Regulating the secretion of biological factors

Abstract

A method of externally reprogramming how a cell behaves and what biological factors it secretes, for example, by applying an external (controllable) stimulus to achieve the desired result of producing a target biological factor.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 449,971, filed March 4, 2023, which is expressly incorporated herein by reference in its entirety.

[0002] Inventive embodiments of the present disclosure generally relate to methods for externally reprogramming how cells behave and which biological factors are secreted from the cells, for example, by applying external (controllable) stimuli to achieve a desired outcome, such as producing a target biological factor. [Background technology]

[0003] Cells must constantly communicate with each other to maintain homeostasis in dynamic environments in order to maintain cellular phenotype and function. One way cells communicate with each other is through the secretion of vesicles (e.g., extracellular vesicles). Lipid bilayer vesicles with diameters ranging from 30 to 100 nm are called exosomes and traditionally transport proteins (e.g., enzymes), oligonucleotides (e.g., DNA, mRNA, ncRNA, microRNA, lipids, and other components or biomarkers). Vesicles with diameters ranging from 50 to 1,000 nm are called microvesicles and are capable of transporting larger cargo loads than exosomes. Vesicles with diameters ranging from 500 to 2,000 nm are called apoptotic bodies and are capable of transporting larger cargo such as proteins, enzymes, growth factors, cytokines, oligonucleotides, lipids, and other cargo, as well as organelles. These vesicles are often secreted from one cell and taken up by another. Exosomes are one of the most common vesicles secreted by cells and are present in biological fluids such as serum, plasma, urine, semen, cerebrospinal fluid (CSF), saliva, tears, and breast milk. Exosomes are stable in these fluids and can cross the blood-brain barrier. Therefore, exosomes are being investigated as therapeutic agents for the delivery of various drugs, as well as biomarkers for various diseases, cancer, aging, and injury, and as prognostic indicators of response to treatment.

[0004] Exosomes can be taken up by neighboring cells or can traverse the body and be taken up by cells distant from their secreted or originating cells. Exosomes can be secreted by epithelial cells, endothelial cells, mesenchymal stem cells (MSCs), macrophages, dendritic cells, tumor cells, neurons, oligodendrocytes, reticulocytes, mast cells, platelets, cancer cells, B cells, T cells, and astrocytes. Several researchers have explored and developed methods to stimulate exosome secretion from target cells and isolate exosomes for therapeutic and diagnostic development. Most production methods rely on temporarily "starving" target cells by exposing them to specific media formulations depleted of serum and other protein components. Furthermore, cells are grown in two dimensions (2D) on culture plastic or in bioreactors on microcarriers. However, exosome production (let alone quality) and the ability to scale the production of specific types of exosomes carrying targeted cargo remain challenging.

[0005] Thus, there remains a need in the art for methods and / or platforms that can increase exosome production in MSCs while allowing for modulation of exosome secretion from MSCs in culture (e.g., by providing controllable stimuli to the MSCs to produce a desired outcome). Summary of the Invention [Means for solving the problem]

[0006] One or more embodiments of the present invention may address one or more of the above-mentioned problems. Some embodiments according to the present invention provide a method for characterizing a therapeutically desirable component or component profile of a secretome composition secreted from a cell type of interest. The method includes the steps of (i) seeding a first tissue-mimicking three-dimensional (3D) hydrogel block (TMHB) comprising a macrostructure defined by a continuous polymeric matrix material and a network of microporous channels and / or chambers extending throughout the continuous polymeric matrix material with a first cell type of interest; and (ii) simulating in vitro cell growth and proliferation of the first cell type of interest by providing the first cell type of interest with a first culture medium and allowing the first cell type of interest to proliferate throughout the first TMHB and secrete a secretome composition. (iv) collecting the secretome composition; (v) subjecting the secretome composition to a stratification operation based on molecular weight cutoff values ​​to provide a plurality of distinct layers, each characterized by a different range of component molecular weights; (vi) characterizing and / or identifying one or more individual components present with each layer of interest; and (v) testing each layer of interest for relative therapeutic utility by either in vivo or ex vivo analysis to identify the layer of interest that provides the greatest therapeutic utility as the therapeutic component profile.

[0007] In another aspect, the present invention provides a method for tailoring a component profile of a natural secretome composition secreted from a cell type of interest, the method comprising the steps of: (i) seeding a first tissue-mimicking three-dimensional (3D) hydrogel block (TMHB) comprising a macrostructure defined by a continuous polymeric matrix material and a network of microporous channels and / or chambers extending throughout the continuous polymeric matrix material with a first cell type of interest; (ii) providing a first medium to the first cell type of interest; and (iii) culturing the first cell type of interest in a manner that more closely tracks a target component or target secretome composition having a target component profile (e.g., a therapeutically useful component or profile thereof that has already been identified). The method includes: (iv) introducing at least one external stimulus configured to modify the native secretome composition secreted from the cell type, the at least one external stimulus comprising one or more controllable parameters (e.g., adding a coating to the interface between the continuous polymer matrix material and the network of microporous channels and / or chambers to adjust the oxygen concentration, pH, temperature, electromagnetic properties, and fluid dynamics within the first TMHB); (iv) allowing the first cell type of interest to proliferate throughout the first TMHB and secrete the secretome composition; and (iv) collecting the secretome composition modified by the external stimulus, which differs from the native secretome composition.

[0008] DETAILED DESCRIPTION OF THE INVENTION The present invention will now be described more fully hereinafter with reference to the accompanying drawings, in which some, but not all, embodiments of the invention are shown. Indeed, the invention may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will satisfy applicable legal requirements. Like reference characters refer to like elements throughout. The drawings are as follows: [Brief explanation of the drawings]

[0009] [Figure 1]1 shows two separate IPHB-type TMHBs according to some embodiments of the present invention. [Figure 2] 1 illustrates three interlocked IPHBs according to some embodiments of the present invention. [Figure 3] 1A-1C show top and side views of an IPHB according to some embodiments of the present invention, illustrating a continuous polymer matrix material and a network of microporous channels and / or chambers extending throughout the continuous polymer matrix material and exposed at the surface of the IPHB. [Figure 4] 1 illustrates an IPHB according to some embodiments of the present invention, which includes an interlocking male component on the top surface and an interlocking female component protruding into the bottom surface. [Figure 5] 1 illustrates another IPHB according to some embodiments of the present invention, which includes an interlocking male component on the top surface and an interlocking female component protruding into the bottom surface. [Figure 6] 1 shows the migration of cells from a first IPHB to a second IPHB when the first and second IPHBs are interlocked with each other, according to some embodiments of the present invention. [Figure 7A] ~ [Figure 7H]Figure 7 shows the characterization of ASC populations. This figure shows the characterization of the initial P1 population of ASCs. Figure 7A shows spindle-shaped / mesenchymal-like adherent cells with trilineage differentiation potential. Figure 7B shows the expression of CD73 / 90 / 105 / 271 CD surface markers (scale bar = 100 lm). Negative markers CD34 and CD45 showed less than 5% positivity. Data not shown). Figure 7C shows gene expression of key markers by MSC phenotyping array. Figure 7D shows a photograph of a 1 cm3 3D-printed tissue-mimicking X-Block in a 6-well culture vessel. The X-Block was printed with a defined microarchitecture, which significantly increased the volume-to-surface area ratio through the formation of a macrostructure. Furthermore, the X-Block was fabricated to mimic the mechanical and viscoelastic properties of native adipose tissue. Figure 7E shows the quantification of in situ cell number within the 3D system (X-Block). ASCs were then extracted from 2D or 3D and replated in 2D for analysis after 1 week (post-P2 for 2D) and 2 weeks (post-P3 for 2D) and assessed for population doubling time (Figure 7F), metabolic activity (Figure 7G), and mitochondrial activity (membrane potential) (Figure 7H). The black dashed line indicates the initial P1 population. Error bars are s.e.m. Significance is indicated by *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001 (n=4). [Figure 8]Figure 1 shows ASC growth curves for 2D and 3D cultures to determine the ASC-CM harvest date. ASCs cultured in the 3D system were slightly delayed in reaching the growth phase, where they were evenly distributed throughout the hydrogel microarchitecture, likely due to an initial migration phase. However, they soon surpassed the relative cell numbers in 2D and ultimately demonstrated higher proliferation rates in 3D. Therefore, we pre-assessed cell numbers for 2D and 3D cultures to determine the optimal harvest date for ASC-CMs to standardize the relative ratio of medium per cell. Based on previous literature, ASC-CM harvest in 2D cultures was desirable when the culture was 60–80% confluent, and we chose to harvest ASC-CMs on days 6–8. To account for the difference in cell numbers between 2D and 3D cultures, we adjusted the medium volume accordingly. [Figure 9A] ~ [Figure 9I]This shows that higher molecular weight secretome fractions are the primary drivers of wound healing activity in KCs. Figure 9A is a schematic diagram of the ASC-CM centrifugal filtration step by molecular weight cutoff (kDa). The solution in the upper chamber was considered a concentrate within that range, and the remaining filtrate in the lower chamber was used for the next molecular weight filtration step. Figure 9B shows the ASC-CM protein content by molecular weight fraction. Figure 9C shows representative morphology images of KCs after treatment with KC-GM supplemented with various ASC-CM concentrates [scale bar = 100 lm]. Figure 9D shows a representative image panel of KCs treated with ASC-CM fractions from 2D or 3D and stained with Hoechst (blue) and vimentin (cyan) [scale bar = 50 lm]. Figure 9E shows image quantification of vimentin immunolabeling in KCs (relative to baseline control KCs). Figure 9F shows qRT-PCR analysis of p16ink4a (a senescence marker) in KCs after treatment with fractionated ASC-CM for 24 hours. The dashed line represents baseline control KCs. Figure 9G shows the relative change in metabolic activity (by Presto Blue). Figure 9H shows the relative change in proliferation (by PicoGreen). Figure 9I shows the relative change in migration (by scratch assay). Significance is indicated by *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001 (n=4). [Figure 10] Image analysis of KC senescence after treatment with molecular weight-stratified ASC-CM. Representative images of KCs after treatment with KC-GM supplemented with various ASC-CM molecular weight concentrations for 24 hours. β-galactosidase activity was used as a surrogate measure of senescence. The lower molecular weight fraction of ASC-CM appeared to induce senescence in the KC population, confirming the qRT-PCR data for p16 previously reported. [Figure 11A] ~ [Figure 11D]This figure shows that ASC populations in tissue-mimicking systems promote EV secretion. In Figure 11A, ASC-CM was processed for EV collection / isolation / purification from 2D and 3D cultures. EV enrichment was assessed by protein content (QuickDrop / BCA / Bradford) of the EV fraction (left) and particle count by NTA (right). In Figure 11B, the relative composition of EVs within ASC-CM was then quantified based on the protein content of the "100 kDa" fraction (left) and total (i.e., "full") secreted protein content (right). In Figure 11C, analysis of EV size distribution in 2D and 3D was assessed using NTA data. In Figure 11D, cumulative frequency distributions were generated to determine the percentage of measured particles within the standard exosome range (25-250 nm). The teal-blue patterned bars indicate the "within exosome size" range, while the black bars are too large to be exosomes. Significance is indicated by *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001 (n=4). [Figure 12A] ~ [Figure 12E]This shows that ASC-EVs in the tissue-mimicking system contain a more potent re-epithelialization stimulus. Figure 12A is a schematic diagram of ASC-CM filtration through a 100 kDa centrifugal filter and subsequent isolation of EVs / exosomes from the "100 kDa" concentrate in the upper chamber. Isolated 2D-EVs (yellow circles) and 3D-EVs (blue circles) were then re-applied to the "<100 kDa filtrate" sample. The effects of the "<100 kDa filtrate" of ASC-CM from 2D (silhouettes) and 3D (patterns), with and without EVs, were evaluated for their ability to modulate KC metabolic activity (Figure 12B), proliferation activity (Figure 12C), and migration activity (Figure 12D). Figure 12E shows the evaluation of KC morphology changes after "<100 kDa filtrate" treatment in the absence (top row) and presence (bottom row) of EVs [Scale bar = 100 lm]. Significance is indicated as *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001 (n=4). [Figure 13A] ~ [Figure 13B] Figure 13A shows 3D-EV-enhanced expression of basal and suprabasal cytokeratins in a dose-dependent manner. Figure 13A shows reconstructed images (red) of KCs stained for cytokeratins, including keratin 5 (top row), keratin 10 (middle row), and keratin 16 (bottom row). KCs treated with KC-GM served as a control for baseline expression (left row), while 2D-EV (middle row) and 3D-EV (right row) at the highest dose of 250 μg / mL were the treatment groups (scale bar = 100 μm, inset scale bar = 20 μm). In Figure 13B, ASC-EV treatment of KCs at various doses from 2D (silhouette) and 3D (pattern) images was evaluated by qRT-PCR analysis of K5, K10, and K16. GAPDH was used as an internal control. Each value is expressed as a fold change relative to the baseline control KCs, indicated by the dashed line. Significance is indicated as *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001 (n=3). [Figure 14A] ~ [Figure 14E]These figures show that EMT and epidermal regeneration of KCs exhibit a dose-dependent response to ASC-EVs. In Figure 14A, the effects of ASC-EVs at various doses were assessed from 2D (silhouettes) and 3D (patterns) images on their ability to modulate the metabolic (top row), proliferative (middle row), and migratory (bottom row) activities of KCs. Figure 14B shows representative images of KC morphology and cytoskeletal changes after ASC-EV treatment. The inset is a bright-field image. Staining includes Hoechst-labeled (blue), phalloidin-labeled (green), and DiI-labeled (red) exosomes. The white arrowheads point to the formation of actin caps (phalloidin, green). [Scale bar = 100 lm]. Figure 14C shows qRT-PCR analysis of CCND1, VIM, FLG, TWIST1, CDH1, and CDH2. GAPDH was used as an internal control. Each value is expressed as a relative fold change to baseline control KCs, indicated by the dashed line. Figure 14D shows the relative ratio of N-cadherin (CDH2) to E-cadherin (CDH1) RNA expression. Figure 14E shows a tabulation of all genes assessed by qRT-PCR, along with their respective R values ​​and relative direction of correlation. Significance is indicated by *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001 (n=3). [Figure 15] Figure 1 shows a comparison of morphological changes in KCs with and without DiI-labeled EVs. ASC-EVs were isolated from both 2D and 3D systems, labeled with the lipophilic dye DiI (red), and administered to KC-GM at 250 μg / mL for KC treatment and morphological assessment. To assess perinuclear localization of EVs, the images show KC morphology under transmitted light (top row), low-magnification (20x) fluorescent images of KCs (with phalloidin (green) and Hoechst (blue) showing cell sheet formation in the 3D-EV-treated group), and high-magnification (60x) images of DiI-labeled EVs (red). Control KCs (left row) were compared with 2D-EV-treated (middle row) and 3D-EV-treated (right row). Scale bar for 20x images = 100 μm; scale bar for 60x images = 25 μm. [Figure 16] Morphological changes in KCs after treatment with molecular weight-stratified ASC-CM are shown. Representative morphology images of KCs after treatment with KC-GM supplemented with various ASC-CM molecular weight concentrations for 24 hours are shown. Only the "full" fraction and the "over 100 kDa" fraction induced similar morphological changes in KCs, including spindle cell formation, stratification, and collective cell sheet formation. KC morphological changes were observed at various scales in both 2D and 3D ASC-CM. Furthermore, nuclear area and circularity were assessed using CellProfiler. KCs treated with the "full" fraction or the "under 100 kDa" fraction exhibited nuclei with large surface areas and less circular shapes (0.5 on a 0-1 scale is equivalent to a circle). As a result, some degree of cell flattening, and therefore likely nuclear flattening, is likely. [Figure 17A] ~ [Figure 17D] Characterization of ASC phenotype and matrix coatings is shown. This is the characterization of the initial P1 population of ASCs. Figure 17A shows spindle-shaped / mesenchymal-like adherent cells with trilineage differentiation potential. Figure 17B shows positive CD surface marker expression of CD73 / 90 / 105 / 271 and negative CD34 / 45 expression (scale bar = 100 μm). Figure 17C shows a photograph of a 1 cm3 tissue-mimicking X-Block inserted into a 6-well culture vessel. The textured appearance of the hydrogel is the result of a porous architecture traversing the entire hydrogel. In Figure 17D, ASCs were cultured on 2D and 3D surfaces coated with type I collagen, fibronectin, and fibrin to assess phenotypic and morphological changes. [Figure 18]Fluorescence images of ASCs cultured within a 3D hydrogel system are shown. ASCs were seeded and cultured within the 3D hydrogel system, fixed with 4% PFA, and labeled with Hoechst (blue), phalloidin (green), or mitotracker (magenta). A low-magnification (4x) image of labeled ASCs within the hydrogel system (leftmost panel) is combined with a high-magnification (20x) image focused on a single pore structure within the hydrogel. Images were acquired using a Nikon AXR confocal imaging system, and z-stack reconstruction was performed. [Figure 19A] ~ [Figure 19E] Figure 19A shows that induced diabetic keratinocytes (idKCs) exhibited reduced epidermal regeneration activity. Based on previous literature, Figure 19A shows a schematic diagram of the process for inducing a diabetes-like phenotype in KCs. This process was performed in parallel with healthy KCs from the same donor to allow direct comparison of idKCs with KCs from the same donor. KCs at P1 were seeded in separate culture flasks, and treatment with 25 mM NaCl induced diabetes after 10 days. Figure 19B shows morphology images of healthy KCs and idKCs. There is a clear shift toward more elongated cell shapes in the idKC population (scale bar = 100 μm). The functional effects of diabetes induction on the epidermal activity of idKCs (greenish-blue bars) compared with that of healthy control KCs (black bars) were assessed by metabolic (Figure 19C), proliferation (Figure 19D), and migration (Figure 19E) changes in the idKC population. Representative images of scratch assays in KCs and idKCs at 24 hours are shown. The white line indicates the edge of the original wound. The yellow area highlights other areas that did not heal [scale bar = 50 μm]. Significance is indicated by *p<0.05 or ***p<0.001. [Figure 20A] ~ [Figure 20D]Figure 20 shows that matrix substrates alter ASC secretion of factors that regulate the functional activity of epidermal regeneration in idKCs. The effects of ASC-CM from coated 2D (silhouette) and 3D (pattern) systems (or uncoated controls) were assessed for their ability to regulate the metabolic activity (Figure 20A), proliferation activity (Figure 20B), and migration activity (Figure 20C) of idKCs. Functional activity data are shown as the change compared to baseline control idKCs cultured in keratinocyte growth medium (KC-GM). Migration data are shown as the percentage of area recovered. The dashed line indicates the idKC control. Figure 20D shows qRT-PCR analysis of CCND1, CDKN2A, IL1B, EGFR, FLG, and TWIST1. GAPDH was used as an internal control. Values ​​are expressed as fold change relative to baseline control idKC expression, indicated by the dashed line, using the ΔΔCt method. "NC" indicates no coating, "Col 1" indicates type I collagen, "Fn" indicates fibronectin, and "Fib" indicates fibrin. Significance is indicated by *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001. [Figure 21] Matrix-dependent regulation of the ASC secretome enhances the epidermal phenotype of idKCs. This shows qRT-PCR analysis of CDH1 (E-cadherin), CDH2 (N-cadherin), K5, K10, and K16. GAPDH was used as an internal control. Values ​​are expressed as fold change relative to baseline control idKCs, indicated by the dashed line, using the ΔΔCt method. "NC" indicates uncoated, "Col 1" indicates type I collagen, "Fn" indicates fibronectin, and "Fib" indicates fibrin. Significance is indicated by *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001. [Figure 22A] ~ [Figure 22D]These results demonstrate that matrix coating within the tissue-mimicking system enhances the relative secretion of regenerative compounds from ASCs. In Figure 22A, ASC-CM collected from each group was evaluated for total protein concentration using QuickDrop, BCA, and Bradford (Coomassie) assays. The figure shows the QuickDrop data. In Figure 22B, a total of 12 ELISAs were performed on ASC-CM samples. Only seven contained protein concentrations high enough to exceed the detection limit of the ELISA. Of those seven, five key factors are shown in the figure. The table in Figure 22C shows the relationship between the relative concentration (pg") of each protein tested and the total amount of protein secreted (μg"). An X" indicates that the sample was below the detection limit of the ELISA. In Figure 22D, ASCs were assessed for gene expression changes of key markers (28 selected) by wound healing qRT-PCR array (84 total targets) and displayed in a heat map. A fold change of 10 or more was indicated as the largest increase in fold change (dark blue). Selected markers are related to secretory activity from ASCs, and some were aligned with proteins of interest for ELISA. The endogenous control genes utilized by the array were GAPDH, ACTB, and B2M. "NC" indicates no coating, "Col 1" indicates type I collagen, "Fn" indicates fibronectin, and "Fib" indicates fibrin. Significance is indicated by *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001. [Figure 23A] ~ [Figure 23F]We demonstrate that ASCs exposed to type I collagen in 3D enhance epidermal regeneration in idKCs via secreted EVs. In Figure 23A, ASC-EVs were isolated from 2D (silhouette) and 3D (pattern) ASC-CM, and the relative concentration of EVs per sample was analyzed via protein content. In Figure 23B, the amount of EVs relative to total secreted protein was calculated to identify relative compositional changes in each group. idKCs treated with KC-GM containing 150 μg / ml EVs were evaluated for metabolic activity (Figure 23C), proliferation activity (Figure 23D), and migration activity (Figure 23E). In Figure 23F, qRT-PCR analysis of CCND1, CDKN2A, FLG, K5, K10, and K16 was performed to assess expression changes in idKCs. GAPDH was used as an internal control. Each value is expressed as a fold change relative to baseline control idKC expression, indicated by the dashed line, using the ΔΔCt method. "NC" indicates no coating, "Col 1" indicates type I collagen, "Fn" indicates fibronectin, and "Fib" indicates fibrin. Significance is indicated by *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001. [Figure 24] Quantification of soluble proteins in ASC-CM is shown. Twelve ELISAs were used to quantify specific protein compounds known to be natively important for epidermal regeneration, including EGF, HB-EGF, IGF-1, FGF-2, FGF-7 (KGF), TGF-β1, MMP-1, MMP-2, TIMP-1, TIMP-2, IL-1β, and IL-1Ra. Of these, only eight samples were above the detection limit, seven of which were above the detection limit for both 2D and 3D. MMP-9 was not detected in the 2D (leftmost column) samples but was detected in the 3D (rightmost column) samples. "NC" indicates uncoated, "Col 1" indicates type I collagen, "Fn" indicates fibronectin, and "Fib" indicates fibrin. Significance is indicated by *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001. [Figure 25A] ~ [Figure 25F] We demonstrate that ASCs exposed to type I collagen in 3D enhance epidermal regeneration in idKCs via secreted EVs. In Figure 25A, ASC-EVs were isolated from 2D (silhouette) and 3D (pattern) ASC-CM, and the relative concentration of EVs per sample was analyzed via protein content. In Figure 25B, the amount of EVs relative to total secreted protein was calculated to identify relative compositional changes in each group. idKCs treated with KC-GM containing 150 μg / ml EVs were evaluated for metabolic activity (Figure 25C), proliferation activity (Figure 25D), and migration activity (Figure 25E). In Figure 25F, qRT-PCR analysis of CCND1, CDKN2A, FLG, K5, K10, and K16 was performed to assess expression changes in idKCs. GAPDH was used as an internal control. Each value is expressed as a fold change relative to baseline control idKC expression, indicated by the dashed line, using the ΔΔCt method. "NC" indicates no coating, "Col 1" indicates type I collagen, "Fn" indicates fibronectin, and "Fib" indicates fibrin. Significance is indicated by *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001. [Figure 26] Characterization of the EV population within ASC-CM is shown. The isolated EV fraction was quantified by NTA to determine particle number corresponding to concentration. Significance is indicated by *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001. [Figure 27]Figure 1 shows the quantification of relative antioxidant activity within ASC-CM. ASC-CM was collected from the 2D culture system (silhouette) and the 3D culture system (pattern), and antioxidant activity was assessed using a total antioxidant capacity (TAC) assay kit. Briefly, antioxidant activity was assessed by assessing the reduction of copper(II) relative to copper(I), using the naturally occurring antioxidant uric acid as a control standard for this kit. Therefore, the antioxidant activity of ASC-CM was measured as "mM equivalents" of uric acid. DETAILED DESCRIPTION OF THE INVENTION

[0010] The present invention will now be described more fully hereinafter with reference to the accompanying drawings, in which some, but not all, embodiments of the invention are shown. Indeed, the invention may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will satisfy applicable legal requirements. As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0011] The disclosed invention generally relates to methods and / or platforms that can manipulate the component profile of a secretome composition of a cell type of interest (e.g., adipose-derived mesenchymal stem cells (ASCs)). In this regard, individual components of a secretome composition may include, for example, various growth factors, proteins, cytokines, extracellular vesicles (EVs), chemokines, hormones, digestive enzymes, antibodies, extracellular proteinases, morphogens, toxins, and antimicrobial peptides. The desire to manipulate the component profile of a secretome composition of a cell type of interest may be pursuant to a further aspect of the present invention, in which one or more components of the secretome composition component profile may be identified or characterized as having a particular therapeutic effect. In this regard, some embodiments of the present invention may mimic in vivo cellular secretion in an ex vivo environment and subsequent characterization of the resulting component profile of the secretome composition of a cell type of interest, and identification of one or more components believed, shown, or otherwise known to provide a therapeutic effect, thereby at least in part forming a target component profile of the secretome composition with increased and / or decreased amounts of one or more components and / or relative ratios between one or more components.

[0012] In this regard, some embodiments of the present invention may provide detailed protocols that can be developed using one or more tissue-mimicking three-dimensional (3D) hydrogel blocks (TMHBs) to externally reprogram how cells behave and which biological factors are secreted from cells. A TMHB may comprise a macrostructure defined by a continuous polymeric matrix material and a network of microporous channels and / or chambers containing a first cell type of interest extending throughout the continuous polymeric matrix material. According to some embodiments of the present invention, TMHBs may be interlocked with one another to provide a 3D continuous scaffold for cell proliferation and growth that more closely resembles the native environment of one or more specific cell types of interest. Interlocking TMHBs are sometimes referred to herein as interlocking porous hydrogel blocks (IPHBs). Some embodiments of the present invention enable the development of highly specific protocols that produce desired results for producing one or more target biological factors (e.g., components of a secretome composition). Such protocols can then be used to scale the production of desired biological factors (e.g., for therapeutic use). Additionally, some embodiments of the present invention allow for the development of protocols to specifically control the biological factors secreted by various cell types cultured in TMHB (e.g., for the production of therapeutics, diagnostic markers, and prognostic markers).

[0013] The TMHB comprises a hydrogel containing a network of microchannels and / or micropods that permeate the entire volume of the TMHB. The shape of the TMHB may be such that its macrostructure is a jigsaw puzzle piece or similarly shaped macrostructure that allows for the interlocking of TMHBs (e.g., IPHBs) utilizing male-female connectors. The IPHBs may interlock with each other similar to the way Lego blocks snap together to build larger structures. When IPHBs join together, the microchannels on their surfaces at least partially align, allowing for the free movement of fluids, gases, and cells between two or more IPHBs.

[0014] TMHBs are modular, whether interlocking or not. Microchannels can be made static or dynamic, contracting or expanding based on the media formulation applied to the TMHB. Hydrogels can be formulated to tailor various mechanical, physical, and chemical properties to suit different cell types, e.g., to mimic specific tissues. For example, the hydrogel formulation can be tailored to adjust stiffness and elasticity. Additionally, the shape and pattern of the microchannels can be adjusted to alter fluid flow and cell migration. Additionally or alternatively, coatings, peptides, and other growth factors can be applied to the hydrogel's internal surface (e.g., the interface between the continuous polymer matrix material and the network of microporous channels and / or chambers) to tailor the hydrogel's "stickiness" for cell adhesion (e.g., affinity for adhesion of a cell type of interest). Furthermore, IPHB-type TMHBs can be interconnected in various configurations to control and direct cell migration and fluid flow. For example, IPHBs can be joined in a linear configuration to promote linear cell growth, or cells can be organized in a lattice to promote the formation of a cellular network. IPHBs can be chained or networked in any horizontal or vertical configuration to fabricate custom microfluidic devices. Cells grow on the inner surface of the microchannel, and all biological factors secreted by the cells enter the intraluminal space. The culture medium can be easily aspirated, collected, and filtered from the microchannel to isolate exosomes. This process allows end users to easily collect exosomes or other biological factors for various applications. Because IPHBs can be joined in various configurations and types, the profile, quality, and quantity of biological factors secreted by target cells can be easily altered by modifying the microenvironment and stimuli applied to a single cell type or multiple cell types.In response to external stimuli and injury, cells secrete biological factors as a means of communication, which can be readily utilized in accordance with some embodiments to manipulate or tailor the component profile of the secretome composition of a cell type of interest.

[0015] As noted throughout, conjugating IPHB-type TMHBs together in multiple configurations allows for the alteration of the profile, quality, and quantity of biological factors (e.g., exosomes) secreted by cells cultured within the IPHB. Other known platforms are unable to control the quality and quantity of exosomes secreted, let alone modulate the exosome profile. Furthermore, many competing platforms lack the ability to be easily tailored to various cells or stimuli to induce changes in exosome secretion profiles. IPHB-type TMHBs offer numerous opportunities to manipulate how cells grow and behave within the IPHB platform. Conversely, all other platforms focus on promoting mass exosome production without considering the mechanical, physical, and chemical substrates on which exosome-producing cells are cultured. For example, most other platforms focus on creating media formulations that promote exosome production while ignoring the actual form of the substrate on which cells are cultured to produce MSCs.

[0016] In this regard, parameters of TMHB, whether IPHB type or not, that can be readily manipulated individually or in combination to produce a desired biological factor output include: the composition of the hydrogel (e.g., biological and / or synthetic material); the mechanical properties of the hydrogel (e.g., stiffness, elasticity, ductility, viscoelasticity, compression, tension, and torsion); the structure of the hydrogel (e.g., microchannel shape or cross-section (e.g., circular, triangular, square, polygonal), starting diameter size of the microchannel (prior to swelling or exposure to cells) (e.g., 200 microns, 300 microns, 400 microns, 500 microns, 600 microns, 700 microns, 800 microns, and any micron value between 200 and 800 microns), uniform starting diameter size of the microchannel (e.g., all microchannels are the same diameter), variable starting diameter size of the microchannel (e.g., microchannels may have different diameters), variable diameter pattern of the microchannel (e.g., microchannels Defined patterns of microchannels with different diameter sizes (e.g., the central microchannel and the microchannel most distal from the central microchannel can be the same diameter, while all other microchannels have different diameters), randomly variable microchannel diameters (e.g., microchannels with random diameter differences, with no discernible pattern of microchannels with the same diameter but different diameters), and microchannel spacing (e.g., diffusive and osmotic gradients can be manipulated by increasing or decreasing the spacing between microchannels within the hydrogel). Hydrogel swelling based on medium composition (e.g., basal medium salinity, basal medium pH, basal medium sugar content, basal medium protein content). And physical parameters of hydrogel swelling (e.g., liquid temperature, environmental pressure, environmental humidity). Additional parameters of TMHBs, whether IPHB type or not, that can be easily manipulated individually or in combination to produce a desired biological factor output include:Cellular response to media formulations (e.g., basal media, growth factor composition, presence of serum, presence of animal components, presence of human components, presence of antibiotics, presence of antifungals), and environmental stimuli (e.g., extracellular matrix coatings (animal, human, recombinant) on hydrogels, including, but not limited to, collagen, laminin, elastin, hyaluronic acid, gelatin, fibronectin, fibrin, heparan sulfate, tricalcium phosphate, and decellularized tissue). Additional parameters relevant to cellular response to media formulations include oxygen composition (hypoxic and normoxic), pH, temperature, salinity, electromagnetic field, presence of other cell types, static fluid, moving fluid (e.g., velocity of fluid moving through microchannels, waveform of fluid moving through microchannels, hydrostatic pressure, and duration fluid is moving), presence of toxins in the media, presence of viruses in the media, presence of bacteria in the media, presence of yeast in the media, and presence of fungi in the media. Additional parameters related to cellular response to a media formulation include modification of cells by physical action (e.g., exposure to a blunt or sharp object (e.g., a scalpel cutting through a hydrogel), chemical action (e.g., changes in pH and / or salinity and / or toxicity), mechanical action (e.g., compression, tension, and / or shear stress)). Additional parameters related to cellular response to a media formulation include modification of cells by biological action, including, but not limited to, introduction of one or more cell types with conjugated IPHB, introduction of growth factors into the media, introduction of DNA, mRNA, siRNA, shRNA, microRNA, introduction of exosomes and other biological factors, introduction of proteins, introduction of growth factors, introduction of enzymes, introduction of cytokines, and introduction of peptides. Additional parameters related to cellular response to a media formulation include modification of cells by electrical action (e.g., electroporation, acoustic action, and sonoporation).

[0017] In this regard, some embodiments of the present invention provide methods and / or platforms based on the ability to simultaneously control all desired parameters in a cell culture environment with TMHB, whether IPHB type or not.

[0018] Some embodiments of the present invention provide a method for characterizing a therapeutically desirable component or component profile of a secretome composition secreted from a cell type of interest, the method comprising the steps of: (i) seeding a first tissue-mimicking three-dimensional (3D) hydrogel block (TMHB) comprising a macrostructure defined by a continuous polymeric matrix material and a network of microporous channels and / or chambers extending throughout the continuous polymeric matrix material with a first cell type of interest; and (ii) simulating in vitro cell growth and proliferation of the first cell type of interest by providing the first cell type of interest with a first culture medium and allowing the first cell type of interest to proliferate throughout the first TMHB and secrete a secretome composition. (iv) collecting the secretome composition; (v) subjecting the secretome composition to a stratification operation based on molecular weight cutoff values ​​to provide a plurality of distinct layers, each characterized by a different range of component molecular weights; (vi) characterizing and / or identifying one or more individual components present with each layer of interest; and (v) testing each layer of interest for relative therapeutic utility by either in vivo or ex vivo analysis to identify the layer of interest that provides the greatest therapeutic utility as the therapeutic component profile.

[0019] According to some embodiments of the present invention, the method may include characterizing at least a majority (e.g., each) of the individual components present in the layer of interest that provides the greatest therapeutic benefit, and may also include identifying at least one frequently occurring component that is absent or present in low amounts in each layer of interest from the one or more individual components present in the layer of interest that provides the greatest therapeutic benefit, and identifying the at least one frequently occurring component as a therapeutically desirable component.

[0020] In another aspect, the present invention provides a method for tailoring a component profile of a natural secretome composition secreted from a cell type of interest, the method comprising the steps of: (i) seeding a first tissue-mimicking three-dimensional (3D) hydrogel block (TMHB) comprising a macrostructure defined by a continuous polymeric matrix material and a network of microporous channels and / or chambers extending throughout the continuous polymeric matrix material with a first cell type of interest; (ii) providing a first culture medium to the first cell type of interest; and (iii) culturing the first cell type of interest to more closely track a target component or target secretome composition having a target component profile (e.g., a therapeutically useful component or profile thereof that has already been identified). The method includes: (iv) introducing at least one external stimulus configured to modify the native secretome composition secreted from the target cell type, the at least one external stimulus comprising one or more controllable parameters, for example, adding a coating to the interface between the continuous polymer matrix material and the network of microporous channels and / or chambers to adjust the oxygen concentration, pH, temperature, and fluid dynamics within the first TMHB; (iv) allowing the first target cell type to proliferate throughout the first TMHB and secrete the secretome composition; and (iv) collecting the secretome composition modified by the external stimulus, which differs from the native secretome composition.

[0021] As described above, TMHBs can be of the IPHB type, and these TMHBs can be interlocked with each other to enable continuous 3D growth of various cells and / or tissues. Hydrogels are insoluble polymer matrices that can be designed to retain up to 96% water content by mass (e.g., up to 40, 50, 60, 70, 80, 90, and 95% water content by mass). Various polymers can be used individually or in combination to create unique hydrogels. By crosslinking the polymers with light, temperature shift, or chemical reaction, hydrogels can be tailored to exhibit different mechanical properties, diffusion gradients, osmotic gradients, chemical formulations, and structures (e.g., pores and fibers of various shapes and sizes). Hydrogels can also be degradable or non-degradable. Because of their multifunctional capabilities, hydrogels are used in a variety of applications, such as soft contact lenses, which provide optics to correct a patient's vision. Hydrogels have been used as dressings in wound care applications and as bioinks in life science applications to create unique structural scaffolds for microfluidic experiments or to provide substrates for culturing cells on or in.

[0022] According to some embodiments of the present invention, IPHBs may be joined or interlocked with one another via at least one interlocking male component and at least one interlocking female component. For example, at least one interlocking male component of a first IPHB is configured to be received into at least one corresponding interlocking female component of a second IPHB. In addition to the joining of IPHBs via their 3D macrostructure, the microstructure (e.g., voids) of a network of microporous channels and / or chambers may also be present. Most hydrogels are solid materials. However, by introducing voids and microchannels into hydrogels, it is possible to direct the movement of liquids, gases, and cells in order to expand the hydrogels and form a continuous matrix. This feature is particularly useful according to some embodiments of the present invention, because the microstructure (e.g., voids) of the network of microporous channels and / or chambers allows a second medium to be used to interlock IPHBs (e.g., hydrogels) to create a larger or expanded material for cell and / or tissue growth.

[0023] Hydrogels can be formed by cross-linking any synthetic polymer, biological polymer, tissue component (derived from humans, animals, plants, or combinations thereof), or combinations thereof, in the presence of water using a free radical-mediated reaction (e.g., photoreaction, chemical reaction) or a reaction as a result of a temperature change.

[0024] According to some embodiments of the present invention, IPHBs allow cells to grow in a more native physiological environment than culture in 2D plastic cell culture vessels. For example, IPHBs may be tailored or configured to mimic the native tissue environment in which specific cells develop and grow, which differs from plastic cell culture vessels and other, non-customizable, non-modular technologies. Furthermore, some embodiments of the present invention allow combinations of multiple similar or dissimilar IPHB (e.g., hydrogel) matrices to be bonded together to form an expanded, continuous hydrogel matrix for cell production and / or biopharmaceutical production. For example, similar or dissimilar hydrogels (e.g., IPHBs) may be bonded together to create unique and custom microenvironments for cell growth, which differs from other cell culture vessels or technologies. Furthermore, IPHBs may enable the formation of spheroids and organoids without the development of necrotic cores within the IPHB's microchannel network. For example, according to some embodiments of the present invention, IPHB allows spheroids and organoids to unfold and form sheets, tubes, cylinders, and other sophisticated structures that allow for uniform diffusion of nutrients and gases to cells within the IPHB (e.g., hydrogel). Advantageously, for example, IPHB allows for uniform exchange of nutrients and gases for healthy cell growth, which is different from other technologies characterized by mass cell production. According to some embodiments of the present invention, IPHB may be modified to accommodate a wide variety of cell types. According to some embodiments of the present invention, IPHB allows cells to secrete extracellular matrix to create a natural microenvironment that promotes cell growth, migration, viability, and function. Furthermore, IPHB advantageously eliminates the need for cell subculture. Furthermore, IPHB is easy to use because it may be provided in a preformed format and does not require extensive modification of temperature, pH, or chemical exposure for use.The IPHB may enable users to achieve, for example, one or more of the following: growing custom cell cultures, growing multiple cell types in parallel or sequentially, combining cell cultures to create complex tissues, mass-producing cells without ever stopping cell production, producing cells using the same substrate from benchtop to the end of clinical trials and for industrial production, using less media and consumables than current technologies, and reducing or eliminating human touchpoints in cell production, thereby reducing the risk of human error and contamination. According to some embodiments of the present invention, the IPHB provides a modular platform for any of the above applications.

[0025] The IPHB may include a three-dimensional (3D) macrostructure defined by a continuous polymer matrix material and a network of microporous channels and / or chambers extending throughout the continuous polymer matrix material. The 3D macrostructure may include a top surface, a bottom surface, and a thickness defined by at least one lateral edge extending from the top surface to the bottom surface, and the 3D macrostructure includes at least one interlocking male component and at least one interlocking female component. According to some embodiments of the present invention, at least one interlocking male component of a first IPHB is configured to be received by a corresponding at least one interlocking female component of a second IPHB.

[0026] For example, Figure 1 illustrates two separate IPHBs 1 according to some embodiments of the present invention. Each of these IPHBs includes a top surface 12, a bottom surface 14, and at least one side edge 16. The particular IPHB 1 illustrated in Figure 1 includes at least one interlocking male component 50 and at least one interlocking female component 60. Figure 2 illustrates three interlocked IPHBs 1 according to some embodiments of the present invention. Each IPHB 1 illustrated in Figure 2 includes a first interlocking male component 51, a second interlocking male component 52, a first interlocking female component 61, and a second interlocking female component 62.

[0027] 3 shows top and side views of an IPHB 1, according to some embodiments of the present invention, illustrating a continuous polymer matrix material 10 and a network of microporous channels and / or chambers 30 extending throughout the continuous polymer matrix material and exposed at the surface of the IPHB. As shown in FIG. 3, the seeded IPHB may allow cells 33 to grow and migrate three-dimensionally throughout the network of microporous channels and / or chambers 30.

[0028] According to some embodiments of the present invention, the at least one interlocking male component may include a first interlocking male component extending outward from at least one side edge. For example, the at least one side edge may include a first side edge and a second side edge, wherein the at least one interlocking male component includes a first interlocking male component extending outward from the first side edge and a second interlocking male component extending outward from the second side edge. The interlocking male component extending outward from the side edge may be configured, for example, to interlock or mate with a corresponding interlocking female component of another IPHB to form an expanding, continuous 3D scaffold system in which the interlocked or mate IPHBs expand outward in the xy plane. According to some embodiments of the present invention, the at least one interlocking male component may also include a third interlocking male component extending outward from the top surface. In this regard, the interlocking male components extending outward from the top surface are configured to interlock or join with corresponding interlocking female components located on the bottom surface of another IPHB, for example, to form an expanding, continuous 3D scaffold system in which the interlocked or joined IPHBs extend in the z-direction, perpendicular to the xy-plane. According to some embodiments of the present invention, for example, multiple IPHBs may be interlocked or joined with each other in the xy-plane and stacked together in the z-direction. As described above, the at least one interlocking female component may include a first interlocking female component extending inward from at least one side edge toward an interior portion of the 3D macrostructure. For example, the at least one side edge may include a third side edge and a fourth side edge, wherein the at least one interlocking female component includes a first interlocking female component extending inward from the third side edge toward an interior portion of the 3D macrostructure and a second interlocking female component extending inward from the fourth side edge toward an interior portion of the 3D macrostructure. In such an exemplary embodiment, the first side edge and the third side edge may define a first pair of opposing side edges, while the second side edge and the fourth side edge may define a second pair of opposing side edges.According to some embodiments of the present invention, as described above, the at least one interlocking female component may include a third interlocking female component extending inward from the bottom surface toward an interior portion of the 3D macrostructure. In this regard, multiple IPHBs may be interlocked or joined to one another in the xy plane and stacked together in the z direction.

[0029] According to some embodiments of the present invention, the interlocking feature of IPHBs allows for the custom formation of continuous 3D scaffolds for the growth of various cells and / or tissues, where the number of specific cells seeded and / or cultivated in the continuous 3D scaffold is not limited. Such flexibility in the relative positioning and interlocking of various IPHBs allows for the custom growth of multiple cell types, which may form complex interfaces between different cell types. For example, the interlocked IPHBs defining a continuous 3D scaffold (e.g., a continuous network of microporous channels and / or chambers extending throughout the interlocked IPHBs) may comprise 1 to 20 different cell types and / or tissue types grown simultaneously, e.g., at least about any of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 different cell types and / or tissue types grown simultaneously, and / or up to about any of 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, and 10 different cell types and / or tissue types grown simultaneously.

[0030] For example, Figure 4 illustrates an IPHB 1 according to some embodiments of the present invention, which includes an interlocking male component 53 on the top surface 12 and an interlocking female component 63 that protrudes into the bottom surface 14 of the IPHB. In this regard, the interlocking male component 53 on the top surface 12 and the interlocking female component 63 that protrudes into the bottom surface 14 of the IPHB may be configured to engage and interlock with another IPHB, as described herein.

[0031] According to some embodiments of the present invention, the 3D macrostructure other than the at least one interlocking male component and the at least one interlocking female component may define a cube, a square prism, or a triangular prism. According to some embodiments of the present invention, the 3D macrostructure other than the at least one interlocking male component and the at least one interlocking female component may define a polygonal prism having 3 to 12 side edges, e.g., at least about 3, 4, 5, 6, 7, and 8 side edges, and / or up to about 12, 11, 10, 9, and 8 side edges. According to some embodiments of the present invention, each side may include either an interlocking male component and / or an interlocking female component. Alternatively, some of the side edges may be free of interlocking male and female components.

[0032] According to some embodiments of the present invention, the at least one lateral edge includes a first lateral edge, a second lateral edge, and an arcuate lateral edge disposed between and adjacent the first lateral edge and the second lateral edge. For example, the first lateral edge may include at least one interlocking male component extending outward from the first lateral edge, and the second lateral edge may include at least one interlocking female component extending inward from the second lateral edge toward an interior portion of the 3D macrostructure. In this regard, each IPHB may define a pie-like shape that, when assembled or interlocked with one another, forms a circle (e.g., a cylinder if each IPHB has a thickness). Such a configuration of IPHBs may be desirable for use with circular culture wells. Additionally or alternatively, the at least one interlocking male component may include a second interlocking male component extending outward from the top surface, and / or the at least one interlocking female component may include a second interlocking female component extending inward from the bottom surface toward an interior portion of the 3D macrostructure. In this regard, multiple IPHBs may be stacked on top of each other in the z-direction to form a thicker cylinder or semi-cylinder. According to some embodiments of the present invention, the 3D macrostructure other than the at least one interlocking male component and the at least one interlocking female component may define a semi-cylinder, e.g., a one-eighth cylinder to a one-half cylinder, e.g., a one-eighth cylinder, a quarter cylinder, a one-third cylinder, or a half cylinder.

[0033] For example, FIG. 5 illustrates a semi-cylindrical IPHB 100 according to some embodiments of the present invention, which includes an interlocking male component 153 on the top surface 112 and an interlocking female component 163 that protrudes into the bottom surface 114 of the IPHB. The semi-cylindrical IPHB 100 illustrated in FIG. 5 includes a first interlocking male component 150 extending from the second side end 118 and a first interlocking female component 160 that protrudes into the second side end 116. The first side end 116 and second side end 118 of the IPHB 100 illustrated in FIG. 5 are joined by an arcuate side end 119 at one end of the IPHB. In this regard, the interlocking male component 153 on the top surface 112 and the interlocking female component 163 that protrudes into the bottom surface 114 of the IPHB may be configured to engage and interlock with another IPHB, as described herein.

[0034] According to some embodiments of the present invention, at least one interlocking male component may occupy or cover between about 5% and about 50%, e.g., at least about any of 10, 15, 20, and 25%, and / or up to about any of 50, 45, 40, 35, 30, and 25%, of the macroscopic surface area of ​​the surface (e.g., side edge, top, or bottom) from which it protrudes. Additionally or alternatively, at least one interlocking female component may occupy between about 5% and about 50%, e.g., at least about any of 10, 15, 20, and 25%, and / or up to about any of 50, 45, 40, 35, 30, and 25%, of the macroscopic surface area of ​​the surface (e.g., side edge, top, or bottom) from which it penetrates.

[0035] According to some embodiments of the present invention, the bottom surface may have a rougher texture than the top surface. For example, the top surface may be relatively smoother than the bottom surface, which may have a textured structure. For example, the textured structure of the bottom surface may facilitate the flow of culture medium or wash medium throughout the IPHB by providing structural spacers that facilitate the drainage of culture medium or wash medium from the IPHB. The textured surface of the bottom surface may include, for example, a plurality of small protrusions, e.g., individual bumps or ridges that function as short spacers. However, the plurality of small protrusions may be significantly smaller in size than the at least one interlocking male component, e.g., at most about one-tenth the size of the at least one interlocking male component. In this regard, these small protrusions may generally not have any interlocking function, according to some embodiments of the present invention.

[0036] According to some embodiments of the present invention, the macroscopic surface area that can be included on the top surface of the IPHB is about 0.25 cm 2 ~Approx. 25cm 2 For example, at least about 0.25, 0.5, 0.75, 1, 1.5, 2, 5, 8, 10, and 12 cm 2 and / or about 25, 22, 20, 18, 15, and 12 cm 2 Additionally or alternatively, the base may include a macroscopic surface area of ​​approximately 0.25 cm 2 ~Approx. 25cm 2 For example, at least about 0.25, 0.5, 0.75, 1, 1.5, 2, 5, 8, 10, and 12 cm 2 and / or about 25, 22, 20, 18, 15, and 12 cm 2 Additionally or alternatively, the thickness of the 3D macrostructure may be between about 0.5 cm and about 3 cm, such as at least about any of 0.5, 0.75, 1, 1.25, and 1.5 cm, and / or at most about any of 3, 2.5, 2, and 1.5 cm.

[0037] As described above, each of the at least one interlocking female component may be configured to receive at least one corresponding interlocking male component of a second IPHB. In this regard, multiple IPHBs may be joined or interlocked with one another in a sequential, additive manner to extend the 3D scaffold as needed along the xy plane and along the z direction. For example, cell growth can continue in the z direction on multiple interconnected IPHBs by stacking additional layers of IPHBs on top of the first layer of IPHB.

[0038] According to some embodiments of the present invention, the continuous polymeric matrix material may be non-degradable. In this regard, cells and / or tissues produced within the IPHB may need to be removed from the internal network of microporous channels and / or chambers for subsequent analysis, purification, or development. Additionally or alternatively, the continuous polymeric matrix material may be selectively degradable. For example, the hydrogel formulation may be made biodegradable, for example, by inserting enzyme-sensitive sequences or utilizing compounds derived from the native matrix. For example, the continuous polymeric matrix material may comprise a selectively degradable hydrogel material comprising one or more degradable polymers (e.g., one or more biopolymers derived from living organisms). The one or more biopolymers derived from living organisms may comprise, for example, polynucleotides, polysaccharides, polypeptides, or any combination thereof. According to some embodiments of the present invention, the one or more biopolymers may include collagen, gelatin, laminin, alginate, glycosaminoglycans, oligonucleotides (e.g., DNA, RNA), carbohydrates, lipids, cellulose, alginate, and proteins, which may be gently degradable, for example, by the use of protein-specific enzymes, ionic solvents, mild detergents, weak acids, or peroxides to break the biopolymer chains. According to some embodiments of the present invention, the one or more biopolymers may include degradable monomers, including esters (e.g., hydroxybutyrate, lactic acid, glycolic acid, and caprolactone), anhydrides (e.g., adipic acid and sebacic acid), sugars (e.g., cellulose, alginate, pectin, dextrin, chitosan, hyaluronic acid, chondroitin sulfate, and heparin), proteins, nucleotides (DNA, RNA), peptides (e.g., collagen, gelatin, silk, and fibrin), urethanes, phosphates, carbonates, and vinyl chlorides. According to some embodiments of the present invention, the selectively degradable hydrogel material may further comprise a synthetic polymer (eg, polyester, polyanhydride, polycarbonate, polyurethane, polyphosphate, or combinations thereof).The continuous polymer matrix material, according to some embodiments of the present invention, may comprise a 3D cross-linked polymer network, a non-cross-linked polymer network, or a combination thereof.

[0039] The continuous polymer matrix material may comprise a swellable hydrogel material, as described above. The swellable hydrogel material may comprise a radical-mediated reaction product of at least a first monomer comprising an acrylate or methacrylate functional group and a second monomer or oligomer comprising at least two free-radically polymerizable functional groups. For example, the at least two free-radically polymerizable functional groups may, independently of one another, comprise an acrylate or methacrylate group, an allyl group, an alkynyl group, a vinyl nitrile, a vinyl ether, a vinyl ester, a vinyl amide, a styrene group, a maleate group, a fumarate group, or a norbornene group. According to some embodiments of the present invention, at least one of the first monomer or the second monomer comprises a polyethylene glycol functionality (e.g., —O(C2H4O) n H) (wherein n has a value of 1 to 100), polypropylene glycol functionality (e.g., —O(C3H6O) nH), where n has a value of 1 to 100, and / or glycerol functionality incorporated into the backbone of the monomer and / or grafted onto the monomer as a side chain or side chain component. By way of example only, at least one of the first or second monomers may include 2-hydroxyethyl acrylate (HEA), poly(ethylene glycol) methyl ether acrylate (MPEGA), N-methylacetamide (NMA), or poly(ethylene glycol) diacrylate (PEGDA). Non-limiting examples of non-degradable monomers that may be utilized in hydrogel materials, according to some embodiments of the present invention, may include polyolefins (e.g., ethylene, propylene), styrenes, nylons (e.g., amides), and / or acrylics. According to some embodiments of the present invention, non-limiting examples of degradable monomers that can be used in hydrogel materials include esters (e.g., hydroxybutyrate, lactic acid, glycolic acid, and caprolactone), anhydrides (e.g., adipic acid and sebacic acid), sugars (e.g., cellulose, alginate, pectin, dextrin, chitosan, hyaluronic acid, chondroitin sulfate, heparin), proteins, nucleotides (e.g., DNA, RNA), peptides (e.g., collagen, gelatin, silk, fibrin), urethanes, phosphates, carbonates, and vinyl chloride. Additionally or alternatively, a third monomer, including a crosslinker, can be incorporated into the continuous polymer matrix material. Additionally or alternatively, the swellable hydrogel material can include one or more natural polymers, such as plant-derived polymers (e.g., cellulose polymers) and animal-derived polymers.

[0040] According to some embodiments of the present invention, the continuous polymeric matrix material may mimic a native tissue of interest (e.g., a mammalian tissue of interest) by comprising one or more physical properties of the native tissue of interest that are within about 20% (e.g., within about 15%, 10%, 8%, 5%, 3%, or 1%) of the native tissue of interest, the one or more physical properties of interest including softness, internal texture, porous configuration (e.g., microchannel size and geometric distribution), swellability, and tensile strength. For example, the one or more physical properties may include elastic modulus and / or compressibility, storage modulus at 1 Hz, loss modulus at 1 Hz, and / or protein / chemical coating (e.g., collagen types I, II, III, and IV; laminin types I and II; hyaluronic acid; gelatin; fibrin; fibronectin; etc.). By way of example only, native adipose tissue has a storage modulus of 50-100 kPa at 1 Hz, a loss modulus of 10-20 kPa at 1 Hz, and an elastic modulus and / or compressibility of 3 kPa. In this regard, for example, an IPHB has a storage modulus of approximately 110 kPa at 1 Hz, a loss modulus of approximately 22 kPa at 1 Hz, and an elastic modulus and / or compressibility of approximately 3 kPa. For example, by varying the specific chemical composition and / or degree of cross-linking, one or more physical and / or mechanical properties of the resulting continuous polymer matrix material can be tailored to mimic or reflect those properties associated with the native tissue of interest. Additionally or alternatively, the IPHB's network of microporous channels and / or chambers and / or outer surface topography / texture can be manipulated. While most of these surfaces may be smooth, grooves, bumps, ridges, depressions, and other surface irregularities may be introduced to alter the flow of liquids or gases through the network of microporous channels and / or chambers. Such surface irregularities can, for example, introduce flow turbulence that helps to slow the flow of liquids or gases throughout the network of microporous channels and / or chambers.By way of example only, the surface irregularities may be significantly smaller in size than the average diameter of the network of microporous channels and / or chambers, for example, up to about one-quarter to about one-tenth the size of the average diameter of the network of microporous channels and / or chambers.

[0041] According to some embodiments of the present invention, the continuous polymer matrix material is formed by additive manufacturing techniques (e.g., 3D printing or digital photosynthetic printing). In this regard, the network of microporous channels and / or chambers is constructed to mimic the morphology of the natural tissue of interest, for example, by varying the geometry and dimensions of the network of microporous channels and / or chambers to reflect the morphology of the natural tissue of interest. For example, the morphology of the natural tissue of interest can be readily determined by one skilled in the art, and this morphology can be duplicated by 3D printing or digital photosynthetic printing operations to form an IPHB having a network of microporous channels and / or chambers that mimics the morphology of the natural tissue of interest.

[0042] According to some embodiments of the present invention, the average diameter of the network of microporous channels and / or chambers may be from about 100 to about 800 microns, e.g., at least about any of 100, 120, 150, 180, 200, 220, and 250 microns, and / or at most about any of 800, 780, 750, 720, 700, 680, 650, 620, 600, 580, 550, 520, 500, 480, 450, 420, 400, 380, 350, 320, 300, 280, and 250 microns. Additionally or alternatively, the network of microporous channels and / or chambers may comprise at least about 40% by volume of the 3D macrostructure, for example, at least about any of 40, 50, 60, and 70% by volume of the 3D macrostructure, and / or up to about any of 90, 85, 80, 75, and 70% by volume of the 3D macrostructure.

[0043] According to some embodiments of the present invention, the interface between the network of microporous channels and / or chambers and the continuous polymer matrix material (whether the TMHB is an IPHB type or not) may include a coating of a compatibilizer selected to promote the attachment of primary cells of interest. This coating may be applied after formation of the IPHB. By way of example, the coating comprising a compatibilizer may include a biological coating, such as collagen type I (e.g., human mesenchymal stem cells [derived from adipose tissue, bone marrow, or umbilical cord], human neonatal dermal fibroblasts, human adult dermal fibroblasts, human keratinocytes, human myocytes, human osteoblasts, human bone cells, human chondrocytes, bovine myocytes, porcine hepatocytes, porcine chondrocytes, equine muscle-derived stem cells), laminin type I (e.g., human induced pluripotent stem cells, mouse dorsal root ganglia), hyaluronic acid (e.g., porcine hepatocytes, human adult dermal fibroblasts), gelatin (e.g., human mesenchymal stem cells [derived from adipose tissue, bone marrow, or umbilical cord], human neonatal dermal fibroblasts, human adult dermal fibroblasts), or the like. cells, human keratinocytes, human myocytes, human osteoblasts, human bone cells, human chondrocytes, human T cells (CD8+), human T cells (CD4+), human macrophages, bovine myocytes, porcine hepatocytes, porcine chondrocytes, porcine osteocytes, equine muscle-derived stem cells), fibrin (e.g., human keratinocytes), fibronectin (e.g., human mesenchymal stem cells [derived from adipose, bone marrow, umbilical cord], human neonatal dermal fibroblasts, human adult dermal fibroblasts, human keratinocytes, human osteoblasts, human osteocytes, human chondrocytes), or any combination thereof.

[0044] In another aspect, the present invention provides a scaffold system including a plurality of IPHBs (e.g., IPHBs described and illustrated herein). According to some embodiments of the present invention, for example, the plurality of IPHBs includes a first IPHB including a first interlocking male component and a second IPHB including a second interlocking female component, where the second interlocking female component is configured to receive the first interlocking male component. According to some embodiments of the present invention, for example, the first interlocking male component may be inserted into the second interlocking female component, in which case the first IPHB and the second IPHB are each provided in a swollen state, thereby enhancing interlocking between the first IPHB and the second IPHB. In this regard, the swollen state may be provided by the degree of absorption of a liquid (e.g., water or a culture medium). When the first and second IPHBs are swollen during the interlocking state, the frictional force between the mating interlocking male and female components increases, increasing the force required to separate the IPHBs.

[0045] According to some embodiments of the present invention, a first IPHB comprises a first network of microporous channels and / or chambers, and a second IPHB comprises a second network of microporous channels and / or chambers, and when the first and second IPHBs interlock to define a collective continuous network of microporous channels and / or chambers, a first portion of the first network of microporous channels and / or chambers at least partially overlaps a first portion of the second network of microporous channels and / or chambers. In this regard, the density of microporous channels at the surface of the IPHBs is sufficiently great, as described above, such that the overlap of at least a portion of the microporous channels at the surface of each IPHB allows the formation of a continuous collective continuous network of microporous channels and / or chambers extending throughout each interlockable IPHB. As described above, multiple IPHBs may be interlocked with each other along the xy plane and / or along the z direction.

[0046] According to some embodiments of the invention, a first IPHB may be seeded with a first primary cell and a second IPHB may be seeded with a second primary cell, the first primary cell being different from the second primary cell. As noted above, each IPHB may be seeded with distinct and / or unique primary cells, or may be seeded with a combination of multiple primary cells. Alternatively, for large-scale production of a given cell of interest, each IPHB may be seeded with the same primary cell.

[0047] By way of example only, one or more TMHBs (whether IPHB or not) can be seeded with any of the following to allow for cell growth: (1) human stem cells, such as human Wharton's jelly cells (MSCs), human bone marrow-derived mesenchymal stem cells (MSCs), human adipose-derived mesenchymal stem cells (MSCs), human skin-derived induced pluripotent stem cells (iPSCs), human blood cell-derived induced pluripotent stem cells (iPSCs), human CD4+ T cells, and human CD8+ T cells; (2) primary mammalian cells, such as HepG2 cells (liver cancer cells), human adult dermal fibroblasts (primary cells), human neonatal dermal fibroblasts (primary cells), human adult keratinocytes (primary cells), mouse dorsal root ganglia (primary neurons), bovine myocytes (primary cell line), primary porcine hepatocytes, porcine chondrocytes, porcine osteocytes, equine muscle-derived stem cells (primary MSCs), primary snail cells, and human macrophages. (3) Immortalized mammalian cell lines, such as UB-OC2 cells (mouse cochlear epithelium), human myoblastic leiomyoma (muscle tumor), PC3 (prostate cancer), CHO (Chinese hamster ovary cells), HEK293 (human embryonic kidney cells), SHSY5Y (neuronal tumor), PANC-1 (human pancreatic cancer), HeLa (cervical cancer), A549 (lung cancer), and A673 (muscle cancer), and (4) primary plant cells, such as rosemary, tobacco, and tomato.

[0048] 6 illustrates the migration of cells 33 from a first IPHB1 to a second IPHB2 when the first and second IPHBs are interlocked with each other, according to some embodiments of the present invention. As shown in FIG. 6, the first IPHB1 may be seeded with cells 33 and then mated with an empty (e.g., cell-free) second IPHB2. Once mated, cells 33 migrate from the first IPHB1 into the second IPHB2 until an equilibrium state is reached. After an equilibrium state is reached with both IPHBs, cells 33 continue to grow.

[0049] Example The present disclosure is further illustrated by the following examples, which should not be construed as limiting in any way, i.e., the specific features shown in the following examples are illustrative only and not limiting.

[0050] Example #1: Secretome stratification and tissue-mimicking systems reveal the crucial role of exosomes in enhancing epidermal regeneration Background and Overview Recent studies have demonstrated that the secretome of adipose-derived mesenchymal stem cells (ASCs) offers a unique approach to understanding and treating wounds, including the crucial process of re-epithelialization (epidermal regeneration), orchestrated by keratinocytes. However, 2D culture techniques significantly alter ASC secretory dynamics, leading to ambiguity in understanding which secreted compounds (e.g., growth factors, cytokines, exosomes, and ROS) may drive the re-epithelialization process. In this example, we utilize a tissue-mimicking 3D hydrogel system (e.g., the TMHB system) to enhance the retention of the regenerative ASC phenotype and provide an opportunity to highlight secretome differences between 2D and 3D cultures. Cultivation of ASCs in the tissue-mimicking system enhanced protein secretion by up to 50%. Subsequently, stratification of the ASC-secretome by molecular weight and the presence or absence of extracellular vesicles (EVs) was performed. This approach revealed the following: The ability of the ASC-secretome to regulate re-epithelialization functions, including migration, proliferation, differentiation, and morphology, was found within the ">100 kDa" fraction, with 3D ASC-secretome providing the greatest improvement. ASC-EV secretion was enhanced two-fold in 3D and demonstrated dose-dependent effects on several key re-epithelialization functions. Notably, ASC-EVs induced significant morphological changes in keratinocytes, resembling natural wound healing. This included the formation of stratified cell sheets. However, only 3D-EVs promoted collective migration of cell sheets, whereas 2D-EVs contained anti-migration stimuli. Similarly, 3D-EVs provided more effective stimuli for epithelial-to-mesenchymal migration in keratinocytes. This example demonstrates how the critical culture environment influences the regenerative potential of the ASC-secretome. Furthermore, we reveal the important role of EVs in regulating epidermal regeneration and discuss their translatability for future clinical therapies.

[0051] 1. Introduction Skin is the largest organ in the human body, providing a critical external barrier to help prevent dryness and infection, while also providing protection from mechanical, UV, and physical damage. Skin repair and regeneration is a dynamic series of events that occur in the context of tissue injury, requiring a diverse array of interrelated cell populations that communicate through mechanical, physical, and biochemical cues. Paracrine compounds secreted by resident cell populations play a critical role in orchestrating appropriate cellular and molecular signaling pathways during the wound healing process. Ultimately, the goals of physiological wound healing are to promote appropriate cell migration and proliferation into the wound environment, stimulate neotissue formation through deposition / remodeling of extracellular matrix (ECM) components, and regulate the biophysical and biomechanical structure of cells (e.g., cytoskeletal reorganization to promote migration and / or wound contraction).

[0052] During physiological wound healing, cells undergo phenotypic transformation to enhance their regenerative capacity in order to restore anatomical homeostasis. In particular, a fundamental step in proper wound healing is adequate wound "closure," also known as re-epithelialization, which helps protect the wound and deeper tissue structures. The re-epithelialization process is carried out by epidermal keratinocytes, and insufficient keratinocyte activity results in prolonged wound closure and an increased risk of adverse outcomes (e.g., chronic wound development, the need for surgical intervention or amputation, and overall worsening of the patient's morbidity). Proper re-epithelialization of keratinocytes depends on keratinocytes regulating their own cytoskeletal and junctional proteins to enhance migration and / or proliferation. Paracrine activity from surrounding cell populations in the epidermis, dermis, hair follicles, and subcutaneous tissue plays a key role in orchestrating this phenotypic transformation of keratinocytes. However, there remains a critical need to develop therapies aimed at enhancing keratinocyte functionality suitable for reducing the progression of chronic wounds.

[0053] Recent research into stem cell-derived therapies has emerged as a potential avenue for future regenerative treatments for wounds. More specifically, mesenchymal stem / stromal cells (MSCs) are multipotent progenitor cells found in various tissue sources within the human body (including the dermis, fat, and hair follicles) that contain inherent regenerative capabilities due to the compositional plasticity of their secretory profiles. Recent studies have shown that MSCs may exhibit diverse therapeutic potential to modulate several tissue regeneration processes and enhance wound healing. In particular, recent studies suggest that the adaptive secretory properties of MSCs, rather than their multipotency, may drive many of the regenerative effects seen in previous in vitro and in vivo studies. Thus, MSC-derived acellular by-products offer a cell-free alternative to current cell-based therapies and have demonstrated the ability to modulate wound healing activities, including enhancing the reepithelialization activity of keratinocytes.

[0054] Interestingly, previous clinical studies investigating the utility of biological compounds such as growth factors and cytokines have shown mixed results. However, this may be due to the more complex nature of wounds (especially chronic wounds), which require a heterogeneous environment in which factors balance each other, rather than the presence or absence of a single compound (e.g., a single growth factor). Furthermore, growing evidence suggests that extracellular vesicles (EVs) (e.g., exosomes) are more stable and may play an important role in native tissue signaling and regeneration. EVs consist of vesicle-like particles released from cells and include apoptotic bodies, microvesicles (MVs), and exosomes. EV activity and quality depend on the dynamic array of bioregulatory compounds present within them, which can change depending on external stimuli and cell phenotype. In particular, the secretome of adipose-derived MSCs (ASCs) has demonstrated the ability to enhance the rate of wound healing and tissue regeneration in vivo. However, to date, it remains unclear which fractions of MSC-derived secreted factors drive which regenerative wound healing activities (e.g., enhanced keratinocyte migration and proliferation activity observed in previous studies). This is likely due, in part, to the inconsistent and inefficient use of standard 2D culture modalities. As a result, the phenotype and regenerative capacity of MSC populations are inconsistent, and the nature of the MSC secretome is complex and diverse. Therefore, investigation of more robust tissue-mimicking 3D systems (e.g., TMHB) may provide a platform for standardizing MSC culture, generate more rigorous data related to the regenerative mechanisms of MSC-derived biopharmaceuticals, and offer opportunities to enhance and / or tailor their regenerative potential.

[0055] It is important to understand that the microenvironment of our body's native tissues provides a range of biomechanical and biophysical cues that regulate the fate and behavior of cells within the environmental niche. Native 3D tissue environments typically exhibit a range of viscoelasticity, which influences mechanotransductive and biochemical pathways. MSCs have been shown to strongly rely on their 3D interactions, including the role of focal adhesion sites, cell-cell interfaces, applied forces, and matrix stiffness, to retain their "stem-like" phenotype and regenerative capacity. Thus, the native MSC niche is more accurately represented by 3D in vitro systems than 2D, thereby enhancing the efficacy and reproducibility of future regenerative therapies and providing a better understanding of physiological signaling. In particular, most current 3D hydrogel systems (e.g., TMHB systems) rely on manipulating the dense crosslinked network to modulate the hydrogel's mechanical properties, which significantly limits the ability of molecules to easily diffuse throughout the system. In contrast, the novel microarchitecture-designed 3D hydrogel systems in this study (e.g., the TMHB system) acted as "pseudovasculature" conduits and did not impede mass transport like conventional injected / molded hydrogel systems, thereby improving the collection of secreted by-products in addition to sufficient nutrient exchange within the hydrogel and preventing focal necrosis.

[0056] Barriers associated with the translatability of cell-based regenerative therapies (e.g., the depletion of regenerative potential of unhealthy autologous cell populations and inadequate ex vivo expansion systems) have led to increased interest in non-cellular regenerative therapies. While data on MSC-derived acellular products are promising, most previous experiments using MSCs in wound healing have utilized standard 2D culture techniques, which are known to induce differentiation and senescence. Thus, traditional 2D culture modalities can result in heterogeneous and less regenerative MSC populations, leading to impurities and / or inconsistent secreted products, which subsequently limit the potential clinical utility of MSC therapies. Consequently, there is growing interest in developing more efficient 3D expansion systems.

[0057] In this study, we utilize a tissue-mimetic 3D hydrogel system (e.g., the TMHB system) that mechanically resembles native adipose tissue for culturing ASCs (Table 1 below). [Table 1]

[0058] We previously demonstrated the utility of this novel system in protecting ASC populations from senescence and loss of a "stem-like" phenotype, resulting in a more regenerative cell population capable of regulating wound healing activity. An additional benefit of this system is its unique microarchitecture design, which does not impede mass transport and allows efficient collection of secreted by-products. Thus, the system operates like a "bioreactor" that generates biological by-products from the cell population. In this study, we stratify ASC-conditioned medium (ASC-CM) based on molecular weight cutoff and EV / exosome particle content to compare and identify which fractions of ASC-CM drive specific wound healing activities previously observed in keratinocytes. Uniquely, we compare and contrast the effects of 2D and 3D ASC-CM. We hypothesize that the culture conditions of our tissue-mimicking system will provide a more effective measure of how ASCs respond to native responses and reveal functional differences between 2D and 3D ASC-CMs. Furthermore, we question previous studies suggesting that the exosome fraction of "stem-like" cell populations (such as ASCs) is essential for driving many of the functional benefits seen in keratinocyte wound healing activity.

[0059] 2. Materials and Methods 2.1. Cell culture This study utilized human adipose-derived mesenchymal stem cells (ASCs) (Lonza, Lot #18TL212639, 23-year-old female, Black) and human keratinocytes (KCs) (Lonza, Lot #18TL318559, 62-year-old male, Caucasian). ASCs were cultured in RoosterNourish MSC-XF (RoosterBio, Cat. #KT-016) as growth medium (MSC-GM) and then switched to RoosterCollect EV-Pro (RoosterBio, Cat. #K41001) as serum-free, low-particulate medium. DermaLife K Keratinocyte Medium Complete Kit (Lifeline Cell Technologies, Maryland, USA, #LL-0007) was used for KC culture.

[0060] 2.2. Three-dimensional (3D) printed hydrogel cell culture system 3D hydrogel systems (e.g., TMHB systems) can be up to 1 cm 3The bioprinted cell culture and expansion system, called X-Block (Ronawk, Kansas, USA), contains unique macroarchitecture and microarchitecture designs that enable mass transport and nutrient exchange. The hydrogel was printed using specific predefined microstructures, which created hollow / porous regions that created continuous microchannels with a diameter of 300 lm. The microporous components account for 44% of the hydrogel's total volume. Furthermore, the integrated microarchitecture design significantly increases the volume-to-surface area ratio, enhancing cell proliferation and migration. X-Block hydrogel (Ronawk Inc.) is fabricated using a proprietary blend of biodegradable substrates containing biologically native binding epitopes for cell adhesion. For culture, the 3D hydrogel was placed in a glass 6-well culture plate. Cells were then dropped onto the hydrogel surface and allowed to migrate into the microarchitecture for 15 minutes, after which the hydrogel was submerged in culture medium. To consider any potential role of substrate effects in 3D, a thin coating of bioink was utilized as a 2D culture control.

[0061] 2.3. Mechanical properties of tissue-mimicking hydrogels A subset of 3D hydrogels was bioprinted at a z-height (thickness) of 1.2 mm, while retaining all other dimensional and structural characteristics of this unique architectural design of the full-size hydrogel. The hydrogels were analyzed using a Dynamic Mechanical Analyzer (RSA3, TA Instruments) set up to assess mechanical and viscoelastic properties (Table 1). A 5 mm biopsy punch was used to isolate the circular hydrogel samples to prevent force concentration points. Two 5 mm punches were obtained from each hydrogel, and a total of four hydrogels were evaluated over a total of eight runs (n ​​= 4). In addition to frequency sweep analysis over the range of 0.1 to 10 Hz, dynamic mechanical analysis (DMA) was performed using dynamic cylindrical compression analysis. The compression speed was 0.005 mm / s. Frequency sweep analysis helps evaluate the viscoelastic properties of a given material by identifying the relationship between a given compression frequency range and the storage modulus (E0) and loss modulus (E00) of the material. The compression frequency is increased stepwise, and the viscoelastic response at each given frequency is recorded. The onset of viscoelastic modulus at 1 Hz is generally observed due to the similarity of this frequency range to most soft tissues in physiological conditions, including human fat.

[0062] 2.4. Expansion of ASC and KC "Passage 1 (P1)" ASCs and KCs were seeded onto 2D plastic and cultured to a maximum of 80% confluency before subculturing (i.e., passage). Cell subculturing was performed by removing the medium, washing three times, and incubating with 0.05% Trypsin / EDTA (Lonza, Cat. #CC-3232) for 5 minutes at 37°C. The trypsin was neutralized with serum-based medium, and the cells were centrifuged at 500g for 5 minutes, pelleted, and resuspended for reseeding onto new 2D tissue culture plastic vessels or for use in experimental assays. Because the increased surface area of ​​a single 3D hydrogel made subculturing unnecessary during the time course of this study (see details in previous studies), cells in 2D and 3D were cultured at a maximum of 1500 cells / cm to allow for similar comparisons between 2D and 3D cultures. 2 For experimental assays, KCs were seeded at the same seeding density of up to 7500 cells / cm. 2 After initial characterization of ASCs in P1, ASCs subcultured in P2 were seeded in 3D or re-plated in 2D and cultured for 1 week before analysis or subculture. 2D ASCs subcultured for 1 week were re-plated for an additional week to obtain data for P3 (2 weeks). Meanwhile, 3D ASCs were allowed to be continuously cultured in the 3D system without subculture (P3 equivalent / 2 routines) (n=4).

[0063] 2.5. Assessment of ASC phenotype Initial assessment of the ASC (at P1) trilineage differentiation potential (adipogenic, chondrogenic, and osteogenic) was performed by culturing in differentiation medium according to the manufacturer's protocol, as previously described. Adipogenic differentiation was performed using the hMSC Adipogenic Differentiation BulletKit™ (Lonza, Cat. #PT-3004). Chondrogenic differentiation was performed using the hMSC Chondrogenic Differentiation Medium BulletKit™ (Lonza, Cat. #PT-3003) supplemented with TGF-β3 (Lonza, PT-4124) at a concentration of 10 ng / mL. Osteogenic differentiation was performed using the hMSC Osteogenic Differentiation Medium BulletKit™ (Lonza, Cat. #PT-3002). Unless otherwise noted, all primary antibodies were obtained from Abcam (Cambridge, UK). The initial cell population at P1 was assessed for ASC "stem-like" phenotype. This was performed by positive immunolabeling for CD73 / 90 / 105, as previously described. Briefly, ASCs at P1 were seeded in 2D, fixed with 4% paraformaldehyde, washed three times, blocked with 1% donkey serum, and immunolabeled for CD34 (ab81289), CD45 (ab40763), CD90 (ab181469), and CD105 (ab231774). CD73 (Cat. #41-0200) was obtained from Invitrogen (Waltham, MA). Cells were counterstained with immunofluorescent nuclear markers: Hoechst 33342 (Invitrogen, Cat. #H3570) and, in some samples, Alexa Fluor 488 Phalloidin (ThermoFisher, Cat. #A12379). Furthermore, ASCs from P1 (baseline control), P2 (1 week), and P3 (2 weeks) as 2D cultures and P2 ASCs from 3D cultures cultured for 1 week (P2 equivalent) or 2 weeks (P3 equivalent) were cultured at RT. 2The expression of 84 MSC- and MSC-associated genes was assessed using the Profiler™ PCR Array for Human Mesenchymal Stem Cells (Qiagen, Cat. #330231, PAHS-082ZC-24). RNA was isolated and purified using the RNeasy Mini Kit (Qiagen). Only RNA with a 260 / 280 ratio greater than 1.8 was used in this study. Cycle threshold values ​​(Ct values) were recorded and analyzed using the ΔΔCt method. Endogenous control genes utilized in the array (n = 3) were glyceraldehyde-3-phosphate dehydrogenase (GAPDH), beta-actin (ACTB), and beta-2-microglobulin (B2M).

[0064] 2.6. Functional characterization of cells by plate reader assay Both ASCs and KCs were assessed using a plate reader assay. The assay was performed according to the manufacturer's instructions, as previously described. Briefly, plated cells were analyzed for PicoGreen fluorescence at 435 / 535 nm (n=4) to quantify DNA as a surrogate measure of proliferation. Prestoblue fluorescence was acquired at 560 / 590 nm (n=4) and expressed as the mean relative fluorescence units (RFU) of Prestoblue per Hoechst signal (350 / 460 nm) to obtain approximate metabolic activity per cell. Similarly, Mitotracker™ Red CMXRos (Invitrogen, Cat. #M7512, 500 nM) fluorescence at 570 / 605 nm (n=4) was used to assess mitochondrial activity / membrane potential. Finally, the population doubling rate (PDR) of ASCs extracted from 2D or 3D and then re-plated in 2D (n=5) was assessed by taking the PicoGreen cell counts on days 1 and 4 and using the formula [(t) / ((log(n / n)) × 3.32], where t=time, n=cell number at the final time point, and n=initial cell number.

[0065] 2.7. Assessment of ASC proliferation and viability in 2D and 3D To assess ASC proliferation in the hydrogel system, 2D and 3D systems were seeded at the same seeding density and cell number was assessed at 1 or 2 weeks. For 2D samples at 2 weeks, ASCs underwent one additional passage event. At each time point, ASCs were detached (2D) or isolated by hydrogel degradation (3D) and replated overnight (up to 16 hours) in 2D cultures and assessed for readhesion, cell number (PicoGreen), metabolic activity (PrestoBlue), and mitochondrial activity (MitoTracker).

[0066] 2.8. Isolation of ASC-conditioned medium When conditioned medium (CM) from ASC cultures was required, the MSC-GM medium was removed, the cells were washed three times, and serum-free MSC medium was added for another 24-hour wash. After the 24-hour wash, fresh serum-free medium was added, followed by three consecutive days of collection at 24-hour intervals between days 6 and 8. Due to variability in proliferation rates between 2D and 3D, to standardize medium consumption, collection days were based on pre-determined cell proliferation data to establish the medium / cell ratio. 2D confluency ranged from 60 to 80%. ASC-CM was collected only from the 1-week time point, before any additional subculture events in 2D were performed. Collected ASC-CM was centrifuged at 1500×g for 10 minutes to remove cell debris, filtered through a 0.22 μm filter using a Steriflip filter, and stored at -80°C for long-term storage until use. To ensure sufficient volume for all experiments and to completely eliminate potential variability between ASC-CM from different days, ASC-CM from each day (day 6, 7, or 8) was pooled for each replicate (n=4) to create a "batch" mixture.

[0067] 2.9. Stratification of ASC-CM into molecular weight fractions The collected ASC-CM was subjected to a series of centrifugal filtrations using filters with molecular weight cutoffs of 100 kDa, 30 kDa, 10 kDa, and 3 kDa, according to the manufacturer's instructions. Each centrifugation step was performed at 4000 G for 30-45 minutes. The concentrate in the upper chamber was saved, and the filtrate in the lower chamber was collected and used for the next molecular weight filtration / centrifugation step. This process was repeated until all the retentate was less than 3 kDa. This resulted in ASC-CM concentrates (up to 50-100-fold) for each molecular weight range.

[0068] 2.10. ASC-CM Protein Quantification ASC-CM samples were quantified by QuickDrop absorbance at 280 nm, BCA, and Bradford (Coomassie) total protein analysis. Samples were then used for downstream analysis by protein quantification. Protein quantification was performed using the Pierce™ BCA Protein Assay Kit (Invitrogen, Cat. #23225), Pierce™ Coomassie "Bradford" Protein Assay Kit (Invitrogen, Cat. #23200), and QuickDrop (Molecular Devices, SpectraMax QuickDrop Micro-Volume Spectrophotometer) by absorbance at 280 nm. Relative protein content was calculated and back-calculated to determine the relative concentration in the medium prior to the molecular weight concentration step (e.g., concentrating 10 mL of medium to 500 μL resulted in a 20-fold increase in concentration). The relative protein content of each molecular weight fraction was calculated relative to the "full" ASC-CM before stratification. Assays were performed in technical and biological replicates (n=4).

[0069] 2.11. ASC-CM extracellular vesicle (EV) production EVs were isolated by centrifuging the ASC-CM through a 100 kDa centrifugal filter (as described above) at 4000 g for 30 minutes, followed by washing with PBS and recentrifuging through a 100 kDa filter at 4000 g for 5 minutes, for a total of three washes. EVs were precipitated overnight from the remaining ">100 kDa" concentrate using an ExoQuick-TC kit (SBI, Cat. #EXOTC10A-1) according to the manufacturer's instructions. EVs were resuspended in PBS, and an aliquot was removed and used to quantify protein content. This was performed as an indirect measure of EV content using QuickDrop, the BCA method, and the Bradford (Coomassie) assay. Relative EV protein fractions were compared to the total protein content in the ">100 kDa" and "full" ASC-CM fractions. Furthermore, the purified EV samples were then evaluated by Nanoparticle Tracking Analysis (NTA, Malvern Panalytical, Nanosight LM10) to further calculate both the concentration and size distribution of particles extracted from ASC-CM along with exosomes, which typically range from 25 to 250 nm. Unused (unexposed to cells) serum-free ASC medium was subjected to the same treatment and used to establish / calculate baseline EV / particle levels, which were negligible. Because the number of EV particles in the serum-free control ASC medium was negligible, full workup was not necessary; only comparative analysis between 2D and 3D was adequately performed.

[0070] 2.12. Functional activity of KCs after ASC-CM treatment ASC-CM was used as a "supplement" to keratinocyte growth medium (KC-GM) and administered at a 2:1 ratio based on the initial volume of ASC-CM (i.e., 20 mL of ASC-CM was concentrated to 200 μl and added to 10 mL of KC-GM). KCs were plated in 2D and allowed to acclimate and reach the desired confluency (>24 h). KC-GM was then removed, cells were washed, and ASC-CM was applied for 24 h. Experimental assays for metabolic, mitochondrial, proliferative, or migratory activity were then performed according to the manufacturer's protocol. Metabolic activity was assessed using Presto Blue (as described above). Proliferative activity was assessed using PicoGreen (as described above). Mitotracker was used to assess mitochondrial activity (as described above). Migratory activity was assessed using a scratch "wound" assay. KC scratch assays were performed to evaluate changes in wound size / area. This was done as a surrogate measure of KC migration after wounding within a confluent monolayer of KCs (n = 4). Images of migration were acquired using an ImageXpress Micro XLS Imaging System (Molecular Devices), and the percentage of closed wound area at 24 hours was calculated by ImageJ analysis.

[0071] 2.13. KC Aging Senescence of KCs was performed by immunofluorescent labeling of β-galactosidase activity using the CellEvent™ Senescence Green Detection Kit (Invitrogen, Cat. #C10850) according to the manufacturer's protocol. KCs from P2 or P3 were used for this study. KC-GM was removed from the KC culture and supplemented with ASC-CM stratification concentrate for 24 hours. KCs in KC-GM were used as a baseline measurement of senescence. After 24 hours, KCs were fixed and stained. Hoechst 33342 was used as a counterstain to identify nuclei. Senescence characterization was performed in quadruplicate (n=4). Five fields of view were imaged per biological replicate, resulting in a total of 20 measurements per sample.

[0072] 2.14. Protein expression of KCs Immunolabeling of cells was used for both ASCs and KCs. Briefly, cells were washed three times with HBSS, fixed with 4% PFA, and washed three times with HBSS. Cells were incubated for at least 1 hour in blocking buffer, which consisted of 2% donkey serum in HBSS with or without 0.1% Triton-X. Cells were then incubated with primary antibodies overnight at 4°C. Unless otherwise noted, all primary antibodies were obtained from Abcam and included vimentin (ab8978), keratin 16 (ab76416), keratin 10 (ab76318), and keratin 5 (ab52635). The following day, cells were washed three times with blocking buffer, then secondary antibodies were applied for 1 hour, followed by another three washes with HBSS. Cells were counterstained with immunofluorescent nuclear markers. The marker used was Hoechst 33342, and in some samples, Alexa Fluor 488 Phalloidin. The secondary antibody was donkey-derived and procured from Invitrogen (n=3).

[0073] Western blotting was performed as previously described. Briefly, cell lysates were prepared using RIPA buffer with a protease inhibitor cocktail (Cat. #P8340). Protein concentrations were obtained by absorbance at 280 nm using a QuickDrop filter, analyzed by SDS-PAGE with proteins running on a 4-12% NuPAGE Bis-Tris gel (Invitrogen, Cat. #NP0321BOX), transferred to a PVDF membrane, and immunoblotted. All primary antibodies were sourced from Abcam and included vimentin, CD9 (ab263019), CD63 (ab134045), CD81 (ab109201), Tsg101 (ab125011), GAPDH (ab8245), β-tubulin (ab6046), keratin 16, keratin 10, and keratin 5. The secondary antibody was from donkey, and the blocking buffer, sourced from Invitrogen, consisted of 2% donkey serum in HBSS.

[0074] 2.15. Gene expression in KCs RNA was isolated and purified as described above. Briefly, the RNeasy Mini Kit (Qiagen) was used according to the manufacturer's instructions. Untreated KCs cultured in KC-GM served as a control for KC analysis. The purity of cDNA samples was assessed using a QuickDrop spectrophotometer (Molecular Devices). A 260 / 280 absorbance ratio of >1.8 was considered pure. RT-qPCR was performed for CDKN2A, CTNNB1, CDH1, CDH2, FLG, K10, K16, K5, TWIST1, VIM, and CCND1 using individual qPCR primers (Qiagen, Cat. #330001). GAPDH was also used as an endogenous control for these samples. A complete list of GeneGlobe IDs is shown in Table 2. [Table 2]

[0075] 2.16. In vitro tracking of EVs EVs previously isolated from ASC-CM were fluorescently labeled with the lipophilic membrane dye DiI (Invitrogen, Cat. #D282) at a concentration of 1 μM. The labeled EVs were washed with PBS and recentrifuged through a 100 kDa filter at 3500 g for 15 minutes to remove excess dye. DiI-labeled EVs were added to KC-GM, which was then plated onto KCs for 24 hours. The medium was removed, and KCs were washed twice, fixed, counterstained with Hoechst 33342 and Alexa Fluor 488 Phalloidin, and observed under a fluorescent microscope.

[0076] 2.17. Administration of EVs for comparison of 2D and 3D quality Next, EVs isolated from 2D and 3D samples were resuspended at known concentrations and added to KC-GM to achieve final EV protein concentrations of 5, 25, or 250 μg / mL in KC-GM. EV concentrations were calculated based on the average of all three protein assays (QuickDrop, BCA, and Bradford) (n=4).

[0077] 2.18. Imaging Analysis Image processing and analysis were performed with ImageJ and CellProfiler. All sets of images and image analysis were automated with a standardized pipeline to ensure uniform processing across all similar image sets.

[0078] 2.19. Statistical analysis All data were reported as means with standard errors of the means (sem). ASC population characterization analyses for metabolic, functional, and phenotypic data were evaluated using one-way ANOVA. All secretome stratification data were analyzed using two-way ANOVA. Extracellular vesicle (exosome) characterization was assessed using an unpaired Student's t-test. Exosome administration studies were further evaluated using two-way ANOVA. Unless otherwise noted, a minimum of four replicates (n = 4) were used. Data were tested for normality using the Shapiro-Wilk test and the Kolmogorov-Smirnov test and plotted using a QQ plot. Analysis was performed using GraphPad Prism 9.4.2 software (La Jolla, CA), with p < 0.05 considered significant. Images were processed using CellProfiler™ and ImageJ.

[0079] 3. Results 3.1. Evaluating the role of mesenchymal stem cells in wound healing 3.1.1. Characterization of ASC populations According to the International Society for Cellular Therapy, the minimum requirements for a cell population to be considered MSC are that it is adherent (Figure 7A), exhibits trilineage differentiation potential (Figure 7B), and expresses MSC "stem-like" surface markers, including positive staining for CD73 / 90 / D105 (Figure 7B). Similarly, ASCs were subsequently cultured in 2D or 3D for 1 week and assessed for MSC phenotypic gene expression relative to the initial ASC population (Figure 7C). ASCs in 3D culture (Figure 7D) showed stronger retention of key MSC and MSC-like markers, while cells in 2D showed a significant reduction (Figure 7C). Furthermore, an in situ population doubling experiment was performed to assess the relative cell numbers in 2D and 3D and determine the optimal ASC-CM collection for future experiments (Figure 7E). Based on previous literature, ASC-CM was collected when 2D ASCs showed 60–80% confluency, which was identified as days 6–8 (Figure 8).

[0080] We isolated ASCs in our tissue-mimicking 3D hydrogel system (e.g., the TMHB system) and replated them in 2D to further assess functional changes in proliferative, metabolic, or mitochondrial activity compared to conventional 2D culture. The population doubling time (PDT) of ASCs in 3D was shorter than that of ASCs replated in 2D, indicating that cells could be expanded more quickly (Figure 7F). Furthermore, the metabolic (Figure 7G) and mitochondrial (Figure 7H) health and activity of replated ASCs showed no significant differences between 2D and 3D systems over a 2-week time course.

[0081] 3.2. Differentiation of wound healing interventions by molecular weight stratification of the secretome 3.2.1. Higher molecular weight secretome fractions are the primary drivers of wound healing activity in KCs ASC-CM collected from 2D and 3D cultures was stratified by molecular weight (Figure 9A), and each fraction was analyzed for total protein content (Figure 9B) and its ability to modulate the functional activity of KCs. ASCs in the tissue-mimicking system secreted up to 40–50% more total protein than their 2D counterparts. In this case, the "full" and "100 kDa" fractions were significantly larger in 3D cultures compared to 2D cultures (Figure 9B). For total protein, the "100 kDa" fraction accounted for up to approximately 49% and 53% in 2D and 3D cultures, respectively. All other fractions tested retained comparable relative protein content in 2D and 3D cultures.

[0082] Our results thus far indicate that the ASC secretome can modulate KC morphology to become more spindle-like and stratified, potentially indicating that potential changes in KC phenotype may be important and relevant to the wound healing ability of ASC-CM. While control KCs (cultured in KC-GM) retained a more homogeneous, rounded morphology, the 100 kDa fraction of ASC-CM promoted the morphological transformation of KCs from both 2D and 3D cultures toward stratified cell sheets with spindle-like cells (Figure 9C). KCs treated with the lower molecular weight fraction of ASC-CM appeared to exhibit smaller cell size and a rounded morphology, without cell sheets. Interestingly, KCs treated with the higher molecular weight fractions of ASC-CM (the "100 kDa" fraction and the "100-30 kDa" fraction) showed a significant increase in the expression of vimentin, a marker of mesenchyme and migration (Figures 9D-E). Conversely, KCs treated with the lower molecular weight fractions in either 2D or 3D showed increased expression of the senescence-associated markers p16 and β-galactosidase (Figures 9F and 10).

[0083] KC metabolic, proliferative, and migratory activity was used as a surrogate measure of in vitro wound healing activity. Previous studies have shown that "full" ASC-CM from this 3D system has advantages over 2D. The "100 kDa" ASC-CM fraction in both 2D and 3D showed a slight tendency toward increased activity compared to "full" in enhancing KC metabolism (Figure 9G) and proliferation (Figure 9H). While only the "100 kDa" 3D group appeared to enhance KC migratory activity (Figure 9I), the 2D "100 kDa" group showed a slight decrease compared to "full." Furthermore, the "100-30 kDa" fraction appeared to promote KC migratory activity in both 2D and 3D (Figure 9I). The relative activities of the lower molecular weight fractions of ASC-CM are comparable in enhancing KC metabolic and proliferative activity when comparing 2D and 3D groups with their respective controls. However, the lower molecular weight fractions exhibit a lower ability to enhance the functional activity of KC than the entire higher molecular weight fractions (ie, "100 kDa" and "full").

[0084] 3.3. ASC-EVs drive key regenerative wound healing pathways in keratinocytes 3.3.1. ASC populations in tissue-mimicking systems promote EV secretion The quantity and size distribution of EV production and characterization within ASC-CM were evaluated between 2D and 3D culture systems. Total EV production was more than two-fold increased in 3D compared to 2D (Figure 11A). Similarly, the relative secretome / composition of EVs to proteins was approximately 2:1, with EVs accounting for up to 14% (3D) or 7% (2D) of secreted proteins (Figure 11B). EVs were then characterized using exosome markers and nanoparticle tracking analysis (NTA) to determine whether exosomes were the predominant EV type being produced (e.g., EV size distribution was calculated (Figure 11C)). Further validation of NTA revealed an up to two-fold increase in particle number in 3D ASC-CM (Figure 11A), with more than 90% of particles exhibiting a size distribution within the exosome range (25–250 nm, Figures 11C and 11D). Notably, the average size of particles from 3D ASC-CM tended to be slightly smaller than that from 2D, but this was not significant.

[0085] 3.3.2. ASC-EVs in tissue-mimicking systems contain stronger re-epithelialization stimuli To isolate and observe the role of ASC-derived EVs / exosomes in regulating KC re-epithelialization activity, ASC-CM was divided into a >100 kDa (EV-containing) fraction and a <100 kDa (filtrate) fraction. Next, EVs / exosomes were extracted from the >100 kDa fraction, separated from soluble proteins within the >100 kDa fraction, and used to administer 2D and 3D ASC-CM "filtrate" fractions (Figure 12A). Both 2D and 3D ASC-CM "filtrate" had comparable effects on KC metabolic activity (Figure 12B), proliferation activity (Figure 12C), and migration activity (Figure 12D). The addition of 2D-EVs resulted in a slight increase in KC metabolic and proliferative activity, but only 3D-EVs significantly increased these activities (Figures 12B-12C). Notably, the migratory activity of KCs was significantly enhanced when ASC-CM filtrate was treated with 3D-EVs, whereas 2D-EVs significantly reduced the migratory ability of ASC-CM filtrate (Figure 12D).

[0086] Comparing the morphology of KCs treated with "filtrate" or "filtrate + EV," KCs displayed the previously observed morphological changes. While KCs treated with ASC-CM "filtrate" showed minimal morphological changes, the addition of ASC-EV from 2D or 3D resulted in KCs with morphology comparable to that previously observed after treatment with "full" or "100 kDa" fractions (Figure 12E).

[0087] 3.4. EV administration assessment reveals that some KC wound healing responses are dose-dependent 3.4.1. 3D-EV-enhanced expression of basal and suprabasal cytokeratins in a dose-dependent manner EV treatments of 5 μg / mL, 25 μg / mL, and 250 μg / mL were performed to further understand the functionality and quality of ASC-EVs and to assess whether KC phenotypes responded in any dose-dependent manner by immunolabeling (Figure 13A) and qRT-PCR (Figure 13B). Immunolabeling and Western blotting showed that ASC-EV-treated KCs had different levels of K5 (basal layer), K10 (suprabasal layer), and K16 (wound healing-responsive) cytokeratins. Only K16 showed increased expression levels after treatment with the highest dose of 2D-EV (250 μg / mL), whereas K10 was significantly decreased after 2D-EV treatment. Treatment with the highest dose of 3D-EV demonstrated the ability to significantly increase the expression of all three cytokeratins at the protein and RNA levels (Figures 13A-13B). Furthermore, only K16 showed a dose-dependent response to 2D-EV, whereas K5, K10, and K16 all showed a dose-dependent response to 3D-EV (Figure 13B).

[0088] 3.4.2. KC EMT and epidermal regeneration show dose-dependent responses to ASC-EVs The metabolic and migratory activities of KCs tended to decrease when treated with 2D-EVs (R2 = 0.42 and 0.98, respectively), but tended to increase when treated with 3D-EVs (R2 = 0.76 and 0.96, respectively). Meanwhile, KC proliferative activity showed a positive dose response to both 2D-EVs (R2 = 0.97) and 3D-EVs (R2 = 0.96) (Figure 14A). Notably, KC morphological changes showed a dose-dependent response. With increasing EV administration, KCs exhibited increased cytoskeletal regulation through spindle-like transformation, cell clustering, cell sheet formation, and actin cap formation (Figures 14B and 15). Notably, morphological changes were observed in both 2D-EVs and 3D-EVs, but were more pronounced with 3D-EV treatment.

[0089] Furthermore, to determine whether KC gene expression showed comparable changes to functional and proteomic assessments, we assessed several key gene markers related to KC wound healing activity and phenotype (Figure 14C). Expression of the key junction / migration markers CDH1 (E-cadherin) and CDH2 (N-cadherin) did not show any significant dose-dependent response to ASC-EVs, whereas the highest dose of 3D-EVs did result in a significant decrease in CDH1 expression (relative to 2D-EVs). On the other hand, expression of the proliferation marker CCND1 (cyclin D1) did show a dose-responsive response with increasing concentrations of ASC-EVs from both 2D and 3D (Figure 14C). Similarly, the expression of FLG (filaggrin), a marker of suprabasal differentiation (granular layer), and VIM (vimentin), a marker of KC migration, both showed a treatment response to ASC-EVs, with 250 μg / mL 3D-EVs resulting in a significant increase compared to 2D-EVs. On the other hand, VIM (vimentin), a marker of KC migration, showed no treatment response (Figure 14C). TWIST1, a marker of epithelialization and EMT-like responses, also showed a treatment response to ASC-EVs, with the effect of 3D-EVs being stronger (Figure 14C). Considering the relative expression of both CDH1 and CDH2, KCs showed a significant increase in CDH2 (N-cadherin) expression (Figure 14D). The relative correlation of each gene expression marker is shown in Figure 14E.

[0090] 4. Discussion The diverse patient populations and wound types, as well as the wide variety of tissue types and cell populations involved in wound healing, highlight the need to tailor therapies for each application. Recent studies into harnessing the dynamic secretome of MSCs have demonstrated unique opportunities to advance the fields of regenerative medicine and personalized wound healing modalities. Thus far, the secretome of MSC-like populations has been shown to promote various wound healing functions, including modulating fibrotic activity in fibroblasts, promoting angiogenic activity in endothelial cells, and increasing keratinocyte migratory activity.

[0091] Although preliminary data on MSC biopharmaceuticals are promising, more effective tailoring of regenerative biological therapies will require differentiating the roles / activities of MSC secretome components to create modalities for reproducibly generating effective products. Several studies have begun to explore the utility of several MSC secretome components, including examining the effects of single components (e.g., specific growth factors (e.g., VEGF) or exosome production). Other studies have focused on the effects of bulk (i.e., full) conditioned medium from MSCs. Ultimately, the ideal composition of factors will depend on the application, but it is the diverse milieu of MSC-secreted factors that provides a dynamic balance of factors that can be applied to various clinical scenarios.

[0092] However, to date, most research on MSC biopharmaceuticals has utilized 2D culture systems, and studies specifically using 3D systems have focused on using systems (e.g., hydrogels) for cell delivery and / or production of specific secretory products via "priming stimuli" (e.g., fibrin hydrogels enhance VEGF secretion). Therefore, current data are incomplete and stifled by the detrimental effects of traditional 2D systems on MSC phenotype, viability, and regenerative capacity. The non-physiological environment of 2D systems significantly hinders the standardization and reproducibility of MSC-based therapies. Similarly, many current 3D systems (e.g., spheroids and microcarriers), while more effective than 2D systems in various applications, lack tissue-mimetic properties and / or exhibit limited proliferation, which leads to cellular heterogeneity and hinders the efficiency of large-scale expansion, which will be essential for future clinical therapies.

[0093] Thus, in this example, we utilized a tissue-mimicking 3D hydrogel system (e.g., the TMHB system) that we previously demonstrated enhanced retention of non-senescent MSC-like populations compared to standard 2D cultures. When designing a tissue-mimicking system, there are several important viscoelastic properties of tissue to consider, including compressive, storage, and loss moduli, which are compared in Table 1 between our hydrogel system and previously published data for adipose tissue.

[0094] Two key advantages of this system are its unique architectural design, which allows for the easy collection of secretory by-products. Unlike previously studied injected / molded 3D hydrogels in the literature, which lack defined microchannels and structures, the microarchitecture and macroarchitecture provide a unique opportunity for continuous cell expansion within the hydrogel system, allowing for the easy and continuous collection of secretory by-products such as EVs. The microchannels help circumvent the diffusion constraints inherent in traditional hydrogel systems, increasing the efficiency of secretory by-product collection. Furthermore, as a result of the tissue-mimicking environment, MSC-like populations retain adaptive and regenerative capabilities, allowing them to respond to stimuli more appropriately, as they would in vivo. To support and highlight the relative benefits of the 3D system for MSC phenotype, RNA arrays were performed, showing that ASCs exhibited significant retention of key MSC and MSC-like markers in 3D compared to 2D. The refinement of the ASC phenotype within this adipose-like microenvironment results in a secretome composition that enhances regenerative responses compared to 2D. This is likely due, in part, to increased secretion of pro-regenerative factors from 3D cells combined with increased secretion of anti-regenerative factors (e.g., ROS and pro-senescence factors (Figure 9F)) from 2D cultured cells.

[0095] The ability to achieve targeted, rapid degradation of the hydrogel matrix allowed for easy extraction of ASCs for RNA analysis. To confirm and highlight the relative benefits of our 3D system for MSC phenotypes, we performed RNA arrays and demonstrated that ASCs exhibited significant retention of key MSC and MSC-like markers in 3D compared with 2D (Figure 1C and Table S1). Thus, surface markers associated with the MSC-like phenotype, including NT5E (CD73), THY1 (CD90), ENG (CD105), ALCAM (CD166), and NGFR (CD271), retained a similar gene expression profile to primary (P1) ASC populations within the 3D hydrogel. In contrast, the 2D system resulted in a sharp decline in their expression. Furthermore, ASCs in 3D showed increased retention of the pluripotency markers PPARG (adipogenesis), RUNX2 (osteogenesis), and SOX9 (chondrogenesis), as well as retained or increased expression of several markers associated with "stemness" (including LIF, OCT4, HGF, ZFP42, and NOTCH1). Notably, CASP3 expression was significantly decreased in 3D-ASCs. CASP3 is a key marker for cell health and the induction of apoptotic cell death.

[0096] Although we initially utilized the same seeding density, variations in cell proliferation were expected, as evidenced by examining the relative proliferative activity of ASCs extracted from 2D and 3D culture systems through population doubling times (PDTs) (Figure 7F). Therefore, we pre-assessed cell numbers for 2D and 3D cultures to determine the optimal harvest date for ASC-CMs to standardize the relative ratio of medium per cell. Furthermore, both 2D and 3D ASCs were placed in P2 immediately after seeding and remained in culture for 8 days in preparation for this study. Therefore, any differences noted are a reflection of the culture environment and not the effects of subculture. Based on previous literature, ASC-CMs were harvested in 2D cultures at 60-80% confluency.

[0097] Previous studies have demonstrated the positive impact of the MSC secretome on KC wound healing activity. However, to date, data are limited, and the adaptive and dynamic nature of the secretome has led to mixed results regarding which components may drive specific activities (e.g., migration and proliferation). Therefore, we performed functional and phenotypic assessments of KCs as an indirect surrogate measure and quantification of MSC secretome components from either 2D or 3D culture systems. Molecular weight stratification was used due to its ability to compartmentalize compounds into defined fractions and standardize consistent processing methods, as well as the minimally invasive nature of this method, which does not require solvents or other agents to precipitate and / or sequester proteins from conditioned media. Notably, ASCs cultured in our 3D culture system showed enhanced secretion of protein compounds compared to 2D (Figure 9B). Immediately after stratification, only the "100 kDa" fraction significantly increased in 3D compared to 2D counterparts. This suggests that the enhanced protein production in 3D is a result of the "100 kDa" fraction, which consists primarily of larger growth factors / cytokines, matrix-associated proteins, and EVs.

[0098] Next, we evaluated the functional activity of KCs to assess the secretome activity of ASC-CMs per molecular weight fraction. The functional benefits of ASC-CMs from the "100 kDa" fraction showed the greatest positive impact on KC activity, comparable to the effects and benefits seen in the "full" medium group, including KC morphological, metabolic, proliferative, and migratory activities. In contrast, lower molecular weight fractions had smaller effects on KC functional activity. Closer examination revealed that the effects of lower molecular weight fractions from both 2D and 3D were comparable, but there was a significant decrease in the effect from the 3D "100 kDa" fraction (Figure 9G-I). This is potentially due to the increased benefit from the 3D "100 kDa" fraction rather than a negative impact of the lower molecular weight 3D compounds.

[0099] Interestingly, both the 2D and 3D "full" and "100 kDa" groups induced significant morphological changes in the KC population, which resembled more flattened, stratified, and spindle-shaped cells forming cell sheets. In contrast, the lower molecular weight fractions had minimal effects on KC morphology. Furthermore, further evaluation of KC nuclear size revealed an increase in nuclear surface area only in the "full" and "100 kDa" groups (Figure 16), potentially due to the cell flattening observed in the morphological changes resulting in a decrease in cell height. Future studies will explore the relationship between KC morphological changes and wound healing activity; however, some KCs are thought to undergo epithelial-to-mesenchymal transition (EMT) and suprabasal differentiation during reepithelialization, which may be the primary cause of the morphological changes observed.

[0100] Previous studies have shown that senescent cell populations, including MSCs, can restore the regenerative activity of surrounding cell populations in several ways, including the secretion of senescence-inducing factors. In particular, we previously demonstrated that ASCs cultured in this tissue-mimicking system delayed the induction of ASC senescence, whereas 2D culture rapidly induced senescence. Thus, ASCs cultured in 2D may secrete protein and non-protein factors associated with senescence and impaired cell health, which may induce KC senescence (Figures 7F and 10). Analysis of KCs after ASC-CM treatment showed that the "10-3 kDa" molecular weight fraction from 2D was more likely to induce senescence in the KC population, and the "30-10 kDa" fraction from 2D also showed a tendency to increase. Interestingly, similar fractions in 3D did not induce senescence. Because the relative amounts of proteins secreted in the lower molecular weight fractions were comparable between 2D and 3D, the induction of senescence is not a result of protein quantity but rather is due to either protein quality / composition or non-protein factors (e.g., reactive oxygen species (ROS)). In particular, it is thought that an imbalance in some secretory factors can promote the progression of cellular senescence in a paracrine manner. Furthermore, the senescence-associated secretory phenotype (SASP) of MSC-like populations has previously been shown to induce senescence in other cells (potentially via ROS or inflammatory factors in the 10-30 kDa range). However, the dynamic nature of the SASP warrants further investigation into the exact mechanism of senescence induction.

[0101] To further investigate the mesenchyme-like morphological changes and enhanced migratory activity of KCs treated with high-molecular-weight ASC-CM fractions, we assessed vimentin and found a correlation with KC migratory activity. In 3D, vimentin expression was significantly increased in KCs treated with the "100 kDa" and "100-30 kDa" fractions compared with lower-molecular-weight fractions, particularly at the outer edge of the cell sheet. In contrast, in 2D, only the "100-30 kDa" fraction was enhanced, and the "100 kDa" fraction in 2D showed a reduced ability to promote KC migration and express vimentin. This suggests that components within the "100 kDa" fraction likely promote KC migratory activity in 3D ASC-CM, and that secretion of these factors is reduced / negated in 2D cultures. Furthermore, there may be a secondary set of factors within the 100-30 kDa fraction that also promote KC migratory activity, similarly in 2D and 3D. These data suggest that the 100 kDa and 100-30 kDa fractions of the ASC secretome contain compounds that regulate KC migratory activity. Thus, culture of ASCs in a tissue-mimicking system may enhance the secretion of positive 100 kDa factors, or 2D culture may promote the secretion of negative 100 kDa factors while leaving migratory factors in the 100-30 kDa range unchanged. This is the first demonstration of the divergent effects of molecular weight-specific secretome fractions and provides insight into the functional significance of specific bioregulatory fractions. For example, whether a "reversal" of the pro-regenerative migration stimulus within the 100 kDa range is possible depends on the culture system used. Furthermore, previous studies have shown that increased vimentin expression at the leading edge of epidermal migration is a critical step towards native epithelial regeneration, whereas our data show that treatment with the "100 kDa" fraction of the ASC secretome can promote this physiological wound healing response in KCs.

[0102] To investigate whether EVs within the "100 kDa" fraction were driving the observed functional changes in KCs, further stratification of the "100 kDa" component was performed based on the observed benefits of the "100 kDa" fraction. Culturing ASCs in tissue-mimicking systems, including adjusting the relative ratio of (secreted) EVs to proteins, resulted in increased production of EV particles. This indicates that ASC secretome dynamics tend to favor the secretion of EV particles over soluble proteins when in a more tissue-mimicking (i.e., native) environment, with a >100% increase in secreted EVs to protein in 3D compared to 2D. Furthermore, the relative size distribution of EVs from ASCs in 3D showed a tendency toward slightly smaller sizes, potentially suggesting that EVs are more likely to be exosomes (25-250 nm) in nature than MVs (100-1000 nm) or apoptotic bodies (200-2000 nm). This was further supported by the presence of exosomal proteins CD9, CD63, CD81, and Tsg101. To our knowledge, this is the first time we have uncovered a shift in MSC secretion dynamics toward promoting the secretion of EVs / exosomes from MSC-like populations in a tissue-mimicking system over soluble proteins (Figure 12B), providing potential insight into native tissue signaling preferences. In contrast, previous studies have only examined the overall amount or effect of EVs / exosomes in 3D systems, without comparing the relative total amount of secreted proteins with 2D or controls.

[0103] Next, we evaluated the role of EVs / exosomes from both 2D and 3D cultures in their ability to restore / improve the functional wound-healing activity of KCs. This was done by utilizing <100 kDa filtrate administered with or without ASC-EVs to determine whether the benefits seen in the "full" and "100 kDa" fractions were due to EVs. Because EVs are primarily located within the "100 kDa" fraction, the "<100 kDa filtrate" group plus ASC-EVs, according to stratification studies, resembles "full" medium but is depleted of soluble "100 kDa" proteins not derived from EVs. Previous molecular weight stratification data suggested that the "100 kDa" fraction resulted in a slight increase in KC metabolic and proliferative activity compared to the "full" group in both 2D and 3D ASC-CMs. We found that when EVs were removed and the "<100 kDa filtrate" was administered, both 2D-EVs and 3D-EVs were able to restore the metabolic activity of KCs seen in the "full" group. On the other hand, the effect of the "<100 kDa filtrate" on KC proliferation was significantly enhanced only when administered together with 3D-EVs. Notably, KCs in the "<100 kDa filtrate + 3D-EV" group showed a significant increase in proliferation activity, suggesting that the contents of 2D-EVs may not have the same proliferation potential as the contents of 3D-EVs. Therefore, the previous proliferation benefit observed with the "<100 kDa" fraction in the 2D case may be non-EV-derived, or EV stimulation may not be as robust.

[0104] Furthermore, when comparing the effects of 2D-EVs and 3D-EVs on KC migratory activity, striking differences become apparent. As previously observed with the "100 kDa" fraction, there is a "reversal" of migration signaling between 2D-EVs and 3D-EVs, with 3D-EVs enhancing KC migration, whereas 2D-EVs inhibit KC migratory activity. These data support the stratification data suggesting that the "100 kDa" fraction of 2D ASCs does not contain the same pro-migratory stimuli as their 3D counterparts, but rather that 2D-EVs may contain potential anti-migratory stimuli for KCs. One possible explanation for this could be that ASCs release anti-migratory (and possibly anti-proliferative) signals within EVs as a result of an adaptive response of ASCs within the 2D monolayer to the blockage of crowding and contact. Conversely, 3D culture likely results in a more regenerative ASC population and significantly increases the surface area for migration, thereby promoting pro-migration and pro-proliferation signaling between cells. Notably, the effects of the "<100 kDa filtrate" from both 2D and 3D have comparable effects on KC functional activity. This suggests that the difference in activity after "full" 3D ASC-CM treatment is likely the result of the "<100 kDa" fraction, and that the difference in "<100 kDa filtrate" between 2D and 3D is likely to be minimal, which is further supported by the changes in secretory activity seen in Figure 9B.

[0105] Previous studies have shown that EV content can vary depending on cell type, tissue source, or donor, but often ignore culture conditions. This study demonstrates that EVs derived from the exact same tissue and donor exhibit a shift in regenerative capacity when harvested from ASCs in a tissue-mimicking culture system. This suggests that the ability of primary ASCs to retain a more robust and regenerative phenotype likely results in ASCs with pro-regenerative properties, and subsequent production of EVs and their contents can drive wound healing processes such as epithelialization. In contrast, traditional 2D culture results in a rapid decline in ASC phenotypic characteristics, resulting in a decline in EV potency, as highlighted in the EV administration experiments in this study. Thus, as noted above, not only was the quantity of EVs produced in 3D increased, but the quality was also improved.

[0106] Interestingly, closer examination of the stratification and EV analysis reveals that the morphological changes exhibited by KCs after treatment with "full" or "100 kDa" ASC-CM potentially depend not only on the migratory capacity of KCs but also on the ASC-EV fraction. This can be seen when comparing the equivalent relative migration of KCs after treatment with the "100 kDa" and "100-30 kDa" fractions from 3D, but only the "100 kDa" fraction induces morphological changes. Furthermore, the "<100 kDa filtrate" fraction fails to dramatically alter KC morphology, even when containing the "100-30 kDa" migratory stimulus, whereas the addition of 2D-EVs or 3D-EVs results in the same morphological changes seen within the "100 kDa" fraction (Figure 12E). It is also important to note that both 2D-EVs and 3D-EVs induce varying degrees of KC morphological changes, even though 2D-EVs appear to contain anti-migration stimuli. Similarly, the dependence of KC morphological changes on ASC-EVs is further supported by DiI labeling and in vitro tracking data of ASC-EVs, showing that the most significant morphological changes undergone by KCs are those occurring in the largest proportion of EVs.

[0107] Finally, EV administration studies were performed to further understand ASC-EV functionality at comparable doses and whether KCs exhibit any dose-dependent wound healing responses to EVs and / or changes in KC phenotype. Native expression of K5 (and K14) is typically thought to decrease during wound healing, while K16 (and K6) is thought to increase. K10 is rather an indicator of suprabasal differentiated KCs, which, in combination with increased expression of FLG, may indicate KCs in the granular layer. Suprabasal K10-expressing KCs are thought to switch to K16 / K6 expression during the wound healing process. K16 / K6 expression is thought to play an essential role in the collective cell migration of KCs. In this example, 3D-EVs appeared to significantly enhance the expression of all cytokeratin markers in a dose-dependent manner. In contrast, 2D-EVs resulted in decreased K10 expression and the formation of sporadic aggregates of K5, in contrast to the more homogenous cellular distribution seen in 3D-EV-treated KCs, possibly implying enhanced K5 protein turnover and remodeling in 2D-EV-treated KCs.

[0108] Further assessment, involving functional and RNA analysis, demonstrated a positive dose-dependent correlation between the metabolic, proliferative, and migratory activities of KCs treated with 3D-EVs. In contrast, treatment with 2D-EVs resulted in a negative correlation with KC migratory activity and a positive correlation with proliferative activity. This data further demonstrates that an inhibitory stimulus for KC migration was found within ASC-EVs from 2D cultures. While both 2D-EVs and 3D-EVs were able to promote dose-dependent morphological changes in KCs, 3D-EVs contained a more potent stimulus, with a low dose of 3D-EV (5 μg / mL) having an effect equivalent to that of a high dose of 2D-EV (250 μg / mL).

[0109] It is important to note that key morphological changes within KCs appear to occur in stages after ASC-EV treatment. First, KCs begin to cluster, forming small satellite-like colonies that eventually coalesce into larger colonies. Next, KCs begin to change their cellular morphology from rounded to more irregular, spindle-like, with rearrangements of cytoskeletal structures and increased cellular elongation. Finally, KCs begin to adhere tightly to each other and integrate, forming a continuous cell sheet with a more flattened cell population. The correlation between EV / exosome uptake and nuclear actin cap formation within KC cell sheets was an intriguing and unexpected finding, which likely played a direct role in the aforementioned morphological flattening and increased nuclear surface area. Furthermore, actin cap formation promotes nuclear flattening and is a known regulator of gene expression, often associated with mechanotransduction and epigenetic responses. Broadly speaking, both 2D-EVs and 3D-EVs were able to elicit this response of KCs to varying capacities. However, where 3D-EVs branched, their respective capacities to stimulate KC migratory activity were enhanced. This is the first demonstration of branching ASC-EV migratory activity and its possible association with KC morphological changes. To our knowledge, this is the first in vitro demonstration of substantial EV-induced KC morphological changes (e.g., actin cap and cell sheet formation) and their potential association with KC wound-healing activity. Therefore, EV-induced KC morphological changes require further study.

[0110] Interestingly, the RNA profiles of KCs treated with ASC-EVs revealed diverse phenotypic profiles. Both 2D-EVs and 3D-EVs showed dose-dependent responses in several key KC genes (including K10, K16, FLG, TWIST1, and CCND1). However, the enhancement of KC wound-healing activity was more pronounced at the functional and RNA levels after 3D-EV treatment than 2D-EVs. In particular, 3D-EVs were able to promote increased expression of proliferation and basal-like phenotypes (CCND1 and K5), differentiated suprabasal-like phenotypes (K10 and FLG), wound-healing phenotypes (K16), and more migratory phenotypes (K16 and TWIST1). Furthermore, KCs treated with 2D-EVs showed clear disruption and transformation of K5 protein (Figure 14A), suggesting a potential transitional period in which KCs were losing their basal-like phenotypes. Furthermore, upon closer examination, 3D-EV-treated KCs exhibited an EMT-like transition accompanied by increased TWIST1 expression and an increased N-cadherin to E-cadherin ratio, which was further supported by increased vimentin production, morphological changes, and enhanced migration. Collectively, these data suggest that 3D-derived ASC-EVs can more effectively stimulate KCs to form stratified, collective cell sheets composed of EMT-like migratory KCs that form the leading edge of the cell sheet, while maintaining cell-cell adhesion within the cell sheet and the expression of cytokeratins associated with suprabasal differentiated (K10), migratory (K16), and proliferative (K5) phenotypes.

[0111] In particular, collective cell-sheet migration is a proposed mechanism for native KC re-epithelialization, and thus far, this process has been incompletely recapitulated in vitro. It is believed that 2D monolayer cultures of KCs fail to recapitulate the appropriate differentiation and morphological changes necessary to promote KC collective cell-sheet migration activity. In contrast, our data suggest that EVs may, in fact, be the missing link and key to promoting this process as it is thought to occur in vivo. To our knowledge, this is the first time that ASC-EVs have been shown to broadly promote multiple important functional phenotypes in KCs, not just migration and / or proliferation, and provides new insights into the potential native in vivo signaling dynamics of wound repair, warranting further study. More specifically, this is the apparent ability of KCs to express mesenchymal-like markers (vimentin, N-cadherin, TWIST1) after EV treatment, while simultaneously retaining the expression of important basal or suprabasal epithelial-like cytokeratin markers.

[0112] 5. Conclusion In conclusion, this study highlights the importance of understanding the role of the culture environment on MSC-like cells, their phenotype, and their ability to secrete regenerative compounds by directly comparing the regenerative functionality of ASC secretomes from both 2D culture and tissue-mimicking systems (e.g., TMHB systems). It is important to note that MSC-like populations are dynamic and highly adaptable to their environment; therefore, secretomes and / or exosomes from one population may vary depending on the system utilized to generate biopharmaceuticals. Therefore, to generalize the effects of specific MSC-derived secretome components, the effects must be evaluated across systems. Furthermore, this study demonstrates that EV exosomes may be a key driver of epidermal regenerative activity within ASC secretomes and, when generated within a tissue-mimicking system, can enhance the formation of collective migratory KC cell sheets, an essential process during native epidermal regeneration. Due to their enhanced stability compared to protein biopharmaceuticals (growth factors) and their diverse / dynamic composition, EVs / exosomes offer a unique approach to clinically improve current wound healing modalities. Therefore, EVs / exosomes can serve as adjuvant therapy by being integrated into current wound dressings and / or injectable hydrogel systems to modulate therapy and enhance wound outcomes. Finally, although further research may be needed to explore the potential clinical benefits that MSC-derived EVs / exosomes may offer, this study helps provide new insights into the direct role of EVs / exosomes in KC wound healing and epidermal regeneration.

[0113] Example #2: Modulating the secretome composition of mesenchymal stem cells to enhance specific functions of epidermal regeneration: an in vitro diabetes model Background and Overview Wound healing consists of a dynamic series of events that are highly dependent on paracrine factors for proper progression through each phase of wound healing. Inappropriate progression through each phase of wound healing is associated with insufficient regeneration of the epidermis (i.e., re-epithelialization) of the wound and subsequent development of chronic wounds (e.g., diabetic ulcers) as the patient's pathological condition worsens. Recently, studies of the dynamic secretome of adipose-derived mesenchymal stem cells (ASCs) have offered promise for enhancing the wound healing response of chronic diabetic wounds. However, currently utilized 2D culture techniques are known to significantly alter the regenerative phenotype of ASCs. In this example, we utilized the TMHB system as a means of culturing ASCs. We then assessed the ability of the ASC secretome to enhance epidermal regeneration activity after exposing ASCs to 2D and 3D "wound priming stimuli." The priming stimuli consisted of coating 2D and 3D systems with wound matrix proteins, type I collagen, fibronectin, and fibrin. To understand the potential benefits of ASC secretome in the context of diabetic wound healing, we exposed keratinocytes (KCs) to supraphysiological glucose levels to induce a diabetic-like phenotype (idKCs). Compared to KCs, idKCs exhibited a 52% and 23% decrease in proliferation and migration, respectively. Analysis of the ASC secretome was then performed. ASC-conditioned medium from tissue-mimicking cultures (ASC-CM) showed a greater than 50% increase in protein secretion and a two-fold increase in EV secretion compared to 2D cultures. Interestingly, different priming stimuli did not alter the total amount of secreted proteins or EVs in the tissue-mimicking system. However, evaluation of specific soluble proteins by ELISA revealed significant differences in key epidermal regeneration factors (e.g., EGF, IGF-1, FGF-2, MMP-1, TIMP-1, and TGFβ-1). Furthermore, the relative effects of ASC-EVs from 2D and 3D systems on the epidermal regeneration functionality of idKCs were significantly different, with EVs from 3D collagen cultures providing the most significant benefit to idKC activity.Collectively, this data supports the use of tissue-mimicking cultures to enhance the adaptive and secretory activity of MSC-like populations for the generation of tailored biopharmaceuticals via priming stimuli for specific wound healing applications.

[0114] 1. Introduction Wound repair and regeneration are complex and dynamic series of events that occur in the context of tissue injury and involve a diverse array of cell populations. To effect the restoration of tissue function and anatomical homeostasis, tightly regulated cellular and molecular signaling cascades promote cell migration and proliferation, extracellular matrix deposition / remodeling, and cytoskeletal regulation of local wound-healing cell populations. However, many patients and wound types may be prone to inadequate healing and prolonged wound healing responses, which may ultimately result in progression toward a chronic wound phenotype.

[0115] The epidermal barrier of skin is responsible for maintaining the viability of deeper tissue structures and allowing proper wound healing processes to occur. Therefore, an essential step in wound healing after injury is the process of re-epithelialization (epidermal regeneration), which is carried out by epidermal keratinocytes with the goal of restoring the skin's external barrier properties. Proper re-epithelialization requires keratinocytes to undergo a phenotypic transition. This phenotypic transition involves changing cytoskeletal and junctional proteins (e.g., cell-to-cell and cell-to-matrix junctions) to become more migratory and proliferative in order to refill and "close" the wound. Furthermore, keratinocytes must change their keratin expression from a more proliferative basal type (keratins 5 and 14) to expressing keratins associated with suprabasal differentiation (keratin 10) and an active wound response (keratin 16). Feedback and paracrine signaling from surrounding cell populations help orchestrate proper re-epithelialization, including critical crosstalk between dermal fibroblasts, dermal fat, and other epidermal keratinocytes. However, this intercellular communication can become dysregulated by an imbalance in paracrine signaling, resulting in persistently non-healing wounds that fail to close and often progress to significant limb disease.

[0116] Chronic wounds are often the result of concurrent conditions such as diabetes, with up to 15% of diabetic patients suffering from ulcerative wounds, with a recurrence rate of over 50%. Diabetic wounds inherently exhibit an inappropriate balance and composition of bioactive compounds within the tissue, such as depleted growth factor bioavailability, unbalanced proteolytic activity, and an inflammatory cytokine profile. Furthermore, elevated glucose levels are known to result in increased oxidative damage and glycation of cellular proteins. This leads to the widespread abnormal progression of several wound healing processes, including the proper differentiation of keratinocytes and functional activities related to epidermal regeneration. In particular, keratinocytes isolated from diabetic wounds have been shown to lose their original ability to change their keratin expression pattern and migrate properly. Therefore, diabetic wounds are associated with a long-term cycle of inappropriate intercellular signaling and insufficient neoplasia. Consequently, failure to close wounds can lead to polymicrobial infection, desiccation, and reinjury of diabetic wounds, which remains the leading cause of non-traumatic lower limb amputations, with an associated 5-year mortality rate of 55-70% after amputation, second only to lung cancer. In this study, we exploited the principle of impaired wound healing capacity in diabetic keratinocytes by generating an induced diabetic-like keratinocyte population through prolonged exposure to high glucose levels, thereby generating functional impairment within keratinocytes.

[0117] The desolate and complex nature of chronic wounds and their tissue microenvironments has also led to a lack of focused and effective therapeutic interventions, and insufficient reepithelialization remains a major limitation of current chronic wound treatments. Therefore, there remains a critical need to develop therapies that can help circumvent the decline in proper keratinocyte functionality and restore the wound's ability to properly reepithelialize, preventing / reversing the progression of complex chronic wounds. Bioactive compounds (e.g., growth factors (GFs) and extracellular vesicles (EVs)) orchestrate tissue repair processes and preserve immune regenerative capabilities. Furthermore, enhanced proteolysis, radial diffusion, and a lack of biological diversity from single GF administration in clinical trials to date have led to mixed results in therapies targeting GFs for chronic wounds. However, the diverse yet balanced milieu of GFs, antioxidants, extracellular vesicles (EVs), immunomodulatory cytokines, proteases, and protein inhibitors secreted by adipose-derived mesenchymal stem cells (ASCs) may offer a unique approach that circumvents the limitations of single GF and / or autologous cell therapies for developing targeted therapies to treat chronic wounds.

[0118] Currently, the most common treatment modalities used clinically for chronic wound treatment are wet dressings and serial surgical debridement. However, early studies of ASCs in in vivo animal models and clinical trials have demonstrated their potential to enhance the rate at which wounds close and improve diabetic wound outcomes. Recent studies have shown that a key component of ASCs' inherent regenerative capacity is their secretome. ASC populations and their secretomes are highly diverse and adaptive, allowing them to rapidly respond to various environmental stimuli (e.g., within the wound environment) in a context-specific manner. Notably, direct injection of both ASCs and ASC-secreted compounds into chronic wounds has previously been shown to improve in vivo outcomes. Interestingly, hypoxic priming of ASCs has been shown to upregulate the secretion of angiogenic compounds, and priming with inflammatory cytokines has been shown to upregulate the secretion of anti-inflammatory and mitogenic compounds from ASCs. Furthermore, fibrin, matrix compounds, and native wound healing stimuli have been used as delivery vehicles for ASCs and have shown the ability to improve overall wound outcomes, including chronic wounds. Additional matrix-derived biomaterials have also been investigated to control the biological activity of MSC populations, including type I collagen and fibronectin, both of which are intricately intertwined within native wound tissue and have shown the ability to promote wound healing. Thus, ASCs exhibit an innate ability to respond to environmental stimuli by modulating their secretory activity. Previous studies focusing on delivery vehicles such as fibrin have also highlighted that matrix-derived substrates may also be essential in modulating the wound healing capacity of ASCs. Collectively, this data suggests that the wound environment contains various stimuli (e.g., hypoxia, inflammation, and matrix compounds) that alter the adaptive secretion of paracrine factors from ASCs, which may promote wound healing.

[0119] Previous data on MSC-derived biopharmaceuticals and cell therapies have shown promise; however, to date, they have relied almost exclusively on 2D culture expansion to achieve sufficient amounts of biological products (e.g., cells or secretomes). In particular, biochemical and biomechanical cues exposed to cells within the native tissue microenvironment are important for regulating cell phenotype and activity, particularly in mesenchymal stem cell (MSC) populations such as ASCs. Accordingly, culturing ASCs in non-physiological and rigid 2D culture systems has been shown to promote a significant decline in ASC phenotype and viability. As a result, 2D-cultured ASCs lose their inherent adaptive and regenerative properties, resulting in inconsistent and contaminated secreted products. In contrast, culturing ASCs in softer 3D systems, which more closely resemble native tissue organization, has been shown to retain the native ASC phenotype longer and result in more robust secretion of regenerative compounds. Therefore, recent research has focused on developing 3D tissue-mimicking systems that enhance the culture of ASC populations and allow for easy tuning of their properties in order to modulate ASC phenotypic activity (e.g., through the introduction of exogenous priming stimuli). To date, most data on ASC priming has involved 2D culture systems or 3D spheroids and focused on the enrichment of specific soluble factors secreted by ASCs (e.g., increased VEGF secretion). However, data on understanding "functional" changes to the entire ASC secretome in the context of chronic wounds is still being explored. Based on previous clinical trials and the literature, we hypothesized that treatment with a complete, balanced ASC secretome would enhance keratinocyte function and display regenerative activity through the additive and synergistic effects of all secreted compounds (e.g., GFs, EVs, antioxidants, antiprotease enzymes), but that in certain circumstances, specific compounds within the secretome may provide more potent stimulation.

[0120] In patients with chronic (e.g., diabetic) wounds, the regenerative capacity of autologous ASCs to enhance wound healing is often not as robust as that seen in healthy ASCs due to their health status. Therefore, ASC-derived acellular by-products offer an acellular alternative to current cell-based regenerative therapies and demonstrate immunoparacrine effects, including modulation of epidermal regeneration. In this study, we utilized tissue-mimicking 3D hydrogel systems (e.g., TMHB systems) that mechanically resemble native adipose tissue for ASC culture and compared them with conventional 2D culture. To assess the modulation of the ASC secretome to enhance keratinocyte epidermal regeneration activity, both the 2D and 3D systems were coated with various matrix-derived substrates native to wound tissue. This study aims to provide useful insights into the additive and / or synergistic effects of priming allogeneic ASCs with various substrates while controlling their respective mechanical environments in a tissue-mimicking 3D system (e.g., TMHB systems). Thus, a more robust population of ASCs within the same 3D mechanical environment but exposed to different matrix compounds present in the wound could potentially provide new insights into the native signaling response to wound tissue, as well as demonstrate the ability to produce acellular biopharmaceuticals tailored for specific applications.

[0121] 2. Materials and Methods 2.1. Cell culture This study utilized human adipose-derived mesenchymal stem cells (ASCs) (Lonza, Lot #18TL212639, 23-year-old female, Black) and human keratinocytes (KCs) (Lonza, Lot #18TL318559, 62-year-old male, Caucasian). ASCs were cultured in RoosterNourish MSC-XF (RoosterBio, Cat. #KT-016) as growth medium (MSC-GM), and then switched to RoosterCollect EV-Pro (RoosterBio, Cat. #K41001) as serum-free, low-particulate medium. DermaLife K Keratinocyte Medium Complete Kit (Procured from Lifeline Cell Technology, Maryland, USA, #LL-0007) was used for KC culture. Conventional T-150 flasks were used for expansion until a maximum of 80% confluency was reached, allowing for subculture (i.e., subculture) of ASCs and KCs. A functional diabetic phenotype was induced in "passage 1 (P1)" keratinocytes (KCs) by administering KC-GM with a high dose (25 mM) of glucose for 10 days to achieve an "induced diabetic" phenotype (idKC) of keratinocytes. Initial characterization of the ASC phenotype at P1 was performed, and P2 ASCs were utilized intact for this study to eliminate any influence of subculture. To allow for similar comparisons between 2D and 3D cultures and conditioned media, 2D and 3D cells were seeded at the same density (maximum 1500 cells / cm). 2 and 200 μL medium / cm 2 ) The TMHB system was seeded at a maximum of 1 cm 3This is a 3D-printed cell culture and expansion system called X-Block (Lonau, Kansas, USA), which contains a unique microarchitecture design running throughout the TMHB, allowing for mass transport and nutrient exchange. For ASC culture, the TMHB was placed in a glass 6-well culture plate. Coating of the 2D and 3D systems was performed 24 hours before cell seeding. Cells were then dropped onto the surface of the hydrogel and allowed to migrate and disperse within the porous microarchitecture. Substrate coating included a thin coating of type 1 collagen (Corning, Cat. #354265) at a concentration of 1 μg / mL, a thin coating of fibronectin (Corning, Cat. #356008) at a concentration of 1 μg / mL, or a thin coating of fibrin at a concentration of 10 μg / mL. Fibrin was prepared. This was accomplished by coating with 10 μg / mL fibrinogen (Sigma, Cat. #F3879) for 24 hours, followed by three washes with HBSS and the addition of 10 units of thrombin (Sigma, Cat. #T6884) for 30 minutes. Uncoated controls were used in both 2D and 3D. All samples were then gently submerged in HBSS three times to wash away any residual substrate.

[0122] 2.2. Assessment of ASC phenotype Initial assessment of the ASCs' (P1) trilineage differentiation potential for adipogenesis, chondrogenesis, and osteogenesis was performed by culturing them in differentiation medium according to the manufacturer's protocol. Adipogenic differentiation was performed using the hMSC Adipogenic Differentiation BulletKit™ (Lonza, Cat. #PT-3004), and adipogenesis was assessed by the Oil Red O assay (ScienCell, Cat. #0843). Chondrogenic differentiation was performed using the hMSC Chondrogenic Differentiation Medium BulletKit™ (Lonza, Cat. #PT-3003), supplemented with TGF-β3 (Lonza, PT-4124) at 10 ng / mL, and chondrogenesis was assessed by the Alcian Blue assay (ScienCell, Cat. #8378). Osteogenic differentiation was performed using the hMSC Osteogenic Differentiation Medium BulletKit™ (Lonza, Cat. #PT-3002), and bone formation was assessed by Alizarin Red S assay (ScienCell, Cat. #0223). Additionally, assessment of the ASC "stem-like" phenotype at P1 was performed for positive immunolabeling of CD73 / 90 / 105 and negative immunolabeling of CD34 / 45. All primary antibodies were from Abcam (Cambridge, UK) unless otherwise noted. Briefly, ASCs were seeded in 2D for 24 hours, fixed with 4% paraformaldehyde for 15 minutes, washed three times with HBSS, blocked with 2% donkey serum, and immunolabeled for CD34 (ab81289), CD45 (ab40763), CD90 (ab181469), and CD105 (ab231774). CD73 (Cat. #41-0200) was obtained from Invitrogen (Waltham, MA). Hoechst 33342 (Invitrogen, Cat. #H3570) and Alexa Fluor 488 Phalloidin (ThermoFisher, Cat. #A12379) were used as counterstains.ASCs were cultured in 2D or 3D systems for 8 days and then extracted for proteome and phenotype analysis. Immediately after extraction, ASCs were pelleted by centrifugation, lysed with 0.5% Triton-X, and assayed using the Picogreen Quant-iT™ dsDNA Assay Kit (Invitrogen, Cat. #P7589) according to the manufacturer's protocol.

[0123] 2.3. Isolation of ASC-conditioned medium ASC-conditioned medium (ASC-CM) was collected by removing the MSC-GM medium. The cells were then washed three times with HBSS and serum-free MSC medium was added for another 24-hour wash. After the 24-hour washout, fresh serum-free medium was added, followed by three consecutive days of collection with 24-hour intervals between days 6 and 8 of culture. The medium / cell ratio was standardized for all groups. The collected ASC-CM was centrifuged at 1500×g for 10 minutes to remove cell debris, filtered through a 0.22 μm filter, and stored at -80°C for long-term storage until use. To ensure sufficient volume for all experiments and completely eliminate potential variability between ASC-CM collected on different days, ASC-CM from each day (day 6, 7, or 8) was pooled for each replicate (n=3) to create a "batch" mixture.

[0124] 2.4. Production of ASC-CM extracellular vesicles (EVs) EVs were isolated by centrifuging the ASC-CM at 4000g for 30 minutes through a Vivaspin 20 MWCO 100,000kDa centrifugal cutoff filter (Cytiva, Cat. #28932363), followed by washing with PBS and recentrifuging through a 100kDa filter at 4000g for 5 minutes, for a total of three washes. EVs were precipitated overnight from the remaining >100kDa concentrate using an ExoQuick-TC kit (SBI, Cat. #EXOTC10A-1) according to the manufacturer's instructions. EV samples (n = 3) were then resuspended in PBS, and aliquots were removed and used to quantify relative protein content. This was performed using the Pierce™ BCA Protein Assay Kit (Invitrogen, Cat. #23225), quantification by absorbance at 280 nm using a QuickDrop (Molecular Devices, SpectraMax QuickDrop Micro-Volume Spectrophotometer), and an indirect measurement of EV content using the Pierce™ Coomassie "Bradford" Protein Assay Kit (Invitrogen, Cat. #23200). Purified EV samples were also evaluated by Nanoparticle Tracking Analysis (NTA, Malvern Panalytical, Nanosight LM10) to further assess particle concentration and size distribution.

[0125] 2.5. ELISA of ASC-CM soluble proteins Twelve Quantikine ELISAs (enzyme-linked immunosorbent assays) (R&D Systems) were purchased and used to quantify the concentrations of 12 key protein factors involved in wound healing and epidermal regeneration in ASC-CM: epidermal growth factor (EGF), heparin-binding EGF (HB-EGF), insulin-like growth factor (IGF-1), fibroblast growth factor 2 (FGF-2), keratinocyte growth factor (FGF-7), transforming growth factor beta 1 (TGF-β1), interleukin-1 beta (IL-1β), interleukin-1 receptor antagonist (IL-1Ra), promatrix metalloproteinase (MMP-1), matrix metalloproteinase 9 (MMP-9), tissue inhibitor of metalloproteinase 1 (TIMP-1), and tissue inhibitor of metalloproteinase 2 (TIMP-2). The assay was performed according to the manufacturer's protocol and was performed only on soluble proteins contained in the collected ASC-CM. If the protein concentration was below the detection limit of the ELISA on the first attempt, the ASC-CM protein was concentrated 10-fold and the assay was repeated. If the protein was still not detected, the protein of interest was deemed too low in abundance to be quantified.

[0126] 2.6. ASC-CM Antioxidant Composition The antioxidant activity of collected ASC-CM samples was assessed using a total antioxidant capacity (TAC) assay kit (Cell Biolabs, Cat. #STA-360) according to the manufacturer's instructions. The TAC kit evaluates antioxidant activity based on the reduction of copper(II) relative to copper(I) and utilizes the naturally occurring antioxidant uric acid as a control standard. Therefore, antioxidant activity was measured in mM uric acid equivalents. Control serum-free MSC medium was used to assess the baseline antioxidant activity of medium not exposed to cells. Assays were performed in technical replicates and biological triplicates (n = 3).

[0127] 2.7. Functional activity of KCs and idKCs after ASC-CM treatment For each assay, KCs and idKCs were plated on 2D culture plates and allowed to acclimate and reach appropriate confluency (>24 h). KC-GM was then removed, the cells washed, and plated with ASC-CM for 24 h. Experimental assays for metabolic, mitochondrial, proliferative, or migratory activity were then performed according to the manufacturer's protocol. ASC-CM was used as a "supplement" to keratinocyte growth medium (KC-GM) at a 2:1 ratio (ASC-CM to KC-GM). Briefly, plated cells were analyzed for PicoGreen fluorescence acquired at 435 / 535 nm (n=3) to quantify DNA as a surrogate measure of proliferation. Prestoblue fluorescence was acquired at 560 / 590 nm (n=3) and expressed as the average relative fluorescence units (RFU) of Prestoblue per Hoechst signal (350 / 460 nm) to obtain approximate metabolic activity per cell. A KC scratch assay was performed to assess changes in wound size / area. This was performed as a surrogate measure of KC migration after wounding within a confluent monolayer of KCs (n = 3). Images of migration were acquired using an ImageXpress Micro XLS Imaging System (Molecular Devices), and the percentage of closed wound area at 24 h was calculated using ImageJ analysis. EVs isolated from both 2D and 3D coated and uncoated samples were then added to KC-GM to achieve a final EV protein concentration of 150 μg / mL. EV concentration was calculated using the previously performed protein quantification, and idKCs were then evaluated for functional changes in phenotype and epidermal regeneration capacity (metabolism, proliferation, migration) to assess variability in EV quality / composition from each group.

[0128] 2.8. Proteome analysis Immunolabeling of specific intracellular proteins was performed on both ASCs and KCs to detect specific phenotypic surface markers (ASCs) and cytokeratin markers (KCs). The immunolabeling was performed similarly up to the time the primary antibody was added. Briefly, cells were washed three times with HBSS, fixed with 4% PFA, and washed three times with HBSS. Next, cells were incubated for at least 1 hour in a blocking buffer containing 2% donkey serum in HBSS with or without 0.1% Triton-X. Primary antibodies were then suspended in the blocking buffer and applied to the cells overnight at 4°C. Unless otherwise noted, all primary antibodies were obtained from Abcam and included N-cadherin (ab98952), E-cadherin (ab40772), keratin 5 (ab52635), keratin 10 (ab76318), keratin 16 (ab76416), CD34, CD45, CD73, CD90, and CD105. CD73 (Cat. #41-0200) was obtained from Invitrogen (Waltham, MA). The following day, the primary antibody solution was removed, and cells were washed three times with blocking buffer. Secondary antibodies were then applied for 1–2 h, followed by three washes of the final set with HBSS. Cells were counterstained with the nuclear marker Hoechst 33342. Secondary antibodies were donkey, not mouse or rabbit, and were obtained from Invitrogen. Other proteomic assessments included Western blotting as previously described. Briefly, KC cell lysates were prepared using 1x RIPA buffer with a protease inhibitor cocktail (Cat. #P8340, 1:100). Protein concentrations were obtained by absorbance at 280 nm using a QuickDrop filter and analyzed by SDS-PAGE by running proteins on 4-12% SurePage Bis-Tris gels (Genescript), transferred to PVDF membranes, and immunoblotted.All primary antibodies were sourced from Abcam and included CD9 (ab263019), CD63 (ab134045), CD81 (ab109201), Tsg101 (ab125011), GAPDH (ab8245), β-tubulin (ab6046), N-cadherin, E-cadherin, keratin 5, keratin 10, and keratin 16. Secondary antibodies were derived from donkey and used as previously described. Blocking buffer consisted of 2% donkey serum in HBSS.

[0129] 2.9. Quantitative real-time PCR (qRT-PCR) expression analysis RNA was isolated and purified using the RNeasy Mini Kit (Qiagen). Only RNA with a 260 / 280 ratio greater than 1.8 was used in this study. Cycle threshold values ​​(Ct values) were recorded and analyzed using the ΔΔCt method. qRT-PCR analysis was performed for ASCs using a wound healing assay and for KCs using individually selected primers. For ASCs, ASCs cultured in both 2D and 3D (with or without coating) were collected during the experimental assay after ASC-CM collection and analyzed by RT-PCR. 2The expression of 84 wound healing and wound healing-related genes was assessed using the Profiler™ PCR Array for Human Wound Healing (Qiagen, Cat. #330231, PAHS-121ZC-24). The endogenous control genes used in the array were glyceraldehyde-3-phosphate dehydrogenase (GAPDH), beta-actin (ACTB), and beta-2-microglobulin (B2M). For KCs, untreated KCs and idKCs cultured in KC-GM served as controls in each group. The purity of the cDNA samples was assessed using a QuickDrop spectrophotometer (Molecular Devices). A 260 / 280 absorbance ratio of >1.8 was considered pure. Individual qPCR primers (Qiagen, Cat. #330001) were purchased and used to perform RT-qPCR for CDKN2A, CDH1, CDH2, FLG, KRT5, KRT10, KRT16, TWIST1, EGFR, VIM, TWIST1, IL1B, and CCND1. GAPDH expression was used as an endogenous control for all samples. All qRT-PCR samples were performed in triplicate (n=3). The above procedure was performed using a qTower from Analytical Jena. 3 A ThermoCycler was used. SYBR Green was used as the fluorophore. A complete list of GeneGlobe IDs for KC is shown in Table 3. [Table 3]

[0130] 2.10. Statistical analysis All data were reported as means with standard deviations. Comparative functional analysis of early-induced diabetes patients was evaluated by one-way ANOVA. All other statistics, including keratinocyte ASC-CM assay, ASC-CM protein, EV quantification, ELISA, and RNA analysis, were evaluated by two-way ANOVA. Unless otherwise noted, a minimum of three biological replicates (n = 3) were used. Data were tested for normality using the Shapiro-Wilk test and the Kolmogorov-Smirnov test and plotted using a QQ plot. Analysis was performed using GraphPad Prism 9.4.2 software (La Jolla, CA), with p < 0.05 considered significant. ImageJ was used for image processing.

[0131] 3. Results 3.1. Characterization of adipose-derived mesenchymal stem cell populations 3.1.1 Characterization of ASC phenotype and matrix coating To assess their "MSC-like" phenotype, ASCs were first evaluated to confirm that they were adherent (Figure 17A), exhibited trilineage differentiation potential (Figure 17B), and expressed MSC "stem-like" surface markers, including positive staining for CD73 / 90 / D105 / CD271 and negative staining for CD34 / 45 (Figure 17B). Photographs of the TMHB system were taken to visualize the macrostructure and porous architecture (Figure 17C). Next, 2D and 3D systems were coated with different matrix-derived proteins, and ASCs were cultured for 8 days. ASCs were imaged throughout the culture to observe morphological changes between 2D and 3D systems and with different substrate coatings (Figure 18).

[0132] 3.2 High glucose supplementation induces a “functional” diabetic phenotype in the KC population 3.21. Induced diabetic keratinocytes (idKCs) exhibit reduced epidermal regeneration activity To achieve a KC phenotype that functionally exhibited similar properties to native diabetic keratinocytes, long-term culture with supraphysiological glucose levels was performed (Figure 19A). Next, KC populations (with or without 25 mM glucose exposure) were evaluated for changes in epidermal functional activity. While the cell size of both healthy KCs and idKCs remained relatively similar, the cell shape of idKCs showed a slight shift toward a more elongated / elliptical morphology compared to the more rounded shape of KCs (Figure 19B). Metabolic activity (Figure 19C), proliferative activity (Figure 19D), and migratory activity (Figure 19E) were all assessed for healthy KCs and idKCs. While the metabolic activity of KCs and idKCs was comparable, the proliferative and migratory activities of idKCs showed a significant decrease within 24 hours, with a maximum decrease of 52% and 23%, respectively.

[0133] 3.3. Substrate-dependent effects on the ASC secretome have diverse effects on idKC functionality 3.3.1 Matrix substrate modulates ASC secretion of factors that regulate the functional activity of regenerating epidermis Next, we evaluated the additive and / or synergistic benefits of coating materials in 3D hydrogel systems compared with 2D systems by assessing the effect of ASC-CM on the epidermal regeneration functionality of idKCs. ASC-CM from cells cultured in the coated tissue-mimicking system showed a stronger tendency to enhance idKC metabolic activity compared with the 2D ASC-CM group, with only 3D-Col (1.79-fold) and 3D-Fib (1.81-fold) resulting in significant improvements in idKC metabolic activity compared with their respective 2D-coated counterparts (Figure 20A). Interestingly, the uncoated 3D ASC-CM group remained comparable to all 2D groups in enhancing idKC proliferation activity, whereas all 3D coatings significantly improved idKC proliferation capacity compared with the conventional 2D-NC sample (Figure 20B). Furthermore, ASC-CM from 3D-Col (1.36-fold) and 3D-Fib (1.13-fold) significantly improved idKC proliferation compared to their respective 2D-coated counterparts, 2D-Col (0.88-fold) and 2D-Fib (0.74-fold) (Figure 20B). Similarly, while 2D coating did not significantly enhance ASC-CM-enhanced idKC migration, ASC-CM from 3D-Col produced the greatest improvement in idKC migration. Furthermore, 3D-Col was the only ASC-CM group that significantly altered idKC migration compared to the control group, resulting in a maximum wound closure of 73% and 62%, respectively, at 24 hours (Figure 20C).

[0134] Further evaluation of idKC activity was performed by assessing gene expression (Figure 20D). Cyclin D1 (CCND1) expression was evaluated to assess changes in idKC cell cycle and proliferation. In this case, both fibronectin and fibrin coating demonstrated an improved ability to enhance CCND1 expression at 24 h, with no difference between 2D and 3D. Similarly, EGFR and IL1B (IL-1β) expression in idKC showed minimal differences with ASC-CM treatment between 2D and 3D, with only ASC-CM treatment from 3D-Col resulting in a significant decrease in IL1B expression compared to 3D-Fn. In contrast, EGFR expression showed a tendency to decrease in all 3D groups, while both 2D and 3D groups showed an increase in IL1B expression compared to baseline expression in idKC. Furthermore, all 3D ASC-CM samples demonstrated the ability to significantly reduce the expression of p16ink4a (CDKN2A), a marker of senescence, compared to their 2D ASC-CM counterparts and baseline expression in idKCs. Furthermore, both 3D-Col and 3D-Fn ASC-CM significantly increased the expression of the suprabasal marker filaggrin (FLG) in idKCs, while ASC-CM from 3D-Col and 3D-Fib increased the expression of the migration marker TWIST1 in idKCs (Figure 20D).

[0135] 3.3.2 Matrix-dependent regulation of the ASC secretome enhances the epidermal phenotype of idKCs Assessment of idKC cytokeratins and cell-binding proteins was evaluated to identify changes in the epidermal phenotype of idKCs after treatment with ASC-CM by qRT-PCR. Gene expression changes by qRT-PCR were used to identify changes in the epidermal phenotype of idKCs after treatment with ASC-CM (Figure 21). In particular, E-cadherin (CDH1) expression was significantly enhanced in idKCs treated with all 3D ASC-CM groups, with 3D-Fn providing the most significant stimulation. All 3D coating groups provided varying levels of enhancement, while 2D-coated samples did not provide significant differences between coating groups. Conversely, N-cadherin (CDH2) expression was significantly reduced after treatment with all 3D ASC-CM groups, while 2D ASC-CM did not have a significant effect on CDH2 expression in idKCs. Interestingly, 2D-Col ASC-CM resulted in decreased expression of K5 in idKCs, whereas 3D-Col resulted in increased expression compared to the other 3D ASC-CM groups. K16, a marker of migration and wound response, was significantly enhanced in idKCs treated with ASC-CM from 2D-Col and 3D-Col, with 3D-Col resulting in the most significant increase of all groups. Meanwhile, ASC-CM from 3D-Fn and 3D-Fib resulted in a significant decrease compared to 3D-NC. K10, a marker of suprabasal differentiation, showed minimal differences between 2D and 3D ASC-CM treatments and between the different coatings, but all groups showed decreased expression compared to baseline idKCs.

[0136] 3.4. Matrix coating of tissue-mimicking systems enhances the relative secretion of regenerative compounds from ASCs 3.4.1. Enhanced secretion of trophic, immunomodulatory, and degradative proteins involved in epidermal regeneration The total protein content in the ASC-CM of each group was analyzed. 2D-NC (1.34 μg / mL) contained significantly less protein than all other groups. Culture in 3D-NC significantly enhanced the overall secretory activity of ASCs. Secretion of protein compounds increased by up to 48%, which is comparable to previously reported data. Interestingly, coating in the 2D group increased the concentration of secreted proteins to the same level as in all 3D groups. In contrast, 3D ASC-CM showed no difference between coated and uncoated groups (Figure 22A). Specific secreted protein markers known to regulate epidermal regeneration were assessed by ELISA, and their respective concentrations were quantified (Figures 22B-22C), validating ASC gene expression of specific secreted factors (Figures 22D and 23). Coating in the 2D group had no effect on regulating EGF and TGF-β1 secretion. On the other hand, 2D coatings were able to regulate the secretion of IGF-1, FGF-2, and MMP-1, and 2D-Fb and 2D-Fn significantly increased the secretion of several factors. 3D culture significantly enhanced the secretion of EGF, IGF-1, FGF-2, MMP-1, MMP-9, TGF-β1, and TIMP-1 compared to the 2D group. 3D-Fib had the most significant effect on FGF-2 secretion. Furthermore, all coatings significantly reduced the secretion of EGF and TGF-β1 in 3D. On the other hand, all coatings significantly increased the secretion of IGF-1 and MMP-1 in 3D compared to 3D-NC (Figures 22B and 24). Interestingly, 3D culture significantly reduced MMP-1 secretion in 3D-Fn but significantly increased it in 3D-Col compared to their respective 2D counterparts. Finally, both 2D and 3D coating significantly increased TIMP-1 secretion, with similar patterns for TIMP-1 and MMP-1 secretion (Figures 22B and 24). Protein secretion from ASCs was further validated by assessing gene expression in wound healing arrays. Heatmaps show that ASC expression of several secreted proteins in 3D was increased compared to the 2D group.Coating in 2D had minimal effect on ASC activity (Figure 22D). A complete list of relative gene expression across the array is shown in Table 4. [Table 4]

[0137] 3.5. Coating the tissue-mimicking system with type I collagen enhances the epidermal regeneration capacity of ASC-EVs 3.5.1 ASCs exposed to type I collagen in 3D enhance epidermal regeneration of idKCs via secreted EVs Next, we evaluated the quality of ASC-EVs with respect to their ability to modulate idKC activity. ASCs in the TMHB system showed enhanced extracellular vesicle (EV) secretion compared to 2D in both uncoated and coated samples (up to a 1.89-fold increase in 3D). In contrast, substrate coating in 2D and 3D did not alter the relative concentration of EVs in ASC-CM for each group (i.e., within-group comparison) (Figure 25A). Furthermore, when controlling for total protein secreted in ASC-CM, the relative composition of EV proteins to total secreted proteins was significantly greater in the 3D ASC-CM group compared to their respective 2D counterparts, with the exception of fibrin (Figure 25B). Analysis of EV size distribution by NTA showed that over 95% of all six particles analyzed were in the 25-250 nm range, approximately 2-3 times larger in 3D than in 2D, consistent with the EV protein quantification data (Figure 26). Furthermore, the presence of exosomal proteins CD9, CD63, CD81, and Tsg101 suggests that EVs are of exosomal origin.

[0138] To assess the role of ASC-EVs in the previously observed benefits of ASC-CM treatment, idKCs were treated with KC-GM medium containing isolated EVs from each of the 2D and 3D groups at a concentration of 150 μg / mL. Next, idKCs were evaluated for epidermal functional activity through assessment of metabolic activity (Figure 25C), proliferative activity (Figure 25D), and migratory activity (Figure 25E). Notably, when comparing each 3D-coated group with their 2D counterparts, EVs from 3D-Col were the only 3D-EVs that significantly enhanced all three idKC functions (metabolic activity, proliferative activity, and migratory activity) (Figures 25C–E). More specifically, 3D-Col EVs induced the greatest increase in idKC proliferative activity (approximately 2-fold increase compared to 2D-Col) (Figure 25D). On the other hand, both 3D-Col and 3D-Fn EVs resulted in a significant increase in idKC migration compared to their 2D counterparts (Figure 25E).

[0139] Next, gene expression analysis of idKCs after 24 hours of ASC-EV treatment (relative to control idKCs) was performed to further validate the functional and phenotypic changes in idKCs (Figure 6F). 2D coating was able to produce ASC-EVs with enhanced expression of K5, K16, and FLG compared to the uncoated group. 2D-Col showed a significantly higher tendency for increased expression of K16 and FLG (Figure 25F). In contrast, 3D-NC significantly improved the expression profile of idKCs compared to 2D-NC for all markers except K5. Notably, 3D coating significantly reduced CDKN2A expression compared to all 2D-EV groups, demonstrating its ability to modulate ASC-EV activity and improve the expression of several idKC markers. Furthermore, 3D-Col was the most consistent group to significantly affect gene expression in idKCs and was the only 3D-EV group to significantly enhance the expression of all three keratins, K5, K10, and K16, compared to their 2D counterparts (Figure 25F).

[0140] 4. Discussion The incomplete or incomplete closing of wounds caused by trauma, surgery, acute or chronic diseases, and radiation-induced tissue damage often results in non-healing chronic wounds, which affect millions of people every year. Diabetic wounds, in particular, are one of the most common chronic wounds, currently affecting up to 15% of all diabetic patients in the United States. With the aging population and increasing prevalence of diabetes, the prevalence of diabetic chronic wounds is expected to increase. Therefore, therapies tailored to diabetic wounds are a highly researched topic. However, to date, there are few studies specifically focused on improving epidermal regeneration capacity in diabetic wounds. Therefore, there remains a critical need to develop therapies that can prevent the decline in proper keratinocyte functionality and restore proper epidermal regeneration capacity to wounds, thereby preventing further progression of diabetic chronic wounds.

[0141] Early studies of MSC populations in in vitro and in vivo wound models demonstrated their ability to promote wound healing and regulate wound regeneration in several key wound-healing cell populations. Furthermore, in vivo animal models and clinical trials in diabetic wound models demonstrated the ability of ASCs to directly enhance wound healing, increasing the rate at which wounds "close" and improving overall outcomes. Injections of ASCs and ASC-laden wound dressings for the treatment of diabetic ulcers have shown promise. However, to enhance the therapeutic benefit of ASC therapy, it appears essential to retain ASCs within the wound site using a wound dressing or other delivery method. This is thought to be, in part, a result of the proximity of "wound tissue stimuli" to the ASCs, rather than the radial diffusion and cell death typically seen in injected cell therapies, ultimately allowing ASCs to continuously adapt and respond to the wound tissue environment in which they develop.

[0142] Although promising, cell-based therapies certainly have their limitations and can be highly variable depending on the baseline viability and robustness of the ASC source. This is often a disadvantage in patients presenting with chronic wounds. Recent research into the adaptive and secretory properties of ASC populations in response to priming stimuli offers a unique opportunity to develop regenerative wound therapies that can circumvent many of the limitations of autologous cell therapies. The regenerative potential observed in previous ASC therapies is thought to be due, in part, to the secretion of various paracrine compounds from ASCs, including antioxidants, anti-inflammatory compounds, anti-protein compounds, and targeting EV compounds. However, to date, most therapies have explored the use of 2D-cultured MSC populations, which are known to result in reduced viability, terminal differentiation, and loss of regenerative capacity, thereby significantly hindering the translatability of potential MSC-derived regenerative therapies.

[0143] Based on previous studies demonstrating the ability of 2D-cultured ASCs to enhance wound healing activity, this study utilized a unique tissue-mimicking 3D system and compared it with a conventional 2D system. 3D systems (e.g., the TMHB system) are more physiological for ASC populations because they mechanically resemble native adipose tissue. Therefore, this study uniquely investigated the effects of multiple wound matrix coatings on ASC secretome functionality while controlling for mechanical inputs from 2D and 3D. By utilizing various matrix-derived proteins used as priming stimuli in both 2D and 3D systems, we altered ASC secretory activity and subsequently directly assessed the role of the wound matrix in modulating the epidermal regenerative potential of the ASC secretome. Based on previous studies demonstrating how this tissue-mimicking system can help maintain a more regenerative ASC population, we hypothesized that a more robust ASC population would respond better to priming stimuli compared to 2D cultures and, accordingly, secrete higher concentrations of specific regenerative factors. This was supported in multiple ways in this study, including the increased secretion of soluble proteins (Figure 22A), EVs (Figure 25A), and antioxidants (Figure 27) by ASCs in 3D systems compared to 2D systems. Priming in 3D systems resulted in greater diversification of the ASC secretome's functionality and composition compared to the 2D secretome, which exhibited less overall diversity among different coatings.

[0144] The matrix coating in this study was selected based on the following principles. Fibrin has a native role in wound healing and has been shown to improve wound outcomes in vivo. Fibrin has also demonstrated the ability to further enhance the wound healing outcomes of MSC-based treatments in vivo, including complex diabetic wounds, by promoting the secretion of several specific soluble factors, including VEGF, EGF, and FGF. Fibronectin is an essential component of the extracellular matrix and basement membrane. It directly interacts with collagen during wound healing and exhibits rapid turnover during cutaneous wound healing due to frequent damage to the basement membrane. Finally, type I collagen is the most abundant matrix protein in tissues, including skin, and plays a major role in wound healing and epidermal regeneration. In particular, type I collagen accounts for a significant portion of dermal collagen exposed upon wounding and is thought to provide a primary migration stimulus for wound-healing cells. Furthermore, several studies have investigated the benefits of encapsulating MSC populations in type I collagen and demonstrated improved wound healing outcomes. Therefore, these three matrix proteins were chosen because they provide a diverse array of substrates involved in various components of native wound healing. Future studies will explore the role of other substrates, as well as heterogeneous mixtures of substrates, combined with additional environmental or biochemical stimuli (e.g., hypoxia), as a means of generating tissue-mimicking in vitro systems capable of producing factor compositions tailored for wound application.

[0145] Previous studies have shown that hyperglycemia in native diabetic patients can induce a phenotypic transition in KCs, altering their barrier functionality and simultaneously inhibiting their proper migratory and proliferative activities. Furthermore, studies have shown that high glucose exposure to KCs can induce a "functional" diabetic KC phenotype, and similar cellular changes, including reduced proliferation and migration, are observed in KCs from patients with diabetic wounds (Figure 19). This study utilized an inducible system to allow for parallel non-diabetic control KC populations. However, primary KCs from diabetic patients do not guarantee functional impairment in wound healing, and non-diabetic controls from the same patients are not possible.

[0146] ASC-CM assays of idKCs revealed that coating with type I collagen in a tissue-mimicking system significantly enhanced the epidermal regeneration functionality of ASC secretomes compared to fibrin or fibronectin in both 2D and 3D (Figures 20A-20C). Interestingly, ASC-CM from 3D-Fib induced stronger metabolic and proliferation stimulation in healthy KCs than 3D-Col (Figure 17). These data provide new insights, suggesting that healthy and diabetic KCs may have different sensitivities regarding which factors are required to achieve epidermal regeneration, and further highlight the importance of tailored therapy for each wound application. The four most significantly increased differentiation markers in idKCs treated with 3D ASC-CM were E-cadherin, filaggrin, twist1, and k16, all of which are responsive genes essential for epidermal regeneration. Notably, ASC-CM from both 3D-Col and 3D-Fn resulted in the greatest upregulation of filaggrin and E-cadherin, key proteins involved in suprabasal epidermal barrier function and cell sheet formation (Figure 20D). In contrast, only 3D-Col ASC-CM significantly increased the expression of basal layer marker (keratin 5), suprabasal differentiation marker (keratin 10), and migration or wound-responsive marker (keratin 16) in idKCs compared with the other coatings (Figures 21A-21C). 2D-Col was the only one able to promote increased K16 expression compared with the other 2D coatings, albeit to a lesser extent. Collectively, these data suggest that ASC-CM from 3D-Col exhibited the greatest overall ability to enhance the diverse and regenerative KC phenotype, which may be related to the improved barrier formation and functional activity observed in idKCs, and was the only group to significantly enhance idKC migration capacity from their baseline state (Figure 20C).

[0147] ASC-CM from all 3D groups showed no increase in the production of key signaling factors compared with their 2D counterparts. Interestingly, although 3D-Col resulted in the greatest ability to enhance the functional activity of idKCs, 3D-Col did not result in the greatest secretion of any of the major soluble proteins thought to be essential for epidermal regeneration functionality (including EGF, IGF-1, FGF-2, or FGF-7 (KGF)). This may suggest that the relative composition and equivalent balance among several factors may be important, that idKCs may depend on other proteins not examined, or that non-protein factors (e.g., EVs) may play a more important role in the regenerative activity of idKCs. Notably, the concentration of EVs secreted from ASCs was increased in each coating group in 3D compared with their 2D counterparts, but there was no noticeable difference between the 3D groups. This suggests that the increased EV production was likely dependent on the tissue-mimicking environment rather than the coating. This data further supports the notion that ASCs retain a higher propensity for regenerative secretory activity in 3D, while exogenous factors and / or coating substrates can be used to fine-tune and adjust the relative composition of the secretome.

[0148] To examine whether the EV fraction was driving any of the functional changes observed in idKCs, we administered 150 μg / ml of EVs to KC-GM, allowing us to assess the functional quality of EVs on an EV-to-EV basis. Controlling for total EV levels, idKCs exhibited variable functional changes after ASC-EV treatment within 24 hours. Both the metabolic and proliferative activities of idKCs were significantly enhanced by 3D-Col compared to the other ASC-EV groups. Furthermore, both 3D-Col and 3D-Fn significantly enhanced the migratory capacity of idKCs. Thus, 3D-Col was the only 3D coating that enhanced the functional activity of idKCs compared to their 2D counterparts across all three assays.

[0149] Interestingly, the effect of ASC-EV treatment on the metabolic and proliferative activities of idKCs showed a similar pattern to the data from "full / complete" ASC-CM treatment (Figure 20). This suggests that idKC metabolic and proliferative activities may be significantly influenced by EV entities rather than secreted protein factors. Conversely, 3D-Fn had no significant effect on idKC migratory activity when treated with "full / complete" ASC-CM (Figure 20). Although migratory activity was significantly enhanced by 3D-EV, the relative migration amount at 24 h was less with ASC-EV treatment alone (up to 44% with 3D-Col) compared with "full / complete" ASC-CM (up to 75% with 3D-Col). This suggests that there is likely an additive or synergistic effect between ASC-EVs and non-EV soluble factors in enhancing idKC migratory activity. Collectively, these data revealed that the quality and composition of EV contents varied depending on the substrate, with 3D-Col-derived EVs showing the greatest tendency to consistently enhance epidermal regeneration functionality in idKCs. Furthermore, the effect of EV administration on idKC gene expression indicated that 3D-EVs were able to modulate idKC gene patterns more significantly than 2D-EVs. For example, the expression of CCND1 and CDKN2A in idKCs significantly changed after treatment with 2D-EVs and 3D-EVs. Therefore, ASC-EVs may play a major role in regulating the relative progression of idKC cell cycle progression, as demonstrated by the increased expression of the proliferation marker CCND1 after 3D-Col and 3D-Fib EV treatment, and the significant decrease in the senescence marker CDKN2A in all 3D-EVs. The significant decrease in CDKN2A expression compared to control idKCs suggests that ASCs in the tissue-mimicking system may secrete EVs containing senescence-protective compounds. Next, the suprabasal differentiation marker FLG showed a similar expression pattern between ASC-EVs from 2D- and 3D-coated groups, whereas 3D-NC resulted in a significant increase in expression compared with 2D-NC.Thus, while the tissue-mimetic properties of the 3D system may be broadly advantageous in modulating FLG expression, this advantage of the 3D system is negated by the addition of matrix-derived compounds, which appear to have a stronger effect on EV functionality in the direction of altering FLG. This was demonstrated by the comparable idKC expression profiles after treatment with either 2D-EVs or 3D-EVs from coated samples. Finally, ASC-EVs were able to modulate the expression of idKC keratins. 3D-Col was the only 3D-coated group to significantly increase all three keratin markers compared to their respective 2D counterparts, including the major "wound-responsive" keratin K16. These data are comparable to the expression analysis performed on "full / intact" ASC-CM in Figure 21, where 3D-Col was superior to all other groups in promoting K16 expression. The superior idKC functionality after treatment with 3D-Col EVs may be related to its ability to modulate K16 activity in idKC, although this requires further investigation.

[0150] The EV data in this study potentially suggest that KCs may become more dependent on signaling factors present within EVs when they exhibit a diabetic-like phenotype. To our knowledge, this is the first time we have uncovered the divergent effects of ASC-derived EVs based on matrix coating and a potential shift in sensitivity to external signaling molecules when KCs exhibit a diabetic-like phenotype. While the underlying kinetic mechanisms require further extensive study, one possible explanation is that signaling kinetics may shift in diabetic KCs to favor the uptake of EV-like particles as a result of altered cell surface receptors. This could potentially be due to cellular and protein damage caused by supraphysiological glucose levels and elevated oxidative activity, which may alter the sensitivity of diabetic KCs to external signaling factors. Furthermore, the ASC secretome effects from the 2D system show significantly less heterogeneity in functional effects on idKCs compared to coated 3D-ASC secretomes. This indicates that there is not only a coating / material effect but also possibly a combined effect with the 3D hydrogel system on ASC phenotype and functionality. Furthermore, the coating effect on the ASC secretome can be further enhanced because ASCs in 3D are in a more tissue-mimicking environment and therefore can retain their intrinsic adaptability and respond more appropriately to coating stimuli.

[0151] Although there are limitations in this study in its current form that require further investigation, these do not ultimately detract from the overall value of the findings. ASC populations may exhibit variability in regenerative properties between donors, which may result in altered secretory activity among different ASC populations. However, the study design, which allowed for comparison between 2D and 3D tissues and between different coatings, ensured that any donor-specific effects were comparable across groups. Similarly, the effects of ASC-CM were tested only in an "induced diabetic" KC model. This allowed for parallel testing of healthy KCs, but there may still be differences between KC populations isolated from diabetic chronic wounds at various stages of chronic wound progression. Furthermore, future studies of other wound-healing cell populations are needed to establish a more holistic picture of the role of ASC secretome components in diabetic wound healing. While the use of in vitro analyses alone can be limiting, the holistic perspective gained by utilizing a wide range of functional, protein, and RNA assays provided data that can be used to plan future in vivo studies and provide a framework for tailoring the composition of the ASC secretome. In particular, the similarities between the in vitro diabetic model used in this study and the "functional" phenotype of KCs isolated from diabetic wounds have already been established in previous studies. Further research is also needed into the dynamic and interrelated roles of the utilized substrate / coating material and the X-Block hydrogel system. The authors speculate that the phenotype of ASCs shifts when exposed to the coating material, preserving their inherent adaptability; however, the extent to which this occurs is unknown and requires further study. Finally, although beyond the scope of this study, further studies of the dynamic array of secretome compounds, including protein mass spectrometry, as well as the contents within EVs (e.g., proteins, ROS, nucleic acids, miRNAs) should be conducted to better understand how specific environmental conditions shift secretome composition.It is possible that the same key protein compounds that drive healthy epidermal regeneration also promote epidermal regeneration in diabetic KCs, but that they were not detected by ELISA because they were packaged within EVs rather than secreted as soluble proteins.

[0152] This study highlights the value of utilizing tissue-mimicking environments for cell culture and evaluates their role in maintaining more regenerative MSC-like populations for the development of regenerative therapies. More specifically, the various 3D ASC-CM populations were able to exhibit different functional capacities, whereas the 2D ASC-CM populations exhibited minimal differences between the various coatings, supporting the hypothesis that ASCs are able to adapt to environmental stimuli more easily in tissue-mimicking systems. The authors demonstrated the tunable roles that dimensionality, mechanism, and substrate binding play in influencing ASC secretory activity and the resulting functional changes observed in the ASC secretome. While these parameters represent only a small part of a larger understanding, they demonstrate that in vitro systems attempting to control MSC-like populations and their secretomes must consider numerous parameters to truly understand and control their activity. Various other parameters to consider include the incorporation of environmental (e.g., hypoxia), chemical (e.g., growth factors / cytokines), mechanical (e.g., dynamic compression), or biological (e.g., co-culture) stimuli, as well as the addition of specific drugs to better understand the role of the secretome in drug responses. Thus, this study offers insight into the future development and potential of tunable, pre-fabricated noncellular by-products for many soft tissue wound healing applications, and also provides an opportunity to more effectively assess the responses of native cells to various stimuli.

[0153] 4. Conclusion Finally, previous studies have shown that ASCs can be primed by exposure to various compounds and environmental conditions to enhance specific functions in vivo (e.g., hypoxic priming to enhance angiogenic activity). In contrast, this study provides insight into the comparative effects of matrix-derived compounds on ASC secretome functionality with respect to epidermal regeneration in idKCs. The potential benefits of tailored ASC biopharmaceuticals for epidermal regeneration have yet to be demonstrated. This study reveals the role of the ASC secretome in regulating key epidermal regeneration signaling pathways in idKCs, including their ability to alter keratin expression, promote suprabasal differentiation, and improve barrier formation. More specifically, we evaluated the role of ASC-EVs within the ASC secretome and demonstrated that they are potential key drivers of functional responses in idKCs, suggesting a dynamic shift in signaling toward preferring EV-like compounds when KCs exhibit a diabetic-like phenotype. Furthermore, this study provides insight into the crucial role of tissue-mimicking cultures in achieving robust MSC-like populations that retain a more dynamic secretory phenotype and can more easily adapt to changing environmental conditions. In contrast, 2D cultures exhibited significantly less change when exposed to various coated substrates. Thus, by introducing predetermined exogenous stimuli into tissue-mimicking systems, it is possible to adjust and fine-tune the composition of secreted products, providing opportunities to generate an array of diverse functional biopharmaceuticals for various applications.

[0154] These and other modifications and variations to the present invention may be practiced by those skilled in the art without departing from the spirit and scope of the present invention, which is more particularly set forth in the appended claims. Moreover, it will be understood that aspects of the various embodiments may be interchanged in whole or in part. Moreover, those skilled in the art will recognize that the foregoing description is merely illustrative and is not intended to limit the invention, as further set forth in such appended claims. Therefore, the spirit and scope of the appended claims should not be limited to the exemplary description of the forms contained herein.

Claims

1. 1. A method for characterizing a therapeutically desirable component or component profile of a secretome composition secreted from a cell type of interest, comprising: (i) seeding a first tissue-mimicking three-dimensional (3D) hydrogel block (TMHB) comprising a macrostructure defined by a continuous polymeric matrix material and a network of microporous channels and / or chambers extending throughout said continuous polymeric matrix material with a first cell type of interest; (ii) simulating in vitro cell growth and proliferation of the first cell type of interest by providing the first cell type of interest with a first medium and allowing the first cell type of interest to grow throughout the first TMHB and secrete the secretome composition; (iv) collecting the secretome composition; (v) subjecting the secretome composition to a stratification operation based on molecular weight cutoff values ​​that provide a plurality of different layers, each characterized by a different range of component molecular weights; (vi) characterizing and / or identifying one or more individual components present with each layer of interest; (v) testing each layer of interest for relative therapeutic benefit by either in vivo or ex vivo analysis to identify the layer of interest that provides the greatest therapeutic benefit as the therapeutic moiety profile; A method comprising:

2. The method of claim 1 , further comprising the step of characterizing at least a majority (e.g., each) of the individual components present in the layer of interest that provide the greatest therapeutic benefit.

3. 3. The method of claim 2, further comprising identifying at least one frequently occurring component that is absent or present in small amounts in each of the layers of interest from the one or more individual components present in the layers of interest that provides the greatest therapeutic benefit, and identifying the at least one frequently occurring component as the therapeutically desirable component.

4. 1. A method for modulating the component profile of a natural secretome composition secreted from a cell type of interest, comprising: (i) seeding a first tissue-mimicking three-dimensional (3D) hydrogel block (TMHB) comprising a macrostructure defined by a continuous polymeric matrix material and a network of microporous channels and / or chambers extending throughout said continuous polymeric matrix material with a first cell type of interest; (ii) providing a first culture medium to a first cell type of interest; (iii) introducing at least one external stimulus configured to modify the natural secretome composition secreted from a cell type of interest so as to more closely track a target component or a target secretome composition having a target component profile, wherein the at least one external stimulus comprises one or more controllable parameters (e.g., adding a coating to the interface between the continuous polymer matrix material and the network of microporous channels and / or chambers, adjusting oxygen concentration, pH, temperature, and fluid dynamics within the first TMHB); (iv) allowing the first cell type of interest to proliferate throughout the first TMHB and secrete the secretome composition; (iv) collecting a secretome composition modified by the external stimulus that differs from the native secretome composition; A method comprising:

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