Bioprinting strategies for generating cell therapy delivery systems using continuous liquid interface production

Abstract

A method of producing a therapeutic device, such as a therapeutic implant, for delivery of one or more therapeutic agents is described. An exemplary method includes printing a bioresin that includes one or more live cells and a biocompatible polymer resin using an additive manufacturing process, such as continuous liquid interface production (CLIP), to provide a three-dimensional device where one or more live cells are encapsulated in cured polymer and remain viable, e.g., so that they can proliferate and spread. Exemplary devices containing anticancer drug-secreting live cells are described. Also described are related therapeutic methods, including methods of treating cancer.

Description

Attorney Docket No. 4210.0546WOBIOPRINTING STRATEGIES FOR GENERATING CELL THERAPY DELIVERY SYSTEMS USING CONTINUOUS LIQUID INTERFACE PRODUCTIONCROSS REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims the benefit of United States Provisional Patent Application Serial No. 63 / 715,171, filed November 1, 2024, the disclosure of which is incorporated herein by reference in its entirety.GOVERNMENT SUPPORT CLAUSE

[0002] This invention was made with government support under Grant No. CA269974 awarded by National Institutes of Health. The government has certain rights in the invention.TECHNICAL FIELD

[0003] The presently disclosed subject matter relates to methods of preparing therapeutic agent delivery devices using additive manufacturing processes, such as continuous liquid interface production (CLIP). Exemplary devices comprise therapeutic live cells encapsulated in biocompatible polymeric scaffolds and are printed from a resin formulation comprising the cells. The presently disclosed subject matter further relates to methods of treating disease using the devices and to the devices themselves.BACKGROUND

[0004] Cell therapies are a class of therapeutic modality which have been leveraged for treating a variety of pathologies. Living cells can respond dynamically to their environment, thus enabling the possibility for a safer and more potent therapeutic effect. While cell therapies are thought to have high potential for clinical success, to date many fail in clinical testing due to poor efficacy and safety concerns, which are typically related to inadequate delivery of the treatment cells to the target site. Presently utilized treatmentAttorney Docket No. 4210.0546WO therapies typically require repeated infusions of systemically delivered cell therapies to achieve minimum therapeutic concentrations of the treatment cells at the target site, resulting in unstable pharmacokinetic profiles as well as adverse effects from off-target toxicity. Local delivery of cell therapies is thought to enhance interactions with the cells and their therapeutic target; however, even when utilizing presently known techniques for local delivery of therapeutic cells, it is still common for such cells to suffer from, among other things, poor transplantation efficiency. Furthermore, rapid clearance of the therapeutic cells from the implantation site is often observed using presently known local delivery techniques.

[0005] Accordingly, there is an ongoing need to develop effective and reliable devices and methods for delivering therapeutic cells to a target site. There is also an ongoing need to provide reliable methods of producing such devices.SUMMARY

[0006] This summary lists several embodiments of the presently disclosed subject matter, and in many cases lists variations and permutations of these embodiments. This summary is merely exemplary of the numerous and varied embodiments. Mention of one or more representative features of a given embodiment is likewise exemplary. Such an embodiment can typically exist with or without the feature(s) mentioned; likewise, those features can be applied to other embodiments of the presently disclosed subject matter, whether listed in this summary or not. To avoid excessive repetition, this Summary does not list or suggest all possible combinations of such features.

[0007] In some embodiments, the presently disclosed subject matter provides a method of producing a device for delivery of one or more therapeutic agents to a target site of a subject in need thereof, the method comprising: preparing a bioprinting resin, wherein the bioprinting resin comprises a biocompatible polymer resin and one or more therapeutic agents, wherein said one or more therapeutic agents comprise a live cell and / or a microorganism; and forming and curing the bioprinting resin via an additive manufacturing process, thereby producing the device for delivery of the one or more therapeutic agents,Attorney Docket No. 4210.0546WO wherein the device comprises the one or more therapeutic agents encapsulated in cured biocompatible polymer. In some embodiments, the additive manufacturing process comprises a continuous liquid interface production (CLIP) process.

[0008] In some embodiments, the one or more therapeutic agents comprise a live cell, optionally wherein the live cell is selected from the group comprising an anticancer drugsecreting cell, a stem cell, a chimeric antigen receptor (CAR)-T cells, a T cell, a macrophage, and a natural killer cell. In some embodiments, the anti cancer drug-secreting cell is a cell that secretes tumor necrosis factor (TNF)-related apoptosis inducing ligand (TRAIL) and / or wherein the anticancer drug-secreting cell is a fibroblast or a stem cell. In some embodiments, the stem cell is a neural stem cell or a mesenchymal stem cell. In some embodiments, the one or more therapeutic agents comprise a bacteria cell, a yeast cell, or an oncolytic virus. In some embodiments, the one or more therapeutic agents comprise a live cell and a non-living component, optionally wherein the non-living component comprises one of the group comprising a polypeptide, a nucleic acid, an antibody, an exosome, a nanoparticle, a microparticle, and a small molecule.

[0009] In some embodiments, the biocompatible polymer resin comprises one or more of the group comprising a functionalized gelatin, a functionalized alginate, a functionalized hyaluronic acid, a functionalized polyethylene glycol (PEG), a functionalized collagen, and combinations thereof, optionally wherein the biocompatible polymer resin comprises one or more of the group comprising gelatin methacryloyl (GelMA), methacrylated alginate, methacrylated hyaluronic acid, methacrylated PEG, acrylated PEG, methacrylated collagen, and combinations thereof.

[0010] In some embodiments, the live cell and / or microorganism proliferates within the device for a period of time after formation of the device. In some embodiments, the live cell and / or microorganism is viable and therapeutically active after formation of the device, optionally wherein the live cell and / or microorganism remains viable for at least 30 days in vivo. In some embodiments, a concentration of one or more of the one or more therapeutic agents in the device is adjustable during formation of the device via the additive manufacturing apparatus.Attorney Docket No. 4210.0546WO

[0011] In some embodiments, formation of the device is controlled to form a desired shape, optionally a disc shape, a spherical shape, or a porous shape. In some embodiments, the desired shape of the device is selected to improve structural integrity and / or functionality of the device following in vivo implantation. In some embodiments, the desired shape of the device is customized to fit a resection cavity for implantation and / or administration. In some embodiments, the desired shape of the device is customized to improve the viability and / or migration of a live cell and / or microorganism encapsulated in the device. In some embodiments, the desired shape of the device is customized to optimize a degradation rate of a degradable material in the device, to optimize encapsulation of the one or more therapeutic agents, and / or to optimize a release rate of one or more therapeutic agents from the device. In some embodiments, the desired shape of the device is customized to reduce foreign body response against the device.

[0012] In some embodiments, the presently disclosed subject matter provides a method of treating a disease in a subject, the method comprising providing a subject in need of treatment for the disease and administering to the subject a device prepared according to the method comprising: preparing a bioprinting resin, wherein the bioprinting resin comprises a biocompatible polymer resin and one or more therapeutic agents, wherein said one or more therapeutic agents comprise a live cell and / or a microorganism; and forming and curing the bioprinting resin via an additive manufacturing process, thereby producing the device, wherein the device comprises the one or more therapeutic agents encapsulated in cured biocompatible polymer.

[0013] In some embodiments, administration comprises surgical implantation of the device at a desired site in the subject, optionally wherein administration comprises post-surgical implantation of the device in the subject after removal of a tumor from the subject. In some embodiments, the subject is suffering from or susceptible to a cancer, optionally wherein the cancer is selected from the group comprising glioblastoma, ovarian cancer, colorectal cancer, pancreatic cancer, liver cancer, melanoma, breast cancer, and leukemia. In some embodiments, administering the device to the subject treats the cancer, optionally wherein administering the device delays tumor regrowth and / or increases expected subject lifespan.Attorney Docket No. 4210.0546WO

[0014] In some embodiments, the presently disclosed subject matter provides a therapeutic composition, the therapeutic composition comprising a device prepared according to a method comprising: preparing a bioprinting resin, wherein the bioprinting resin comprises a biocompatible polymer resin and one or more therapeutic agents, wherein said one or more therapeutic agents comprise a live cell and / or a microorganism; and forming and curing the bioprinting resin via an additive manufacturing process, thereby producing the device, wherein the device comprises the one or more therapeutic agents encapsulated in cured biocompatible polymer. In some embodiments, the therapeutic composition is formulated for administration and / or implantation in a subject in need of treatment, optionally a subject suffering from or susceptible to a cancer.

[0015] Accordingly, it is an object of the presently disclosed subject matter to provide a method of producing a device for delivery of one or more therapeutic agents, as well as related methods of treating disease and related devices. These and other objects are achieved in whole or in part by the presently disclosed subject matter. Further, objects of the presently disclosed subject matter having been stated above, other objects and advantages of the presently disclosed subject matter will become apparent to those skilled in the art after a study of the following description, Drawings and Examples.BRIEF DESCRIPTION OF THE DRAWINGS

[0016] One or more example embodiments of the disclosed devices are described herein, with reference to the accompanying drawings, in which:

[0017] FIG. 1 is a schematic illustration of an exemplary strategy for 3D bioprinting gelatin methacryloyl (GelMA) scaffolds using a continuous liquid interface production (CLIP) apparatus.

[0018] FIGS. 2A and 2B are graphs showing the average quantity of normal human diploid fibroblast (NHF1) cells encapsulated in exemplary disc-shaped CLIP -bioprinted scaffolds (FIG. 2A) and the viability of the encapsulated cells in the scaffolds over time based on bioluminescence imaging (BLI) signals (total flux measured in photons perAttorney Docket No. 4210.0546WO second (p / s)). (FIG. 2B). Scaffolds were printed in using a GelMA bioresin comprising a cell concentration of 4 x 106cells / mL. For FIG. 2A, n=18 per batch. For FIG. 2B, n=3.

[0019] FIG. 3 is a series of fluorescence images of green fluorescent protein (GFP)- positive NHF1 cells over time in exemplary bioprinted scaffolds as described for FIG. 2B.

[0020] FIGS. 4A and 4B show aspects pertaining to the ability to scale production of cell-laden scaffolds using CLIP bioprinting. FIG. 4A is a graph showing the BLI signal (p / s) of NHF1 cells in disc-shaped CLIP bioprinted scaffolds printed from GelMA bioresins comprising various concentrations of cells. FIG. 4B is a graph showing the BLI signal (p / s) of NHF1 cells in disc-shaped CLIP bioprinted scaffolds as a function of scaffold volume (in cubic millimeters (mm3)). n=3 for both FIGS 4A and 4B.

[0021] FIGS. 5A and 5B show aspects pertaining to viability and drug release from CLIP bioprinted scaffolds printed from GelMA bioresins comprising various cell concentrations (2 x 106cells / mL, 3 x 106cells / mL, 4 x 106cells / mL, or 5 x 106cells / mL). FIG. 5A is a graph showing the viability of bioprinted tumor necrosis factor (TNF)-related apoptosis inducing ligand (TRAIL) secreting NHF 1 (NHF1TRAIL) cells over time based on fluorescence intensity (in arbitrary units (AU)) from a PrestoBlue assay (n=3). FIG. 5B is a graph showing ELISA-based quantification of TRAIL (nanograms (ng)) secreted from the bioprinted NHFlTRAIL-laden scaffolds over time (n=3).

[0022] FIGS. 6A-6E show aspects of the efficacy of the NHF11RAILcell-laden bioprinted CLIP scaffolds in killing glioblastoma (GBM) cells from different GBM cell lines after in vitro treatment of the cells with TRAIL-conditioned media produced by coculture of the media with the scaffolds for 72 hours. FIG. 6A is a graph showing the BLI signal (p / s) of GBM8Fluccells after treatment with TRAIL-conditioned media from scaffolds prepared from bioresins with different concentrations of NHF1TRAILcells (n=3). Data from cells treated with media co-cultured with an acellular CLIP scaffold (CLIP) and from untreated GBM8FLUCcells (Untreated) is provided as a control. FIG. 6B is a graph showing the BLI signal (p / s) of 1 x 10'' LN229Fluccells after treatment with TRAIL- conditioned media (n=3) from media co-culture with a NHF1TRAILcell laden bioprinted CLIP scaffold (CLIP / TRAIL). FIG. 6C is a graph showing the BLI signal (p / s) of 1x104Attorney Docket No. 4210.0546WOLN229Fluccells after treatment with TRAIL-conditioned media from co-culture with the cell laden scaffold (n=3). FIG. 6D is a graph showing the BLI signal (p / s) of IxlO5U87Fluccells after treatment with TRAIL-conditioned media from co-culture with the cell laden scaffold (n=3). FIG. 6E is a graph showing the BLI signal (p / s) of IxlO4U87Fluccells after treatment with TRAIL-conditioned media from co-culture with the cell laden scaffold (n=3). For FIGS. 6B-6E, “Untreated” refers to untreated LN229Fluc or U87Fluc cells, “CLIP” refers to cells treated with media cultured with an acellular CLIP scaffold, and “TRAIL” refers to cells treated with media cultured with TRAIL.

[0023] FIGS. 7A-7C show aspects related to molecular characterization of the cells exposed to conditions related to CLIP bioprinting. FIGS. 7A-7C are graphs showing the quantification of relative expression levels of messenger RNA (mRNA) for Superoxide Dismutase 1 (SOD1) (FIG. 7A), Heme Oxygenase 1 (HM0X1) (FIG. 7B), and DNA ligase 4 (LIG4) (FIG. 7C) via quantitative reverse transcription polymerase chain reaction (qRT- PCR) of untreated NHF1 cells (Control), NHF1 cells immediately after exposure to the CLIP UV light source under the same conditions involved in an exemplary CLIP bioprinting process (CLIP), or NHF1 cells immediately after exposure to a sublethal intensity of UV light (UV low) for 4 minutes.

[0024] FIG. 8 shows aspects related to persistence of encapsulated cells in bioprinted CLIP scaffolds in vivo. More particularly, FIG. 8 is a graph of the fold change in the BLI total flux of NHF1F1UCcells over time when administered to a murine brain resection cavity in PBS (triangles) or in a bioprinted CLIP scaffold (circles). n=4 for both treatment groups.

[0025] FIGS. 9A and 9B show aspects related to efficacy of the encapsulated cells of the bioprinted CLIP scaffolds in vivo in mice bearing partially resected GFM8 tumors created by implantation of GBM8FLUCcells. Scaffolds were implanted the same day as tumor resection, 11 days following tumor cell implantation. FIG. 9A is a graph showing the fold change in BLI total flux over time for mice treated with PBS (PBS, n=4), acellular CLIP scaffolds (CLIP, n-4), or bioprinted CLIP scaffolds containing NHF1TRAILcells (CLIP / TRAIL, n=5). FIG. 9B is a graph showing the Kaplan-Meier survival curvesAttorney Docket No. 4210.0546WO(probability of survival versus days post tumor resection) for the GBM tumor-bearing mice in the different treatment groups described for FIG. 9A.

[0026] FIG. 10 is a schematic drawing of an exemplary porous scaffold design used in a bioresin printability test according to an aspect of the presently disclosed subject matter.

[0027] FIGS. 11A-11D show graphs of relative cell viability (expressed as a percentage (%)) of NHF1GFP'FLcells in various bioresin component formulations. FIG. HA shows a graph of relative cell viability in formulations comprising different concentrations (w / w%) of polyethylene glycol diacrylate (PEGDA) with a molecular weight of 6,000 (PEGDA6K) or 10,000 (PEGDA10K), and either with or without UV exposure. FIG. 11B shows a graph of relative cell viability in formulations comprising different concentrations (w / w%) of GelMA, either with or without UV exposure. FIG. 11 C shows a graph of relative cell viability in formulations comprising different concentrations (w / w%) of the photoinitiator LAP, with or without UV exposure. FIG. 1 ID shows a graph of relative cell viability in formulations comprising different concentrations (w / w%) of a photoabsorber, with or without UV exposure.

[0028] FIGS. 12A and 12B show aspects of the printability and in vitro cell viability of different bioresins. FIG. 12A is a series of images showing printing resolutions from different bioresins, including G (5 w / w% GelMA); P6K (10 w / w% PEGDA6K); and P10K (10 w / w% PDGDA10K). All bioresins were prepared with 0.25 w / w% LAP in mixed Dulbecco’s phosphate-buffered saline (DPBS) and Dulbecco’s Modified Eagle Medium (DMEM). FIG. 12B shows a graph of the BLI signal (p / s) from NHF1GFP'FLladen scaffolds (small disc shaped, 5 mm in diameter, 1 mm in height) prepared from G, P6K and P10K bioresin formulations.

[0029] FIGS. 13A and 13B show aspects of bioresin printability and CLIP scaffold properties. FIG. 13A is a composite image showing printing resolution from different bioresins, including G-P6K (5 w / w% PEGDA6K, 2.5 w / w% GelMA) and G-P6K-PA (2.5 w / w% GelMA, 5 w / w% PEGDA6K, 0.5 w / w% PA, where PA is the photoabsorber Ecamsule). Both formulations were prepared with 0.25 w / w% LAP in DPBS and DMEMAttorney Docket No. 4210.0546WO1 : 1 mixed solution). FIG. 13B is a graph showing the degradation rate (percentage (%) versus time in hours (hr)) of a CLIP hydrogel scaffold printed with G-P6K-PA.

[0030] FIGS. 14A-14C show aspects related to the impact of material composition on in vitro NHF 1GFP’FLcell viability and in vivo NHF1GFP’FLcell persistence after intraperitoneal implantation of cell-laden, CLIP bioprinted scaffolds. FIG. 14A is a graph showing the BLI signal (p / s) from NHF1GFP'FLbioprinted scaffolds (small disc, 5 mm diameter, 1 mm height) printed from G-P6K and G-P6K-PA bioresins on the day of printing (Day 0) and on Days 3 and 10 after printing. FIG. 14B shows representative fluorescence images of GFP-expressing fibroblasts (NHF 1GFP'FLcells) in the scaffolds described for FIG. 14A. The scale bars in the lower left of each image represents 5 mm. FIG. 14C is a graph showing the relative BLI signal of NHF1GFP'FLcells in bioprinted scaffolds prepared from different bioresins (G-P6K and G-P6K-PA) implanted in the intraperitoneal (I.P.) cavity of mice over time (in days). For comparison, data for free NHF 1GFP'FLcells injected into the I.P. cavity (no scaffold) is also shown.

[0031] FIGS. 15A and 15B show aspects of the characterization of 3D CLIP bioprinted scaffolds using a G-P6K-PA bioresin containing NHF 1GFP'FLcells. The bioresin was printed in batches, each batch containing a 4x7 array of scaffolds (diameter 5 mm; height 1 mm) on the printing platform of a CLIP apparatus. FIG. 15A is a graph comparing total BLI signal (p / s) from NHF1GFP'FLcells in bioprinted scaffolds from three separate batches of scaffolds printed with G-P6K-PA containing a cell concentration of 5 x 106cells / mL, n=26 scaffolds per batch. FIG. 15B is a graph of the total BLI signal (p / s) from NHF 1GFP'FLbioprinted scaffolds printed from the G-P6K-PA bioresin as a function of time (1, 6, 12, 18, 26, and 33 days after printing, n=4~6).

[0032] FIGS. 16A-16D show aspects related to the scalability of CLIP bioprinting. GFP-positive NHF1 cells were bioprinted using a G-P6K-PA bioresin with various cell densities / concentrations (Low Density: 5 x 106cells / mL; Middle Density: 1.25 x 107cells / mL; High Density: 2.5 x 107cells / mL) to provide different bioprinted scaffold sizes (Small: diameter 5 mm; Medium: diameter 7.5 mm; Large: diameter 10 mm). FIG. 16A is graph showing the total BLI signal (p / s) of NHF1 cells bioprinted from a G-P6K-PAAttorney Docket No. 4210.0546WO bioresin at various cell densities and different scaffold sizes. FIG. 16B is a graph showing the absolute longitudinal BLI signal (p / s) of NHF1 cells bioprinted from a G-P6K-PA bioresin at various cell densities as a function of time. Scaffold size is small (diameter 5 mm) for all groups. FIG. 16C is a graph showing relative longitudinal BLI signal of NHF1 cells bioprinted from a G-P6K-PA bioresin at various cell densities as a function of time. Scaffold size is small (diameter 5 mm) for all groups. FIG. 16D is a series of representative fluorescent images of GFP-positive NHF1 cells bioprinted from a G-P6K-PA bioresin at a cell density of 5 x 106cells / mL with various strut width (top), graphs showing corresponding BLI signal (p / s) over time (middle) and graphs showing BLI signal divided by scaffold volume (p / (s x mm3)) (bottom).

[0033] FIGS. 17A-17D show aspects of the impact of scaffold 3D structure on in vitro NHF1GFP’FLcell proliferation. FIG. 17A shows a schematic of the design of scaffolds of different porosities. All scaffolds were printed as squares with an outer diameter of 5 mm x 5 mm x 1 mm (length x width x thickness) but included different numbers and sizes of hollow / pore regions to vary scaffold porosity from 0% to 49% and scaffold volume from 25 mm3to 12.75 mm3. FIG. 17B is a graph showing Day 0 BLI signal from NHF 1GFP'FLporous scaffolds prepared from a G-P6K-PA bioresin. FIG. 17C is a graph showing Day 0 printing efficiency (Day 0 BLI signal / Total solid volume) from NHF 1GFP FLporous scaffolds prepared from a G-P6K-PA bioresin. FIG. 17D is a series of graphs showing total BLI signal (p / s) from NHF 1GFP'FLporous scaffolds prepared from a G-P6K-PA bioresin on the day of printing (Day 0) and on days 2, 5, 8, 11, 16, and 25 after printing. (n=4).

[0034] FIGS. 18A-18E are a series of graphs showing the application of CLIP bioprinting on various types of cells. FIGS. 18A-18E show the BLI signal (p / s) versus time (days) after printing of scaffolds prepared from G-P6K bioresins comprising: (FIG. 18A) 1.25 x 106cells / mL human mesenchymal stem cells (hMSCmCherry'FL); (FIG. 18B) 5 x 106cells / mL human neural stem cells (HB 1.F3GFP'FL); (FIG. 18C) 5 x 106cells / mL human induced spheroidal neural stem cells (hiNeuroSmCherry'FL); (FIG. 18D) 1.25 x 106Attorney Docket No. 4210.0546WO cells / mL human astrocyte cells (hAstrocytemCherry'FL); and (FIG. 18E) 1.5 x 107cells / mL human ovarian cancer cells (ES2mCherry‘FL).

[0035] FIGS. 19A-19H show aspects relating to bioprinting and characterizing therapeutic porous scaffolds prepared with GFP-expressing and TRAIL-secreting human neural stem cells (hiNeuroS-GFP-TRAIL cells) from a G-P6K-PA bioresin formulation. FIG. 19A is a schematic showing the 3D design and dimensions of scaffolds with different porosities (0%, 27%, and 54%). Scaffold outer dimension: Dimensions = 9.5 mm x 9.5 mm x 1 mm, height = 1 mm. Strut thickness = 0.5 mm. FIG. 19B is a graph showing Day 0 (day of printing) viability of cell-laden scaffolds containing hiNeuroS-GFP-TRAIL cells bioprinted with a 1.5 x 107cells / mL density G-P6K-PA resin formulation into scaffolds of different porosities. Cell viability was measured with the PrestoBlue assay right after bioprinting the scaffolds. n=3 for each porosity. FIG. 19C is a graph showing the Day 0 printing efficiency of the scaffolds described for FIG. 19B. Printing efficiency is calculated by dividing the PrestoBlue assay absorbance from FIG. 19B by scaffold design solid volume. n=3 for each porosity. FIG. 19D is a graph showing the viability of cell-laden scaffolds described for FIGS. 19B and 19C measured with the PrestoBlue assay over 3 days. n=3 for each porosity (0%, 27%, or 54% porosity) and time point (Day 0, 1, and 3). FIG. 19E is a graph showing the printing efficiency (PrestoBlu assay absorbance divided by scaffold volume) of the cell-laden scaffolds described for FIGS. 19B and 19C calculated over 3 days. n=3 for each porosity and time point. FIG. 19F is a graph showing in vitro human ovarian cancer cell (ES2-mCherry-Fluc) killing from media collected after 24 hours of conditioning with Day3 hiNeuroS -TRAIL bioprinted scaffolds of different porosities (0%, 27%, or 54%) or with media collected after 24 hours of conditioning with 2 x 106hiNeuroS-TRAIL cells only (no scaffold) as positive control. FIG. 19G is a graph showing the quantification of hiNeuroS-GFP-TRAIL cells released from the bioprinted scaffolds over time. N=3 for each porosity and time point. FIG. 19H is a graph showing TRAIL quantification from 1 mL of media conditioned for 24 hours with the bioprinted scaffolds on each day after bioprinting up to Day 8. N=3 for each porosity and time point.Attorney Docket No. 4210.0546WO

[0036] FIG. 20 shows aspects of the in vivo efficiency of CLIP-bioprinted hiNeuroS- TRAIL cell-laden scaffolds in a mouse metastatic ovarian cancer model. FIG. 21 is a graph showing the fold change in BLI total flux over time for mice treated with (from left to right for each group of 4 bars) PBS ( = 8), non-therapeutic NFH1 cell laden bioprinted CLIP scaffolds with 0% porosity (CLIP, n = 8), bioprinted hiNeuroS-TRAIL scaffolds with 0% porosity (hiNeuroS-TRAIL@CLIP, OP, n = 7), and bioprinted hiNeuroS -TRAIL scaffolds with 27% porosity (hiNeuroS-TRAIL@CLIP, 27P, n = 8).

[0037] The drawings are not necessarily to scale. The drawings are merely representations, not intended to portray specific parameters of the disclosure. The drawings are intended to depict example embodiments of the disclosure, and therefore are not to be considered as limiting in scope.DETAILED DESCRIPTION

[0038] The presently disclosed subject matter will now be described more fully. The presently disclosed subject matter can, however, be embodied in different forms and should not be construed as limited to the embodiments set forth herein below and in the accompanying Examples. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the embodiments to those skilled in the art.

[0039] All references listed herein, including but not limited to all patents, patent applications and publications thereof, and scientific journal articles, are incorporated herein by reference in their entireties to the extent that they supplement, explain, provide a background for, or teach methodology, techniques, and / or compositions employed herein.I. Definitions

[0040] While the following terms are believed to be well understood by one of ordinary skill in the art, the following definitions are set forth to facilitate explanation of the presently disclosed subject matter.

[0041] All technical and scientific terms used herein, unless otherwise defined below, are intended to have the same meaning as commonly understood by one of ordinary skillAttorney Docket No. 4210.0546WO in the art. References to techniques employed herein are intended to refer to the techniques as commonly understood in the art, including variations on those techniques or substitutions of equivalent techniques that would be apparent to one of skill in the art. While the following terms are believed to be well understood by one of ordinary skill in the art, the following definitions are set forth to facilitate explanation of the presently disclosed subject matter.

[0042] In describing the presently disclosed subject matter, it will be understood that a number of techniques and steps are disclosed. Each of these has individual benefit and each can also be used in conjunction with one or more, or in some cases all, of the other disclosed techniques.

[0043] Accordingly, for the sake of clarity, this description will refrain from repeating every possible combination of the individual steps in an unnecessary fashion. Nevertheless, the specification and claims should be read with the understanding that such combinations are entirely within the scope of the invention and the claims.

[0044] Following long-standing patent law convention, the terms "a", "an", and "the" refer to “one or more” when used in this application, including the claims. For example, the term "a cell" refers to one or more cells, e.g., one of more of the same or different cells. Similarly, the phrase “at least one”, when employed herein to refer to an entity, refers to, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 45, 50, 75, 100 or more of that entity.

[0045] “Optional” or “optionally” means that the subsequently described event, circumstance, or material may or may not occur or be present, and that the description includes instances where the event, circumstance, or material occurs or is present and instances where it does not occur or is not present.

[0046] Ranges may be expressed herein as from "about" one particular value, and / or to "about" another particular value. When such a range is expressed, also specifically contemplated and considered disclosed is the range from the one particular value and / or to the other particular value unless the context specifically indicates otherwise. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will beAttorney Docket No. 4210.0546WO understood that the particular value forms another, specifically contemplated embodiment that should be considered disclosed unless the context specifically indicates otherwise.

[0047] For example, term “about,” when referring to a value or to an amount of a composition, dose, sequence identity (e.g., when comparing two or more nucleotide or amino acid sequences), mass, weight, temperature, time, volume, concentration, percentage, etc., is meant to encompass variations of in some embodiments ±20%, in some embodiments ±10%, in some embodiments ±5%, in some embodiments ±1%, in some embodiments ±0.5%, and in some embodiments ±0.1% from the specified amount, as such variations are appropriate to perform the disclosed methods or employ the disclosed compositions. 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 unless the context specifically indicates otherwise. Finally, it should be understood that all of the individual values and sub-ranges of values contained within an explicitly disclosed range are also specifically contemplated and should be considered disclosed unless the context specifically indicates otherwise. For example, 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.90, 4, and 5. The foregoing applies regardless of whether in particular cases some or all of these embodiments are explicitly disclosed.

[0048] The term “comprising”, which is synonymous with “including”, “containing”, or “characterized by”, is inclusive or open-ended and does not exclude additional, unrecited elements and / or method steps. “Comprising” is a term of art that means that the named elements and / or steps are present, but that other elements and / or steps can be added and still fall within the scope of the relevant subject matter.

[0049] The phrase “consisting essentially of’ limits the scope of the related disclosure or claim to the specified materials and / or steps, plus those that do not materially affect the basic and novel character! stic(s) of the disclosed and / or claimed subject matter. For example, a pharmaceutical composition can “consist essentially of’ a pharmaceutically active agent or a plurality of pharmaceutically acitive agents, which means that the recited pharmaceutically active agent(s) is / are the only pharmaceutically active agent(s) present in the pharmaceutical composition. It is noted, however, that carriers, excipients, and / or otherAttorney Docket No. 4210.0546WO inactive agents can and likely would be present in such a pharmaceutical compostion and are encompassed within the nature of the phrase “consisting essentially of.”

[0050] As used herein, the phrase “consisting of’ excludes any element, step, or ingredient not specifically recited. It is noted that, when the phrase “consists of’ appears in a clause of the body of a claim, rather than immediately following the preamble, it limits only the element set forth in that clause, other elements are not excluded from the claim as a whole.

[0051] With respect to the terms “comprising”, “consisting of’, and “consisting essentially of’, where one of these three terms is used herein, the presently disclosed and claimed subject matter can include the use of either of the other two terms. For example, a composition that in some embodiments comprises a given active agent also in some embodiments can consist essentially of that same active agent, and, indeed, can in some embodiments consist of that same active agent.

[0052] As used herein, the term “and / or” when used in the context of a listing of entities, refers to the entities being present singly or in combination. Thus, for example, the phrase “A, B, C, and / or D” includes A, B, C, and D individually, but also includes any and all combinations and subcombinations of A, B, C, and D.

[0053] The term “additive manufacturing” (also known as “rapid prototyping” or “3D printing”) as used herein refers to a process by which a part, e.g., as defined from a computer-aided design (CAD) file, is generated, traditionally, in a layer-by-layer fashion. Compared to conventional plastic molding manufacturing processes like injection molding and extrusion, 3D printing provides a plethora of design freedom and enables relatively rapid fabrication of customized objects with complex geometries. One advantage of 3D printing is the ability to directly translate a concept design into an end product in a convenient, cost-efficient manner. Exemplary additive manufacturing processes include, but are not limited to, stereolithography, multijet modeling, binder jet technique, fused deposition modeling (FDM) or fused filament fabrication (FFF), selective laser melting (SLM), selective laser sintering (SLS), digital light processing (DLP), top-down SLA DLP, intelligent liquid interface (ILI) using wettable membrane technology, powder bed andAttorney Docket No. 4210.0546WO inkjet head 3D printing (3DP), electron-beam melting (EBM), selective heat sintering (SHS), stereolithography (SLA), and continuous liquid interface production (CLIP).

[0054] The terms “anticancer drug” and “anticancer compound” as used herein refer to molecules that have ability to kill cancer cells and / or to reduce cancer cell growth, as well as molecules that can trigger a patient’ s immune system to inhibit cancer cell growth and / or kill cancer cells. As used herein anticancer drugs can be naturally occurring compounds or synthetic small molecules, proteins, or nucleic acids. Exemplary anticancer drugs include chemotherapeutic drugs (drugs that kill fast-growing cells), such as alkylating agents, antimetabolites, drugs that inhibit DNA replication or RNA transcription, miotic inhibitors, topoisomerase inhibitors, antibodies that target cancer cells, tyrosine kinase inhibitors, and immunotherapy agents (e.g., checkpoint inhibitors). For certain cancers (e.g., breast and prostate cancers), hormone therapy agents, such as aromatase inhibitors and androgen receptor inhibitors can be used as anticancer drugs.

[0055] The term “biocompatible” as used herein refers to materials and any metabolites or degradation products thereof that are generally non-toxic to animals and that do not cause significant adverse effects to animals.

[0056] The term “biodegradable” as used herein refers to polymeric materials that can break down over time when exposed to enzymes or other chemicals or conditions (e.g., pH) present at one or more locations (e.g., a tissue, an organ, the gastro-intestinal tract, the blood stream, etc.) in a biological organism (e.g., a human or other mammal).

[0057] The term “cancer” as used herein refers to diseases caused by uncontrolled cell division and / or the ability of cells to metastasize, or to establish new growth in additional sites. The terms “malignant”, “malignancy”, “neoplasm”, “tumor”, “cancer” and variations thereof refer to cancerous cells or groups of cancerous cells.

[0058] Particular types of cancer include, but are not limited to, skin cancers (melanoma), connective tissue cancers (e.g., sarcomas), adipose cancers, breast cancers, head and neck cancers, lung cancers (e.g., mesothelioma), stomach cancers, pancreatic cancers, ovarian cancers, cervical cancers, uterine cancers, anogenital cancers (e.g., testicular cancer), kidney cancer, bladder cancer, colorectal cancers (i.e., colon cancers orAttorney Docket No. 4210.0546WO rectal cancers), prostate cancers, central nervous system (CNS) cancers, retinal cancer, blood cancers (e.g., leukemias, multiple myeloma), neuroblastomas, and lymphoid cancers (e.g., Hodgkin’s and non-Hodgkin’s lymphomas).

[0059] The term “metastatic cancer” refers to a cancer that has spread from its initial (primary) site in a patient’s body.

[0060] The term “disease” refers to a state of health of an animal wherein the animal cannot maintain homeostasis.II. Three-Dimensional (3D) Printing for Drug Delivery Systems

[0061] 3D printing (3DP) is a useful strategy for developing highly optimized, and even personalized, drug delivery systems. Many cell therapy delivery devices have been developed with 3DP for a variety of clinical applications, including, for example, tissue regeneration, wound healing, and cancer, among others where the local delivery of the therapeutic cells in a protective matrix promotes cell viability, persistence, and therapeutic function in vivo. In some of these examples, cells are seeded and cultured on the external surfaces of a printed structure. However, this strategy, known as external seeding, usually involves time-intensive post-processing steps to prepare the printed part for seeding cells. Further, seeding protocols must be developed and optimized for unique systems or cell types and can include lengthy post-seeding culture periods to grow the cells to therapeutic densities prior to in vivo implantation. Moreover, the seeding process can involve manual handling of the material, which can introduce significant variability in seeding among parts, especially if the parts are seeded one at a time.

[0062] In contrast to 3DP, therapeutic cells can be combined with the resin prior to printing to generate parts containing cells embedded or encapsulated within the printed structure. This strategy, known as 3D bioprinting (3DBP), remedies many of the previously mentioned disadvantages associated with external seeding. Using 3DBP techniques, the therapeutic cells and / or other compounds are incorporated into printed parts in a single step, eliminating the resource- and time-intensive post-print seeding stage that must be performed for 2DP techniques. Additionally, encapsulated cells can be entrapped in a wider array of materials, as seeding does not rely entirely upon cell-material interactions. In someAttorney Docket No. 4210.0546WO3DBP systems, particularly those based on photopolymerization, arrays of identical parts can be produced in a single print, increasing overall production efficiency. However, lightbased 3DBP, including methods based on vat polymerization, digital light processing, and stereolithography, incurs several challenges related to cytotoxicity. Cells are exposed directly to unreacted resin components, all of which can cause cytotoxicity at high concentrations, including monomers with reactive functional groups, (i.e., acrylates), photoinitiators, and, in some cases, light absorbing compounds. Systems that utilize light, particularly wavelengths below 405 nm, to catalyze photopolymerization are also known to suffer from UV-induced cellular damage. As a result, it is imperative not only to ensure that the therapeutic cells remain viable post-bioprinting (e.g., after the 3DBP process is complete), but that the therapeutic cells remain functionally and genetically stable after exposure to such various sources of potential cellular damage.

[0063] Continuous liquid interface production (CLIP) is a light-based monolithic 3DP strategy that employs controlled oxygen inhibition of photopolymerization to allow continuous synthesis of a solid 3D part from a liquid photoactive resin. See for example, U.S. Patent No. 9,498,920, incorporated herein by reference in its entirety. See also, Tumble ston et al.. Science. 2015; 347: 1349-1352. After creating a custom 3D structure using computer-aided design (CAD), this 3D structure is created in an inverted manner, with its base layer polymerized onto the upper build platform, which rises as the printing, or part generating, process proceeds. The window through which UV light is displayed to cure the resin at distinct foci is also permeable to oxygen, which inhibits free radical polymerization in a thin layer above the window known as the “dead zone.” This “dead zone” acts as a continuous source of liquid resin from which the 3D structure can be constructed. This promotes more rapid and efficient printing compared to similar 3DP methods, such as stereolithography, in which intermediate processing steps are required due to the layer-by-layer nature of the printing strategy.

[0064] CLIP has been utilized for various biomedical applications, including, for example, to generate 3D hydrogels externally seeded with anticancer cells for the treatment of glioblastoma (GBM). However, there are no known previous uses of CLIP forAttorney Docket No. 4210.0546WO bioprinting structures containing live cells. According to an aspect of the presently disclosed subject matter, several advantages of CLIP are utilized in a bioprinting process. For example, the speed and efficiency of CLIP can be advantageous in that cells in a CLIP resin formulation undergo limited exposure to unreacted resin and UV light, thus minimizing potential cytotoxicity. Moreover, CLIP can generate arrays of multiple parts in a single printing session, thereby permitting rapid production of cell-laden 3D printed structures with high levels of consistency. This reduces the total amount of post-processing time needed to generate structures ready for in vivo use.III. Devices for Delivery of Therapeutic Agents and Related Methods

[0065] Disclosed herein are examples of preparing therapeutic agent delivery devices, particularly therapeutic cell delivery devices, using additive manufacturing processes. In some embodiments, the presently disclosed subject matter provides a novel bioprinting strategy using CLIP that illustrate that CLIP is compatible for 3DP with live cells. The stability of the cells post-printing was evaluated using several functional and molecular characterization assays. In an illustrative example, the utility of CLIP 3DBP was demonstrated for use in generating constructs loaded with cells engineered to constitutively secrete the anticancer protein TRAIL against a model of GBM resection in mice. In other illustrative examples, bioprinted cell-laden scaffolds were prepared from different bioresins and having different 3D architectures, thereby demonstrating the ability to tailor a variety of features, such as the mechanical and swelling properties of the 3D-printed scaffolds. Further, the utility of CLIP 3DBP is demonstrated for generating constructs for intraperitoneal (I.P.) implantation and with a variety of clinically relevant stem cell lines. In vivo performance of the bioprinted scaffolds following I.P implantation showed that the CLIP-bioprinted scaffolds showed significantly prolonged cell persistence compared to directly injected cells. A CLIP-bioprinted scaffold laden with stem cells engineered to secrete TRAIL controlled ovarian tumor cell growth in a mouse model of ovarian cancer.

[0066] Accordingly, in some embodiments, the presently disclosed subject matter provides a method of producing a device for delivery of one or more therapeutic agents. The method can include providing a polymer resin (e.g., a biocompatible polymer resin)Attorney Docket No. 4210.0546WO comprising the one or more therapeutic agents and forming and curing the polymer resin via an additive manufacturing process, thereby simultaneously providing the polymeric scaffold of the device and encapsulating the one or more therapeutic agents in the cured polymer. By “encapsulated” is meant herein that the therapeutic agents are interspersed between polymer chains throughout the thickness of the polymer scaffold as opposed to being merely absorbed to an outer surface of the scaffold. In some embodiments, the device is configured for administration to a subject, such as a human or another mammal. In some embodiments, the device is configured to deliver the one or more therapeutic agents to a target site in the subject. For example, the device is configured to be administered via implantation at a target site of interest in the subject, such as a particular organ or tissue associated with a disease state. In some embodiments, the device can be configured for implantation to a tumor or at a tumor resection site.

[0067] In some embodiments, the additive manufacturing process used in forming and curing the device is a CLIP process. CLIP methodology and related apparatuses are described, for example, in U.S. Patent No. 9,498,920, the disclosure of which is incorporated herein by reference in its entirety. In some embodiments, the provided polymer resin is a resin that comprises compounds (e.g., monomeric compounds, oligomers, or polymers) that can undergo photopolymerization and / or free radical polymerization reactions. The resin can also include an initiator compound (i.e., an “initiator”) capable of generating a free radical, e.g., upon exposure to UV light. Typically, the polymer resin is a liquid that solidifies when components in the resin react with one another via photopolymerization or free radical polymerization reactions to form larger polymer chains and / or polymer chains with a higher degree of cross-linking between individual polymer chains. This solidification process can also be referred to as “curing”. In some embodiments, the curing can involve the application of heat as well as light (e.g. UV light).

[0068] Suitable photopolymerizable and free-radical polymerizable components are known in the art. See, for example, U.S. Patent Nos. 9,498,920 and 8,232,043, the disclosures of each of which are incorporated herein by reference in their entireties.Attorney Docket No. 4210.0546WOSuitable polymerizable resin components include, but are not limited to, acrylics, methacrylics, acrylamides, styrenics, olefins (i.e., alkenes), cyclic alkenes, maleic anhydride, alkynes, and carbon monoxide. In some embodiments, the resin components comprise a functionalized oligomer or polymer, wherein the term “functionalized” refers to the presence of one or more (typically two or more) photopolymerizable and / or free radical polymerizable terminal and / or pendant groups to an individual “parent” polymeric or oligomeric molecule. For example, a functionalized polymer can refer to a polyethylene glycol (PEG) chain functionalized at each end with a vinyl group (i.e., a -CH=CH2 group), an acryloyl group (i.e., a -C(=0)-CH=CH2 groups) or a methacryloyl group (i.e., a -C(=O)- C(CH3)=CH2 group). Functionalized polymers also include polymers, e.g., natural polymer such as polysaccharides or proteins, that have monomeric units with pendant groups or side chains that have been functionalized with photopolymerizable and / or free radical polymerizable groups. For instance, polysaccharides can be functionalized via addition of photopolymerizable and / or free-radical polymerizable groups to hydroxyl or carboxylate groups in one or more monosaccharide unit in the polysaccharide. Proteins can be functionalized via addition of such groups to hydroxyl, thiol, carboxylate, or amino group- containing side chains.

[0069] In some embodiments, the one or more therapeutic agents of the presently disclosed devices comprise a therapeutic bioagent, such as a live cell and / or a microorganism (e.g., a bacteria cell, a yeast cell, or a virus) where the live cell and / or microorganism is capable of acting as a therapeutic agent. Accordingly in some embodiments, the method of producing the device for the delivery of one or more therapeutic agents comprises: providing a bioprinting resin comprising a biocompatible polymer resin and one or more therapeutic agents, wherein the one or more therapeutic agents comprise a live cell and / or a microorganism; and forming and curing the bioprinting resin via an additive manufacturing process, thereby producing the device for delivery of the one or more therapeutic agents, wherein the device comprises the one or more therapeutic agents encapsulated in cured biocompatible polymer. As used herein, the term “bioprinting resin” refers to a biocompatible polymer resin containing a live cell and / orAttorney Docket No. 4210.0546WO microorganism and can also be referred to here as a “bioresin”, a “bioresin formulation” or a “bioprinting ink”. In some embodiments, the additive manufacturing process comprises or consists of a CLIP process.

[0070] In some embodiments, the one or more live cell and / or microorganism is a single celled microorganism, such as a bacteria cell or a yeast cell. The bacteria or yeast cell can be engineered to secrete a therapeutic compound, e.g., an anticancer drug. Microorganisms for use in the presently disclosed bioresins also include viruses. For instance, in some embodiments, the microorganism for inclusion in the bioresin is an oncolytic virus, i.e.., a virus that can preferentially infect and kill cancer cells. Examples of oncolytic viruses include, but are not limited to, the oncolytic adenovirus Hl 01 and the oncolytic herpes viruses known as talimogene laherparepvec (T-Vec) and Herpes simplex virus mutant 1716 (HSV1716).

[0071] In some embodiments, the bioresin comprises a live cell. For example, the live cell can be a mammalian (e.g., human) cell or another animal cell or a bacteria or yeast cell. Suitable mammalian (e.g., human) cells include, but are not limited to stem cells, macrophages, T cells, natural killer (NK) cells, chimeric antigen receptor (CAR)-T cells, and / or cells that provide (e.g., secrete) a therapeutic compound (e.g., an anticancer drug) or other anticancer agent. In some embodiments, the live cell is a cell that is naturally capable of exerting an anticancer effect (e.g., by secreting an anticancer drug, such as a protein that results in cancer cell death). In some embodiments, the cell is a cell that was genetically engineered or otherwise modified to provide (e.g., secrete) an anticancer drug or anticancer agent. For example, the live cell can be a cell engineered to secrete TRAIL. Other examples of anticancer drugs that cells can be engineered to secrete include, but are not limited to, cytokines (e.g., interleukins, such as interleukin- 12 (IL- 12), interleukin-2 (IL-2), or interleukin- 18 (IL-18), and interferons (e.g., IFN-oc, IFN-P or fFN-y)) and prodrug enzymes (e.g., cytosine deaminase, or thymidine kinase). Cells can also be engineered to deliver oncolytic viruses, exosomes (e.g., exosomes that carry anticancer RNAs or other anticancer drugs), and nanoparticles (e.g., nanoparticles that are loaded with and can release anticancer drugs, such as traditional chemotherapeutic drugs like paclitaxel,Attorney Docket No. 4210.0546WO gemcitabine, and doxorubicin). In some embodiments, the cell that is engineered to provide an anticancer drug or agent is a fibroblast or a stem cell. In some embodiments, the cell that is engineered to secrete TRAIL is selected from a fibroblast and a stem cell (e.g., a neural stem cell or a mesenchymal stem cell). In some embodiments, the cell is an astrocyte. In some embodiments, the live cell is a cell known in the field for use in a cellbased immunotherapy, such as, but not limited to a NK cell (e.g., a CAR-NK cell), a T cell (e.g., a CAR-T cell or a T-cell receptor (TCR)-T cell), a tumor-infiltrating lymphocyte (TILs), a macrophage (e.g., a CAR-macrophage (CAR-M cell). In some embodiments, the cell can be engineered to express a detectable agent, such as a dye (e.g., a fluorescent dye). For example, in some embodiments, the cell is engineered to express both an anticancer drug and a detectable agent.

[0072] In some embodiments, the one or more therapeutic agents include a single type of cell and / or microorganism. In some embodiments, the one or more therapeutic agents include two or more different types of cells and / or microorganisms. Thus, for example, the one or more therapeutic agents can include an anticancer drug-secreting cell and a cell for cell-based immunotherapy or can include two different types of anticancer drugsecreting cells.

[0073] In some embodiments, the one or more therapeutic agents further comprise a non-living component (i.e., in addition to the live cell and / or microorganism). For instance, in some embodiments, the one or more therapeutic agent can include a live cell and a nonliving component. More particularly, the non-living component can be an additional therapeutic agent that is not present in and / or secreted by the live cell and / or microorganism. Exemplary non-living components include, for example, polypeptides, antibodies, exosomes, nanoparticles, microparticles, and small molecules (e.g., synthetic molecules with a molecular weight of less than about 800 Daltons or less than about 600 Daltons). In some embodiments, the small molecule is a traditional chemotherapeutic drug (e.g., paclitaxel). In some embodiments, the non-living component is a component that comprises a moiety that targets a cancer cell, such as a ligand that binds to a receptor on a cancer cell.Attorney Docket No. 4210.0546WO

[0074] In some embodiments, the biocompatible polymer resin comprises a functionalized natural polymer (e.g., a functionalized protein or a functionalized polysaccharide), a functionalized biocompatible synthetic polymer, or a combination thereof. Exemplary nature polymers for use in the instantly disclosed resins and scaffolds include, but are not limited to, gelatin, alginate, collagen, chitin, starch, cellulose, and hyaluronic acid. Exemplary biocompatible synthetic polymers include, but are not limited to, polyethylene, polypropylene, polyglutamic acid (PGA), polyaspartic acid (PAA), polylactic acid (PLA), polyvinylpyrrolidine (PVP), polyhydroxyalkanoates, and PEG. In some embodiments, the polymer resin includes at least one polymeric component based on a polymer that is hydrophilic or amphiphilic. The biocompatible polymer resin can include a single type of functionalized oligomer or polymer or two or more different types of functionalized oligomer or polymer. Exemplary functionalized biocompatible polymer resins include, but are not limited to, functionalized gelatin, functionalized alginate, functionalized hyaluronic acid, functionalized PEG, functionalized collagen, and combinations thereof. In some embodiments, the biocompatible polymer resin comprises one or more of gelatin methacryloyl (GelMA), methacrylated alginate, methacrylated hyaluronic acid, methacrylated PEG, acrylated PEG, and methacrylated collagen. In some embodiments, the biocompatible polymer resin comprises or consists of GelMA. In some embodiments, the biocompatible polymer resin comprises a functionalized PEG (e.g., an acrylated PEG). In some embodiments, the biocompatible polymer resin comprises GelMA and an acrylated PEG (e.g., a diacrylated PEG). In some embodiments, the functionalized PEG has a weight average molecular weight between about 1,000 and about 15,000. In some embodiments, the functionalized PEG has a weight average molecular weight of about 5,000 to about 12,000.

[0075] In some embodiments, the polymer prepared by curing the resin (i.e., the cured biocompatible polymer) is swellable (e.g., in water or a biologically relevant fluid). In some embodiments, the cured biocompatible polymer is biodegradable. For example, in some embodiments, the cured biocompatible polymer can include one or more ester orAttorney Docket No. 4210.0546WO amide bonds that can undergo hydrolysis in a biological environment, such as due to the action of an enzyme or pH conditions present in a biological environment.

[0076] In some embodiments, the bioprinting resin comprises a biocompatible polymeric resin comprising an UV radical initiator and one or more live cells. In some embodiments, the UV radical initiator is present in the bioprinting resin at a concentration of less than about 1% w / w or less than about 0.5% w / w. In some embodiments, the UV radical initiator is present at a concentration of about 0.05% w / w to about 0.25 % w / w. In some embodiments, the bioprinting resin further comprises a photoabsorber, e.g., to reduce negative effects of UV exposure to the live cells but without significantly reducing curing of the polymeric resin components. In some embodiments, the photoabsorber is present at a concentration of about 1% w / w or less. In some embodiments, the photoabsorber is present at a concentration of about 0.1% w / w to about 0.5% w / w.

[0077] As described in the examples herein below, in some embodiments, the live cells and / or microorganism encapsulated in the device can proliferate within the device (e.g., in vitro, such as when the device is kept in contact with a suitable cell media) after the formation of the device. In some embodiments, after the device is produced, the cell density of the device can increase by at least about 100% or more, at least about 125% or more, at least about 150% or more, or at least about 175% or more. In some embodiments, the cell density of the device increases by about 175% within about 1 to 2 weeks after the device is formed. In some embodiments, the live cells or microorganisms encapsulated in the device remain viable and / or therapeutically active for a period of time in vitro (e.g., in contact with a cell media) after the device is formed. In some embodiments, the live cells or microorganisms remain viable and / or therapeutically active for at least one week in vitro, at least two weeks in vitro, at least three weeks in vitro, or for at least one month (i.e., at least 30 days) in vitro.

[0078] In some embodiments, the concentration of the one or more therapeutic agents in the device is controlled via control of the concentration of the agent in the bioresin used to prepare the device. Thus, for example, the concentration of one or more of the one or more therapeutic agents in the device can be adjusted during formation of the device toAttorney Docket No. 4210.0546WO provide a desired initial cell density in the device. In addition, the use of additive manufacturing (e.g., CLIP) allows for control of the shape of the device (i.e., the shape of the polymeric scaffold). Accordingly, the instantly disclosed devices can be formed with any desired and / or suitable shape (e.g., any geometry or size). Suitable device shapes include, but are not limited to, spheres, hemispheres, discs, cones, pyramids, ellipsoids, cylinders, cubes, rectangular prisms, torus shapes, five-sided prisms, and three-sided prisms. In some embodiments, the shape is irregular. The device can be porous or non- porous. The term “porous” with regard to device / scaffold shape is meant herein to refer to a device that contains one or more holes or “hollows” in the design of the device, i.e., where the hollows were provided by not printing bioresin in some portions within the outer dimensions of the device. In some embodiments, each hollow can be surrounded on four sides by a “strut” or wall of polymer. Examples of porous scaffold design are shown, for instance, in FIGS. 10, 17A, and 19A. In some embodiments, the desired / selected shape of the device is selected from the group including a disc, a sphere, and a porous shape.

[0079] The shape of the delivery device (i.e., the desired shape) can be selected to improve the structural integrity and / or functionality of the device. For instance, the shape of the device can be selected so that the device is customized to fit a particular target site, such as a resection cavity in a subject being treated for cancer. Thus, the device can be configured to directly delivery the one or more therapeutic agents to a desired site in a subject. In some embodiments, the desired shape is selected to improve the viability and / or migration of a live cell and / or microorganism encapsulated in the device. For example, the device thickness or the porosity and / or strut thickness can be tailored to provide desired cell viability and / or cell migration. In some embodiments, the thickness and / or porosity of the device is controlled to achieve a desired release rate for the one or more therapeutic agents, a desired amount or rate of swelling, and / or a desired degradation rate for the device (i.e., when the device comprises a degradable polymer). In some embodiments, the desired shape (e.g., the thickness and / or porosity of the shape) can be customized to optimize encapsulation of the one or more therapeutic agents. In some embodiments, the desiredAttorney Docket No. 4210.0546WO shape can be customized to reduce foreign body response against the device, e.g., by providing a device shape that has a faster degradation rate.

[0080] In some embodiments, the presently disclosed subject matter provides a method of treating a disease in a subject. In some embodiments, the disease is cancer. In some embodiments, the method comprises providing a subject in need of treatment and administering to the subject a device for delivery of one of more therapeutic agents as described herein, e.g., a device prepared by forming and curing a bioresin using an additive manufacturing process, e.g., CLIP. In some embodiments, the method comprises preparing a bioprinting resin, forming and curing the bioprinting resin using an additive manufacturing process (e.g., CLIP) to provide a device comprising one or more therapeutic live cells and / or microorganisms encapsulated in a polymer scaffold; and administering the device to the subject in need thereof. In some embodiments, the method further comprises contacting the device with a suitable cell media for a period of time (e.g., one or more hours or days) prior to administering the device. Thus, in some embodiments, the method comprises preparing the device and allowing the one or more therapeutic live cells or microorganisms to proliferate for a period of time prior to administration. In some embodiments, the period of time is about 12 hours to about 14 days or about 1 day to about 7 days. In some embodiments, the period of time is about 1 to 3 days.

[0081] In some embodiments, administrating the device to the subject comprises surgically implanting the device at a desired site in the subject, such as in a tissue or organ associated with the disease. In some embodiments, administration comprises surgical implantation of the device in a tumor. In some embodiments, administration of the device comprises surgical implantation at a tumor site during a surgery to remove all or part of the tumor. In some embodiments, administration comprises post-surgical implantation of the device in the subject in a tumor resection cavity after removal of a tumor from the subject.

[0082] The methods of the presently disclosed subject matter are particularly useful in warm-blooded vertebrates. More particularly, the presently disclosed subject matter concerns treatment of disease in mammals and birds. For example, the methods and devices can be used for treatment of humans as well as those mammals of importance toAttorney Docket No. 4210.0546WO humans dues to being endangered, of economic importance (e.g., animals raised on farms for consumption by humans), or social importance (e.g., animals kept as pets or in zoos). Thus, in some embodiments, the subject is selected from the group including a cat, a dog, swine, rodents (e.g., rats, mice, guinea pigs, etc.), ruminants (e.g., cattle, oxen, sheep, giraffes, deer, goats, bison, camels) and horses. In some embodiments, the subject is a subject suffering from or susceptible to a cancer. For example, the subject can be a subject with a genetic marker associated with an increased risk of cancer, a family history of cancer, a history of exposure to a cancer-causing agent, or a subject previously treated for a cancer who is at risk of recurrence. The cancer can be any form of cancer. In some embodiments, the cancer is selected from the group including, but not limited to, glioblastoma, ovarian cancer, colorectal cancer, pancreatic cancer, liver cancer, melanoma, breast cancer, and leukemia.

[0083] In some embodiments, administering the device to the subject treats the cancer, such as by delaying tumor regrowth, reducing the size of a tumor, reducing the risk of tumor recurrence, and / or increasing the lifespan of the subject, i.e., as compared to a comparable subject that did not receive treatment or who received treatment with live cells that were not provided in a device as disclosed herein.

[0084] In some embodiments, the presently disclosed subject matter provides a therapeutic composition (e.g., for treatment of cancer or another disease) wherein the therapeutic composition comprises a device for the delivery of one or more therapeutic agents as described herein. In some embodiments, the therapeutic device comprises a CLIP bioprinted polymeric scaffold comprising one or more live cells and / or microorganisms encapsulated with the polymeric scaffold. The therapeutic composition can be formulated for administration (e.g., implantation) to a subject in need of treatment. In some embodiments, the composition is for administration to a human. In some embodiments, the composition is for administration to a subject suffering from or susceptible to a cancer (e.g., a glioblastoma or an ovarian cancer). In some embodiments, the therapeutic composition comprises a device that is customized to fit into a tumor resection cavity in the subject.Attorney Docket No. 4210.0546WO

[0085] In some embodiments, the presently disclosed subject matter provides a method of producing a 3D polymeric scaffold comprising one or more live cells encapsulated in the scaffold, wherein the method comprises: providing a bioprinting resin comprising one or more live cells and a polymer resin; and forming and curing the bioprinting resin to provide the 3D polymeric scaffold. The live cells can be any live cells. For example, the live cells can be bacteria cells, yeast cells, or mammalian cells (e.g., astrocytes, fibroblasts, stem cells, or cancer cells). In some embodiments, the cells are stem cells or cancer cells. The scaffold can be provided for use, for example, as an in vitro device for studying and / or proliferating the cells. For instance, in some embodiments, the scaffold can be used as a 3D organoid or disease model. In some embodiments, the scaffold can be used for tissue regeneration.EXAMPLES

[0086] The following Examples have been included to provide guidance to one of ordinary skill in the art for practicing representative embodiments of the presently disclosed subject matter. In light of the present disclosure and the general level of skill in the art, those of skill can appreciate that the following Examples are intended to be exemplary only and that numerous changes, modifications, and alterations can be employed without departing from the scope of the presently disclosed subject matter. For instance, the methods, systems, and products disclosed herein were produced using the exemplary materials and / or cell lines; however, the subject matter disclosed herein is not in any way limited to these example materials and / or cell lines.EXAMPLE 1General Methods for Examples 2-3

[0087] Materials and Cell Lines: Gelatin methacryloyl (GelMA, bloom strength = 300, degree of methacrylation = 45-55%) was purchased from Cellink. UV radical initiator lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) was purchased from TCI Chemicals. NHF1 cells were obtained from W. Kauffman (University of North CarolinaAttorney Docket No. 4210.0546WOSchool of Medicine) and were hTERT immortalized. LN229 and U87 cells were obtained from the American Type Culture Collection. NHF1, LN229, and U87 cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM, Gibco) containing 10% fetal bovine serum and 1% penicillin-streptomycin, hereby referred to as standard culture medium. GBM8 cells were obtained from H. Wakimoto (Massachusetts General Hospital) and cultured in filtered Neurobasal Medium (Gibco) containing 1.5% L-glutamine, 2% B27 supplement, 0.5% N2 supplement, 2 pg / mL heparin, 20 ng / mL epidermal growth factor, 20 ng / mL fibroblast growth factor, and 0.5% anti-anti. All cells were cultured at 37 °C and 5% CO2.

[0088] Lentiviral Transduction: Cells were treated with 8 pg / mL polybrene and the lentiviruses for 24 hours at 37 °C and 5% CO2. NHF1 cells were transduced with lentiviruses encoding for green fluorescent protein (GFP) and firefly luciferase (Flue), hereby referred to as NHF 1F1UC, or GFP and TRAIL, hereby referred to as NHF 1GFPor NHFlTRAiL, respectively. LN229, U87, and GBM8 cells were transduced with lentiviruses encoding mCherry (mCh) and Flue, hereby referred to as LN229Fluc, U87Fluc, and GBM8F1UC. All lentiviruses were purchased from the Duke Viral Vector Core.

[0089] CLIP Bioprinting: CLIP bioprinting was performed using the SI CLIP prototype printer (Carbon) utilizing a 385 nm LED UV light source. The cylindrical scaffold designs were created in TinkerCAD and exported as STL files, which were sliced at 1 pm using the Carbon printing software. Unless otherwise noted, all studies were performed with cylindrical scaffolds of diameter = 3.75 mm and height = 1.5 mm.

[0090] The bioprinting resin was prepared by first mixing 10% (wt) GelMA, 0.5% (wt) LAP, and 89.5% (wt) DMEM at 45°C and 250 RPM until fully dissolved. Next, the GelMA solution was mixed thoroughly in a 1 : 1 volume ratio with 8xl06cells / mLNHFl suspension in standard culture medium by pipetting, resulting in final bioresin concentrations of 5% GelMA, 0.25% LAP, and 4xl06cells / mL. Scaffolds were printed at a continuous speed of 48 mm / hr, with light intensity of 1 mW / cm2. Excess resin was removed via gentle washing with deionized water, and scaffolds were immediately placed individually into 12-well plates containing 1 mL fresh standard culture media in each well. Scaffolds were culturedAttorney Docket No. 4210.0546WO at 37°C and 5% CO2, with media changes occurring at 24 hours post-printing, and every subsequent 48 hours.

[0091] Quantification ofNHFl Seeding in Bioprinted Scaffolds: NHF1F1UCseeding in bioprinted scaffolds was calculated using a genomic DNA extraction kit (ThermoFisher, KI 82002). DNA content was extracted per the manufacturer’s instructions from cell suspensions containing 5xl04to 2xl06NHF1F1UCto create a standard curve (n = 3). DNA concentrations were quantified using the Qubit Fluorometric Quantification system (ThermoFisher). To quantify the number of cells encapsulated per scaffold (n = 18 per batch), scaffolds were digested individually with proteinase K at 37 °C for one hour. Next, DNA was extracted and quantified, and NHF1 cell count per scaffold was determined based on the standard curve.

[0092] Bioluminescence and Presto Blue Viability Assays: Bioluminescence was used to compare cell densities in scaffolds printed with different NHF 1F1UCconcentrations or dimensions. I hour after printing, the scaffolds were placed in a black-walled, clear- bottomed 96-well plate and submerged in 15 mg / mL D-luciferin dissolved in IX PBS (Revvity, 122799). The samples were incubated in the luciferin solution for 5 minutes prior to bioluminescence imaging using the in vivo imaging system (IVIS) Spectrum. To measure NHF 1F1UCviability in the scaffolds over time, scaffolds cultured for various time points between 0-14 days were incubated in luciferin and imaged in the same manner.

[0093] Presto Blue reagent (Invitrogen, A13261) was diluted to IX in IX PBS. 100 pL of the diluted reagent was added to a black-walled, clear-bottomed 96-well plate, and NHF lTRAIL-laden scaffolds cultured for various time points between 0-14 days were added to the wells (n = 3). Wells containing only diluted reagent were used as background controls. The scaffolds were incubated in the reagent for 45 minutes at 37 °C and 5% CO2. Next, the scaffolds were removed from the well plate and the fluorescence of the reagent was measured using SpectraMax M-series plate reader at an excitation of 560 nm and emission of 590 nm. Results were plotted as absolute fluorescence intensity at each time point with background fluorescence values subtracted.Attorney Docket No. 4210.0546WO

[0094] In vitro TRAIL Release: Bioprinted scaffolds were prepared as described above with NHF1TRAILcells and cultured in 12-well plates in 1 mb standard culture media for 1 week at 37°C and 5% CO2. After periods of 1 day, 3 days, and 7 days, 500 pL of media was collected and tested using a human TRAIL enzyme-linked immunosorbent assay (ELISA) (Invitrogen, BMS2004) according to the manufacturer’ s protocol to determine the concentration of TRAIL secreted into the media. Absorbance was measured using a SpectraMax M-series plate reader and compared to a standard curve.

[0095] In vitro Co-Culture Killing Assays: All co-culture assays were conducted using four treatment groups: untreated control, acellular GelMA scaffold, 4xl05plated NHF ITRAILanj 4xio5NHF 1TRAILin bioprinted scaffolds, prepared as described elsewhere herein. Each treatment was added to a 6-well plate containing 2 mL of standard culture media and incubated at 37 °C and 5% CO2 for 72 hours. After 72 hours, 500 pL of conditioned media from each treatment group was added to a 24-well plate containing tumor cells, which had been plated at a density of IxlO5cells / well (GBM8Fluc) or IxlO5, 5xl04, and IxlO4cells / well (LN229Flucand U87Fluc) the day prior to treatment. The tumor cells were treated for 24 hours before tumor viability was measured via the IVIS Spectrum by incubating the cells with 150 pg / mL luciferin in standard culture media with a 2-minute incubation period prior to image acquisition.

[0096] Quantification of Reactive Oxygen Species (ROS): To quantify ROS generated during bioprinting, a cellular ROS sensor was used (Abeam, ab 186027) according to the manufacturer’s instructions. NHF1TRAILcells were incubated with IX PBS containing 0.2% (vol / vol) dye for 30 minutes at 25 °C on an orbital shaker in the dark. Next, cells for evaluation were prepared for CLIP bioprinting, UV curing (positive control), or incubation on ice (negative control). Cells were suspended in standard culture media at a concentration of 8xl06cells / mL and placed on ice until processing. CLIP bioprinting was performed as described above. After printing and washing, the scaffolds were placed individually in a black-walled, clear-bottomed 96-well plate containing 100 pL DMEM per well. For UV- cured cells, the cell suspension was diluted 1 : 1 with 2X concentrated printing resin, then added to a black-walled, clear-bottomed 96-well plate in 100 pL aliquots. The samplesAttorney Docket No. 4210.0546WO were cured in the plate in an LED UV oven (365 nm, 0.6 mW / cm2) at 100% intensity for 4 minutes. Immediately after curing, 100 pL DMEM was added to each well. Untreated cells added in 100 pL aliquots to a black-walled, clear-bottomed 96-well plate containing 200 pL DMEM per well. After treatment, the samples were immediately imaged using a SpectraMax M-series plate reader at an excitation of 520 nm and emission of 605 nm to measure the fluorescence intensity of the ROS dye. A second reading was performed to measure the GFP signal in each well at an excitation of 488 nm and emission of 507 nm. The ROS dye measurement for each well was normalized to the GFP signal in each well to control for the number of cells contained in each sample.

[0097] Comet Assay: DNA damage from bioprinting was assessed for the examples prepared as described herein using a comet assay kit (Abeam, ab238544) according to the manufacturer’s instructions. NHF 1FIUCcells were suspended in standard culture media at a concentration of 4xl06cells / mL and placed on ice until processing. Negative control cells were maintained on ice. Cells treated with the CLIP light source were loaded into the printing reservoir and exposed to UV under the same conditions of CLIP bioprinting as described above. Positive control cells were loaded into petri dishes and placed in an LED UV oven (365 nm, 90 mW / cm2) at 100% (UV high) or 5% (UV low) intensity for 4 minutes. After treatment, cells were collected and resuspended in cold IX PBS at a concentration of IxlO6cells / mL. Cells were mixed at a 1 : 10 volume ratio with agarose and pipetted onto a previously prepared comet assay slide containing a thin base layer of gelled agarose. The slides were kept at 4 °C for 20 minutes to allow the cell-containing layer to form a gel. Next, the slides were immersed in pre-chilled lysis buffer for 1 hour at 4 °C, followed by immersion in pre-chilled alkaline solution for 30 minutes at 4 °C. Slides then underwent electrophoresis in alkaline solution for 15 minutes at 1 volt / cm. Slides were washed twice with deionized water, immersed in cold 70% ethanol for 5 minutes, then air dried. Finally, slides were stained with vista green DNA dye and imaged on a fluorescent microscope. Comets were analyzed using the ImageJ OpenComet plugin, and tail moment was calculated for at least 125 cells per treatment group.Attorney Docket No. 4210.0546WO

[0098] Quantitative reverse transcription polymerase chain reaction (qRT-PCR): qRT-PCR: was performed on three treatment groups: untreated control, UV cured samples, and CLIP 3DBP samples. Control cells were plated in cell-culture flasks and cultured at 37 °C and 5% CO2. UV-cured samples were prepared as described above with NHFlFluccells using a LED UV oven (365 nm, 90 mW / cm2) at 5% intensity for 4 minutes. Cured samples were then cultured in 12-well plates in 1 mL standard culture media at 37 °C and 5% CO2. CLIP 3DBP scaffolds were prepared as described above with NHF 1F1UCcells and cultured in 12-well plates in 1 mL standard culture media at 37 °C and 5% CO2. At days 1 and 7, control cells were harvested from tissue culture plates using trypsin, washed with IX PBS, and pelleted. For the UV-cured and CLIP groups, 8 scaffolds were pooled per sample per time point. Scaffolds were incubated with trypsin in a 15 mL conical tube on a rotating mixer then centrifuged to release encapsulated cells. Released cells were then washed with IX PBS and pelleted. Total RNA was extracted from cells using an RNA extraction kit per the manufacturer’s protocol (Invitrogen, 12183020), and converted to cDNA (Biorad, 1708890). qRT-PCR was performed using the Applied Biosystems QuantStudio 3 Real- Time PCR system with pre-designed KiCqStart SYBR Green primers (KSPQ12012) for glyceraldehyde-3 -phosphate dehydrogenase (GAPDH), superoxide dismutase 1 (SOD1), heme oxidase 1 (HM0X1), and DNA ligase 4 (LIG4) with PowerUp SYBR Green master mix (Applied Biosystems, A25742). The thermal protocol was 1 cycle at 50 °C for 2 minutes, 1 cycle at 95 °C for 2 minutes, and 40 cycles at 95 °C for 15 seconds, followed by 60 °C for 1 minute. Relative mRNA expression was quantified using the AACT method using untreated control cells as the biologic control and GAPDH as the endogenous control for each time point.

[0099] In vivo NHBlFlucPersistence in Bioprinted Scaffolds: Female, athymic nude mice (Animal Studies Core, University of North Carolina at Chapel Hill) aged 6-8 weeks were used for all efficacy evaluations. The animals were first anesthetized using 3% inhaled isoflurane, then placed into a stereotactic frame. The surgical site was prepared using antiseptics betadine and 70% isopropyl alcohol. The skull was exposed with a small incision, and a craniotomy was performed using a microdrill to remove a small portion ofAttorney Docket No. 4210.0546WO the parietal skull plate, 3 mm in diameter, between the bregma and lambda points in the right hemisphere of the brain. Bleeding was controlled using cold saline, and the wound was closed using Vetbond (3M, 1469SB) after bleeding had subsided. Post-operative pain management was performed using subcutaneous injection of 5 mg / kg mel oxicam, 24- and 48-hours post-surgery. 5 days prior to the implantation surgery, bioprinted scaffolds (d = 2.55 mm, h = 1.2 mm) were prepared as described above and cultured for 5 days. 1 week after the craniotomy, implantation of cells or scaffolds was performed. The mice were prepared for surgery as described above, and the surgical site was reopened. The dura mater was removed using a 25 G needle, and a vacuum pump was used to make a mock resection cavity approximately 3 mm in diameter and 2 mm in height. Bleeding was controlled with cold saline and an absorbable gelatin sponge sold under the tradename GELFOAM® (Pfizer) when needed. After bleeding had subsided, IxlO5NHF 1FIUCcells suspended in 3 pL IX PBS or encapsulated in bioprinted scaffolds (n = 4) were implanted into the cavity. The wound was closed with Vetbond and pain was managed via 5 mg / kg subcutaneous mel oxicam 24- and 48-hours post-surgery. Serial BLI imaging was performed for one month using the IVIS spectrum with 150 mg / kg intraperitoneal injection of D-luciferin in IX PBS to evaluate in vivo NHF 1F1UCpersistence in bioprinted scaffolds.

[0100] In vivo efficacy of NHF1TRAILin bioprinted scaffolds against a GBM8 resection model: Craniotomies were performed as described above. 1 week after the craniotomy, mice were prepared for tumor cell injections. The wound was reopened and 5xl04GBM8F1UCcells suspended in 3 pL IX PBS containing 10% Matrigel were injected into the brain parenchyma using a stereotactic auto-injector. The injections were performed at a rate of 1 pL / min at stereotactic coordinates 2.5, -0.5, -0.5 from the bregma, and avoiding the lateral ventricles. The syringe was slowly removed 2 minutes after the injection was complete to avoid reflux of the cell suspension. The wound was then closed with Vetbond and post-operative pain was managed via 5 mg / kg subcutaneous mel oxicam injection, 24- and 48-hours after surgery. 5 days prior to the implantation surgery, bioprinted scaffolds (d = 2.55 mm, h = 1.2 mm) were prepared as described elsewhere herein and cultured for 5 days. 11 days after tumor implants, the mice were prepared for tumor resection andAttorney Docket No. 4210.0546WO implantation of therapeutic cells (in this example, stem cells). Prior to surgery, mice were randomized into groups exhibiting statistically similar mean total flux values based on tumor BLI imaging performed immediately preceding resection. The wound was reopened, and tumors were resected using a vacuum pump and fluorescence imaging as guidance. Next, after bleeding had subsided, 1.75xl05NHF1TRAILcells encapsulated in bioprinted scaffolds were implanted into the resection cavity (n = 5). Acellular CLIP scaffolds and PBS vehicle injection served as negative controls (n = 4). The wounds were closed using Vetbond, and post-operative pain was managed via 5 mg / kg subcutaneous meloxicam injection, 24- and 48-hours after surgery. BLI imaging with the IVIS Spectrum and 150 mg / kg D-luciferin in IX PBS via intraperitoneal injection was used to track tumor volume. Mice were euthanized when more than 20% of their original body weight was lost or if other pain-related symptoms, such as dehydration, hunched position, tremors, and low body temperature, were observed.

[0101] Statistical Analysis: In the examples presented and discussed herein, all results are presented as a mean value ± standard error of the mean. Batch-to-batch consistency data, in vitro bioluminescence viability data, in vitro co-culture killing data, ROS assay data, comet assay data, and qRT-PCR data were analyzed via one-way ANOVA with Tukey’s Multiple Comparisons test. In vivo persistence data was analyzed via Student’s t test. Kaplan-Meier survival curves were analyzed using the Log-Rank (Mantel-Cox) test. For all analyses, “ns” indicates not significant, * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, and **** indicates p < 0.0001. Statistical analyses were conducted using Prism GraphPad (version 9).EXAMPLE 2Fabrication of Bioprinted GelMA Scaffolds

[0102] FIG. 1 schematically shows how 3DBP GelMA scaffolds were fabricated using a CLIP bioprinting strategy. FIGs. 2A and 2B are graphical plots showing results associated with these 3DBP GelMA scaffolds. The data shown in FIGS. 2A and 2B was obtained by evaluating the same 3 batches of 3DBP scaffolds, with each batch having n=18 scaffoldsAttorney Docket No. 4210.0546WO per batch. FIG. 3 shows representative fluorescence images of GFP-positive NHFls over time in such 3DBP GelMA scaffolds / constructs.

[0103] GelMA is a widely used biopolymer in light-based bioprinting systems, known for its biodegradability, cell adhesion properties, and non-immunogenicity. A cell-laden bioresin containing 5% (w / w) GelMA in DMEM with 0.25% (w / w) LAP as a photoinitiator was created by mixing a solution of 10% GelMA and 0.5% LAP with an equal volume of cell suspension containing 8xl06NHF 1F1UCcells / mL in DMEM. In this example, rapid and even distribution of the cells in the solution was observed. Immediately following preparation of the cell-laden bioresin, 3 mL of this cell-laden bioresin was dispensed into the CLIP printing reservoir and disc-shaped scaffolds (d = 3.75 mm, h = 1.5 mm) were generated, as shown in FIG. 1.

[0104] The quantity of cells encapsulated within individual scaffolds printed in accordance with the example shown in FIG. 1 in a single batch was also evaluated. For this purpose, the DNA content of individual scaffolds was compared to a standard curve of DNA concentrations for defined cell counts ofNHFlFluccells. Cell counts were determined for 3 independently printed batches of scaffolds (N = 54, n = 18 per batch). An average of 3.87 ± 0.73xl05NHF 1F1UCcells was found to be encapsulated in each 3DBP scaffold. For each individual batch of 18 3DBP scaffolds, a sum of 6.96 ± 0.73xl06cells is encapsulated in the 3DBP scaffolds, out of the total 12xl06cells that were in the 3DBP bioresin loaded into the printer, demonstrating consistency of bioprinting efficiency among independent, batches. Further demonstrating the consistency of the bioprinting process, the data from was separated by batch and compared for statistical differences, the results of each of the three batches are shown in FIG. 2A. The cell encapsulation per scaffold for batches A (3.94 ± 0.57xl05cells), B (3.70 ± 0.92x10’ cells), and C (3.97 ± 0.67xl05cells) was found to be statistically insignificant from each other, thereby indicating that CLIP 3DBP is a strategy that enables the production of consistently seeded 3DBP scaffolds both within 3DBP scaffolds that are produced in the same batch and also between 3DBP scaffolds produced in different batches.Attorney Docket No. 4210.0546WO

[0105] Next, as shown in FIG. 2B, the viability of encapsulated cells within such 3DBP scaffolds over time was evaluated. Cell viability was quantified with a bioluminescence assay, the results of which are shown in FIG. 2B. FIG. 2B shows that encapsulated cells proliferate within the 3DBP scaffold over time, reaching 174% of the initial live cell density after 14 days in vitro. Fluorescence imaging of GFP-positive NHF 1F1UCcells, examples of which are shown in FIG. 3, confirmed the bioluminescence data shown in FIG. 2B, with increasing cell densities being observed over time (i.e., as a function of time) in culture. The strong GFP signal around the circumference of the 3DBP scaffolds, as shown in FIG. 3, indicates that the cells exhibit a preference to grow on the outer edges of the 3DBP scaffold. Additionally, concentrated loci of bright GFP signal can be observed on days 7 and 14, indicating that cells may be forming clusters as the cell density in the 3DBP scaffold increases over time.

[0106] The ability to scale CLIP bioprinting processes to produce such 3DBP scaffolds was evaluated. Since it has been shown herein that therapeutic cells can be consistently seeded in bioprinted CLIP scaffolds and that the cells survive for at least two weeks postprinting, it is also advantageous to be able to change and / or control the concentration of therapeutic cells within a 3DBP scaffold and / or for dimensions of the 3DBP scaffold to be changed. To verify this, a bioluminescence assay was used to quantify the relative density of live cells within each such 3DBP scaffold. As shown in FIG. 4A, for 3DBP scaffolds of the same size (e.g., 3.75 mm diameter, 1.5 mm height) produced from bioresins with different concentrations of NHF 1F1UC(i.e., ranging from 2-5xl06cells / mL), the bioluminescence signal correlates linearly (R=0.9584) with the bioresin therapeutic cell concentration. See FIG. 4A. Thus, initial seeding of therapeutic cells for 3DBP scaffolds of the same size and printing conditions can be easily scaled based on the quantity of cells suspended in the bioresin.

[0107] Next, exemplary disc-shaped 3DBP scaffolds were created with different dimensions from each other. The exemplary “small” scaffold has a diameter of about 2.55 mm and a height of about 1.20 mm; the “medium” scaffold has a diameter of about 4.50 mm and a height of about 1.80 mm; and the “large” scaffold has a diameter of about 5.25Attorney Docket No. 4210.0546WO mm and a height of about 2.40 mm. The differently sized 3DBP scaffolds were all printed using the same exemplary concentration or density of therapeutic cells, namely, 4xl06NHFlFluc / mL in this example. As shown in FIG. 4B, the bioluminescence signal (“Total Flux”) increases linearly (R=0.9970) as a function of the volume of the 3DBP scaffold, thereby indicating that seeding can be easily adjusted by modifying the scaffold dimensions.

[0108] These initial evaluations were conducted with NHF 1F1UCcells. Further studies were performed using NHF1 cells engineered to secrete an anticancer compound, i.e., TNF-related apoptosis inducing ligand (TRAIL). As shown in FIGS. 5A and 5B, the viability of TRAIL-secreting NHF1 cells within bioprinted constructs (e.g., the 3DBP scaffolds disclosed elsewhere herein) was evaluated and, additionally cell growth patterns were compared to cumulative TRAIL release.

[0109] A Presto Blue viability assay (see FIG. 5A) was used to determine the initial live cell density proliferation rates of CLIP scaffolds printed with different bioresin NHF ITRAIL concentrations. As shown in the results of FIG. 5A, 3DBP scaffolds that were printed with a concentration of 5x106NHF1TRAILcells / mL grew at the fastest rate until day 3. In contrast, the scaffolds printed with a density of 2xl06NHF1TRAILcells / mL exhibited the slowest growth rate until day 3, at which point the growth rate increased through at least day 14. As shown in FIG. 5A, all of the 3DBP scaffolds reached a similar cell density at day 14, regardless of the initial concentration of NHF1TRAILcells in the bioresin; while 3DBP scaffolds printed with a concentration of about 5xl06NHF1TRAILcells / mL showed cell growth to about 2.8 times its initial concentration, 3DBP scaffolds printed with a concentration of about 2xl06NHF1TRAILcells / mL exhibited growth to 5 times its initial concentration.

[0110] The results of the Presto Blue assay were confirmed with fluorescence imaging. At 2X magnification, the overall concentration or density of GFP-positiveNHFlTRAILcells within the scaffold was visualized. A bioresin concentration-dependent trend of scaffold cell density was observed on days 0 and 3, whereas at days 7 and 14, the cell concentrations for different initial bioresin concentrations appear to be substantially similar (e.g., withinAttorney Docket No. 4210.0546WO10% of each other). High GFP signal was observed around the circumference of the scaffold by day 7, indicating that the cells prefer to adhere to and proliferate on the exterior edges of the scaffold. Images at 10X magnification, demonstrated cell morphology over time in the scaffold. Observations of cell morphology changes were the same among scaffolds printed with different bioresin concentrations. At day 0, the cells maintain a spherical morphology immediately post-printing. By day 3, cells begin to take on a flattened, fibroblast-like morphology. After day 7, the cells form flat monolayers within the material, appearing similar to the monolayers that form when the cells are grown in 2D tissue culture plastics, demonstrating cell-material interactions with the GelMA scaffold. Spheroid-like clusters of cells can also be observed at this time, suggesting the influence of cell-cell interactions among encapsulated cells.

[0111] An ELISA assay was also used to quantify the amount of TRAIL secreted into the media for cultured NHF1TRAILbioprinted scaffolds over time, the results of which are shown in FIG. 5B. As shown therein, the trend in cumulative TRAIL release is not dependent on the initial NHF1TRAILbioresin concentration used in forming the 3DBP scaffold. Without being bound to any one theory, one potential explanation for this result is that TRAIL is entrapped within the matrix as it is secreted by encapsulated cells, likely by electrostatic interactions between the positively charged TRAIL protein and negatively charged GelMA backbone. It is likely that the TRAIL accumulation in media is largely owed to the cells growing closer to or on the edges of the scaffold, where the effect of protein-biomaterial interactions is minimized. As cells are seen congregating quickly around the outer surfaces of the scaffold regardless of initial printing concentration, the differences in TRAIL secretion are statistically insignificant. While the average amount of TRAIL released in 7 days is highest for scaffolds printed with the highest initial bioresin cell concentration (8.18 ng total TRAIL released) and lowest for scaffolds printed with the lowest initial bioresin cell concentration (4.82 ng total TRAIL released), the difference between these groups is not believed to be statistically significant. This result could be modified via multiple strategies. First, the resin composition could be altered to reduce electrostatic interactions with the TRAIL protein, so long as any new resin formulationAttorney Docket No. 4210.0546WO does not negatively impact cell viability or function. Second, the structure of the device could be manipulated to exhibit a higher exposed surface area, which would facilitate increased total release of TRAIL protein into the media. However, for degradable materials such as GelMA, a higher exposed surface area will also theoretically increase the degradation rate of the material, which could result in reduced cellular persistence in vivo.

[0112] Functional co-culture tumor killing assays demonstrated that TRAIL concentrations in media conditioned by NHF1TRAILcells embedded in GelMA constructs via CLIP 3DBP were still therapeutically active. Killing of GBM8 cells was significant compared to untreated controls for NHF1TRAILcells printed in GelMA at all initial printing concentrations, though a concentration-dependent trend in killing was observed. However, the differences in killing among the NHFlTRAIL-containing constructs were statistically insignificant, which correlates to the results of the TRAIL release assay.EXAMPLE 3Treatment of GBM with NHFlTRAIL-laden 3DBP CLIP Scaffolds

[0113] The effect of NHF 1TRAIL-laden 3DBP CLIP scaffolds on GBM cells was studied using scaffolds prepared according to the methods described in Examples 1 and 2. Since, as shown herein, NHF1TRAILcells survive and proliferate after bioprinting and, furthermore, since secreted TRAIL from bioprinted scaffolds is detectable in culture media, the therapeutic effect of NHFlTRAIL-laden 3DBP scaffolds on GBMFluccells was evaluated in vitro. First, an assay was performed against GBM8rluctumor cells, a cell line with known sensitivity to TRAIL. The results are shown in FIG. 6A. In this study, it was shown that culturing of GBM8 cells with TRAIL-conditioned media (e.g., media conditioned by exposure to a NHFlTRAIL-laden 3DBP scaffold for 72 hours) resulted in increased tumor killing, the efficacy of which increased as a function of NHF1TRAILconcentration in the bioresin used to produce the 3DBP scaffold. Meanwhile, media conditioned with acellular CLIP scaffolds was generally nontoxic to GBM8Fluccells, demonstrating that tumor killing is driven by TRAIL release.Attorney Docket No. 4210.0546WO

[0114] Studies were also performed to investigate how less TRAIL-sensitive tumor lines, LN229 and U87, would respond to 3DBP NHF1TRAILscaffolds, as well as how this treatment compares to media conditioned by plated NHF1TRAILcells. Accordingly, LN229Flucand U87Fluecells were treated with TRAIL-conditioned media from NHF1TRAILscaffolds printed using a bioresin with concentration of 4xl06NHF1TRAILcells / mL. The effects of the NHF1TRAILscaffolds were compared to the effects of NHF1TRAILcells plated at a matched density to the number of cells encapsulated in 3DBP scaffolds, acellular scaffolds, and media only. It was determined that using conditioned media to treat IxlO5tumor cells resulted in significant killing of LN229Flucby the scaffold-encapsulated NHF 1TRAIL cells, though a more robust effect was observed with plated NHF1TRAILcells, as shown in FIG. 6B. In contrast, as shown in FIG. 6D, the effect of TRAIL on U87Fluccells at the same concentration was found to be antagonistic. The same assay was also performed against lower tumor cell counts to increase the ratio of TRAIL-secreting cells to tumor cells; at a concentration of IxlO4tumor cells, the difference in tumor killing by scaffold-encapsulated or plated NHF1TRAILcells was insignificant for both LN229Fluccells (see FIG. 6C) or U87Fluccells (see FIG. 6E), although robust killing of these tumor cells by these treatment groups was observed compared to the media-only and acellular CLIP controls.

[0115] Although the viability of therapeutic cells after CLIP 3DBP is, as shown elsewhere herein, high, a further study was performed to address the cellular response to bioprinting on the molecular level, using several characterization assays. The ROS generated after bioprinting was quantified using an ROS sensor dye. Dye was incubated with cells prior to treatment, after which the dye reacts with ROS inside the cells and emits a fluorescence signal. It was found that, compared to untreated therapeutic cells, therapeutic cells in CLIP bioprinted constructs exhibited a mild but statistically insignificant increase in ROS immediately after bioprinting. Therapeutic cells cured in the same bioprinting resin using a UV oven with a light wavelength of 365 nm at high intensity was used as a positive control. In these samples, the increase in ROS was statistically significant compared to both the positive control and CLIP 3DBP samples.Attorney Docket No. 4210.0546WO

[0116] A comet assay was also performed to assess the DNA damage induced in the therapeutic cells immediately after exposure to various light sources. In this assay, the therapeutic cells were untreated (control) or exposed to the CLIP light source under the same conditions as the previously described bioprinting protocol (CLIP), a sublethal intensity of 365 nm UV light (UV low) for 4 minutes, or a lethal intensity of 365 nm UV light (UV high) for 4 minutes. The tail moment, defined as the product of the comet tail length and the comet tail intensity, was plotted for at least 125 comets in each treatment group. The tail moments of the control, CLIP, and UV (low) cells were statistically insignificant to one another. In comparison, the tail moment for the UV (high) cells was increased to 3 times the value of the control cells, a statistically significant result compared to all other treatment groups.

[0117] A qRT-PCR assay was used to assess gene expression of several genes implicated in the cellular response to oxidative stress. SOD1 and HM0X1 are antioxidants, whereas LIG4 is a DNA ligase involved in DNA repair. Cells were harvested from tissue culture plates, from scaffolds generated with CLIP 3DBP as described elsewhere herein, or from gels generated by UV curing after 1 or 7 days of in vitro culture, and mRNA expression of the three genes was assessed. For SOD1 (see FIG. 7A), gene expression for the UV or CLIP treatment groups did not exhibit statistically significant changes compared to the control on day 1 or day 7. However, SOD1 expression in CLIP cells was decreased to 0.27-times the control group expression on day 1. While expression increased to 1.76- times the expression of control cells on day 7, there was more variability in the day 7 samples. In contrast, UV cells exhibited 3- and 3.6-times higher expression of SOD1 than control cells at day 1 and day 7, respectively. For HM0X1 (see FIG. 7B), expression was statistically increased for both the UV and CLIP groups (29.4- and 33-times higher, respectively) compared to controls on day 1. On day 7, changes to mRNA expression were statistically insignificant among all treatment groups, though the average expression for the UV and CLIP groups remained higher than controls (4.9- and 4.5-times higher, respectively). LIG4 mRNA expression (see FIG. 7C) was significantly increased for the CLIP group and insignificantly increased for the UV group (82.9- and 58.5-times higher,Attorney Docket No. 4210.0546WO respectively) compared to controls on day 1. Expression was statistically increased in both the UV and CLIP groups (26.6- and 46.2-times higher, respectively) compared to controls on day 7.

[0118] The effects of the bioprinted NHF 1TRAILcells were further characterized in vivo. The ability of the GelMA scaffold to act as a protective barrier for encapsulated NHF 1F1UCcells was studied using a model of mock GBM resection in mice. Scaffolds with dimensions suitable to fit inside a typical murine brain resection cavity (2.55 mm diameter, 1.2 mm height), printed with a bioresin concentration of 4X106NHF 1F1UCcells / mL, and precultured for 5 days were implanted into non-tumor bearing mice with resections taken from healthy brain tissue. Mice dosed with IxlO5NHF 1F1UCcells delivered in saline served as controls. Based on the BLI signal of implanted NHF 1F1UCcells, it was observed that cells delivered in saline were cleared from the brain rapidly, with the fold change in total flux approaching zero within 11 days. See FIG. 8. By contrast, the BLI signal of cells encapsulated in bioprinted scaffolds exhibited a slower rate of decline until day 14, after which the BLI signal began to increase until the study concluded at day 31.

[0119] To confirm that NHF1TRAILcells encapsulated in CLIP -bioprinted GelMA scaffolds remain therapeutically active in vivo, the 3DBP scaffolds disclosed herein were evaluated in mice bearing GBM8 tumors using a model of glioblastoma resection. GBM8 cells were grown in athymic nude mice for 11 days prior to implantation of NHF11RAIL- laden 3DBP CLIP scaffolds, acellular CLIP scaffolds, or PBS. Tumor killing was measured via the fold change in bioluminescence signal over time. See FIG. 9A. While mice in the control groups exhibited immediate and rapid tumor growth post-resection, mice treated with the 3DBP CLIP / NHF 1TRAILscaffolds disclosed herein exhibited a two-week delay in tumor recurrence. This delay resulted in a statistically significant improvement in the survival of the mice receiving the 3DBP CLIP, NHFlTRAIL-laden scaffolds (median = 54 days) compared to the PBS (median = 38 days) and CLIP (median = 44.5 days) treatment groups. See FIG. 9B.

[0120] Enhancing the delivery of cell therapies is important for easing their translation to clinical application. The presently disclosed strategies for 3DBP scaffolds are widelyAttorney Docket No. 4210.0546WO applicable to various cell types and disease states, having the ability to rapidly and reproducibly generate cell-laden structures that can be easily implanted into the patient. Bioprinting is a technique that enables the scalable and consistent production of cell therapy delivery devices, as the encapsulation of cells within the polymeric matrix provides protection from external stimuli experienced by the cells in vivo and during manual handling of the device during printing, post-processing, and surgical implantation. CLIP, which can generate an array of identical devices at once in a highly efficient manner, is thus thought to be particularly useful for this purpose. Thus, as demonstrated elsewhere herein, a novel 3D bioprinting strategy using CLIP has been developed and validated, showing its potential for the reliable manufacturing of therapeutic cell-laden devices.

[0121] Using GelMA, a material with excellent biocompatibility characteristics for 3DBP, a resin formulation was identified, and a printing protocol was established to encapsulate NHF1 fibroblasts in small discs. It was determined that, within and across batches, seeding of therapeutic cells among the individual discs is consistent - a notable improvement upon individually processed, manually seeded scaffolds. See FIG. 2A. It was also demonstrated with a bioluminescence assay that encapsulated cells are viable and can proliferate within the GelMA constructs post-printing. See FIGS. 2B and 3.

[0122] A significant advantage of 3DBP for encapsulation of the therapeutic cells is the ability to readily change the printing protocol or 3D structure to manipulate the quantity and / or concentration of the therapeutic cells and, thus, the effective therapeutic dose, within each cell-laden device.

[0123] The subject matter disclosed herein shows the immense potential for CLIP to be utilized as a biocompatible system for 3DBP. These results demonstrate that cells encapsulated in GelMA constructs via CLIP 3DBP are viable and therapeutically active after CLIP 3DBP. CLIP 3DBP offers the ability to generate structures with high-resolution features. The subject matter disclosed herein can be used to bioprint with a variety cell types. It is thought to be advantageous to use a 3D biocompatible printer with a light engine in the visible light range and / or using a heated reservoir to maintain the cells at physiological temperature, both of which are thought to further improve theAttorney Docket No. 4210.0546WO biocompatibility of the system. Although the delivery of an anticancer cell therapy to treat glioblastoma was used here as an example, the subject matter disclosed herein is also believed suitable for use in tissue engineering, synthesis of organoids, development of 3D disease models, and the delivery of other anticancer cell therapies, among others.EXAMPLE 4Methods and Materials for Examples 5-7

[0124] Methods, Reagents and Cell Lines: Gelatin Methacrylate (GelMA, Type A, 300 Bloom, Porcine Gelatin, degree of methacrylation = 45-65%, #VL3500000502), Polyethylene Glycol Diacrylate (PEGDA) with molecular weights of 6,000 (PEDGA6K, #5339-lGM) and 10,000 (PEDGA10K, 5340-1GM) were all purchased from Advanced Biomatrix. UV radical initiator lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) was purchased from TCI Chemicals (#L0290). Photoabsorber Ecamsule disodium (Cas No.: 90458-75-6) was purchased from GlpBio (#GC61828). Dulbecco's phosphate- buffered saline (DPBS) was purchased from Gibco (#14190-144). D-Luciferin potassium salt was purchased from Revvity (#122799). Type I collagenase from Clostridium histolyticum with >125 CDU / mg solid was purchased from Sigma-Aldrich (#C0130- 100MG). For animal study, absorbable suture Med Vet International SUTURE VICRYL 4-0 CLEAR (J422H) was purchased from Fisher Scientific (#50-118-0841).

[0125] NHF1 cells were obtained from W. Kauffman (University of North Carolina School of Medicine) and were hTERT immortalized. HB1.F3.CD cells were obtained from Dr. Karen Aboody (City of Hope National Medical Center) and generated as previously described9. Both cell lines were cultured using Dulbecco’s Modified Eagle Medium (DMEM. Gibco, #11995-065), containing 10% fetal bovine serum (Coming, #35-O15-CV) and 1% penicillin-streptomycin (Pen Strep, 10,000 units penicillin and 10 mg streptomycin / mL. Gibco, #15140-122,), hereby referred to as standard culture medium.

[0126] Adipose-derived human mesenchymal stem cells (hMSC) were purchased from ATCC (PCS-500-011). hMSC cells were cultured in Dulbecco’s Modified Eagle Medium with low glucose and GlutaMAX™ supplement (Gibco, #10567-014), containing 10%Attorney Docket No. 4210.0546WOFBS and 1% Pen Strep. All cell lines were incubated in 5% CO2 at 37°C and passaged periodically using 0.05% Trypsin-EDTA (Gibco, #25300-054) and centrifugation at l,000*g for 5 min.

[0127] hiNeuroS generation: hiNeuroS cells were generated as previously described (see Jiang et al.. Science Advances. 2021; 7(24): eabfl526). hiNeuroS cells are cultured in ReNcell media (Sigma- Aldrich, #SCM005) and supplemented with doxycycline (Takara, #631311), epidermal growth factor (EGF. Fisher Scientific, #50-990-731), and fibroblast growth factor (FGF. Fisher Scientific, #50-104-7691) every other day. hiNeuroS cell line was incubated in 5% CO2 at 37°C and passaged periodically using centrifugation. Before use, hiNeuroS cells were dissociated into a single-cell suspension with Accutase (Sigma Aldrich, #A6964-100ML).

[0128] Lentiviral transduction: Cells were treated with 8 ug / mL polybrene and the lentiviruses for 24 hours at 37°C and 5% CO2. NHF1 and HB1.F3 cells were transduced with lentiviruses encoding for green fluorescent protein (GFP) and firefly luciferase (Flue), hereby referred to as NHF1GFP’FLand HB1.F3GFP'FL. hMSC and hiNeuroS cells were transduced with lentiviruses encoding mCherry (mCh) and Flue, hereby referred to as hMSCmCherry'FL, hiNeuroSmCherry'FL. All lentiviruses were purchased from the Duke Viral Vector Core.

[0129] Resin toxicity screening: 20,000 NHF1GFP'FLcells were seeded in a blackwalled, clear-bottomed 96-well plate one day before. Materials were prepared into different concentrations with standard culture medium for NHF1 and added to NHF1 cells. To explore influence of UV explore, cells undergo UV exposure with 100% intensity, 365nm UV light for 60s. The cells were then incubated in 5% CO2 at 37°C for Ihr. Then cells were added lOuL luciferin solution (15 mg / mL D-luciferin dissolved in 1XDPBS) for 5 minutes prior to bioluminescence imaging using the in vivo imaging system (IVIS) Spectrum. Viability for each treatment group was calculated as the BLI signal for each treatment group divided by the average of non-treated cells.

[0130] CLIP 3D bioprinting: CLIP bioprinting was performed using the SI CLIP prototype printer (Carbon) utilizing a 385 nm LED UV light source. The cylindricalAttorney Docket No. 4210.0546WO scaffold designs were created in TinkerCAD (tinkercad.com) and exported as STL files which were sliced at 1 pm using the Carbon printing software. For G-P6K-PA formulation, a 2X concentration resin was prepared first by mixing 5 w / w% GelMA, 10 w / w% PEGDA6K, 0.5 w / w% LAP, 1 w / w% Ecamsule, and 83.5 w / w% DPBS at 55°C and 500 RPM until fully dissolved. Next, the GelMA solution was mixed thoroughly in a 1 : 1 volume ratio with 0.75-1 mL 1 x 107- 5 x 107cells / mL NHF1 cell suspension in standard culture medium by pipetting, resulting in final bioresin concentrations of 2.5% GelMA, 5% PEGDA6K, 0.25% LAP, 0.5% Ecamsule, and 5 x 106- 2.5 x 107cells / mL in 1 5~2mL total bioresin mixture. The bioresin mixture were then added to printer cassette. Carefully remove all bubbles within the bioresin. Scaffolds were printed at a continuous speed of 10 mm / hr and light intensity of 12 mW / cm2. Excess resin was removed via gentle washing with deionized water, and scaffolds were immediately placed individually into 48-well plates containing 1.5 mL fresh standard culture medium per well. Scaffolds were cultured at 37°C and 5% CO2 with media changes occurring at 24 hours post-printing, and every subsequent 48 hours. All the other resin formulations and cells were prepared using the same procedure.

[0131] Printability: An array of hollow scaffolds with different hollow sizes (ranging from 1mm to 3mm) and strut widths (ranging from 0.05mm to 1mm) were designed on TinkerCad. See FIG. 10. Detailed dimensions are listed in Table 1, below. Different formulations were prepared as described herein to print the scaffold array. Printing parameters were optimized for each resin to ensure the best printing results for each resin. Pictures were taken for the printed results of each resin.

[0132] TABLE 1. Hollow Scaffold Diameters.Attorney Docket No. 4210.0546WODegradation Study: Scaffolds were cured in molds with dimension of 12.5 mm x 10 mm x 5 mm (L x W x H) using 100% intensity 365 nm UV light. Scaffolds were then taken out and put into INCU-Line digital incubators (VWR) set at 37°C to dry. Initial scaffold dry weight was measured (WO,D). 5ml of 1.5 unit / mL collagenase in DPBS solution was added to scaffolds. Scaffolds were degraded at 37°C under agitation at 100 rpm on an incubating orbital shaker (VWR, #35001). Collagenase solution was changed every 3 days. Scaffolds were washed and collected at certain time points, and then dried in an oven to measure the left-over dry weight (WT,D) after degradation. Degradation rates over time were calculated using the formula:Degradation rate = (WO,D-WT,D) / WO,D

[0133] Swelling test: Scaffolds were cured in molds with dimension of 12.5mm x 10mm x 5mm (L x W x H) using 100% intensity 365nm UV light. Initial scaffold wet weight was measured (Wo,w). Each scaffold was incubated in 5mL DPBS and were swelled at 37°C under agitation at lOOrpm on an incubating orbital shaker. DPBS solution was changed every 3 days. Wet scaffold weight was measured at different time points (WT,W).Attorney Docket No. 4210.0546WOSwell Ratio = (WT,W-WO,W) / WO,W

[0134] Modulus test: Hydrogel think sheets (4cm x 3.5cm x 1mm, L x W x H) were CLIP 3D printed first using G-P6K-PA. Dogbone-shaped hydrogel scaffolds were then cut from the think sheets using a mold with bridge dimensions 12 mm x 2 mm. Samples were loaded into an RSA-G2 dynamic mechanical analysis (DMA) system (TA Instruments) and stretched uniaxially at a constant strain rate of 0.005 s1until breaking point.

[0135] In vitro Bioluminescence viability assay: Bioluminescence was used to compare cell densities in all scaffolds printed with firefly luciferase expressing cells (NHFGFP-FL, hMSCmcherry'FL, HB 1.F3GFP-FL, hiNeuroSmCherry’FL). Shortly after printing, the scaffolds were placed in a black- walled, clear-bottomed 24- well plate in ImL media. IOOUL of 15mg / mL D-luciferin solution were then added to each well. The samples were incubated in the luciferin solution for 20 minutes prior to bioluminescence imaging using the IVIS Spectrum. To measure NHF 1F1UCviability in the scaffolds over time, scaffolds cultured for various time points between 0-28 days were incubated in luciferin and imaged in the same manner.

[0136] Free NHF°FP~FLcell injection into mouse intraperitoneal cavity and persistence imaging: Female, athymic nude mice (Animal Studies Core, University of North Carolina at Chapel Hill) aged 6-8 weeks were used for all studies. The animals were first anesthetized using 3% inhaled isoflurane. NHFGFP'FLcells were collected and resuspended in a final concentration of 2 x 106cells in 300uL DPBS. In total 2 x 106cells in 300uL DPBS were then injected into the i.p. cavity of the mice. Serial BLI imaging was performed for two months using the IVIS spectrum with 150 mg / kg intraperitoneal injection of 15mg / mL D-luciferin in IX DPBS and 25min as the waiting time before imaging.

[0137] In vivo NHFFP~FLbioprinted scaffolds implantation into mouse intraperitoneal cavity: Female, athymic nude mice (Animal Studies Core, University of North Carolina at Chapel Hill) aged 6-8 weeks were used for all studies. The animals were first anesthetized using 3% inhaled isoflurane, then placed into a stereotactic frame. The surgical site was prepared using antiseptics betadine and 70% isopropyl alcohol. A midline incision was made to expose the intraperitoneal cavity.Attorney Docket No. 4210.0546WO

[0138] 3 days prior to the implantation surgery, bioprinted scaffolds (Diameter = 5 mm,Height = 1 mm) were prepared as described above and cultured for 3 days. One bioprinted scaffold was then put into the opened mouse intraperitoneal cavity before the incision was sutured. Post-operative pain management was performed using subcutaneous injection of 5 mg / kg meloxicam directly, 24- and 48-hours post-surgery.

[0139] In vivo NHFGFP~FLpersistence in bioprinted scaffolds: Serial BLI imaging was performed for two months using the IVIS spectrum with 150 mg / kg intraperitoneal injection of 15mg / mL D-luciferin in IX DPBS and 25min as the waiting time before imaging.

[0140] Statistical analysis: All results are presented as mean ± standard error of the mean. Modulus data, batch-to-batch consistency data, in vitro bioluminescence viability data were analyzed via one-way ANOVA with Tukey’s Multiple Comparisons test. In vivo persistence data was analyzed via Student’s t test. For all analyses, ns indicates not significant, * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, and **** indicates p < 0.0001. Statistical analyses were conducted using Prism GraphPad (version 10).EXAMPLE 5Additional Bioresins and Bioresin Blends

[0141] To further explore using CLIP for 3D bioprinting of living cells, further studies were conducted to explore alternative resin formulations that provide high viability of encapsulated cells. The cytotoxicity of various factors was explored during bioprinting, including exposure to monomers, photoinitiator, photoabsorber, and UV exposure. Several biocompatible materials were selected, including GelMA and different molecular weights of PEGDA (6K and 10K). GelMA was selected due to its characteristics of being biocompatible, biodegradable, and for providing an extracellular matrix environment including cell adhesion motifs for proper cell expansion. High molecular weight PEGDA was selected to improve material physical characteristics and printability. LAP was tested as a common water soluble photoinitiator (see Bagheri and Jin, ACS Applied PolymerAttorney Docket No. 4210.0546WOMaterials. 2019; 1(4):593-611). Ecamsule, a water soluble, FDA-approved UV-filter used in sunscreen (see Abid et al.. Journal of Photochemistry and Photobiology A: Chemistry 2017; 332:241-50), was used to absorb excessive scattering UV light during printing to prevent overcuring, was also tested for the first time in a 3D bioprinting system. Material solutions were prepared with concentration gradients and incubate with NHF1GFP'FLcells for an hour to identify resin components and concentrations that result in maintaining 80% cell viability during the mixing and printing process.

[0142] In addition to directly co-culturing cells with the resin components, the cell / material solutions were exposed to 100% intensity 365nm UV light for 60 seconds to investigate the impacts on cell viability from both materials and exposure to UV light. UV light and the formation of free radicals generated by photoinitiators can induce DNA damage and cell apoptosis during printing process (see Sinha and Hader, Photochemical and Biological Sciences. 2002; l(4):225-36; and Xu et al.. Military Medical Research. 2022; 9(l):70). However, it is noted that the UV exposure during CLIP 3D printing system is different from this direct UV exposure. The CLIP 3D printer uses a 385nm light source, with a significantly lower light intensity than the direct UV light source. Further, during 3D printing, the cells within the resin are exposed to UV light for about 5 minutes in the CLIP system, depending on the object dimension to be printed, which is longer than the direct UV exposure. However, while the two UV exposure environments are different, by using the direct UV exposure for initial resin screening, the UV exposure process that cells could undergo in any light-based 3D printing system is mimicked and it is possible to assess how it influences cell viability before the 3D bioprinting process is further developed. The following were explored following UV exposure: (1) the influence of UV light exposure on cell viability, (2) whether free radicals developed from activated photoinitiator LAP induce further toxicity on cells besides the material itself, and (3) whether the photoabsorber Ecamsule could offer protection to the cells against UV light. NHF1GFP'FLviability was measured via a bioluminescence viability assay. A threshold of above 80% cell viability upon resin component exposure is preferred to ensure most cells remain viable before undergoing bioprinting.Attorney Docket No. 4210.0546WO

[0143] In testing PEGDA6K (see FIG. 11 A), cells were about 90% viable in 0.5-5 w / w% solutions compared to non-treated cells, but 100% viable in 10 w / w% PEGDA6K solution, suggesting that PEGDA6K induced low level toxicity, but viability remained well within the 80% threshold defined for testing. When GelMA was tested, cell viability is no less than 100% at all concentrations tested, indicating GelMA induced minimal toxicity up to 10 w / w%. See FIG. 1 IB. When the impact of the combination of these materials was explored with UV exposure, it was found that cell viability decreased 10% for all three reagents at all tested concentrations (see FIGS. 11 A and 1 IB), indicating that UV exposure led to a small level of cell death but did not have a combined cytotoxic effect with the materials. Based on these results, it appears that it is preferable to minimize UV exposure during printing. For developing the bioprinting resin, PEGDA6K, PEGDA10K, and GelMA showed minimal to no detectable cytotoxicity at working concentrations up to 10 w / w%.

[0144] When the photoinitiator LAP was tested, cell viability decreased from nearly 100% to 0% as LAP concentration increased from 0 to 0.5 w / w% (see FIG. 11C), demonstrating dose-dependent cytotoxicity. Following UV exposure, viability declined by less than 10% at 0.01 w / w% compared to non-UV controls; however, higher LAP concentrations produced progressively greater reductions, with viability dropping by over 10% at 0.05 w / w% and exceeding 20% at 0.1-0.2 w / w%. This suggests that, in addition to UV light-induced cell death, the activation of LAP by UV light to produce free radicals could also contribute to cell death. As LAP concentration increases, more free radicals are generated upon constant UV exposure, leading to increased cell death. Therefore, besides the singular cytotoxicity from each component, UV light and LAP can also have a combined cytotoxic effect on cells. However, it is possible that a low concentration of LAP would not be sufficient to generate enough free radicals to initiate polymerization, resulting in the need for a high UV dose, either through stronger UV intensity and / or longer curing time, both of which will also induce more cell damage. Based on the 80% viability threshold, a LAP concentration of 0.25 w / w% was selected for further development of the resin formulation.Attorney Docket No. 4210.0546WO

[0145] For the photoabsorber Ecamsule (i.e., terephthalylidine dicamphor sulfonic acid), cell viability decreased from 100% to 59% as the photoabsorber concentration increased from 0 w / w% to 0.5 w / w%, indicating dose-dependent cytotoxicity at higher concentrations. See FIG. 11D. Upon UV exposure, cell viability dropped by about 10% when no photoabsorber was added to the cell culture media, which is consistent with all other non-treatment groups in previous studies. Interestingly, there were no significant differences in cell viability between UV exposure and non-UV-exposure cells at all tested photoabsorber concentrations, even at a very low photoabsorber concentration of 0.01 w / w%. This suggests that even at low concentrations, Ecamsule can absorb UV light, limiting the cell exposure and preventing cell death. The photoabsorber can improve printability at an appropriate concentration; however, too much photoabsorber in the resin can make it difficult to cure, necessitating an increased UV dose to print properly. While some toxicity associated with higher concentrations of Ecamsule was observed, the 1-hour incubation used in these studies does not reflect actual bioprinting conditions. During bioprinting, cells are only exposed to the resin for a few minutes during mixing and bioprinting. Therefore, reduced cytotoxicity can be expected during bioprinting. Based on these factors, an initial concentration of 0.5 w / w% photoabsorber in the resin was selected to maximize the photoabsorbent effect.

[0146] Based on the cytotoxicity screening results, resin formulations containing different monomers, all formulated with 0.25 w / w% LAP, including G with 5 w / w% GelMA; P6K with 10 w / w% PEGDA6K; and Pl OK with 10 w / w% PEGDA10K were selected for study. Printability (e.g., printing resolution) of these formulations was explored. Using computer-aided design (CAD), an array of hollow scaffold featuring void sizes ranging from 1mm to 3mm and strut width ranging from 0.05mm to 1mm. See FIG. 10 and Table 1 above. This design was then printed on the SI printer using the different resin formulations. Pictures of the printing resolution results are displayed in FIG. 12A. For formulation G, most printed scaffolds exhibited poor edge definition and significant overcuring at the scaffold base. Designs with the finest strut widths (0.05 mm and 0.1 mm) failed to print entirely, indicating limited resolution. For formulation P6K, modestAttorney Docket No. 4210.0546WO improvements in edge sharpness were observed, but overcuring at the scaffold base remained significant. While strut features down to 0.1 mm were occasionally printed, they were poorly resolved, and the 0.05 mm features were still unprintable. In contrast, formulation P10K demonstrated markedly improved print fidelity. Scaffolds printed with P10K exhibited sharper edges and reduced base overcuring. Notably, strut widths down to 0.1 mm were printed with high resolution. However, even with P10K, the 0.05 mm strut features could not be fully resolved, suggesting that this value approaches the resolution limit for the CLIP process using these resin systems.

[0147] After evaluating printability, the influence of resin formulation on in vitro cell viability post-bioprinting was studied. Human fibroblast NHF1GFP‘FLcells were bioprinted into each formulation and cell viability was tracked via BLI for up to 10 days. See FIG. 12B. On Day 0, G scaffolds showed 27-fold higher viability than P6K and 270-fold higher than P10K scaffolds. In G scaffolds, viability increased about 1.5-fold by Day 3, then plateaued at Day 10. This suggested an initial period of viability and proliferation that peaked over the course of 10 days. In contrast, P6K and P10K scaffolds exhibited markedly lower viability throughout the time course, suggesting low initial cell viability and limited longitudinal cell proliferation. Representative GFP fluorescence images at Day 0 and Day 3 confirmed these trends.

[0148] Scaffold swelling behavior also varied by formulation. Although all scaffolds were printed with a 5 mm initial diameter, post-wash and swelling in culture medium led to distinct changes. On Day 0, G scaffolds maintained their original diameter (5.00 mm), while P6K and P10K scaffolds swelled to 6.38 mm and 7.24 mm, respectively. By Day 3, further swelling was observed: G scaffolds expanded to 5.26 mm, P6K to 6.46 mm, and P10K to 7.41 mm. The higher swelling ratio of P10K compared to P6K is consistent with the theoretical expectation that higher molecular weight PEGDA (I OK) yields scaffolds with larger mesh sizes and greater water uptake capacity.

[0149] Without being bound to any one theory, it is possible that the differences observed in initial BLI signals and longitudinal growth may be due to differences in resin viscosity, different resin toxicity, and the presence of RGD cell binding motifs in GelMA.Attorney Docket No. 4210.0546WOIn more viscous resins, such as G, cells remained well-suspended throughout the printing process, enabling more uniform cell distribution and loading. In contrast, in less viscous resins (P6K and P10K), cells tended to sediment over time, particularly during the printing process. Resin toxicity differences might be minimal as 5 w / w% GelMA and 10 w / w% PEGDA6K and PEGDA10K both exhibited nontoxicity to cells in the toxicity screening test. See FIG. 11 A. The limited cell proliferation over time in PEGDA scaffolds was also likely due to the absence of cell-adhesive motifs such as RGD found in gelatin, which supports cell attachment and growth. Cells in the G scaffold expanded initially in the matrix, but this growth gradually slowed, and this proliferation differed from the growth in P6K or P10K matrices. In G scaffolds, the high initial cell density may have led to densitydependent inhibition by Day 3, limiting further growth by Day 10. Conversely, the low initial cell densities in P6K and P10K scaffolds may have failed to reach a critical threshold needed for efficient cell-cell interactions, resulting in negligible proliferation throughout the time course.

[0150] Overall, PEGDA-based materials alone (P6K, P10K) appeared to be suboptimal for cell-laden bioprinting, as they provided limited support for initial cell loading, compared to a GelMA-based formulation. However, while GelMA offers superior bioactivity, its lower print fidelity relative to PEGDA restricts its ability to fabricate scaffolds with high structural precision. Further optimization of the resin composition can be performed to achieve a balance between bioactivity and printing resolution.

[0151] For example, to achieve a balance between bioactivity and printing resolution, a blended resin formulation containing both GelMA and PEGDA 6K, referred to as G- P6K, was developed. To further improve printability, the photoabsorber Ecamsule was incorporated into the formulation, resulting in G-P6K-PA. PEGDA 6K was selected over PGEDA 10K due to solubility constraints when forming co-monomer solutions with GelMA.

[0152] The printability of G-P6K and G-P6K-PA was assessed using the resolution test. See FIG. 13A. G-P6K exhibited sharp edge fidelity and minimal base overcuring. Most scaffold designs printed well, although struts with widths of 0.2 mm and 0.1 mmAttorney Docket No. 4210.0546WO were partially formed, and the 0.05 mm design could not be printed. In contrast, G-P6K- PA demonstrated the highest printing resolution among all tested formulations. Scaffold edges were clean and well-defined with no observable overcuring. The 0.2 mm strut design was printed with excellent resolution, though the 0.1 mm design remained partially printed, and the 0.05 mm design still could not be resolved.

[0153] Printing performance can be influenced by the resin’s monomer composition (i.e., polymer content and acrylate group density) and the inclusion of photoabsorbers. Higher concentrations of acrylate groups result in faster and more complete polymerization, requiring less UV exposure but increasing the risk of overcure, making these formulations more suitable for printing large-scale features. Conversely, resins with fewer acrylate groups polymerize more slowly, requiring longer exposure times, which can help preserve fine features by minimizing overcure. However, if the UV dose is too low, polymerization may not occur at all. The addition of photoabsorbers like Ecamsule reduces UV light penetration depth and scattering, thus improving z-axis resolution. Still, excessive photoabsorber content can limit UV penetration and hinder full curing. Overall, the effective printing resolution of CLIP SI for soft hydrogel systems was found to be approximately 100-200 pm, and printability was significantly enhanced by optimizing monomer ratio and incorporating photoabsorber.

[0154] Hydrogel degradation useful for implants for intraperitoneal (I P.) implantation, as it provides a mechanism for therapeutic cells to migrate from the scaffold and interact with the tumor microenvironment. Controlled degradation also allows for nutrient and oxygen diffusion and sustains therapeutic activity. Furthermore, degradable materials reduce the risk of chronic inflammation and foreign body response (FBR). Persistent or non-degradable implants can provoke FBR, resulting in fibrotic encapsulation and impaired mass transport and cell release. To assess degradation (see Fig. 13B), G-P6K-PA scaffolds were incubated in collagenase. Dry weight was measured at designated timepoints to calculate mass loss. G-P6K-PA scaffolds exhibited -75% mass loss over 14 days. This exceeded the expected degradation based on GelMA content (33.3% of total dry weight), suggesting that the breakdown of GelMA crosslinks released small PEGDA6KAttorney Docket No. 4210.0546WO crosslink blocks from the network. The material contains three crosslinking types: GelMA- GelMA, PEGDA-PEGDA, and hybrid GelMA-PEGDA-GelMA crosslinks. While PEGDA is not enzymatically degradable by collagenase, degradation of the GelMA backbone disrupts hybrid crosslinks and frees PEGDA segments, which are subsequently washed out. This demonstrates how blending degradable and non-degradable polymers provides tunable degradation profiles.

[0155] Swelling is another parameter influencing the scaffold's mechanical stability, porosity, and nutrient transport, factors that directly impact cell viability and therapeutic efficacy. Excessive swelling can compromise implant localization or irritate the peritoneal environment. G-P6K-PA scaffolds showed a swelling ratio of approximately 1.64 times over 20 days. Notably, the majority of swelling occurred within the first few days, stabilizing by Day 3 with 1.45X swelling ratio. This suggests that pre-conditioning the scaffolds before implantation (e.g., implanting after Day 3) would minimize further swelling in vivo, maintaining structural integrity in the I.P. cavity.

[0156] For intraperitoneal implantation, it is desirable that biomaterials exhibit mechanical properties comparable to native peritoneal tissues, which typically have a modulus ranging from 1-15 kPa. See Arda et al., American Journal of Roentgenology. 2011; 197(3):532-6; and Liu et al.. International Journal of Molecular Sciences. 2015; 16(7): 15997-6016. Implanted materials with a higher modulus than tissues can induce irritation and provoke FBR, which can limit therapeutic cargo release and thus therapeutic effect. On the other hand, implants with modulus below 1 kPa often lack mechanical integrity, making them unsuitable for handling and implantation. For I.P. cavity implantation, materials that match organ and tissue modulus have favorable mechanical stability and better integrate with soft tissues, ultimately enhancing the therapeutic efficacy and biocompatibility of the implant. To assess modulus, G-P6K-PA scaffolds were molded into dog-bone shapes and tested for Young’s modulus. The blend exhibited a modulus of -10-15 kPa, falling within the optimal range for I.P. implantation and ensuring both mechanical compatibility and implant stability.Attorney Docket No. 4210.0546WO

[0157] Based on its high printing resolution, controlled degradation, stable swelling behavior, and physiologically relevant modulus, the G-P6K-PA formulation represents an excellent bioresin for 3D bioprinting of scaffolds intended for intraperitoneal therapeutic cell delivery.EXAMPLE 6Bioactivity of Bioprinted Scaffolds for Intraperitoneal Implantation

[0158] Following the development of the G-P6K-PA formulation with favorable printability and mechanical properties, its bioactivity was evaluated by comparing in vitro and in vivo cell behavior using bioprinted scaffolds made from G-P6K and G-P6K-PA. NHF 1GFP'FLcells were bioprinted within both formulations to assess cell viability, proliferation, and persistence.

[0159] An in vitro cell viability test using a BLI assay over 10 days was performed. See FIG. 14A. For G-P6K scaffolds, BLI signals increased 3.53-fold by Day 3 and 4.35- fold by Day 10, indicating robust cell proliferation. G-P6K-PA scaffolds showed a similar trend, with a 3.8-fold increase by Day 3 and a 4.24-fold increase by Day 10, despite slightly lower initial Day 0 BLI signal intensity compared to G-P6K. Although both had lower Day 0 BLI signals than G-only scaffolds, their signal intensities increased over time to comparable levels, suggesting that while the GelMA-PEGDA blend could slightly reduce initial cell loading, it does not impair longitudinal cell viability or proliferation.

[0160] Fluorescence imaging further confirmed these results. See FIG. 14B. Cells in G-P6K scaffolds were distributed evenly and proliferated homogeneously. In contrast, G- P6K-PA scaffolds showed preferential accumulation of viable cells near the scaffold periphery, a trend that persisted over time. Nonetheless, both formulations supported high initial cell loading and sustained in vitro proliferation. Importantly, inclusion of the photoabsorber Ecamsule in G-P6K-PA did not adversely affect cell viability over time.

[0161] Encouraged by the in vitro results, evaluation was performed to determine whether bioprinted scaffolds could enhance in vivo cell persistence in the intraperitonealAttorney Docket No. 4210.0546WO(I.P.) cavity, compared to direct injection. It was hypothesized that the scaffolds could serve as a protective niche for therapeutic cells, shielding them from rapid clearance.

[0162] Accordingly, a surgical protocol was developed to implant bioprinted scaffolds into the mouse I P. cavity. Immunodeficient nude mice were used to minimize immune- mediated clearance of human NHF1GFP‘FLcells. Scaffolds (5 mm diameter, 1 mm thickness) were printed using G-P6K or G-P6K-PA formulations and pre-cultured for 3 days to reach swelling equilibrium, eliminate unreacted components, and allow cell recovery prior to implantation. On the day of surgery, one scaffold was surgically implanted per mouse. For comparison, control groups received a direct injection of 2 x 106NHFIGFP-FLcepssuspended in 300 pL of DPBS. Longitudinal BLI monitoring using the IVIS Spectrum system tracked cell persistence across all groups. See FIG. 14C. Free cells cleared most rapidly, with 50% signal loss within 3 days and more than 90% loss by Day 7. In contrast, G-P6K and G-P6K-PA scaffolds significantly prolonged cell persistence.

[0163] G-P6K-implanted scaffolds initially showed a slight signal drop, followed by a rebound, reaching over 100% of the Day 0 signal by Week 2. By Day 25, 50% of the signal remained, and two mice retained detectable signal through Day 58. G-P6K-PA scaffolds, while also outperforming direct injection, showed a faster signal decline than G-P6K, with 50% loss by Day 7. Still, more than 10% of the original signal persisted through Day 58. Two mice in the G-P6K-PA group died before study completion for unrelated reasons.

[0164] However, heterogeneous cell signal distribution was observed within each scaffold-implanted group, in contrast to the more uniform signal observed in the noscaffold direct injection group. This variability can be influenced by differences in host immune response, as nude mice still possess functional B cells and macrophages, and individual foreign body responses to the implanted scaffolds. These observations suggest that inter-individual immune variability may affect scaffold performance and cell persistence, indicating the need for further optimization of material properties or immune- modulating strategies in future studies.

[0165] Together, these findings demonstrate that both G-P6K and G-P6K-PA bioprinted scaffolds significantly enhance in vivo cell persistence in the intraperitonealAttorney Docket No. 4210.0546WO(I.P.) cavity compared to direct cell injection. This improvement is likely attributed to the physical protection provided by the hydrogel scaffolds, which buffer the encapsulated cells from mechanical stress, enzymatic degradation, and rapid clearance. These results highlight the potential of the presently disclosed 3D bioprinting platform and hydrogel formulations as a promising strategy for long-term, localized delivery of therapeutic cells in intraperitoneal cancer therapy. Incorporation of a photoabsorber to enhance printability did not adversely affect in vitro or in vivo bioactivity, further supporting the feasibility of using this approach to bioprint complex, structurally defined scaffolds tailored for in-cavity implantation. Considering printability, physical characteristics, and the ability to support both \n vitro and in vivo cell persistence, the G-P6K-PA formulation demonstrated the most favorable overall performance for efficient therapeutic cell delivery into the intraperitoneal (I.P.) cavity. Therefore, G-P6K-PA was selected as the resin formulation for further characterization of 3D CLIP bioprinting using NHF1GFP’FLcells.

[0166] The consistency of cell loading across a single batch of bioprinted scaffolds, arranged in a 4 x 7 array (28 disks total, scaffold diameter = 5mm, height = 1mm) was assessed. IVIS imaging and quantification of BLI signal from each scaffold revealed a spatial bias, with higher BLI signals in scaffolds printed near the edge of the platform compared to those in the center.

[0167] To address this variability, the printing speed was increased from 10 mm / hr to 30 mm / hr. This adjustment significantly improved printing outcomes. When printing with a density of 5 x 106cell / mL and using 1.5 mL bioresin, the overall BLI signal increased by 3.88-fold, and the distribution of BLI signal across the array became more uniform. Additional printing parameters are described in Table 2, below. Without being bound to any one theory, this improvement is attributed to the reduced overall UV exposure resulting from the faster printing speed, which likely minimized phototoxic effects on cells and improved post-printing viability.Attorney Docket No. 4210.0546WO

[0168] Table 2. Printing Parameters.

[0169] To further validate the consistency and reproducibility of the CLIP 3D bioprinting process, three independent batches of G-P6K-PA scaffolds were bioprinted. Bioluminescence imaging (BLI) was used to quantify cell viability in each individual scaffold across batches. See FIG. 15 A. No statistically significant differences were observed in mean BLI signals among batch 1 (5.34 ± 2.65 x 107p / s), batch 2 (5.85 ± 2.49x107p / s), and batch 3 (5.55 ± 2.17 x 107p / s), demonstrating reliable and reproducible cell loading across different print runs.

[0170] After confirming batch-level consistency, cell proliferation within bioprinted scaffolds over time was studied. A single batch of 28 scaffolds was printed, and 4 to 6 scaffolds were randomly selected for BLLbased viability quantification on Days 1, 6, 12, 18, 26, and 33. See FIG. 15B. The results revealed a steady increase in cell viability, reaching a 3.5-fold increase by Day 18. From Day 18 to Day 33, the BLI signals plateaued (3.4-fold on Day 26 and 3.3 -fold on Day 33), indicating that the cell population had reached a growth limit. This plateau is consistent with the known phenomenon of densitydependent inhibition22in fibroblasts, where proliferation slows or halts at high cell densities due to contact inhibition and limited space. By Day 18, cells likely reached a density threshold within the scaffold matrix, limiting further expansion.

[0171] Fluorescence imaging of GFP-positive NHF 1GFP FLcells supported the BLI data, showing increasing cell density over time. Morphologically, cells transitioned from rounded shapes in the first week post-bioprinting to elongated, fibroblast-like structures by later time points, suggesting that the CLIP-printed matrix provides a supportive environment for fibroblast attachment and spreading. Interestingly, stronger GFP signalAttorney Docket No. 4210.0546WO intensity at the scaffold periphery was observed compared to the center, indicating preferential cell growth at the edges. This can be due to improved nutrient diffusion, oxygen availability, and waste exchange at the scaffold boundaries. These findings suggest that the internal architecture of bioprinted scaffolds could be further optimized to promote more uniform cell growth and viability throughout the entire construct.

[0172] After establishing a consistent and reproducible CLIP 3D bioprinting protocol, its scalability was further evaluated by varying cell loading densities, scaffold dimensions, and structural designs. Disc-shaped scaffolds were printed using G-P6K-PA resin with NHF 1GFP-FLcepst three different concentrations: low (5X 106cells / mL), medium (1.25X 107cells / mL), and high (2.5X107cells / mL), and across three scaffold sizes: small (5 mm), medium (7.5 mm), and large (10 mm) in diameter. As expected, scaffolds with higher cell concentrations and larger dimensions produced brighter GFP signals and higher BLI intensities. See FIG. 16A.

[0173] To further characterize the effects of cell density on post-printing behavior, both absolute and relative BLI signals were tracked in 5 mm scaffolds over time. See FIGS. 16B and 16C. While absolute BLI signals increased proportionally with initial cell density, the relative proliferation rate decreased. Specifically, low-density scaffolds exhibited the fastest growth, followed by medium-density and then high-density scaffolds. This trend likely reflects early contact inhibition in high-density groups, where cells may have approached the threshold for density-dependent inhibition immediately after bioprinting, limiting further proliferation.

[0174] Interestingly, all cell density groups exhibited a tri-phasic growth pattern. An initial increase in BLI signal was observed within the first 24 hours, followed by a transient plateau or decline, and then a recovery phase with continued proliferation. Without being bound to any one theory, it is believed that this phenomenon can be attributed to a combination of early adaptation to the hydrogel environment, temporary stress from the bioprinting process, and gradual remodeling of the matrix.

[0175] For instance, right after bioprinting, fibroblasts could experience a burst of proliferation due to the fresh environment and the availability of nutrients. This initialAttorney Docket No. 4210.0546WO phase is characterized by high cell viability and rapid growth as the cells adapt to their new surroundings. The cells then experience a decline in cell viability and growth, possibly due to stress and damage from the bioprinting process, leading to a temporary decline in viability. This stress can be mechanical (from the printing process) or chemical (from the bioink components). Nutrient depletion and waste accumulation could also contribute. As cells proliferate, they consume nutrients in the medium. If the medium is not replenished frequently, nutrient depletion can lead to reduced cell viability. Although nutrient depletion and waste accumulation can contribute to reduced viability, these factors were minimized in the presently disclosed system through regular medium changes every other day. Nonetheless, scaffold geometry and lack of porosity likely influenced nutrient diffusion and local microenvironmental conditions, contributing to the observed differences in growth dynamics.

[0176] Continuing, after the stress period, cells can recover and continue proliferating until reaching a relatively constant growth rate. In this stage, cells that survived may have already adapted to the environment and start proliferating again. This recovery phase can be attributed to adaptation, medium changes, and extracellular matrix (ECM) production. Cells adapt to the bioprinted environment, overcoming initial stress factors. Regular changes in the culture medium can replenish nutrients and remove waste products, promoting cell growth. Fibroblasts produce and establish ECM components, which can provide a more supportive environment for cell growth and proliferation.

[0177] During the recovery phase, the scaffold BLI signals keep increasing until eventually reaching saturation. The scaffold materials might undergo degradation over time, providing less matrix for cells to attach and grow. Additionally, the limited volume of the scaffold eventually restricts the space available for cell expansion, explaining the saturation. Nevertheless, the increased BLI signal demonstrates that the culture conditions are not toxic to the cells over time, confirming the biocompatibility of both the original materials and their degraded byproducts. Together, these data indicates that the present systems can be easily scaled up and the cell loading amount altered using the CLIP 3D bioprinting systems, e.g., by altering cell number and scaffold dimensions.Attorney Docket No. 4210.0546WO

[0178] To explore the effects of scaffold design on cell loading efficiency and proliferation, we next evaluated a series of ring-shaped scaffolds, all with a fixed 10 mm outer diameter and cell concentration (5x 106cells / mL), but with varying strut thicknesses ranging from 1 mm to 5 mm (where 5 mm corresponds to a solid disc). See FIG. 16D. Despite substantial differences in printed volume, the Day 0 BLI signals were similar across all designs (see FIG. 16D, top), suggesting that thinner scaffolds, although containing less solid material, encapsulated a comparable number of viable cells. Furthermore, BLI signals steadily increased in all designs over a two-week period, indicating that these architectural variations did not hinder in vitro cell proliferation.

[0179] To assess design-dependent cell-loading efficiency, we calculated printing efficiency by normalizing BLI signal to scaffold volume (p / s / mm3). The 1 mm strut scaffold exhibited the highest efficiency, achieving a 3.7-fold improvement over the full scaffold on Day 0 and a 3.0-fold increase by Day 14. See FIG. 16D, bottom. GFP fluorescence imaging confirmed these results. Notably, except for the 1 mm design, all other scaffold types displayed uneven cell distribution, with brighter fluorescence at the scaffold edges and diminished signal closer to the center. This edge-biased pattern likely resulted from uneven resin replenishment and light scattering during printing. Specifically, during the CLIP process, the growing structure is continuously drawn out of the resin bath, relying on the surrounding liquid to refill the print zone. Central regions can experience less efficient resin replenishment, leading to lower local cell density. Additionally, UV light scattering may concentrate in the scaffold center, increasing localized phototoxicity and reducing cell viability.

[0180] These results highlight that even when using the same resin and cell concentration, structural design can influence cell loading efficiency and spatial distribution of cells within the printed construct. Optimizing scaffold architecture can therefore improve material utilization and provide homogeneous and maximized cell loading, particularly for applications requiring high-throughput production and consistent therapeutic performance.Attorney Docket No. 4210.0546WO

[0181] While a simple and efficient CLIP 3D bioprinting platform was established, given that scaffold architecture can influence factors such as nutrient and oxygen exchange, degradation rate, swelling behavior, and in vivo foreign body response, additional studies were performed to determine how varying porosity levels affect cell loading and growth dynamics. Five scaffold types using G-P6K-PA resin were designed and printed: four porous scaffolds with a consistent strut width (i.e., thickness of between pores) of 0.5 mm but varying pore widths (0.4 mm, 0.625 mm, 1 mm, and 1.75 mm), resulting in porosities of 16%, 25%, 36%, and 49%, respectively; and one fully solid scaffold (0% porosity) as a control. See FIG. 17A. All scaffolds had an overall dimension of 5x5x1 mm. Scaffolds with higher porosity contained fewer square-shaped pores, but each pore had a larger size. See Table 3, below. NHF1GFP'FLcells were bioprinted at a concentration of 5 * 106cells / mL in 2 mL of resin.

[0182] Table 3. Scaffold Design Measurements.

[0183] Immediately after printing, BLI measurements revealed that the 16% porous scaffolds had significantly higher signal intensity (1.26-fold) than the full scaffold, indicating greater viable cell loading. See FIG. 17B. Scaffolds with 25% and 36% porosity also showed moderately higher signals (1.17- and 1.11-fold, respectively), although not statistically significant. In contrast, the 49% porous scaffold showed the lowest BLI signal (0.72-fold of the full scaffold), likely due to reduced total material volume and lower absolute cell number.Attorney Docket No. 4210.0546WO

[0184] To better understand the impact of scaffold architecture on cell loading efficiency, BLI signal was normalized by scaffold solid volume to calculate “printing efficiency”. See FIG. 17C. All porous scaffolds had significantly higher printing efficiency than the full scaffold, with 25% porosity achieving the highest efficiency (1.57-fold), followed by 16% (1.5-fold), 36% (1.48-fold), and 49% (1.41-fold). These results indicate that porosity enhances cell loading efficiency, though excessive porosity reduces total cell content due to diminished printed volume. Without being bound to any one theory, this effect can stem from changes in UV light exposure during printing. As porosity increases, less solid volume will be printed, so total UV exposure per scaffold decreases, leading to reduced phototoxicity and improved cell viability. However, at higher porosity levels, reduced material results in fewer total encapsulated cells. For example, the 49% porous scaffold had a total solid volume of 12.75 mm3compared to 25 mm3for the solid control. Therefore, although porosity supports higher per-volume cell loading, an optimal balance is needed to maintain sufficient absolute cell numbers. Additional studies on total UV exposure, live / dead ratios, and cell distribution across scaffold designs will provide further insights.

[0185] To assess how porosity impacts cell proliferation, BLI measurements were taken over time. All scaffold types showed a consistent growth pattern: a relatively flat proliferation phase during the first two weeks, followed by rapid expansion beginning on Day 18 and continuing through Day 25. See FIG. 17D. Scaffolds with 16% and 25% porosity consistently outperformed the solid scaffold in total BLI signal, while 36% porosity produced similar results. Interestingly, although the 49% porous scaffold started with the lowest signal, it exhibited the fastest growth rate and eventually reached higher signal levels than the other groups.

[0186] When normalized by solid volume, printing efficiency continued to increase with porosity, with the 49% porous scaffold achieving the highest efficiency. These results suggest that porosity enhances not only initial loading but also long-term proliferation, especially under conditions of reduced material-induced stress and improved nutrient diffusion. This finding aligns with earlier observations, where thinner ring struts alsoAttorney Docket No. 4210.0546WO yielded higher efficiency. Additional studies can be performed to further evaluate which porosity offers the best tradeoff between initial cell loading and long-term proliferation to support therapeutic outcomes in vivo. For example, higher initial loading (16-36% porosity) could support stronger early-stage delivery, while higher proliferation (49% porosity) could benefit long-term persistence and functional output.

[0187] Fluorescence imaging of GFP-positive NHF1GFP’FLcells within all scaffold types confirmed BLI trends and offered additional insight into cell distribution. As porosity increased, cells distributed more evenly across the struts. In contrast, solid scaffolds exhibited an edge-dominant pattern, with stronger signals on the periphery than the center. By Day 11, this edge effect was particularly prominent in 16% porous scaffolds, while cell distribution in higher porosity scaffolds (25%, 36%, 49%) appeared more uniform. Over time, scaffolds became increasingly amorphous and softened, particularly in high-porosity groups. This change is likely due to scaffold degradation triggered by NHF1 -secreted ECM-degrading enzymes and active ECM remodeling. These findings are consistent with the previous degradation data on G-P6K-PA materials. Increased porosity correlated with faster scaffold degradation, which may influence in vitro proliferation and in vivo persistence. While a faster degradation rate could reduce acute immune response and foreign body response, and improve nutrient exchange, it could also lead to premature loss of scaffold structure and faster clearance of therapeutic cells. Tuning the degradation rate can balance reduced foreign body response with prolonged therapeutic effect.Exploring how 3D porous architecture affects in vitro cell loading and proliferation is useful in optimizing intraperitoneal cell delivery. Porous designs provide higher cell loading per unit volume and reduce material burden, potentially minimizing immune response and foreign body reaction upon implantation. These advantages can contribute to improved in vivo cell persistence and therapeutic duration. Continued exploration of scaffold architecture, degradation kinetics, and drug release dynamics will inform future designs for effective intraperitoneal therapeutic delivery platforms.Attorney Docket No. 4210.0546WOEXAMPLE 7Bioprinted Scaffolds with Other Cells

[0188] While CLIP 3D bioprinting has been demonstrated as a promising strategy for delivering cells into the I.P. cavity using NHF1GFP'FLfibroblasts as a model system, stem cells also hold significant clinical potential due to their ability to secrete regenerative and anti-cancer factors. Previous studies have shown that therapeutic stem cells secreting anticancer reagents can be effective against I.P. tumors such as ovarian cancer. See Cao et al., Bioconjug Chem. 2017; 28(6): 1767-76; Mooney et al., Stem Cell Research & Therapy. 2021; 12: 1-11; Mooney et al.. Molecular Therapy-Oncolytics. 2019; 12:79-92; and Hammad et al.. Molecular Therapy-Oncolytics. 2020; 18:326-34. However, rapid clearance from the peritoneal cavity remains a major challenge for stem cell therapies. To address this, studies were performed to determine if the present CLIP bioprinting platform could be extended to support various other types of cells, including various types of stem cells, and serve as a delivery system that enhances their viability and persistence.

[0189] To evaluate stem cell compatibility, three different human stem cell types were bioprinted: (1) human mesenchymal stem cells (hMSCmtl'cr'v'1 1'), (2) human neural stem cells (HB1.F3GFP'FL), (3) a lab-derived spheroidal human induced neural stem cell line (hiNeuroSmCherry FL), (4) a human astrocyte cell line (hAstrocytemCherry FL), and (5) a human ovarian cancer cell line (ES2mCherry'FL) Each cell type was suspended in G-P6K resin at appropriate densities: 2.5 x 106cells / mL for hMSC and hAstrocyte, 1 x 107cells / mL for HB1.F3 and hiNeuroS, and 3 x 107for ES2 cells. Each suspension was mixed 1 : 1 with 2x G-P6K resin and bioprinted as described previously. Cell viability was assessed via BLI, and fluorescence imaging was used to monitor cell morphology and growth within the scaffolds. See FIGS. 18A-18E.

[0190] hMSCmtllc"v'I Fcells demonstrated excellent biocompatibility with the bioprinting process. See FIG. 18A. BLI signals increased steadily over 3 weeks, reaching a 12-fold increase by Day 21. Fluorescence imaging confirmed this trend, and cell morphology gradually shifted from rounded to spindle-shaped, characteristic ofAttorney Docket No. 4210.0546WO mesenchymal cells cultured in 2D. These results indicate that hMSCs maintained high viability and proliferative capacity post-bioprinting.

[0191] BLI data for HB1.F3GFP'FLcells revealed a 9-fold increase in signal within the first 6 days, followed by a decline and plateau at approximately 3-fold by Day 31. See FIG. 18B. This suggests that the cells reached a density threshold by Day 6, triggering densitydependent inhibition and subsequent cell death or growth arrest. GFP imaging confirmed cell viability and revealed the formation of 3D cell clusters and spheres beginning on Day 6. These structures persisted and grew over time, with pronounced cell accumulation at the scaffold edges. Notably, although HB1.F3 cells are typically cultured in 2D as adherent cells, their ability to form 3D clusters in the bioprinted scaffolds indicates that the matrix may provide a favorable 3D microenvironment for neural stem cell function.

[0192] The hiNeuroSmClierry’FI' cells exhibited a more dynamic growth pattern. BLI signals increased 25-fold by Day 8, dropped to 7-fold by Day 14, and recovered to 24-fold by Day 21. See FIG. 18C. This fluctuation likely reflects an initial proliferation phase followed by density-dependent inhibition and partial cell death, with subsequent regrowth. Fluorescence imaging showed that although the spheroidal cells were initially dispersed into smaller aggregates or single cells during resin mixing, they re-aggregated into spheroids resembling their morphology in standard culture. Over time, these cells formed large clusters both within the scaffold center and at its periphery, suggesting that the bioprinted matrix supports their natural aggregation and growth behavior.

[0193] Besides fabricating with stem cells for therapeutic purpose, 3D bioprinting is also as a promising approach for fabricating complex biological constructs for tissue engineering and regenerative medicine. Furthermore, as new approach methodologies (NAMs) are emphasized more by FDA to replace animal testing in preclinical drug safety studies, 3D bioprinting shows great potential to fabricate and develop in vitro or ex vivo disease models to better mimic complex 3D biological and mechanical environment for drug screening. Astrocytes, for instance, can be used to develop in vitro brain models, and ES2, an ovarian cancer cell line, can be utilized to develop in vitro cancer organoids. These 2 cell lines were 3D bioprinted using CLIP. Both hAstrocytemChcrry'FLand ES2mChcrry'FLAttorney Docket No. 4210.0546WO demonstrated excellent biocompatibility, exhibited continuous proliferation after 3D bioprinting for 21 days and 28 days. See FIGS. 18D and 18E.

[0194] These data confirm that a variety of cells, including stem cells, normal tissue cells, and cancer cells, can be viably encapsulated and cultured using the CLIP bioprinting platform, expanding the application scenarios to 3D organoid development. By blending natural-based and synthetic-based materials, and, in some examples, further adding a biocompatible photoabsorber, printability, degradation, swelling, and modulus was tuned for a broad spectrum of resin formulations for promising in vitro cell loading, proliferation, and long-term intraperitoneal cavity cell delivery that exceeds free cell injection.

[0195] This is believed to be the first time Ecamsule, a photoabsorber used for cosmetic sunscreen development, has been tested in a 3D bioprinting system, proving its great potential for improving print fidelity and providing protection for cells from excessive UV damage. The present studies also explore how 3D structure influences cell loading and in vitro proliferation. The scalability data provided herein and the ability to bioprint with different cell types highlights the potential for CLIP to be utilized as a biocompatible system for 3D bioprinting with a broad spectrum of resin formulations, cell types, and 3D designs. Different types of stem cells were able to be bioprinted with high viability and proliferation profde. CLIP 3D bioprinting technology holds great potential for the generation of biomimetic 3D organoids for disease modeling and high-throughput drug screening, as well as the delivery of stem cells for tissue engineering and stem cell therapies. 3D bioprinting can be optimized for each cell type. For example, the hiNeuroS stem cells express significantly higher MMP-1 and MMP-3 proteins compared to its parent fibroblast NHF1 cells. Constant expression of MMPs can degrade the biodegradable material the cells bioprinted into, leading to a self-degradable scaffold that behaves differently both in vitro and in vivo compared to NHF1 bioprinting with the same material, e.g., leading to differences in cell behavior, drug release, in vivo interaction with host tissue, and eventually therapeutic outcome. Accordingly, the CLIP 3D bioprinting system provides a versatile platform for great tunability and personalized design of drug delivery platforms and 3D-printed organoids.Attorney Docket No. 4210.0546WOEXAMPLE 8Treatment of Ovarian Cancer with Bioprinted Scaffolds

[0196] TRAIL- secreting neural stem cells, hiNeuroS-GFP-TRAIL, and resin formulation G-P6K-PA were bioprinted with different porosity, 0% (a full scaffold), 27%, and 54%, to explore how scaffold design influences cell viability, proliferation, therapeutic protein release and in vivo therapeutic efficacy over time. See FIG. 19A. Cell viability within bioprinted scaffolds were measured using PrestoBlue assay. Scaffold viability showed no significant difference across all three porosities right after printing, despite different total solid volume. See FIG. 19B. Printing efficiency was calculated and compared, dividing scaffold total viability measured by PrestoBlue by scaffold solid volume. Right after printing, scaffold printing efficiency differed. See FIG. 19C. While there is no significant difference in scaffold viability between 0% and 27% porosity scaffolds, the 54% porosity scaffold showed significantly higher printing efficiency than the other scaffolds, suggesting that by introducing higher scaffold porosity, cell viability per unit volume can be increased. Scaffold viability and printing efficiency were compared over time for up to 3 days after bioprinting. Cells exhibited similar viability level across all porosities. See FIG. 19D. Scaffolds with 0% and 27% porosity had similar printing efficiency over time, while 54% porous scaffolds exhibited significantly higher printing efficiency than 0% porosity scaffolds through 3 days, and higher than 27% porosity scaffolds through 1 -day post-bioprinting. See FIG. 19E.

[0197] GFP fluorescent images of hiNeuroS-GFP-TRAIL cells also indicated continuous proliferation of cells within the scaffolds. Scattered floating cells were observed outside of the scaffolds, suggesting that cells were constantly released from the scaffolds. Release of iNeuroS-GFP-TRAIL out of the scaffolds was quantified over time. The 27% and 54% porous scaffolds released more cells than the 0% porous full scaffolds over time (see FIG. 19G), correlating to the higher surface area of the scaffolds with the higher porosity.

[0198] Additional studies were performed to assess whether the bioprinted hiNeuroS- TRAIL could efficiently express and release TRAIL into the surrounding environment andAttorney Docket No. 4210.0546WO elicit potent anti-tumor activity. Scaffolds were incubated for three days after bioprinting into ImL of ReNCell media for 24hr. The conditioned media was sampled and added to pre-plated ES2 cells, with 24hr conditioned media from free hiNeuroS-TRAIL cells as a positive control. Conditioned media from the bioprinted scaffolds induced very potent ES2 toxicity at a similar level across different porosities, killing almost 95% tumor cells, comparable to conditioned media from free cell. See FIG. 19F. TRAIL release from the bioprinting scaffold was quantified every 24 hours post-bioprinting until Day 8, and it was found that scaffolds with all three porosities release a similar level of TRAIL protein into media over time, suggesting that reduced solid volume didn’t influence the amount of the therapeutics that one bioprinted scaffolds could deliver. See FIG. 19H.

[0199] After developing and quantifying bioprinted scaffolds containing sufficient hiNeuroS-GFP-TRAIL to release adequate TRAIL protein over time, 0% and 27% porous scaffolds were selected for an in vivo therapeutic study against overian cancer due to their better handle-ability for surgical implantation. hiNeuroS'GFP'TRAILcells were first bioprinted as in Figure 19A to provide 0% and 27%> porosity scaffolds. To serve as a non- therapeutic material negative control, non-therapeutic, blank NHF1 fibroblasts were bioprinted under the same conditions into 0% porosity (i.e., “full”) scaffolds to include the influence of metabolic waste from a large number of cells within the scaffolds. 2 days after bioprinting, nude mice were inoculated with 2E5 ES2Fluccells into the intraperitoneal (I.P.) cavity. The next day, all mice were imaged and regrouped, and went through I.P. surgery to receive different treatments, including PBS only, CLIP-produced scaffolds containing non-therapeutic NHF1 cells, or bioprinted hiNeuroS-TRAIL scaffolds with 0% or 26% porosity. Tumor killing was measured via the fold change in bioluminescence (BLI) signal over time. See FIG. 20. While mice in the control groups exhibited immediate and rapid tumor growth, mice treated with both bioprinted hiNeuroS-TRAIL scaffolds exhibited at least 10 times less rapid tumor growth within 2 weeks post scaffold implantation.

[0200] Collectively, the data indicate that increasing scaffold porosity from 0% to 27%, by introducing additional void spaces, did not compromise cell viability, proliferation, TRAIL release, or in vivo therapeutic efficacy. While scaffolds with reduced solid contentAttorney Docket No. 4210.0546WO may elicit a weaker long-term foreign body response, those with moderate porosity (27%) appear to offer an optimal balance, supporting effective therapeutic delivery with potentially fewer immune-related side effects.

[0201] It will be understood that various details of the presently disclosed subject matter may be changed without departing from the scope of the presently disclosed subject matter. Furthermore, the foregoing description is for the purpose of illustration only, not for the purpose of limitation.

Claims

1. Attorney Docket No. 4210.0546WOCLAIMSWhat is claimed is:

1. A method of producing a device for delivery of one or more therapeutic agents to a target site of a subject in need thereof, the method comprising: preparing a bioprinting resin, wherein the bioprinting resin comprises a biocompatible polymer resin and one or more therapeutic agents, wherein said one or more therapeutic agents comprise a live cell and / or a microorganism; and forming and curing the bioprinting resin via an additive manufacturing process, thereby producing the device for delivery of the one or more therapeutic agents, wherein the device comprises the one or more therapeutic agents encapsulated in cured biocompatible polymer.

2. The method according to claim 1, wherein the additive manufacturing process comprises a continuous liquid interface production (CLIP) process.

3. The method of claim 1 or claim 2, wherein the one or more therapeutic agents comprise a live cell, optionally wherein the live cell is selected from the group consisting of an anticancer drug-secreting cell, a stem cell, a chimeric antigen receptor (CAR)-T cells, a T cell, a macrophage, and a natural killer cell.

4. The method of claim 3, wherein the anticancer drug-secreting cell is a cell that secretes tumor necrosis factor (TNF)-related apoptosis inducing ligand (TRAIL) and / or wherein the anticancer drug-secreting cell is a fibroblast or a stem cell.

5. The method of claim 3, wherein the stem cell is a neural stem cell or a mesenchymal stem cell.

6. The method according to claim 1 or claim 2, wherein the one or more therapeutic agents comprise a bacteria cell, a yeast cell, or an oncolytic virus.Attorney Docket No. 4210.0546WO7. The method according to any one of claims 1-6, wherein the one or more therapeutic agents comprise a live cell and a non-living component, optionally wherein the non-living component comprises one of the group consisting of a polypeptide, a nucleic acid, an antibody, an exosome, a nanoparticle, a microparticle, and a small molecule.

8. The method according any one or claims 1-7, wherein the biocompatible polymer resin comprises one or more of the group consisting of a functionalized gelatin, a functionalized alginate, a functionalized hyaluronic acid, a functionalized polyethylene glycol (PEG), a functionalized collagen, and combinations thereof, optionally wherein the biocompatible polymer resin comprises one or more of the group consisting of gelatin methacryloyl (GelMA), methacrylated alginate, methacrylated hyaluronic acid, methacrylated PEG, acrylated PEG, methacrylated collagen, and combinations thereof.

9. The method according to any one of claims 1-8, wherein the live cell and / or microorganism proliferates within the device for a period of time after formation of the device.

10. The method according to any one of claims 1-9, wherein the live cell and / or microorganism is viable and therapeutically active after formation of the device, optionally wherein the live cell and / or microorganism remains viable for at least 30 days in vivo.

11. The method according to any one of claims 1-10, wherein a concentration of one or more of the one or more therapeutic agents in the device is adjustable during formation of the device via the additive manufacturing apparatus.

12. The method according to any one of claims 1-11, wherein formation of the device is controlled to form a desired shape, optionally a disc shape, a spherical shape, or a porous shape.Attorney Docket No. 4210.0546WO13. The method of claim 12, wherein the desired shape of the device is selected to improve structural integrity and / or functionality of the device following in vivo implantation.

14. The method according to claim 12, wherein the desired shape of the device is customized to fit a resection cavity for implantation and / or administration.

15. The method according to claim 12, wherein the desired shape of the device is customized to improve the viability and / or migration of a live cell and / or microorganism encapsulated in the device.

16. The method according to claim 12, wherein the desired shape of the device is customized to optimize a degradation rate of a degradable material in the device, to optimize encapsulation of the one or more therapeutic agents, and / or to optimize a release rate of one or more therapeutic agents from the device.

17. The method according to claim 12, wherein the desired shape of the device is customized to reduce foreign body response against the device.

18. A method of treating a disease in a subject, the method comprising providing a subject in need of treatment for the disease and administering to the subject a device prepared according to the method of any one of claims 1-17.

19. The method of claim 18, wherein administration comprises surgical implantation of the device at a desired site in the subject, optionally wherein administration comprises post-surgical implantation of the device in the subject after removal of a tumor from the subject.Attorney Docket No. 4210.0546WO20. The method of claim 18 or claim 19, wherein the subject is suffering from or susceptible to a cancer, optionally wherein the cancer is selected from the group consisting of glioblastoma, ovarian cancer, colorectal cancer, pancreatic cancer, liver cancer, melanoma, breast cancer, and leukemia.

21. The method of claim 20, wherein administering the device to the subject treats the cancer, optionally wherein administering the device delays tumor regrowth and / or increases expected subject lifespan.

22. A therapeutic composition, the therapeutic composition comprising the device of any one of claims 1-17.

23. The therapeutic composition of claim 22, wherein the therapeutic composition is formulated for administration and / or implantation in a subject in need of treatment, optionally a subject suffering from or susceptible to a cancer.

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