / HA-NB, N / P 20, HA:PEI 5) were mixed with the granular scaffold material.Surfection methods were used, where scaffolds were incubated with the nanoparticles in a 1 : 10 volume for several minutes before decanting, repeated until the entire nanoparticle solution was used. FLIP scaffolds were then annealed using secondary FXIII / Thrombin chemistry prior to topical cell seeding, at 10,000 cells / pL scaffold volume. Following this, cells were transfected via bolus nanoparticles in solution for the surface loaded (soluble) FLIP control, in which nanoparticles are exposed to all the cells without infiltration required. In addition to nanoparticle properties, cell viability and spreading from Live / Dead staining and imaging, and with flow cytometry for viability and reporter transgene expression were characterized. Both magnitude of expression and percent transfected cells were compared. Reporter genes for luciferase (Gaussia and Firefly) and GFP were compared from plate reader, imaging, and flow cytometry analysis. Viability was assessed via metabolic activity assays (PrestoBlue) and flow cytometry (propidium iodide).

[0257] Plasmids encoding VEGF(165) and IL-4 (primary isoform) were directly used for embedded nanoparticles, just as for reporter plasmids. As mentioned in the disclosure, plasmids encoding a dead Cas9 (dCas9) fused with the VP64 activation domain (VP 16 tetramer) were used for endogenous gene activation and expression in a two-plasmid system. Plasmid pJK52- dCas9VP64-GFP and the guide RNA (gRNA) cassette in plasmid pTK59-U6-gRNA-UbC-DsRed were kindly donated from the Gersbach lab at Duke University. Online tools were used to design gRNA sequences targeting upstream the native VEGF and IL-4 genes and restriction cloned into the pTK59 plasmid via complementary phosphorylated oligos. Plasmids were combined in a 2: 1 molar ratio (pTK59:pJK52) and nanoparticles formed as before, lyophilized, and embedded into the hydrogel precursor solution.

[0258] Cells were cultured in the resulting FLIP scaffolds for several days to a month-long period for time study tracking. Media was collected at each timepoint and stored at -80°C. Once all samples were collected, protein secretion was assessed via sandwich ELISA (DuoSet kits, R&D Systems) according to the manufacturer protocol, with absorbance quantified via plate reader and correlated to a standard curve.

[0259] AAV production and qPCR tittering: HEK293T cells were cultured for AAV2 and AAV9 production in DMEM. Cells were transfected as for reporter studies using linear PEI, 25k (PolySciences) and a triple plasmid cocktail of Helper (p5AdDelta6), Rep / Cap (pAAV2 / 2 or pAAV2 / 9n), and insert (pITR-GFP-P2A-GLuc) plasmids. The standard ratio of 2: 1 : 1 (mass basis) was used for the nanoparticle formulation. For 2D production, cells were seeded onto 150mm plates at 5 x 106cells per plate and 80 pg total plasmid (complexed with PEI for N / P 15-20). Cells were incubated for 5-7 days prior to harvesting for AAV. In the case of AAV2, cells were removedfrom scraping and cryolysed using alternating incubation in dry ice chilled water and a heating block. For AAV9, both lysed cells and purified media were compared. In all cases, the resulting solutions were spun down at < 1,000 x g for 5 mins to remove cells (slower to keep viable for time studies and continued culture), and then > 10,000 x g to remove debris. The resulting supernatant was purified using an AAV serotype purification kit (Takara Bio), comprised of an Amicon 100 kDa filter (Millipore) and buffer exchange with PBS. AAV was stored at -80 °C until validated in cell culture.

[0260] For FLIP culture and AAV production, the same process described above was used, but an additional filtration step was performed prior to concentration to remove free-floating microgels or hyaluronic acid that did not pellet with the cells and debris (0.4 pm filter). For the time study, the media was replaced at each time point for the initial volume. Gaussia luciferase and PrestoBlue assays were performed prior to the AAV purification process, using minimal volumes to avoid reducing too much AAV from the final titer.

[0261] Purified AAV samples were quantified via qPCR. The concentrated AAV products were treated using DNAse to remove residual DNA and reduce background reads (Invitrogeri). Primers were designed to flank the ITR regions. Standard qPCR (40-45 cycles) with melt-curve profiling was done using SYBR Green (Invitrogen). Samples were run in triplicate and at four-fold serial dilutions. Standards were prepared using the pITR plasmid and an 8-point serial dilution. Nontransfection controls and blanks were also run. Ct values and resulting viral titers were determined automatically from the software and compared to manual calculations.

[0262] Capsid loading was determined by processing the purified AAV samples using a sandwich ELISA against intact-particle AAV9 (mouse monoclonal and biotin-conjugated, ADK9, PROGEN Biotechnik) and compared titer from standard dilutions to the titers determined from qPCR. Capsid loading was calculated as the ratio of the qPCR for loaded to total ELISA capsid result. To address Amicon concentration and buffer exchange impurities, the AAV particles was further purified using iodixanol gradient centrifugation. Briefly, iodixanol was prepared in PBS and loaded from lowest to highest weight percent based on Table 1 (3:3:4:3 volumetric ratio). Samples were then centrifuged for 133,000 x g for >6 hours before isolating the 25-40% fraction region, sterile filtering, and then concentrating as before. qPCR was used for titering as before.Table 1: Iodixanol Recipe for AAV Purification.

[0263] Statistical analysis: Most parametric studies consist of a one-way ANOVA followed by appropriated post-hoc tests for the comparison of multiple nanoparticle conditions (namely Tukey HSD). P-values are considered significant if p < 0.05. Samples are assessed for normality and homoscedacity prior to analysis and plotting in GraphPad Prism. DOE studies use an axial CCD (non-factorial) planned and processed in JMP using a=1.41. Conditions in all in vitro studies are run in triplicate for an N>3. In all cases, significance (p<) is represented as **** < 0.001, *** < 0.005, ** < 0.01, and * < 0.05.Example 1 FLIP Scaffolds Demonstrate Sustained Expression of Biologies

[0264] First, whether FLIP gives consistent, sustained report gene expression was validated. Loaded FLIP scaffolds were compared to non-loaded and 3D surface (soluble) administration of a complexed plasmid DNA / polyethyleneimine (PEI) nanoparticle payload in the media supernatant [Figure la - Figure lb]. Homogenous distribution of nanoparticles when embedded in FLIP microgels was further demonstrated, and that this does not interfere with the mechanical properties of the bulk precursor hydrogel and annealed porous scaffold [Figure 2a - Figure 2b], Comparing across standard reporter cell types (HEK293, CHO-K1, and DI mouse MSCs), it was determined that soluble delivery gave higher magnitude of Gaussia expression, at 2-5 days posttransfection [Figure 1c - Figure le]. However, embedded nanoparticle FLIP performed better over the long term, with increased rate of transfection over time amounting to the same cumulative protein output, based on culture of DI MSCs. In contrast, soluble delivery dissipates over time due to the lack of sustained release of nanoparticles, which allows for repeated uptake and expression. This observation was likely cell-type specific, as primary cells, namely mouse bone- marrow derived macrophage (BMDM), astrocytes, and human dermal fibroblasts (HDF) did not respond as strongly to embedded scaffolds as they did for soluble delivery [Figure 2c], To have short-term expression in these more difficult-to-transfect cell types, soluble delivery was the better alternative.

[0265] To support that trends observed for reporter transgenes could translate to therapeutic protein expression, nanoparticles were embedded with plasmids encoding either human vascular endothelial growth factor (VEGF) or interleukin 4 (IL-4) into FLIP scaffolds [Figure If], Culturing HEK293 cells for a month-long period, sustained and increased rate of expression was observed from the embedded scaffolds, while soluble administration only persisted within the first week [Figure 1g], However, VEGF gave higher initial expression from soluble, so despite the rapid decline compared to embedded FLIP, the cumulative amounts of VEGF were still higher due to the initial spike in expression. A gene-specific response appeared as IL-4 gave similar initial levels of expression, allowing embedded FLIP to have higher cumulative IL-4 levels over time [Figure 2f], Production of anti-GFP antibodies was initially higher from soluble FLIP than embedded FLIP, but the production decline was greater in soluble FLIP [Figure Ik], Overall, sustained expression was only possible from the embedded FLIP scaffolds.

[0266] While simple plasmid transfection for reporter or therapeutic protein expression is beneficial for production, more complex biologies can require two or more plasmids, with optimization needed for specific complexing and nucleic acid ratios. To demonstrate that the disclosed platform was directly compatible with such applications, two-plasmid loaded FLIP scaffolds were assessed. Epigenetic activation and protein expression were examined using a dualplasmid dCas9 system. Briefly, a dCas9-VP64 encoding plasmid (pJK52-dCas9-GFP) was used in a 1 :2 ratio with a single guide RNA (gRNA) encoding plasmid (derived from pTK59-U6- DsRed) [Figure lh]. Hit-matching was performed to identify suitable gRNA sequences for IL-4 and VEGF promoter regions (CHOP-CHOP, CRISPOR), and among five candidates, the top performing gRNA plasmid was used for embedded FLIP scaffolds. Successful upregulation from loaded FLIP after 48 hours from seeding and transfection for VEGF targeting plasmids in HEK, HDFs, and HUVECs was demonstrated [Figure 2d], This was also consistent for IL-4 in HEK293, human dermal fibroblasts (HDFs), and RAW264.7 mouse macrophage [Figure 2e], although the magnitude of expression varied greatly by cell type. In both cases, expression was consistently lower using the two-plasmid approach compared to directly encoded transgene plasmids. As with the previous confirmation for VEGF and how direct transgene expression gave sustained expression, HEK transgene activation was compared over time using the dCas9 FLIP systems. HEK responded strongly to VEGF activation from embedded FLIP [Figure li] with trends and magnitude rivaling that of even the direct VEGF 165 transgene. Further, while soluble FLIP dissipated in VEGF expression after a week, the embedded scaffolds continued to express after three weeks. However, differing trends for IL-4 was observed, which can be due to the selected expression cell type. HEK did not respond strongly to endogenous IL-4 activation at 48 hours[Figure 2f] and this remained the case over time compared to soluble [Figure 2g], As such, the two-plasmid approach was possible from embedded FLIP scaffolds.Example 2 Static and Suspension FLIP Scaffolds Produced AAV Transfected with Triple Plasmids

[0267] Static FLIP culture. Turning to more advanced gene therapy products, viral vectors often require transfection of two or three plasmids for control and safety during manufacturing, as with lentivirus, adenovirus, and adeno-associated virus. Here, how FLIP scaffolds perform AAV production was assessed. This introduces several complexities, the main one being the tripleplasmid cocktail required for AAV, from the Helper, Replication / Capsid, and transgene insert (ITR) plasmids. It was first assessed whether our lyophilized nanoparticle approach could work for the three plasmids when prepared at a commonly used ratio of 2: 1 : 1 (Helper : Rep / Cap : Insert) [Figure 3a], compared to cells on standard 2D tissue culture plastic (“2D Bolus”). Particles were complexed with PEI and lyophilized in sucrose using the same approach as with the single and dual plasmid methods, scaling based on the total plasmid mass. The lyophilized particles were then loaded at 1 pg / pL in bulk hydrogel and converted to FLIP as before.

[0268] With FLIP AAV production, reporter Gaussia luciferase expression, as well as virus titer was assessed based on genome copies from qPCR, and endpoint infectivity of the collected virus on DI MSCs [Figure 3b], It was confirmed that embedded scaffolds resulted in similar expression to that of 2D Bolus at 48 hours MSCs [Figure 4a], Neither 2D nor FLIP transfection showed signs of toxicity [Figure 4c], In addition, both methods gave comparable levels of GFP+ cells (-30% and -45% respectively) [Figure 4b], While these values were lower than transfection achieved in HEK in 2D and FLIP culture for the single plasmid applications, the results were within tolerance of transfection efficiency for triple AAV plasmids, since greater emphasis on packing of the ITR plasmid than transcribing it. It is worth noting that some of the GFP expression observed can be due to the produced AAV transducing cells within the FLIP scaffold, since any virus in the media could passively diffuse back into the void space. However, this was assumed to be negligible given the media replacement every few days.

[0269] The advantage of embedded FLIP was sustained, viable transfection over time. When extending the AAV production over a month-long period [Figure 3c, Figure 3f], it was observed that FLIP improved the titers and rate of AAV secreted over time, while those from the bolus transfected 2D plate tapered-off after just 5-7 days from transfection. However, cumulative virus titer was similar across 2D and FLIP cultures [Figure 3d], This was expected for 2D given that most AAV manufacturing protocols harvest at Days 3-5 to avoid cell exhaustion or death and recover the most AAV from media. Surprisingly, neither 2D nor scaffold delivery showed asignificant difference in viability over time, with each facing a decline after 23-27 days [Figure 3i - Figure 3j], although embedded FLIP scaffolds did result in greater cell proliferation based on the rate of division [Figure 3h], Further, it was demonstrated through infectivity studies at a fixed multiplicity of infection (MOI) in DI MSCs that the virus collected over each time point did not vary significantly in activity, equivalent to AAV from bolus transfected cells at 48 hours [Figure 3e], unlike those from plate transfection and activity largely dissipating after just one week. Further, across MOI dilutions, the performance for AAV from loaded FLIP scaffolds was on-par with that of AAV produced in suspension from HEK293F or DI -MSCs after five days of culture [Figure 4d-Figure 4e, respectively], although all of these were still lower in infectivity than AAV produced on large 2D tissue culture plastic. It can be possible to improve on the 3D culture device design such that could support continuous media flow to enhance production and avoid this reduction in transduction and loss of AAV, similar to microfluidic devices used in organ-on-a-chip applications. This would be ideal for in-line manufacturing and using FLIP in a cartridge format akin to current downstream processes, which could potentially ease translation to GMP systems.

[0270] Suspension FLIP culture. The success of nanoparticle-loaded FLIP scaffolds was promising, both for embedded and surface loaded scaffold methods; however, all work to this point had been in static 3D culture. Compatibility with current shaker or suspension bioreactor technology was demonstrated to encourage adoption of the FLIP system. FLIP scaffolds can reduce footprint and media requirements relative to those used in 2D cell culture. In suspension culture, there can also be a greater effect from diffusion out of scaffolds under agitation, compared to static 3D culture, and potentially easier purification of the protein or virus product from the media.

[0271] First, the platform for suspension culture was optimized and how the cell and nanoparticle loading scheme performed was determined [Figure 5a], HEK293 culture using both adherent (HEK293T) and suspension (FreeStyle™ HE293F, Thermo') variants were assessed. Common seeding densities were compared at different levels of cell culture: 100 cells / pL (adherent density, based on surface area in cm2), 300 cells / pL (suspension density), and 1,000 cells / pL (3D culture density, used for FLIP and other granular scaffolds) [Figure 6a-Figure 6b], Different loading schemes for FLIP scaffolds, including withing suspension culture are shown in Figure 6f. Each of these densities were compared for cells with and without FLIP based on total cell number, and cultured for each HEK cell type. Cells were loaded into FLIP based on 1 :10 volumetric ratio (30 pL scaffold in 300 pL media) and encapsulated within the scaffold during the annealing step. Cell- loaded FLIP scaffolds were then incubated on shaker plates at 112 RPM in their respective culturemedium. After five days of culture, cells were processed with PrestoBlue for relative metabolic activity (to the average of the cell controls). Cells were also pre-stained with CFSE for proliferation tracking, assessed by flow cytometry once removed from the 3D culture environment (MMP and hyaluronidase degradation).

[0272] FLIP culture for both cell types improved viability compared to shaken conditions, although lower concentrations tended to do better for both metabolic activity and high cell proliferation. Relative to suspension culture at the highest density (1,000 cells / pL, that of 3D seeding), suspension cells and cell-loaded FLIP cultures were consistent across metabolic activity [Figure 6c], Adherent cells had slightly better metabolic activity (within error) over suspension cells, but it is not significant. For viability (PI exclusion), the most viable cells were from the 3D conditions, and for proliferation (CFDA-SE; cell divisions), better growth occurred in the FLIP conditions than those on the plate. Both adherent and suspension cells did well in suspended FLIP conditions at 300 cells / pL, avoiding concerns for over-confluency at the higher densities, but only suspension cells performed well when without FLIP. The impact of relative surface area and gel volume on cell metabolism and proliferation was also investigated. Given that FLIP are granular scaffolds, porosity and local surface area highly impact how cells spread and grow within the scaffold environment, which is not present when culturing cells freely suspended in media. While an ideal seeding density was determined, there could be local diffusion differences for cells in the core of FLIP than those near the surface. A semi -rationale design to sub-divide the larger gels at the typical 1 : 10 volumetric ratio (gel-to-media) used to culture, was investigated. 30 pL gel was broken up into 2-5 gel “clusters” of equal volume (based on the total volume) and cultured adherent HEK at 300 cells / pL scaffold. In addition, since the seeding density likely worked best at 300 cells / pL - 1000 cells / pL, seeding was performed best at 300 cells / pL and at 600 cells / pL. As before, cells were processed at Day 5 for metabolic activity and proliferation. It was determined that the best condition was 3 gels x 10 pL gel volume (per 300 pL solution), at 600 cells / pL, and shaking at 120 RPM for 3-14 days [Figure 6e], Under these conditions, cells gave improved levels of metabolic activity without appearing over-confluent and continued to have high viability [Figure 6e], Conditions without FLIP again performed worse than those with the scaffolds, although between the subdivided gel clusters, there was not as significant a difference compared to the impact from seeding density.

[0273] With the suspension FLIP conditions optimized, reporter plasmid nanoparticle transfection was performed, comparing the current loading methods of embedded and soluble FLIP cultures to simple bolus transfection of the freely suspended cells [Figure 6g - Figure 6j], The effect of gel loading and the cell seeding density was compared on both viability and transgene expressionusing the optimal conditions at 600 cells / pL scaffold and divided into three equal -volume FLIP clusters. It was observed that embedded FLIP gave the best expression among loaded scaffolds [Figure 6g], although better only in metabolic activity compared to soluble [Figure 6h], Loaded FLIP gave better viability than exposing non-FLIP seeded cells to the 3D dose (1 pg / pL), compared to the lower dose used in suspension cell culture (0.1 pg / pL). As such, cells seeded within nanoparticle loaded FLIP scaffolds improved protein production in suspension culture. Toxicity plays more of an effect on transfection, better in FLIP than in suspension without the scaffold. Note that assessment of therapeutic protein production under suspension conditions for IL-4 and VEGF was undertaken, similar to that from static FLIP [Figure 6i - Figure 6j],

[0274] Regarding triple plasmid loaded FLIP for AAV production, encapsulated adherent HEK293T were compared to their suspension equivalent without the scaffold. Nanoparticles were loaded at 1 pg / pL in scaffolds, with cells seeded at 6000 cells / pL scaffold and 0.1 pL scaffold per pL cell culture [Figure 6k], Samples were collected at 48 hours, Week 1, Week 2, and Week 3 to see if the advantage from static FLIP culture held true for suspension culture. As before, strong Gaussia luciferase expression was observed from both embedded and soluble FLIP conditions [Figure 5b], although overall soluble gave the higher reporter expression. Viability was also improved in the transfected FLIP scaffolds than the non-scaffold 2D control [Figure 5c], AAV production was possibly higher from FLIP transfection, despite similar protein expression in all suspension conditions. Further, the results were not significantly different due to higher spread in titers collected across the samples [Figure 5d - Figure 5e], One of the advantages of FLIP is sustained expression due to improved viability, and that both nanoparticle-embedded and surface transfected scaffolds could surpass suspension culture titers over time in both yield and activity [Figure 5f] . Between static and suspension FLIP scaffold methods, it was observed that broadly AAV from static and suspension scaffold cultures gave similar capsid loading to 2D controls (n.s., p > 0.05; Figure 5g). Between static embedded and soluble scaffold gene delivery, there was no significant difference for capsid loading.

[0275] Attempts to use lower-cost alginate (alginic acid) for < 30 mL FLIP scaffolds failed to give sustained transfection [Figure 7a], which did not improve the resulting AAV titers past one week of culture [Figure 7b], It has been demonstrated that the stabilized particles could be used with alginate, although further optimization was still needed to have it be as effective as HA scaffolds. Further, the activity of the produced AAV under the alginate scaffold conditions, decreased in all samples over time [Figure 7c], reinforcing the need for FLIP optimization to have sufficient cell viability and avoid byproduct interference.

[0276] The differences seen in AAV infectivity or even yield could be related to their capsid loading efficiency. The simple purified AAV samples did not appear to be highly loaded across FLIP and non-FLIP scaffold conditions (at most -45%, from ELISA and qPCR titer comparison, highly variable results) [Figure 7d], Gradient centrifugation methods were used to better purify the AAV and to understand the difference between traditional 2D and FLIP production methods. It was observed that trends could be similar to less purified AAV products, from qPCR, but that the total titers were not, since as low as 1 x 106vg / mL was read. High protein background could be present or AAV leakage across the gradient fractions, although from analysis the qPCR peaks did correspond correctly to the gradient run. On possible loading efficiency improvements [Figure 5g], the main observation was that regardless of titer yield, since similar across both 2D, static FLIP, and suspension FLIP cell culture, only AAV from 2D and static FLIP resulted in similar infectivity. Further, both embedded and soluble FLIP scaffold conditions gave no significant difference among the conditions for capsid loading [Figure 5h], but as suspension FLIP had lower capsid loading overall, it further points to pursing the static FLIP system for industry translation.

[0277] However, suspension scaffolds gave lower capsid loading efficiencies from soluble delivery (p < 0.05) compared to embedded delivery. In summary, sustained expression in FLIP could benefit industry use, since the same initial cell population could yield higher AAV titers without the need to stop production after just several days, reducing downtime and improving the product.Example 3 Perfusion Device with FLIP Provide Continuous Production of AAV molecules

[0278] Having determined that the static FLIP system provided the best level and quality of AAV to 2D plate methods, design options were explored for how this would be applied in a manufacturing process. There are several reactor designs that could work for granular scaffolds, including packed-bed reactors (PBR) or fluidized bed reactors, which have had success for metabolic processes using cells cultured on solid, stiff microparticles. With the shift in manufacturing towards both single-use an in-line or continuous flow designs, potential in FLIP was explored to address the limitations of current perfusion bioreactors. Prototype devices were created based on several geometries for a fixed chamber volume, loaded the FLIP with cells intermixed inside and annealed, and then connected to circulating media line via a peristaltic pump [Figure 8a, Figure 9a-Figure 9b], Originally, designs were constructed in Fusion360 (Autodesk) and printed on an Elegoo Mars 2 SLA 3D printer using isopropanol-soluble resin, with a 50 pm resolution to allow for threading and O-ring seals to avoid leakage, plus Leur-lock adapters to connect to the rest of the circulating system [Figure 9c], However, preliminary testing determined that the hydrogels, while optimized for cell culture in 3D devices, could not sustain even low flowrate and resulting pressures without compacting in the PBR design [Figure 9d] . This resulted in a pressure buildup that either ruptured the connecting lines or caused the scaffold to eject into the line and clog downstream. Alternative chemistries that could support stiffer gels can help here, although a design that could provide cells the best local stiffness for viability and transfection could be selected.

[0279] Subsequently, the organ-on-a-chip field, for a cartridge design was explored [Figure 8b], To prepare the perfusion device, a reverse mold was created to cast channels for our PDMS device, with an inlet and outlet for media to circulate. However, to overcome the gel jamming and pressure issue, the haves of channel were separated so that media flows adjacent to FLIP, allowing diffusion across a membrane. With this approach, FLIP microgels were first intermixed with cells, injected into a perfusion vessel, and annealed within the device chamber. Cells were transfected either from nanoparticles embedded within the FLIP or surface loaded via nanoparticles in the circulating media. This design allowed for a closed system without pressure buildup and the ability to run indefinitely, so long as the media reservoir level is maintained. This reservoir chamber was also used as a sampling port to collect media over time to assess protein expression and virus titer. Comparing FLIP with the triple AAV plasmid nanoparticles embedded, reduced Gaussia expression was observed relative to static FLIP culture [Figure 8d], although metabolic activity was improved at earlier timepoints [Figure 8e], With perfusion, similar levels of virus titer across all timepoints were observed [Figure 8f], resulting in the same total amount of virus produced as in static FLIP [Figure 8g], Further, perfusion appeared to produce virus with the same level of infectivity as those produced in static FLIP culture [Figure 8h], Static and perfusion FLIP scaffolds show similar activity initially and through 30 days. As such it remained unclear whether the reduced Gaussia output from perfusion-produced AAV means that there was simply less transcription of the ITR plasmid and more production of loaded AAV. However, between 3D static and perfusion FLIP scaffold methods, it was observed that broadly AAV from static and perfusion scaffold cultures gave similar capsid loading [Figure 8i],

[0280] In summary, perfusion could be the better option as it affords the ability to connect FLIP via an in-line cartridge, keeping the production a closed system and the cells more viable at earlier timepoints. This also was the simpler method for cell culture, since despite needing media circulation, the individual scaffolds do not need to be prepared in 3D wells and media collected via tedious pipetting. Rather, the cell-scaffold mixture could be directly injected into the cartridge when ready to culture, and did not require any interaction during the batch run.Example 4PEG-FLIP Scaffolds Demonstrate Capacity for Biologically Active Molecule Production

[0281] FLIP scaffolds described herein can be prepared with various polymers. PEG hydrogel chemistry can be used to form a PEG hydrogel network. 4-arm PEG-maleimide can be used as a polymer backbone with a L-MMP di-thiol containing peptide used as a crosslinker. The reaction can include a click reaction in the absence or presence of additional catalysts. [Figure 10a], PEG- hydrogels can be shredded for use in FLIP scaffolds disclosed herein. The PEG-hydrogel can be passed through a 70 pm filter using a centrifuge at 18000G for 5 minutes to prepare shredded hydrogel particles (sHMPs) [Figure 10b], The sHMPs can be annealed into a FLIP scaffold by mixing sHMPs with a 4-arm PEG-SH polymer. Click reaction between the particles creates particle interlinking agents [Figure 10c], Figure lOd shows the size distribution of sHMPs. PEG FLIP hydrogel scaffolds can support viable NIH3T3 cells following transfection with DNA polyplexes at a greater level than under standard conditions and 1 or 2 pg / pL DNA [Figure lOe and Figure 10g], Further, NIH3T3 cells transfected within a PEG FLIP scaffold show similar gene expression as measured via a luciferase assay [Figure 10f],Summary of Specific Examples

[0282] As described herein, across several 3D culture schemes it was determined how the loading method influences transfection efficiency and in vitro toxicity for the FLIP scaffold platform, among other properties [Figure 11], Protein and virus production in FLIP used less resources and provided longer-term expression. FLIP allowed for significantly less media and cells required than 2D plate culture, or when scaled-up they could increase production over 2D culture from higher cell or scaffold amounts used. Similar or better yields were observed in FLIP than 2D for static cell culture, assuming a similar production scale to compare yields (e.g., same number of cells, or same volume of culture). Adoption of an in-line cartridge system is contemplated, taking advantage of the better production output from static FLIP culture and adapting for continuous flow to improve viability and unit operation feasibility. The ability to express genes long term from an injectable porous hydrogel opens many applications of nucleic acid delivery, and established a system with more efficient approach to cell product manufacturing. A comparison across the different production schemes is shown in Figure 13.Table 2: Sequences

Claims

CLAIMSWhat is claimed is:

1. A method of producing a biologically active molecule, the method comprising: loading cells into a hydrogel scaffold, wherein the hydrogel scaffold comprises hydrogel particles and nanoparticles complexed with one or more nucleic acids; and maintaining the cells.

2. The method of claim 1, wherein the one or more nucleic acids encodes a biologically active molecule comprising an oligonucleotide, a DNA molecule, an RNA molecule, a virus, or any combination thereof.

3. The method of claim 1, wherein the biologically active molecule comprises an Adeno- associated virus (AAV).

4. The method of claim 3, wherein the AAV comprises AAV1, AAV2, AAV3 (including 3 A and3B), AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV8 bp, AAV7M8, AAVAnc80, AAVrhlO, AAVPHP.B, AAV type rh32.33, AAV type rh8, AAV type rh74, AAV type hu.68, avian AAV, bovine AAV, canine AAV, equine AAV, ovine AAV, snake AAV, bearded dragon AAV, AAV2i8, AAV2g9, AAV-LK03, AAV7m8, AAV Anc80, AAV-PHP.eB, AAV-TT, AAVv66, rAAV2 / l, rAAV2 / 8, rAAV2 / 9, AAV-DJ, AAV-HAE1, AAV-HAE2, AAVM41, AAV- 1829, AAV2 Y / F, AAV2 T / V, AAV2i8, AAV2.5, AAV9.45, AAV9.61, AAV-B1, AAV-AS, AAV9.45A- String, AAV9.45Angiopep, AAV9.47-Angiopep, AAV9.47-AS, AAV-PHP.B, AAV- PHP.eB, AAV-PHP.S, AAV-F, AAVcc.47, or AAVcc.81.

5. The method of claim 1, wherein the one or more nucleic acids encodes a biologically active molecule comprising a peptide, a polypeptide, a protein, an antibody, or any combination thereof.

6. The method of claim 1, wherein the biologically active molecule comprises a therapeutic protein, therapeutic peptide, recombinant protein, monoclonal antibody, recombinant antibody, chimeric antibody, humanized antibody, non-human antibody, multispecific antibody, antibody fragments, or any combination thereof.

7. The method of claim 1, wherein the biologically active molecule comprises human vascular endothelial growth factor (VEGF), interleukin 4 (IL-4), or any combination thereof.

8. The method of claim 1, wherein the cells comprise a human embryonic kidney (HEK) cells,Chinese hamster ovary (CHO) cells, DXB-11 cells, DG-44 cells, baby hamster kidney (BHK) cells, African green monkey kidney cells, Hela cells, adenocarcinomic humanalveolar basal epithelial cells, human lung cells, human hepatoma (Hep) cells, mouse mammary tumor (MMT) cells, TRI cells, MRC5 cells, FS4 cells, mammalian myeloma cells, E. coli cells, yeast cells, or any combination thereof.

9. The method of claim 1, wherein loading the cells comprises topical seeding of the cells into the hydrogel scaffold.

10. The method of claim 11, wherein topical seeding of the cells comprises combining the cells with the hydrogel scaffold at about 5,000 cells / pL scaffold volume to 15,000 cells / pL scaffold volume.

11. The method of claim 1, wherein loading the cells comprise encapsulating the cells in the hydrogel scaffold.

12. The method of claim 13, wherein encapsulating the cells comprises encapsulating cells at about 100 cells / pL scaffold volume to 10,000 cells / pL scaffold volume.

13. The method of claim 1, wherein the hydrogel scaffold comprises hyaluronic acid (HA).

14. The method of any one of claims 1 or 15, wherein the HA further comprise one or more acrylamide functional groups (HA-AC), one or more RGD ligands (RGDSP) (SEQ ID NO: 36), one or more Q-peptides (NQEQVSPLGGERCG) (SEQ ID NO: 2), one or more K-peptides (FKGGERCG) (SEQ ID NO: 3), or any combination thereof.

15. The method of any one of claims 1-14, further comprising, prior to loading the cells, combining polyethyleneimine (PEI), the nucleic acids, and HA comprising one or more norbomene functional groups (HA-NB), at N / P of about 20, and an HA:PEI ratio of about 5.

16. The method of claim 1, further comprising recovering the biologically active molecules.

17. The method of any one of the preceding claims, wherein at least about 1 x 1010genomic copies or at least about 1000 pg / mL of the biologically active molecule is recovered.

18. The method of claim 1, wherein maintaining the cells comprises removing spent medium and adding fresh medium.

19. The method of any one of the claims 1 or 27, wherein maintaining the cells comprises maintaining the cells for at least 14 days.

20. A method of expressing a nucleic acid in one more cells, the method comprising: loading one or more cells into the hydrogel scaffold, wherein the hydrogel scaffold comprises hydrogel particles and nanoparticles complexed with one or more nucleic acids.

21. The method of claim 20, wherein expression of one or more nucleic acid in one or more cells is sustained for at least 14 days.

22. A perfusion device for production of a biologically active molecule, the device comprising:a chamber comprising hydrogel scaffold loaded with cells; a media channel comprising an inlet and an outlet; a membrane separating the chamber from the media channel; a media reservoir; and a peristaltic pump.

23. The device of claim 22, wherein the hydrogel scaffold comprises hydrogel particles and nanoparticle complexed with one or more nucleic acids.

24. The device of claim 22 or 23, wherein the cells comprise human embryonic kidney (HEK) cells, Chinese hamster ovary (CHO) cells, DXB-11 cells, DG-44 cells, baby hamster kidney (BHK) cells, African green monkey kidney cells, Hela cells, adenocarcinomic human alveolar basal epithelial cells, human lung cells, human hepatoma (Hep) cells, mouse mammary tumor (MMT) cells, TRI cells, MRC5 cells, FS4 cells, mammalian myeloma cells, E. coli cells, yeast cells, or any combination thereof.

25. The device of any one of claims 22-24, wherein the hydrogel scaffold comprises hyaluronic acid (HA).

26. The device of any one of the claims 22-25, wherein the HA further comprise one or more acrylamide functional groups (HA-AC), one or more RGD ligands (RGDSP) (SEQ ID NO: 36), one or more Q-peptides (NQEQVSPLGGERCG) (SEQ ID NO: 2), one or more K-peptides (FKGGERCG) (SEQ ID NO: 3), or any combination thereof.

27. The device of any one of the claims 22-26, wherein the nanoparticle complexed with one or more nucleic acid is prepared by combining PEI, nucleic acid vector and HA comprising one or more norbornene functional groups (HA-NB) at N / P of about 20 and an HA:PEI ratio of about 5.

28. The device of claim 27, wherein the nucleic acid encodes a biologically active molecule comprising an oligonucleotide, a DNA molecule, an RNA molecule, a virus, or any combination thereof.

29. The device of claim 28, wherein the biologically active molecule comprises an Adeno- associated virus (AAV).

30. The device of claim 29, wherein the AAV comprises AAV1, AAV2, AAV3 (including 3A and 3B), AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV8 bp, AAV7M8, AAVAnc80, AAVrhlO, AAVPHP.B, AAV type rh32.33, AAV type rh8, AAV type rh74, AAV type hu.68, avian AAV, bovine AAV, canine AAV, equine AAV, ovine AAV, snake AAV, bearded dragon AAV, AAV2i8, AAV2g9, AAV- LK03, AAV7m8, AAV Anc80, AAV-PHP.eB, AAV-TT, AAVv66, rAAV2 / l, rAAV2 / 8,rAAV2 / 9, AAV-DJ, AAV-HAE1, AAV-HAE2, AAVM41, AAV- 1829, AAV2 Y / F, AAV2 T / V, AAV2i8, AAV2.5, AAV9.45, AAV9.61, AAV-B1, AAV-AS, AAV9.45A- String, AAV9.45Angiopep, AAV9.47-Angiopep, AAV9.47-AS, AAV-PHP.B, AAV- PHP.eB, AAV-PHP.S, AAV-F, AAVcc.47, or AAVcc.81.

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