Large-scale generation of spheroids and extracellular vesicles

Abstract

A method for producing spheroids and cellular by-products of spheroids, such as extracellular vesicles, is provided. The method includes providing a bioreactor having a cavity for culturing cells, inserting the cells into the cavity to form spheroids, and perfusing cell culture medium through the cavity to culture the spheroids. The porous scaffold may be dissolvable or indissolvable, and cells are seeded into the pores of the porous scaffold, where they aggregate to form spheroids. The bioreactor is continuously perfused for large-scale spheroid production, allowing for the recovery of spheroids and / or extracellular vesicles or other elements from the cell secretome.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority under 35 U.S.C. § 119 to U.S. Provisional Patent Application No. 63 / 426,908, filed November 21, 2022, the contents of which are relied upon and incorporated by reference herein in their entirety.

[0002] The present disclosure relates generally to methods for producing cellular by-products, including spheroids and / or extracellular vesicles. Specifically, the present disclosure relates to methods for large-scale production of spheroids and / or extracellular vesicles within porous scaffolds. [Background technology]

[0003] In vitro models of cell function have been developed in various fields, including cancer and stem cell research, drug discovery and screening, and regenerative medicine, among others. The goal of these models is to understand how these cells function and interact at the molecular and cellular levels. These in vitro models were developed using two-dimensional ("2D") systems. While these 2D systems have led to important discoveries in these fields, many challenges remain. In 2D systems, cells adhere to rigid surfaces, are geometrically constrained, and assume a flat morphology, which can alter cytoskeletal regulation important for intracellular signaling and ultimately affect cell growth, migration, and apoptosis. These limitations of 2D culture often result in in vitro biological responses that differ significantly from those observed in vivo. This is because these 2D models cannot fully capture the complexity of in vivo tissues and the contributions of the cellular and non-cellular microenvironments.

[0004] Currently, in drug discovery, the standard procedure for screening compounds begins with 2D cell culture-based testing, followed by animal model testing and then clinical trials. Only approximately 10% of compounds successfully progress through clinical development. Many drugs fail during clinical trials (especially during Phase III, the most expensive phase of clinical development), primarily due to lack of clinical efficacy and / or unacceptable toxicity. Some of these failures result from data collected from 2D culture studies, where cellular responses to the drug(s) are altered due to the non-native microenvironment. Due to the high costs associated with drug discovery, there is a growing demand for the ability to eliminate ineffective and / or unacceptably toxic compounds as early as possible in the drug discovery process. There is a need for cell-based in vitro systems that can more realistically mimic in vivo cellular behavior and provide more predictable results than in vivo testing.

[0005] Three-dimensional ("3D") systems are particularly interesting. Compared to 2D cell culture monolayers, 3D cell cultures offer enhanced cell-cell interactions that more closely mimic the natural microenvironment of tissues. Over the past decade, 3D cell cultures have been used to grow a wide variety of cancerous and non-cancerous cell lines into spheroids or 3D cell colonies. Spheroids have been used for 3D tissue modeling in the fields of drug discovery, toxicology, and regenerative medicine. Recent studies suggest that 3D cell cultures, in contrast to 2D cultures, more accurately represent the environment experienced by cells in vivo, and that cellular responses in 3D cultures are more similar to in vivo behavior than cellular responses in 2D cultures. The additional dimensionality of 3D cultures is thought to lead to differences in cellular responses, not only by affecting the spatial organization of cell surface receptors involved in interactions with surrounding cells, but also by providing physical constraints on cells. These spatial and physical aspects of 3D cultures are thought to affect signaling from the outside to the inside of cells, ultimately affecting gene expression and cellular behavior.

[0006] Cell culture systems are also of interest for components produced by cells and secreted within their environment. One component of particular interest (especially with regard to regenerative medicine and other therapies) is extracellular vesicles (EVs). Extracellular vesicles (EVs) are nanoparticles produced by most cell types, and they have powerful therapeutic potential. Current methods used for EV production use adherent cells either on 2D surfaces, which require large culture spaces to meet the concentrations required for therapy, or on 3D surfaces such as microcarriers or other fixed-bed materials under perfusion in bioreactors. However, despite the need for large-scale production of this important by-product of cells, efficient large-scale EV production methods remain elusive.

[0007] Cells that aggregate into 3D structures are called spheroids, and spheroids mimic in vivo tissues better than cells grown in 2D. Generating large numbers of spheroids is advantageous for a variety of reasons, including for testing drugs and for serving as cell factories for generating cellular by-products such as EVs and exosomes. Because EVs are cellular by-products, they can be collected from the fluid surrounding cells and cell aggregates such as spheroids. However, when drug testing against a cell type is desired (or for other applications, such as research into cell function or regenerative medicine), capturing large quantities of the spheroids themselves is advantageous. Spheroids are currently grown on 2D platforms such as microwell plates and flasks. However, the 2D nature of these platforms limits the number of spheroids that can be generated. Furthermore, because EVs are generated by the cells within the spheroids, the limited number of spheroids generated by 2D systems also limits the amount of EVs that can be generated.

[0008] Capturing spheroids from 2D systems is labor-intensive and inefficient because each plate must be handled individually to collect the spheroids from the microwells, and growing spheroids in 2D systems often requires liquid exchange, which makes it difficult to culture the spheroids without disturbing them. Similarly, capturing EVs and other cellular byproducts produced by the spheroids is labor-intensive and inefficient because the liquid must be collected and separated from the spheroids.

[0009] Therefore, there is a continuing need for 3D matrices that enable 3D cell culture to produce spheroids and / or cellular by-products (e.g., extracellular vesicles) on a large scale. Furthermore, adequate delivery of nutrients to the cultured cells is advantageous, as is the eventual harvesting or recovery of the cultured cells and / or cellular by-products. The present disclosure provides a 3D porous scaffold for culturing spheroids and cellular by-products produced by spheroids that solves the above-mentioned problems faced in 2D culture. Summary of the Invention

[0010] According to aspects of the present disclosure, a method for generating cell spheroids is provided herein. The method includes providing a bioreactor having a cavity for culturing cells, inserting the cells into the cavity to form spheroids, and perfusing cell culture medium through the cavity to culture the spheroids. Aspects of the embodiments include a bioreactor including a porous scaffold within the cavity. The porous scaffold is fabricated from interconnected scaffold materials that define pores. In certain aspects, the porous scaffold is a foamed scaffold, which in some embodiments is dissolvable and in other embodiments is indissolvable. The method can further include seeding cells within the pores of the porous scaffold by inserting the cells into the cavity. In some embodiments, the cells aggregate within the pores of the foamed scaffold to form spheroids. The pores can be sized to trap at least some of the spheroids within the pores. In one aspect of the embodiment, the cells do not adhere to the porous scaffold. In embodiments, the porous scaffold does not have a cell adhesion coating. The method can further include collecting at least one of the spheroids or secreted material from the spheroids, where the secreted material can include extracellular vesicles, proteins, or other components of the cell secretome. The method can include digesting any dissolvable foam scaffold by exposing the dissolvable foam scaffold to an enzyme and / or a chelating agent. In some embodiments, perfusing cell culture medium includes continuously passing cell culture medium over the porous scaffold.

[0011] According to aspects of the present disclosure, provided herein is a dissolvable foam scaffold for cell culture. In some embodiments, the dissolvable foam scaffold comprises an ionotropically crosslinked polygalacturonic acid compound selected from at least one of pectinic acid; partially esterified pectinic acid, partially amidated pectinic acid, and salts thereof; and a water-soluble polymer having surface activity and a hydrophilic-lipophilic balance (HLB) of greater than about 10, preferably greater than about 20. Alternatively, the dissolvable foam scaffold may instead comprise alginic acid as the ionotropically crosslinked polymer. In some embodiments, the water-soluble polymer has an HLB of greater than about 22. In some embodiments, the dissolvable foam scaffold does not comprise glycerol, sorbitol, ethylene glycol, propylene glycol, or polyethylene glycol. In some embodiments, the dissolvable foam scaffold comprises glycerol or one of sorbitol, ethylene glycol, propylene glycol, and polyethylene glycol. According to aspects of the embodiment, the dissolvable foam scaffold may contain only a single water-soluble polymer that has surface activity and may not contain any polymers that do not have surface activity. In some aspects of the present disclosure, provided herein are non-dissolvable foam scaffolds for cell culture. Cells may form spheroids within either the dissolvable or non-dissolvable foam scaffold.

[0012] According to embodiments of the present disclosure, a method for forming a dissolvable foam scaffold is provided herein. The method includes: forming a first aqueous mixture by adding a polygalacturonic acid compound selected from alginic acid or pectinic acid; partially esterified pectinic acid, partially amidated pectinic acid, and at least one of their salts to an aqueous solution; forming a second aqueous mixture by adding a surface-active water-soluble polymer and a divalent metal salt to the aqueous solution; combining the first aqueous mixture with the second aqueous mixture to form a combined aqueous mixture; adding a gel-inducing agent to the combined aqueous mixture to form a foamed solution; and introducing gas bubbles into the foamed solution to form a foamed scaffold. The water-soluble polymer has a hydrophilic-lipophilic balance (HLB) greater than about 10 or greater than 20. In embodiments, the HLB of the water-soluble polymer is about 22 or greater. In some embodiments, forming the second aqueous mixture includes adding a water-soluble plasticizer to the second aqueous mixture, but may not include adding a non-surface-active second polymer. According to further aspects of the embodiment, the method does not include adding an emulsifier to any of the first aqueous mixture, the second aqueous mixture, the combined aqueous mixture, and the foaming solution. According to embodiments, the method includes adding a first amount of gel-inducing agent to the aqueous mixture before foaming and adding a second amount of gel-inducing agent to the aqueous mixture during or after foaming. The ratio of the second amount of gel-inducing agent to the first amount of gel-inducing agent is greater than about 2, or about 7 or greater.

[0013] According to embodiments of the present disclosure, a method for culturing cells in a dissolvable foam scaffold is provided herein. The method includes seeding cells into a dissolvable foam scaffold such that the cells enter the pores of the dissolvable foam scaffold, the dissolvable scaffold comprising an ionotropically crosslinked polygalacturonic acid compound selected from at least one of ionotropically crosslinked alginic acid and its salts, or pectic acid; partially esterified pectinic acid, partially amidated pectinic acid, and salts thereof; and a water-soluble polymer having surface activity and a hydrophilic-lipophilic balance (HLB) greater than about 20; and contacting the dissolvable foam scaffold with a cell culture medium. According to various aspects of the embodiments, the dissolvable foam scaffold has 0% by weight of a water-soluble plasticizer and is free of non-surface-active polymers. According to other aspects of the embodiments, the dissolvable foam scaffold may include a plasticizer, including a water-soluble plasticizer.

[0014] According to aspects of the present disclosure, there is provided a method for producing spheroids or spheroid by-products, the method comprising: (a) providing a bioreactor comprising a cavity for culturing cells, an inlet and an outlet to the cavity, and a porous scaffold; (b) inserting cells of a certain cell type into the porous scaffold to form spheroids; and (c) perfusing cell culture medium through the cavity to culture the spheroids. The porous scaffold comprises pores and interconnections (passageways) between the pores. In some embodiments, the step of inserting cells of a certain cell type into the porous scaffold comprises seeding the cells into the porous scaffold. The cells substantially aggregate within the pores of the porous scaffold to form spheroids. In some embodiments, at least 80% of the cells within the pores of the porous scaffold aggregate to form spheroids. In some embodiments, the pores and the passageways between the pores are sized to confine at least some of the spheroids within the porous scaffold. In some embodiments, the method includes recovering at least one of the spheroids or at least one by-product of the spheroids. In some embodiments, the recovered by-product of the spheroids is extracellular vesicles. In some embodiments, perfusing cell culture medium includes continuously passing cell culture medium over the porous scaffold.

[0015] In some embodiments, the porous scaffold is composed of a material that is non-adhesive to cells, or the material of the porous scaffold has been treated to be non-adhesive to cells, or a combination thereof. In some embodiments, the porous scaffold has a cell density of about 1.0 x 10 -5 m / s~approx. 5.0×10 -4 In some embodiments, the porous scaffold is non-adherent to at least 80% of the cells in the bioreactor when perfused at a linear velocity (Darcy flow rate) of 1000 m / s.

[0016] The porous scaffold comprises pores and interconnections (passageways) between the pores that allow fluid to pass between the pores, with several interconnections per pore. In some embodiments, at least 70% of the pores have a pore diameter of about 200 μm to about 1000 μm when measured in a dry scaffold. In some embodiments, at least 80% of the pores have a pore diameter of about 400 μm to about 800 μm when measured in a dry scaffold. In some embodiments, at least 70% of the passageways have a maximum passage width of about 30 μm to about 500 μm when measured in a dry scaffold. In some embodiments, at least 80% of the passageways have a maximum passage width of about 60 μm to about 400 μm when measured in a dry scaffold. In some embodiments, at least 70% of the pores have 5 to 18 passageways per pore. In some embodiments, at least 80% of the pores have about 6 to about 14 passageways per pore.

[0017] In some embodiments, the porous scaffold comprises an ionotropically cross-linked polygalacturonic acid compound selected from ionotropically cross-linked alginic acid or a salt thereof, or pectic acid; partially esterified pectinic acid, partially amidated pectinic acid, and at least one of their salts. In some embodiments, the porous scaffold is dissolvable. In some embodiments having a dissolvable scaffold, the method also includes digesting the dissolvable scaffold by exposing the dissolvable scaffold to an enzyme. In some embodiments, the exposing step may include exposing the scaffold to about 1 U to about 200 U of the enzyme. In some embodiments, the enzyme for dissolving the scaffold comprises a non-proteolytic enzyme. In some embodiments, the non-proteolytic enzyme is selected from the group consisting of pectin-degrading enzymes, pectinases, and alginate lyases. In some embodiments having a dissolvable scaffold, the method also includes exposing the dissolvable porous scaffold to a chelating agent. In some embodiments, the exposing step may comprise exposing the scaffold to about 1 mM to about 200 mM of a chelating agent. In some embodiments, digestion of the dissolvable porous scaffold is complete in less than about 1 hour.

[0018] According to an aspect of the present disclosure, a bioreactor is provided, comprising a cavity for culturing cells, an inlet and an outlet to the cavity, and a porous scaffold within the cavity. The porous scaffold comprises pores and passageways between the pores, is non-adhesive to cells, and is configured to contain spheroids. In some embodiments, at least 75% of the pores have a pore size of about 200 μm to about 1000 μm when measured in a dry scaffold. In some embodiments, at least 75% of the passageways have a maximum passage width of about 30 μm to about 500 μm when measured in a dry scaffold. In some embodiments, at least 75% of the pores have 5 to 18 passageways per pore. In some embodiments, the porous scaffold comprises a composition comprising an ionotropically crosslinked polysaccharide selected from alginic acid and its salts, pectinic acid and its salts, partially esterified pectinic acid and its salts, partially amidated pectinic acid and its salts, or combinations thereof. In one particular embodiment, the ionotropically cross-linked polysaccharide is polygalacturonic acid. In some embodiments, the porous scaffold of the bioreactor is dissolvable. In some embodiments, the porous scaffold of the bioreactor is a foam scaffold. In some embodiments, the porous scaffold is dissolved by pectinase or alginate lyase.

[0019] According to aspects of the present disclosure, a porous scaffold for culturing spheroids or spheroid outgrowths is provided, comprising pores, passages between the pores, and several passages per pore. The porous scaffold has at least 75% of the pores having a pore diameter of about 200 μm to about 1000 μm as measured within the dry scaffold, at least 75% of the passages having a maximum passage width of about 30 μm to about 500 μm as measured within the dry scaffold, and at least 75% of the pores having 5 to 18 passages per pore. The porous scaffold is also non-adhesive to cells and configured to grow spheroids within the pores from cells of a certain cell type. In some embodiments of the porous scaffold, the certain cell type is selected from a primary cell line and an immortalized cell line. In some embodiments, the primary cell line is bone marrow-derived human mesenchymal stem cells, and the immortalized cell line is HEK293T cells.

[0020] According to aspects of the present disclosure, a perfusion bioreactor is provided, comprising a cavity for culturing cells, an inlet and an outlet to the cavity, and a porous scaffold within the cavity. The porous scaffold of the perfusion bioreactor is non-adhesive to cells and comprises pores and passageways between the pores. The porous scaffold further comprises at least 75% of the pores having a pore diameter of about 200 μm to about 1000 μm as measured in the dry scaffold, at least 75% of the passageways having a maximum width of about 30 μm to about 500 μm as measured in the dry scaffold, and at least 75% of the pores having 5 to 18 passageways per pore, wherein the porous scaffold is adapted to grow spheroids or spheroid by-products. In some embodiments, the perfusion bioreactor is configured to retain at least 20% of any cells added to the cavity through the inlet. In some embodiments, the perfusion bioreactor is configured to retain at least 20% of any cells added to the cavity through the inlet. In some embodiments, the bioreactor is configured to retain at least 60% of any cells added to the cavity through the inlet. In some embodiments, the perfusion bioreactor is adapted to produce extracellular vesicles as a by-product of the spheroids. In some embodiments, the extracellular vesicles produced by the perfusion bioreactor are at a density of about 1 x 10 cells. 3 Approximately 1 x 10 EVs per cell 7 In some embodiments, these extracellular vesicles are generated in amounts of about 1.0 x 10 EVs. -5 m / s~approx. 5.0×10 -4 Extracellular vesicles are generated from perfusion at a linear velocity of 1000 m / s (Darcy flow rate). In some embodiments, increasing the perfusion rate increases the number of extracellular vesicles generated per cell. In some embodiments, the number of extracellular vesicles generated per cell using a perfusion bioreactor exceeds the number of extracellular vesicles generated using a static 2D microwell plate.

[0021] In some embodiments, wound healing with extracellular vesicles produced by a perfusion bioreactor exceeds wound healing with extracellular vesicles produced in 2D flasks for 3D culture at 6 hours post-injury as measured by a wound healing assay using HT-1080 cells. In some embodiments, approximately 2 x 10 extracellular vesicles produced by a perfusion bioreactor exceed wound healing with extracellular vesicles produced in 2D flasks for 3D culture at 6 hours post-injury. 9 Wound healing using 2 x 10 extracellular vesicles generated in 2D flasks for 3D culture was measured by a wound healing assay using HT-1080 cells at 6 hours post-injury. 9 with wound closure that is at least 5% better than wound healing with extracellular vesicles.

[0022] According to aspects of the present disclosure, there is provided herein a foamed scaffold product. The foamed scaffold product is formed from a composition comprising an ionotropically crosslinked polygalacturonic acid compound selected from at least one of ionotropically crosslinked alginic acid and its salts or pectic acid; partially esterified pectinic acid, partially amidated pectinic acid, and salts thereof; at least one first water-soluble polymer having surface activity and a hydrophilic-lipophilic balance (HLB) greater than about 20; and 0% by weight of a water-soluble plasticizer. In other embodiments, the water-soluble plasticizer may be present at a value greater than 0% by weight.

[0023] Additional features and advantages will be set forth in the following detailed description, and in part will be readily apparent to those skilled in the art from that description, or may be learned by practicing the embodiments described herein, including the following detailed description, claims, and accompanying drawings.

[0024] It is to be understood that both the foregoing general description and the following detailed description are merely exemplary and are intended to provide an overview or framework for understanding the nature and character of the claims. The accompanying drawings are included to provide a further understanding, and are incorporated into and constitute a part of this specification. The drawings illustrate one or more embodiments, and together with the description, serve to explain the principles and operation of the various embodiments.

[0025] The present disclosure will be more clearly understood from the following description and the accompanying drawings, given purely by way of non-limiting example. [Brief explanation of the drawings]

[0026] [Figure 1] FIG. 1 is a perspective view of a dissolvable foam scaffold according to the present disclosure. [Figure 2] 1 shows an SEM image of the foam scaffold prepared in Example 1. [Figure 3] 1 shows a phase contrast image of spheroids formed from Vero cells within the pores of a foam scaffold prepared in Example 1, according to an embodiment. [Figure 4] 1 shows an SEM image of a foam scaffold prepared in Example 3, according to an embodiment. [Figure 5A] 1 is an illustration of a foam scaffold for spheroid generation in a culture plate, according to an embodiment. [Figure 5B] 1 is an illustration of a foam scaffold for the production of spheroids or cellular products in a perfusion bioreactor, according to an embodiment. [Figure 6A] 1 is a photograph of an uncoated foam scaffold made from composition XP49, according to an embodiment. [Figure 6B] 1 is a photograph of an uncoated foam scaffold made from composition XP64, according to an embodiment. [Figure 6C] 1 is a photograph of an uncoated foam scaffold made from composition XP76, according to an embodiment. [Figure 6D] 1 is a photograph of an uncoated foam scaffold made from composition FMXP005, according to an embodiment. [Figure 6E] 1 is a plot depicting measured scaffold pore size in different scaffolds, according to an embodiment. [Figure 6F] 10 is a plot depicting measured scaffold interconnect size for different scaffolds, according to an embodiment. [Figure 6G]1 is a plot depicting the number of interconnections per pore in different scaffolds, according to an embodiment. [Figure 7A] Fluorescence microscopy images of HEK293T spheroids 1 and 5 days after seeding the scaffolds with HEK293 cells, according to embodiments. The left column shows spheroids in a foam scaffold using the XP64 composition. The right column shows spheroids in a foam scaffold using the XP76 composition. [Figure 7B] 1 shows fluorescence microscopy images of hMSC spheroids 1 and 7 days after seeding the scaffolds with hMSC cells, according to embodiments. The left column shows spheroids in a foam scaffold using the XP64 composition. The right column shows spheroids in a foam scaffold using the XP76 composition. [Figure 8] 7B is a bar graph showing the expansion of HEK293T cells 1 and 5 days after seeding the cells of FIG. 7A and the expansion of hMSC cells 1 and 7 days after seeding the cells of FIG. 7B, according to an embodiment. [Figure 9A] 1 is a graph of cell seeding data in a perfusion bioreactor, according to an embodiment. [Figure 9B] 1 is a bar graph showing the percentage of cells recovered from scaffolds one day after seeding, according to an embodiment. [Figure 10] 10 shows fluorescence microscopy images from two perfusion bioreactors one day after seeding HEK293T cells into foam scaffolds, according to an embodiment. [Figure 11] 10 shows fluorescence microscopy images of different foam scaffold compositions and spheroids after 48 hours of perfusion at perfusion rates of 1 mL / min and 10 mL / min, according to an embodiment. [Figure 12] 12 is a bar graph showing cell counts of cells recovered from the scaffolds of FIG. 11 at 48 hours after the start of perfusion, according to an embodiment. [Figure 13A] 10A-10C are fluorescence microscopy images of hMSC spheroids within a foam scaffold after perfusion at 10 mL / min for 48 hours, according to an embodiment. [Figure 13B] 10 is a fluorescence microscopy image of hMSC spheroids within a foam scaffold after perfusion at 2 mL / min for 48 hours, according to an embodiment. [Figure 13C] 10 is a bright field contrast phase microscopy image of 2D adherent hMSCs on a 2D static surface after 48 hours, according to an embodiment. [Figure 14A] 10 is a graph of the size distribution of EVs / particles generated within 3D foam scaffolds by hMSCs using a perfusion rate of 10 mL / min based on MADLS analysis, according to an embodiment. [Figure 14B] 10 is a graph of the size distribution of EVs / particles generated within 3D foam scaffolds by hMSCs using a perfusion rate of 2 mL / min based on MADLS analysis, according to an embodiment. [Figure 14C] 1 is a graph of the size distribution of EVs / particles generated by hMSCs in a 2D static environment based on MADLS analysis, according to an embodiment. [Figure 15A] 10 is a plot of the concentration of EVs / particles generated within 3D foam scaffolds by hMSCs using a perfusion rate of 10 mL / min based on MADLS analysis, according to an embodiment. [Figure 15B] 10 is a plot of the concentration of EVs / particles generated within 3D foam scaffolds by hMSCs using a perfusion rate of 2 mL / min based on MADLS analysis, according to an embodiment. [Figure 15C] 1 is a plot of the concentration of EVs / particles generated by hMSCs in a 2D static environment based on MADLS analysis, according to an embodiment. [Figure 16] 15A-15C are bar graphs of the number of EVs per million cells for each of the three culture conditions of FIGS. 15A-15C based on MADLS analysis, according to an embodiment. [Figure 17] 1 is a bar graph of the number of EVs per million cells for each of the three culture conditions based on ELISA for CD63-positive EV analysis, according to an embodiment. [Figure 18] Photographs of Western blot analysis for CD81 and TSG101 in EV isolation samples generated by 10 ml / min perfusion 3D foam culture and static 2D culture, according to an embodiment. [Figure 19]10 shows bright field contrast phase microscopy images of an HT1080 wound healing assay at 0 and 8 hours after wounding and treating cells with 2×10 9 CD63 + EVs or without treatment, according to an embodiment. [Figure 20] 1 is a plot of EVs generated by hMSCs under different conditions using analysis by an in vitro Wood's would healing assay, according to an embodiment. [Figure 21A] 1 shows fluorescence microscopy images of hMSC spheroids observed after 48 hours of exposure to EV collection medium by perfusion at 10 mL / min, according to an embodiment. The spheroids were stained with calcein AM before imaging. [Figure 21B] 1 shows bright field contrast phase microscopy images of hMSC spheroids in microcavity vessels observed after 48 hours of exposure to EV collection medium with agitation at 35 rpm, according to an embodiment. [Figure 21C] 1 shows bright field contrast phase microscopy images of 2D adherent hMSCs in a T-75 CellBIND® flask observed after 48 hours of exposure to EV collection medium under static conditions, according to an embodiment. [Figure 22A] 10 is a particle size distribution graph from MADLS analysis of EVs generated by hMSC spheroids after 48 hours of exposure to EV collection medium by perfusion at 10 mL / min, according to an embodiment. [Figure 22B] 10 is a particle size diameter distribution graph by MADLS analysis of EVs generated by hMSC spheroids after 48 hours of exposure to EV collection medium with agitation at 35 rpm, according to an embodiment. [Figure 22C] 10 is a particle size distribution graph from MADLS analysis of EVs generated by 2D-adherent hMSCs after 48 hours of exposure to EV collection medium under static conditions, according to an embodiment. [Figure 22D] 1 is a bar graph of the amount of particles and EVs produced after 48 hours of exposure to EV collection media, according to an embodiment. [Figure 23A]1 is a bar graph of the number of EVs generated by hMSC spheroids and 2D-adherent hMSCs under different conditions after 48 hours of exposure to EV collection media, as analyzed by ELISA for CD63, according to an embodiment. [Figure 23B] 1 shows a Western blot using CD81 and TSG101 markers of EVs generated by hMSC spheroids and 2D-adherent hMSCs under different conditions after 48 hours of exposure to EV collection medium, according to an embodiment. [Figure 24] 10 is a plot of the results of an EV functionality assay using a wound healing assay with HT-1080 cells using EVs generated by hMSCs under different conditions after 48 hours of exposure to control and EV collection media, according to an embodiment. [Figure 25] Fluorescence microscopy images of hMSCs and 2D-adherent hMSCs after 48 hours of exposure to EV collection medium by perfusion at 20, 10, 2, and 0.5 mL / min, according to an embodiment. Spheroids were stained with calcein AM before imaging. [Figure 26A] 1 shows a plot of the mean diameter of EVs obtained in clarified EV-conditioned perfusion medium at the indicated conditions using MADLS analysis, where the EVs were generated by hMSCs from a bioreactor, according to an embodiment. [Figure 26B] 1 shows a plot of total EVs obtained in clarified EV-conditioned perfusion medium at the indicated conditions using MADLS analysis, where the EVs were generated by hMSCs from a bioreactor, according to an embodiment. [Figure 27A] 1 shows a graph of the diameter distribution of EVs obtained from purified EV samples under the indicated conditions using MADLS, where the EVs were generated by hMSCs from a bioreactor, according to an embodiment. [Figure 27B] 1 shows a bar graph of total EVs obtained from purified EV samples under the indicated conditions using MADLS, where the EVs were generated by hMSCs from a bioreactor, according to an embodiment. [Figure 27C]1 shows a bar graph of EVs per million hMSC cells produced, calculated from the total EVs in the purified EV sample and the total number of cells in the bioreactor, according to an embodiment. [Figure 28A] 1 shows a bar graph of the total number of EVs obtained from purified EV samples under the indicated conditions using ELISA CD63 analysis, according to an embodiment. [Figure 28B] 1 shows a bar graph of EVs per million hMSC cells produced, calculated from the total EVs in the purified EV sample and the total number of cells in the bioreactor, according to an embodiment. [Figure 29] 1 shows a bar graph of % levels of wound healing by EVs obtained from purified EV-conditioned perfusion medium at the indicated conditions observed 6 hours after injury using the HT-1080 wound healing assay, according to embodiments. DETAILED DESCRIPTION OF THE INVENTION

[0027] Reference will now be made in detail to the present embodiment(s), example(s) of which are illustrated in the accompanying drawings. Wherever possible, the same reference numbers will be used throughout the drawings to refer to the same or like parts.

[0028] The singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. The endpoints of all ranges reciting the same characteristic are independently combinable and inclusive of the recited endpoint. All references are incorporated herein by reference.

[0029] As used herein, the term "and / or," when used in a list of two or more items, means that any one of the listed items can be used alone, or any combination of two or more of the listed items can be used. For example, if a composition is described as containing components A, B, and / or C, the composition can contain A alone, B alone, C alone, A and B in combination, A and C in combination, B and C in combination, or A, B, and C in combination.

[0030] Unless otherwise stated, the use of individual numerical values ​​is described as an approximation, as if the value were preceded by the word "about" or "approximately." Similarly, numerical values ​​within various ranges specified in this application are described as approximations, as if both the minimum and maximum values ​​within the stated range were preceded by the word "about" or "approximately," unless expressly indicated otherwise. In this manner, variations above and below the stated ranges can be used to achieve substantially the same results as values ​​within those ranges. As used herein, the terms "about" and "approximately," when referring to numerical values, shall have their ordinary meanings apparent to one of ordinary skill in the art to which the disclosed subject matter most closely pertains or to the range or element in question. The amount of spread from an exact numerical boundary depends on many factors. For example, some factors that may be considered include the importance of the element and / or the impact that a given amount of variation will have on the performance of the claimed subject matter, as well as other considerations known to those skilled in the art. As used herein, the use of different amounts of significant digits for different numerical values ​​is not intended to limit how the use of the word "about" or "approximately" serves to spread a particular numerical value or range. Thus, as a general matter, "about" or "approximately" broadens the numerical value. Also, the disclosure of ranges is intended as a continuous range that includes every value between the minimum and maximum values, as well as the broadening of the range provided by the use of the term "about" or "approximately." Consequently, the recitation of ranges of values ​​herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within that range, and each separate value is incorporated herein as if each separate value were individually recited herein.

[0031] As used herein, the words "have," "having," "include," "including," "comprise," "comprising," and the like are used in an open-ended sense and generally mean "including, but not limited to."

[0032] "Optional" or "optionally" means that the subsequently described element, component, or circumstance may or may not occur, such that the description includes instances in which the element, component, or circumstance occurs and instances in which it does not occur.

[0033] As used herein, "shear stress" or "wall shear stress" are interchangeable and refer to the tangential force per unit area exerted on spheroids cultured inside the pores of a 3D scaffold by fluid flow through those pores.

[0034] All scientific and technical terms used herein have meanings commonly used in the art unless otherwise specified. The definitions provided herein are intended to facilitate understanding of certain terms used frequently herein and are not intended to limit the scope of the present disclosure.

[0035] The present disclosure will be described below generally at first and then in detail based on several exemplary embodiments. The features shown in combination with each other in each exemplary embodiment do not necessarily have to be realized. In particular, each feature may be omitted or may be combined in some other way with other features shown in the same exemplary embodiment or other exemplary embodiments.

[0036] Embodiments of the present disclosure relate to methods for generating large quantities of spheroids from isolated cells and for producing high concentrations of extracellular vesicles and other secreted cellular by-products from these spheroids. The embodiments herein provide, for the first time, methods for generating spheroids by perfusion of a cell suspension within a bioreactor equipped with a scaffold, as well as methods for producing EVs and other secreted by-products from cells within the spheroids under perfusion within the bioreactor. Embodiments include forming spheroids within a dissolvable foam scaffold, after which the spheroids can either be recovered from the scaffold and used for other applications or can be used directly within the scaffold for specific applications, such as EV production. In other embodiments, a non-dissolvable scaffold allows for spheroid formation within the non-dissolvable scaffold, after which the spheroids can be used directly within the scaffold for specific applications, such as EV production. According to embodiments, the dissolvable and non-dissolvable foam scaffolds of the present disclosure for growing and culturing spheroids can be used without an external cell adhesion coating on the scaffold, thus preventing cell adhesion to the scaffold. After seeding the cells into the scaffold, the cells form spheroids within the pores of the scaffold. Depending on the cell type and pore size, these spheroids have the ability to grow and, at least in dissolvable scaffolds, the spheroids can be harvested after formation and used for other applications, including, for example, toxicity assays or other applications that would be understood by one of skill in the art.

[0037] Existing methods used to generate spheroids generally involve growing spheroids in plates with multiple cavities treated to prevent cell adhesion. The limited number of cavities on these plates limits the number of spheroids. Furthermore, the large footprint of such plates results in inefficient use of space compared to the 3D scaffolds disclosed herein. The high density of pores within the scaffold results in a much higher density of spheroids than any current method used to generate spheroids. Other methods for generating spheroids do not allow for easy recovery of these spheroids, especially in large quantities.

[0038] Cells cultured into spheroids can produce useful by-products (secretomes) in the extracellular space around the cells. The secretome is the set of proteins and other biological components expressed by cells and secreted into the extracellular space. One component of the secretome is EVs. According to embodiments of the present disclosure, a method is provided that enables the large-scale production of functional EVs by generating spheroids within a porous material and exposing these spheroids to medium perfusion in a bioreactor.

[0039] Advantages of embodiments of the present disclosure include the formation of large-scale spheroids, which can be recovered when dissolvable scaffolds are used. For spheroids, whose growth is partially dependent on pore size, controlling spheroid size by engineering the pore size of the scaffold material can help provide a homogenous population of spheroids, which is important for some downstream applications. Additionally, aspects of the embodiments herein provide the advantage of large-scale EV generation and other cellular secretome or particle generation (e.g., exosomes) from spheroids via perfusion within the scaffold. The amount of EV, particle, and / or secretome produced by spheroids can also be controlled by their dependence on perfusion rate. Furthermore, embodiments of the present disclosure are scalable to enable the production of spheroids, EVs, and other cellular by-products at various scales, including large-scale production. For example, increasing the diameter of the scaffold increases spheroid and EV / secretome production.

[0040] Embodiments of the present disclosure relate to dissolvable and indissolvable foam scaffolds for cell culture, as well as methods for manufacturing dissolvable foam scaffolds. Embodiments of the present disclosure further relate to cell culture methods for adhering cells, cell aggregates, or spheroids on dissolvable and indissolvable foam scaffolds. Furthermore, embodiments of the present disclosure relate to bioreactors and bioreactor systems including dissolvable and indissolvable foam scaffolds. As will become apparent from the discussion below, in some embodiments, the foam scaffolds disclosed herein are described as both dissolvable and insoluble. As used herein, the term "insoluble" refers to a material or combination of materials that is not soluble and remains crosslinked under conventional cell culture conditions, including, for example, cell culture media. Also, as used herein, the term "dissolvable" refers to a material or combination of materials that is digested when exposed to an appropriate concentration of enzyme that digests or degrades the material or combination of materials. The dissolvable and indissolvable foam scaffolds described herein are porous scaffolds with an open pore structure and highly interconnected pores. The pores of the scaffold provide a protected environment for cell culture, supporting cell-cell interactions and the formation of an extracellular matrix in a 3D format. The dissolvable foam scaffold is completely digestible, allowing cells to be harvested without damaging them using protease treatment and / or mechanical harvesting techniques.

[0041] FIG. 1 is a perspective view of a dissolvable foam (porous) scaffold 10 according to the present disclosure. As will be described in more detail below and become more apparent from other figures of the present disclosure, the dissolvable foam scaffold 10 is a porous foam containing an open pore structure. The dissolvable foam scaffold 10 has a porosity of about 85% to about 96% and an average pore size of about 50 μm to about 500 μm. The dissolvable foam scaffold 10 provides a protected environment within the pores of the foam scaffold for the culture of spheroids. Additionally, the dissolvable foam scaffold 10 is also dissolvable when exposed to an appropriate enzyme that digests or degrades the material, facilitating the recovery of spheroids cultured within the scaffold without damaging the cells.

[0042] The dissolvable foam scaffolds described herein comprise at least one ionotropically cross-linked polysaccharide and can be used to culture three-dimensional cell cultures (e.g., spheroids or organoids) and their by-products (e.g., extracellular vesicles and exosomes). Generally, polysaccharides have beneficial properties for cell culture applications. Polysaccharides are hydrophilic, non-cytotoxic, and stable in culture media. Examples include pectinic acid, also known as polygalacturonic acid (PGA), or its salts, partially esterified pectinic acid or its salts, or partially amidated pectinic acid or its salts. Another example of an ionotropically cross-linked polysaccharide that can be used in the scaffolds of the present disclosure includes alginic acid or its salts. Pectinic acid can be formed by hydrolysis of certain pectin esters. Pectin is a cell wall polysaccharide that naturally plays a structural role in plants. Primary sources of pectin include citrus peels (e.g., lemon and lime peels) and apple peel. Pectin is a predominantly linear polymer based on a 1,4-linked alpha-D-galacturonate backbone randomly interrupted by 1,2-linked L-rhamnose units, with an average molecular weight ranging from about 50,000 to about 200,000 daltons.

[0043] The polygalacturonic acid chains of pectin may be partially esterified, e.g., with methyl groups, and the free acid groups may be partially or completely neutralized with monovalent ions such as sodium, potassium, or ammonium ions. Polygalacturonic acid partially esterified with methanol is called pectinic acid, and its salts are called pectinates. The degree of methylation (DM) of high-methoxyl (HM) pectin can be, for example, 60-75 mol%, while the degree of methylation of low-methoxyl (LM) pectin can be 1-40 mol%. The degree of esterification of the partially esterified polygalacturonic acid described herein can be less than about 70 mol%, or less than about 60 mol%, or less than 50 mol%, or even less than about 40 mol%, and all values ​​therebetween. Without being bound by theory, it is believed that a minimum amount of free carboxylic acid groups (unesterified) promotes some degree of ionotropic crosslinking, allowing for the formation of an insoluble, dissolvable scaffold.

[0044] Alternatively, the polygalacturonic acid chains of pectin may be partially amidated. Pectin with partially amidated polygalacturonic acid can be produced, for example, by treatment with ammonia. Amidated pectin contains carboxyl groups (-COOH), methyl ester groups (-COOCH), and amidated groups (-CONH). The degree of amidation may vary, for example, from about 10% to about 40%.

[0045] According to embodiments of the present disclosure, the dissolvable foam scaffolds described herein may comprise a mixture of pectinic acid and partially esterified pectinic acid. Blends with compatible polymers can also be used. For example, pectinic acid and / or partially esterified pectinic acid can be mixed with other polysaccharides, such as dextran, substituted cellulose derivatives, alginic acid, starch, glycogen, arabinoxylan, and agarose. Glycosaminoglycans, such as hyaluronic acid and chondroitin sulfate, or various proteins, such as elastin, fibrin, silk fibroin, collagen, and their derivatives, can also be used. Water-soluble synthetic polymers can also be blended with pectinic acid and / or partially esterified pectinic acid. Exemplary water-soluble synthetic polymers include, but are not limited to, polyalkylene glycols, poly(hydroxyalkyl(meth)acrylates), poly(meth)acrylamides and derivatives, poly(N-vinyl-2-pyrrolidone), and polyvinyl alcohol.

[0046] According to embodiments of the present disclosure, the dissolvable foam scaffolds described herein may further comprise at least one first polymer. The at least one first polymer is water-soluble, nonionotropically crosslinkable, and surface-active. As used herein, the term "surface-active" refers to the activity of an agent to reduce or eliminate surface tension (or interfacial tension) between two liquids, between a liquid and a solid, or between a gas and a liquid. The at least one first polymer may have a hydrophilic-lipophilic balance (HLB) greater than about 8, or even greater than about 10. For example, the at least one first polymer may have an HLB of about 8 to about 40, or about 10 to about 40. The at least one first polymer may have an HLB of about 8 to about 15, or even about 10 to about 12. HLB provides a measure of the degree of lipophilicity or hydrophilicity of a polymer. A higher HLB value indicates greater hydrophilicity, and a lower HLB value indicates greater lipophilicity. Generally, HLB values ​​range from 1 to 40, with the hydrophilic-lipophilic transition often considered to be about 8 to about 10. If the HLB value is less than the hydrophilic-lipophilic transition, the material is lipophilic; if the HLB value is greater than the hydrophilic-lipophilic transition, the material is hydrophilic. In some embodiments, the HLB value of the at least one first polymer is 10 or greater. In some embodiments, the HLB value of the at least one first polymer is 15 or greater. In some embodiments, the HLB value of the at least one first polymer is 20 or greater. In still other embodiments, the HLB value of the at least one first polymer is 10-40, 15-40, 20-40, or 20-30.

[0047] Exemplary first polymers according to embodiments of the present disclosure may be any of cellulose derivatives, proteins, synthetic amphiphilic polymers, and combinations thereof. Exemplary cellulose derivatives include, but are not limited to, hydroxyethyl cellulose (HEC), hydroxypropyl cellulose (HPC), methyl cellulose (MC), hydroxyethyl methyl cellulose (HEMC), and hydroxypropyl methyl cellulose (HPMC). Exemplary proteins include, but are not limited to, bovine serum albumin (BSA), gelatin, casein, and hydrophobin. Exemplary synthetic amphiphilic polymers include, but are not limited to, poloxamers available under the tradename Synperonics® (commercially available from Croda International, Snaith, UK), poloxamers available under the tradename Pluronics® (commercially available from BASF Corp., Parsippany, New Jersey, USA), and poloxamers available under the tradename Kolliphor® (commercially available from BASF Corp., Parsippany, New Jersey, USA).

[0048] The dissolvable foam scaffolds described herein may further comprise at least one second polymer. The at least one second polymer is water-soluble and not surface-active. Exemplary second polymers may be synthetic polymers, semi-synthetic polymers, natural polymers, or combinations thereof. Exemplary synthetic polymers include, but are not limited to, polyethylene glycol, polyvinylpyrrolidone, polyvinyl alcohol, carboxyvinyl polymers, polyacrylic acid, polyacrylamide, N-(2-hydroxypropyl)methacrylamide homopolymers and copolymers, polyvinyl methyl ether-maleic anhydride, and polyethylene oxide / polypropylene oxide block copolymers. Exemplary semi-synthetic polymers include, but are not limited to, dextran derivatives, carboxymethylcellulose, hydroxyethylcellulose and derivatives, methylcellulose and derivatives, ethylcellulose, ethylhydroxyethylcellulose, and hydroxypropylcellulose. Exemplary natural polymers include, but are not limited to, starch and starch derivatives, polymers obtained by microbial fermentation such as curdlan, pullulan, and gellan gum, xanthan gum, dextran, proteins such as albumin, casein, and caseinates, gelatin, seaweed extracts such as agar, alginates, and carrageenan, seed extracts such as guar gum and derivatives and locust bean gum, hyaluronic acid, and chondroitin sulfate.

[0049] The dissolvable foam scaffolds described herein can be crosslinked to increase their mechanical strength and prevent dissolution of the scaffolds when placed in contact with cell culture medium. Crosslinking can be achieved by ionotropic gelation, as described below, which is based on the ability of polyelectrolytes to crosslink to form crosslinked scaffolds in the presence of multivalent counterions. Without being bound by theory, it is believed that the ionotropic gelation of the polysaccharides of the dissolvable foam scaffolds is the result of strong interactions between divalent cations and the polysaccharides.

[0050] It should be understood that the scaffold for forming and producing the spheroids and cellular by-products of the present disclosure is a dissolvable scaffold in some embodiments only. Similarly, non-dissolvable 3D scaffolds having similar pore sizes, interconnect (through-hole) sizes, and number of interconnects per pore as any of the dissolvable 3D scaffolds described herein function to form spheroids and cellular by-products. Such non-dissolvable scaffolds may be fabricated from, for example, polytetrafluoroethylene (PTFE), polyethylene terephthalate (PET), polystyrene (PS), polypropylene (PP), polyethylene (PE), polyester (PE), polyamide (PA), polyvinylidene fluoride (PVDF), acrylamide, and agar, or combinations thereof. In embodiments where the non-dissolvable scaffold material is cell-adhesive, the non-dissolvable scaffold material is non-adhesive or coated with a material that has been otherwise rendered non-adhesive by chemical modification or other known methods for rendering the scaffold material non-adhesive.

[0051] According to embodiments of the present disclosure, the dissolvable and non-dissolvable scaffolds described herein are porous foam scaffolds. The foam scaffolds described herein may have a porosity of about 85% to about 96%. For example, the foam scaffolds described herein may have a porosity of about 91% to about 95%, or about 94% to about 96%. As used herein, the term "porosity" refers to a measure of the open pore volume of a dissolvable scaffold and is referred to in terms of % porosity, where % porosity is the percentage of voids in the total volume of the dissolvable foam scaffold.

[0052] The foamed scaffolds described herein may have an average pore size of about 200 μm to about 1000 μm, as measured in the dry scaffold. Drying of the foamed (porous) scaffold can be accomplished by any method known to those skilled in the art, but is preferably accomplished by freeze-drying. The pore size is the widest distance across the pore and is measured by scanning electron microscopy ("SEM"). In some embodiments, the average pore size in the dry scaffold can be about 250 μm to about 650 μm, or about 300 μm to about 600 μm, or even about 350 μm to about 500 μm, and all values ​​therebetween. In some embodiments, the average pore size, as measured in the dry scaffold, can be about 450 μm to about 850 μm, about 500 μm to about 700 μm, or about 550 μm to about 600 μm. In some embodiments, the average pore size, as measured in a dry scaffold, can be about 300 μm to about 650 μm, about 350 μm to about 550 μm, or about 400 μm to about 525 μm. In some embodiments, the average pore size, as measured in a dry scaffold, can be about 350 μm to about 1000 μm, about 450 μm to about 850 μm, or about 550 μm to about 650 μm. The average pore size is the average of the pore sizes of the pores in the scaffold.

[0053] In some embodiments, at least 50% of the pores have a pore size of about 200 μm to about 1000 μm when measured in a dry scaffold. In other embodiments, at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more (or any value between 50% and 100%) of the pores have a pore size of about 200 μm to about 1000 μm when measured in a dry scaffold. In one particular embodiment, 65% to 85% of the pores have a pore size of about 200 μm to about 1000 μm when measured in a dry scaffold. In another particular embodiment, at least 70% of the pores have a pore size of about 200 μm to about 1000 μm when measured in a dry scaffold. In some embodiments, at least 50% of the pores have a pore size of about 400 μm to about 800 μm when measured in a dry scaffold. In other embodiments, at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more (or any value between 50% and 100%) of the pores have a pore size of about 400 μm to about 800 μm when measured in a dry scaffold. In one particular embodiment, 70% to 90% of the pores have a pore size of about 400 μm to about 800 μm when measured in a dry scaffold. In another particular embodiment, at least 80% of the pores have a pore size of about 400 μm to about 800 μm when measured in a dry scaffold.

[0054] The scaffolds described herein may have a wet density of less than about 0.40 g / cc. For example, the scaffolds described herein may have a wet density of less than about 0.35 g / cc, or less than about 0.30 g / cc, or less than about 0.25 g / cc. The scaffolds described herein may have a wet density of about 0.16 g / cc to about 0.40 g / cc, or about 0.16 g / cc to about 0.35 g / cc, or about 0.16 g / cc to about 0.30 g / cc, or even about 0.16 g / cc to about 0.25 g / cc, and all values ​​therebetween. The scaffolds described herein may have a dry density of less than about 0.20 g / cc. For example, the scaffolds described herein may have a dry density of less than about 0.15 g / cc, or less than about 0.10 g / cc, or less than about 0.05 g / cc. The scaffolds described herein may have a dry density of from about 0.02 g / cc to about 0.20 g / cc, or from about 0.02 g / cc to about 0.15 g / cc, or from about 0.02 g / cc to about 0.10 g / cc, or even from about 0.02 g / cc to about 0.05 g / cc, and all values ​​therebetween.

[0055] Several pore types are possible in foam (porous) scaffolds. Open pores allow cell access on both sides of the scaffold and allow fluid flow and nutrient transport through the dissolvable scaffold. Partially open pores allow cell access on one side of the scaffold, but nutrient and waste mass transport is limited to diffusion. Closed pores have no openings and do not allow access by cells or nutrient and waste mass transport. Both the dissolvable and non-dissolvable foam scaffolds described herein have an open pore structure and highly interconnected pores. Generally, the open pore structure and highly interconnected pores allow cells to migrate into the pores of the dissolvable foam scaffold and also facilitate the mass transport of nutrients, oxygen, and waste products. The open pore structure also influences cell adhesion and migration by providing a large surface area for cell-cell interaction and space for extracellular matrix ("ECM") regeneration.

[0056] Porous scaffolds (dissolvable and indissolvable) of the present disclosure have passageways (interconnections) between pores that allow fluid to flow through the scaffold. In some embodiments, at least 50% of the interconnections (passageways) have a maximum passageway width of about 30 μm to about 500 μm when measured in a dry scaffold. As used herein, passageway width measurements are those measured in a dry scaffold. In other embodiments, at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more (or any value between 50% and 100%) of the interconnections (passageways) have a maximum passageway width of about 30 μm to about 500 μm when measured in a dry scaffold. In one particular embodiment, 65% to 85% of the passageways have a maximum passageway width of about 30 μm to about 500 μm when measured in a dry scaffold. In another specific embodiment, at least 70% of the passageways have a maximum passageway width of about 30 μm to about 500 μm when measured in a dry scaffold. In some embodiments, at least 50% of the passageways have a maximum passageway width of about 60 μm to about 400 μm when measured in a dry scaffold. In another embodiment, at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more (or any value between 50% and 100%) of the passageways have a maximum passageway width of about 60 μm to about 400 μm when measured in a dry scaffold. In one specific embodiment, 70% to 90% of the passageways have a maximum passageway width of about 60 μm to about 400 μm when measured in a dry scaffold. In another specific embodiment, at least 80% of the passageways have a maximum passageway width of about 60 μm to about 400 μm when measured in a dry scaffold. The maximum passage width is the widest cross-sectional area across the passage of the dry scaffold as measured by scanning electron microscopy.

[0057] In some embodiments, the average interconnect diameter (i.e., the average maximum distance across the cross-sectional area of ​​the passage spaces connecting the pores together) is about 150 μm to about 500 μm, about 250 μm to about 400 μm, or about 275 μm to about 375 μm, when measured in a dry scaffold. In some embodiments, the average interconnect diameter is about 30 μm to about 300 μm, about 80 μm to about 250 μm, or about 130 μm to about 200 μm, when measured in a dry scaffold. In some embodiments, the average interconnect diameter is about 70 μm to about 400 μm, about 120 μm to about 350 μm, or about 170 μm to about 300 μm, when measured in a dry scaffold.

[0058] Porous scaffolds (dissolvable and indissolvable) of the present disclosure also have a number of interconnections (passageways) present per pore. This number can generally range from 1 interconnection per pore to 40 interconnections per pore or more. In some embodiments, at least 50% of the pores have 5-18 passageways per pore. In other embodiments, at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more (or any value between 50% and 100%) of the pores have 5-18 passageways per pore. In one particular embodiment, 65%-85% of the pores have 5-18 passageways per pore. In another particular embodiment, at least 70% of the pores have 5-18 passageways per pore. In some embodiments, at least 50% of the pores have a number of passageways per pore ranging from about 6 to about 14. In other embodiments, at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more (or any value between 50% and 100%) of the pores have between about 6 and about 14 channels per pore. In one particular embodiment, between 70% and 90% of the pores have between about 6 and about 14 channels per pore. In another particular embodiment, at least 80% of the pores have between about 6 and about 14 channels per pore. The number of interconnections (channels) per pore can be measured using a scanning electron microscope.

[0059] In some embodiments, the average number of interconnections (passages) per pore is 6 to 20, 8 to 18, or 10 to 14. In some embodiments, the average number of interconnections (passages) per pore is 4 to 16, 6 to 16, or 7 to 11. In some embodiments, the average number of interconnections (passages) per pore is 6 to 12 or 7 to 10. In some embodiments, the average number of interconnections (passages) per pore is 12 to 18, 13 to 17, or 14 to 16.

[0060] In embodiments with dissolvable foam scaffolds, the scaffolds can be dissolved under certain circumstances. The dissolvable foam scaffolds described herein are digested when exposed to appropriate enzymes that digest or degrade materials. Suitable non-proteolytic enzymes for digesting foam scaffolds, recovering cells, or both include pectinolytic enzymes, or pectinases, a heterogeneous group of related enzymes that hydrolyze pectinic substances. Pectinases (polygalacturonases) are enzymes that break down complex pectin molecules into shorter molecules of galacturonic acid. Commercially available sources of pectinases are generally multienzymes, such as Pectinex™ ULTRA SP-L (commercially available from Novozyme North American, Inc., Franklinton, North Carolina, USA), a pectin-degrading enzyme preparation produced from selected strains of Aspergillus aculeatus. Pectinex™ ULTRA SP-L primarily contains polygalacturonase (EC 3.2.1.15), pectin transeliminase (EC 4.2.2.2), and pectinesterase (EC 3.1.1.11). The EC designation is the Enzyme Commission's classification scheme for enzymes based on the chemical reaction they catalyze. Non-proteolytic enzymes suitable for foam scaffold digestion, cell recovery, or both also include alginate lyase (EC 4.2.2.3). Alginate lyase is an enzyme that breaks down alginate into shorter molecules. Commercially available sources of alginate lyase, such as those from Sigma Alrich®, can be used.

[0061] According to some embodiments of the present disclosure, digesting the dissolvable foam scaffold also includes exposing the scaffold to a divalent cation chelator. Exemplary chelators include, but are not limited to, ethylenediaminetetraacetic acid (EDTA), cyclohexanediaminetetraacetic acid (CDTA), ethyleneglycoltetraacetic acid (EGTA), citric acid, and tartaric acid.

[0062] The time to complete digestion of the dissolvable foam scaffolds described herein can be less than about 1 hour. For example, the time to complete digestion of the foam scaffolds can be less than about 45 minutes, or less than about 30 minutes, or less than about 15 minutes, or less than 5 minutes, or less than 1 minute, or from about 1 minute to about 25 minutes, or from about 3 minutes to about 20 minutes, or even from about 5 minutes to about 15 minutes.

[0063] Also disclosed are methods for forming the dissolvable foam scaffolds described herein, according to embodiments of the present disclosure. The methods described herein may include forming a first aqueous mixture, which includes dissolving a polysaccharide in an aqueous solution. The polysaccharide may be one of those described above, such as pectinic acid or a salt thereof, partially esterified pectinic acid or a salt thereof, or partially amidated pectinic acid or a salt thereof, or blends of these polysaccharides.

[0064] The method for forming a dissolvable foam scaffold described herein may further include forming a second aqueous mixture containing a water-insoluble divalent metal salt in an aqueous solution. The metal of the divalent metal salt may include, but is not limited to, magnesium, calcium, zinc, strontium, barium, and similar cations, and combinations thereof. The anion of the divalent metal salt may include, but is not limited to, oxalate, tartrate, phosphate, carbonate, citrate, and similar organic and inorganic anions, and combinations thereof.

[0065] According to embodiments of the present disclosure, forming the second aqueous mixture may further include adding at least one first polymer described above to the second aqueous mixture. Optionally, the methods described herein may further include adding at least one second polymer described above to the second aqueous mixture. According to embodiments of the present disclosure, the at least one first polymer and the at least one second polymer may be added separately to the second aqueous mixture, or may be added together to the second aqueous mixture. When added as a mixture, the mixture may contain about 50% of the at least one first polymer and about 50% of the at least one second polymer. For example, the mixture may contain about 35% to about 65% (and all values ​​therebetween) of the at least one first polymer and about 35% to about 65% (and all values ​​therebetween) of the at least one second polymer.

[0066] According to embodiments of the present disclosure, forming the second aqueous mixture may further include adding at least one water-soluble plasticizer to the second aqueous mixture. The plasticizers described herein are non-toxic and do not affect the solubility of the polysaccharides of the dissolvable foam scaffold. The plasticizer provides flexibility and softness to the resulting foam, making it soft and pliable. Plasticizers described herein may include, but are not limited to, polyhydric alcohols such as glycerol, sorbitol, ethylene glycol, propylene glycol, polyethylene glycol, and combinations thereof. Adding the water-soluble plasticizer to the second aqueous mixture may include adding less than about 55% by weight of the total solid additives added to form the second aqueous mixture. For example, adding the water-soluble plasticizer to the second aqueous mixture can include adding less than about 50% by weight, or less than about 40% by weight, or less than about 30% by weight, or less than about 25% by weight, or about 15% to about 55% by weight, or about 15% to about 50% by weight, or about 15% to about 40% by weight, or about 15% to about 30% by weight, or about 15% to about 25% by weight, and all values ​​therebetween. As used herein, the term "total solid additives added to form the second aqueous mixture" refers to all of the components of the aqueous mixture except water.

[0067] According to embodiments of the present disclosure, forming the second aqueous mixture may further include adding at least one emulsifier to the second aqueous mixture. Emulsifiers described herein may include, but are not limited to, sodium dodecyl sulfate (SDS) and polysorbates, such as polyethylene glycol sorbitan monolaurate (Tween® 20) and polyoxyethylene sorbitan monooleate (Tween® 80) (each commercially available from Croda International, Snaith, UK).

[0068] According to embodiments of the present disclosure, forming the second aqueous mixture may further include adding at least one leachable solid to the second aqueous mixture. Leachable solids described herein include materials that reinforce or generate pores during the formation of the foamed scaffold. The leachable solid may be a non-toxic leachable material, such as, but not limited to, salts, biocompatible monosaccharides and disaccharides, and water-soluble proteins. Exemplary salts include, but are not limited to, sodium chloride, potassium chloride, calcium chloride, sodium tartrate, sodium citrate, and the like. Exemplary biocompatible monosaccharides and disaccharides include, but are not limited to, glucose, fructose, dextrose, maltose, lactose, and sucrose. Exemplary water-soluble proteins include, but are not limited to, gelatin and agarose.

[0069] Each of the above-mentioned materials associated with the second aqueous mixture may be optionally added to the second aqueous mixture, and may be added to the second aqueous mixture in any order, with the possibility that two or more materials may be added to the second aqueous mixture. In one exemplary method, forming the second aqueous mixture includes adding a leachable solid to an aqueous solution containing a divalent metal salt and mixing the aqueous mixture to promote dissolution of the leachable solid in the aqueous mixture. Thereafter, at least one first polymer, at least one second polymer, and / or a water-soluble plasticizer are added to the second aqueous mixture.

[0070] The methods for forming a dissolvable foam scaffold described herein may further include, after forming the first and second aqueous mixtures, combining the second aqueous mixture with the first aqueous mixture to form a combined aqueous mixture. A foam can be formed from the combined aqueous mixture by introducing gas bubbles into the aqueous mixture by mixing, pulsating, stirring, aerating, whipping, injecting, or other mechanical action. The gas can be, for example, but not limited to, air, nitrogen, helium, hydrogen, argon, carbon dioxide, or other inert gas. The subsequent introduction of gas bubbles into the combined aqueous mixture can be carried out for less than about 30 minutes, for example, from about 1 minute to about 30 minutes, or from about 3 minutes to about 25 minutes, or even from about 5 minutes to about 20 minutes. While introducing gas bubbles into the combined aqueous mixture, the methods for forming a dissolvable foam scaffold may further include adding a gel-inducing agent to the combined aqueous mixture. The gel-inducing agent can be an acid that provides buffering and / or a substance that slowly generates acid. Exemplary acids include, but are not limited to, lactic acid lactone, glycolic acid lactone, glucono-delta-lactone, and acid anhydrides.

[0071] The methods for forming a dissolvable foam scaffold described herein may further include coating the dissolvable foam scaffold with an adhesive polymer coating. Coating the dissolvable foam scaffold may include exposing the scaffold to an aqueous solution having an adhesive polymer in the aqueous solution. As previously described, the adhesive polymer may include a peptide. Exemplary peptides may include, but are not limited to, BSP, vitronectin, fibronectin, laminin, type I and type IV collagen, denatured collagen (gelatin), and similar peptides, as well as mixtures thereof. Additionally, the peptide may have an RGD sequence. The coating may be, for example, Synthemax® II-SC (commercially available from Corning, Incorporated, Corning, NY, USA).

[0072] Also disclosed are methods for culturing cells and spheroids within the dissolvable and non-dissolvable foam scaffolds described herein. Any cell type or spheroid type can be cultured within the foam scaffold, including, but not limited to, immortalized cells, primary cells, cancer cells, stem cells (e.g., embryonic or induced pluripotent), and the like. The cells can be mammalian cells, avian cells, fish cells, and the like. The cells can be of any tissue type, including, but not limited to, kidney, fibroblast, breast, skin, brain, ovarian, lung, bone, nerve, muscle, cardiac, colorectal, pancreatic, immune (e.g., B cells), blood, and the like. The cells can be cultured in any form within the bag, including dispersed (e.g., freshly seeded), confluent, two-dimensional, three-dimensional, spheroid, and the like. Culturing cells or spheroids within the foam scaffold can include seeding the cells within the foam scaffold. Seeding the cells within the foam scaffold can include contacting the scaffold with a solution containing the cells. During seeding of cells into the foam scaffold, the cells enter the pores of the foam scaffold.

[0073] Culturing cells and spheroids within the foam scaffold may further include contacting the scaffold with cell culture medium. Generally, contacting the scaffold with cell culture medium includes placing the cells to be cultured within the scaffold in an environment having the medium in which the cells are cultured. Contacting the scaffold with cell culture medium may include pipetting the cell culture medium onto the scaffold, submerging the scaffold in cell culture medium, or continuously passing the cell culture medium over the scaffold. Generally, as used herein, the term "continuous" refers to culturing cells with a consistent flow of cell culture medium into and out of the cell culture environment. Such continuous passing of cell culture medium over the scaffold may include submerging the scaffold in cell culture medium for a predetermined period of time, then removing at least a portion of the cell culture medium after the predetermined period of time, and adding fresh cell culture medium so that the volume of cell culture medium in contact with the dissolvable foam scaffold remains substantially constant. The cell culture medium may be removed and replaced according to a predetermined schedule. For example, at least a portion of the cell culture medium can be removed and replaced every hour, or every 12 hours, or every 24 hours, or every 2 days, or every 3 days, or every 4 days, or every 5 days. Cells can be cultured for a period on the order of hours, days, or weeks. For example, cells can be cultured for 12 hours, 18 hours, 24 hours, 2 days, 4 days, 6 days, 1 week, 2 weeks, or even longer. It should be understood that these are merely examples of culture times, and any predetermined period of time is acceptable.

[0074] Cell culture media can contain, for example, but are not limited to, sugars, salts, amino acids, serum (e.g., fetal bovine serum), antibiotics, growth factors, differentiation factors, colorants, or other desired factors. Exemplary cell culture media include Dulbecco's Modified Eagle's Medium (DMEM), Ham's F12 Nutrient Mixture, Minimum Essential Medium (MEM), RPMI medium, Iscove's Modified Dulbecco's Medium (IMDM), Mesencult™-XF medium, RoosterNourish-MSC, RoosterNourish-MSC XF, etc. Other exemplary cell culture media include those formulated for collecting cell culture by-products, such as defined low-particle media, including extracellular matrix collection media, such as RoosterCollect™-EV medium, RoosterCollect™-EV-CC medium, and RoosterBio M2001 medium.

[0075] Also disclosed are methods for recovering cells (including three-dimensional cellular structures) from the foam scaffolds described herein. In embodiments having a dissolvable foam scaffold, the methods for recovering cells described herein can include digesting the dissolvable foam scaffold by exposing the scaffold to an enzyme. As previously mentioned, suitable non-proteolytic enzymes for foam scaffold digestion, cell recovery, or both, include pectinolytic enzymes, or pectinases, a heterogeneous group of related enzymes that hydrolyze pectinic substances, and alginate lyases. Commercially available sources of pectinases are generally multienzymes, such as Pectinex™ ULTRA SP-L (commercially available from Novozyme North American, Inc., Franklinton, North Carolina, USA), a pectin-degrading enzyme preparation produced from selected strains of Aspergillus aculeatus. Pectinex™ ULTRA SP-L primarily contains polygalacturonase (EC 3.2.1.15), pectin transeliminase (EC 4.2.2.2), and pectinesterase (EC 3.1.1.11). Alginate lyase (EC 4.2.2.3) degrades alginate and alginic acid and is commercially available from Sigma-Aldrich®, among others. EC designations are the Enzyme Commission's classification scheme for enzymes based on the chemical reaction they catalyze.

[0076] Exposing the dissolvable foam scaffold to an enzyme can include exposing the scaffold to an enzyme concentration of about 1 to about 200 U. For example, the method can include exposing the scaffold to an enzyme concentration of about 2 U to about 150 U, or about 5 U to about 100 U, or even about 10 U to about 75 U, and all values ​​therebetween.

[0077] The methods for recovering cells described herein may further include exposing the material to a chelating agent. Exemplary chelating agents include, but are not limited to, ethylenediaminetetraacetic acid (EDTA), cyclohexanediaminetetraacetic acid (CDTA), ethylene glycol tetraacetic acid (EGTA), citric acid, and tartaric acid. Exposing the dissolvable foam scaffold to a chelating agent may include exposing the scaffold to a chelating agent concentration of about 1 mM to about 200 mM. For example, the method may include exposing the scaffold to a chelating agent concentration of about 10 mM to about 150 mM, or about 20 mM to about 100 mM, or even about 25 mM to about 50 mM, and all values ​​therebetween.

[0078] According to aspects of embodiments of the present disclosure, the foam scaffolds (dissolvable or non-dissolvable) of the present disclosure can be used in perfusion bioreactors for three-dimensional cell culture (e.g., spheroids and organoids) and can also be used for the production of cell culture by-products such as extracellular vesicles and extracellular matrix proteins. The perfusion bioreactors of the present disclosure have an inlet for fluid entry into the bioreactor and an outlet for fluid exit from the bioreactor. The perfusion bioreactors of the present disclosure also have a substrate (i.e., a porous scaffold) inside the bioreactor to support the cultured cells. Cells of the desired cell type can be added directly into the foam scaffold (known as "seeding") before placing the foam scaffold in the bioreactor to culture the cells. Alternatively, cells of the desired cell type can be added through an inlet (a location on the bioreactor where fluid can enter the bioreactor) and reach the foam scaffold inside the bioreactor. In either option, the added cells enter the pores present in the foam scaffold and the interconnected spaces between the pores (known as "interconnections"). The pores and interconnections of the foam scaffold accommodate cells, allowing for the formation of spheroids, organoids, or other three-dimensional cell types. In some embodiments, at least 70%, 80%, 90%, or more of the cells within the pores of the porous scaffold aggregate to form spheroids. In one particular embodiment, at least 80% of the cells within the pores of the porous scaffold aggregate to form spheroids. The formed spheroids, organoids, or other three-dimensional cell types remain contained within the pores and interconnect during cell culture in the bioreactor. For three-dimensional cell culture, the foam scaffold is preferably non-adhesive to cells to provide optimal formation of spheroids, organoids, or other three-dimensional cell culture types. Generally, the less adhesion to the foam scaffold, the more likely the cells will adhere to each other and form a three-dimensional culture. Culturing three-dimensional cells can produce a variety of by-products, including extracellular vesicles, proteins, and other secreted materials. Reference to any particular by-product (eg, EV) is not intended to limit the scope of the embodiments with respect to that particular by-product, which is used for illustrative purposes only.It is understood that exosomes and other cellular by-products secreted by spheroids can be cultured using the scaffolds of the present disclosure in place of the EVs described herein.

[0079] 5A-5B show illustrative examples of foam (porous) scaffolds used for three-dimensional cell culture. FIG. 5A shows a porous scaffold fabricated from scaffold material 2500, with pores 2502 formed within scaffold material 2500. The scaffold is placed in tissue culture plate 2506, and cells are seeded onto it. After a culture period, the cells form spheroids 2504 within pores 2502. FIG. 5B shows a similar scaffold for growing spheroids, but this time, scaffold material 2510, with pores 2512, is placed within bioreactor 2516 of a perfusion bioreactor system. Cells are seeded into bioreactor 2516 through inlet 2517, forming spheroids 2514 within pores 2512. Bioreactor 2516 has an outlet 2518, allowing spent fluid to exit the bioreactor. The bioreactor system shown in FIG. 5B is a simplified depiction of a system with a medium conditioning vessel 2520 and fluid flow paths 2522 that allow for perfusion and / or recirculation of medium through the bioreactor 2516. The use of a porous scaffold inside the bioreactor with medium perfusion can increase extracellular vesicle production. The system shown in FIG. 5B is merely an example and is not intended to limit the scope of the embodiments of the present disclosure. For example, instead, cells may be added to the foam scaffold 2500 through the bioreactor outlet 2518, and fluid may exit through the bioreactor inlet 2517. As another example, a medium supply vessel may be added to the perfusion bioreactor system that conditions the medium conditioning vessel. As yet another example, a pump or controller, or both, may be in line between the bioreactor and the medium conditioning vessel.

[0080] According to aspects of embodiments of the present disclosure, two-dimensional and three-dimensional cellular structures (e.g., spheroids, organoids, etc.), as well as by-products of these cells and structures, are cultured and harvested using the foam scaffolds of the present disclosure. The cell expansion from the time the cells are seeded to the time they are harvested can be about 0.2-fold to about 200-fold. In one embodiment, the expansion of cells from a primary cell line (e.g., hMSC cells, etc.) is about 0.2-fold to about 100-fold, about 0.2-fold to about 50-fold, about 0.2-fold to about 30-fold, about 0.2-fold to about 10-fold, or about 0.5-fold to about 5-fold. In another embodiment, the expansion of cells from a non-primary cell line (e.g., HEK293T cells, etc.) is about 5-fold to about 200-fold, about 50-fold to about 150-fold, about 50-fold to about 100-fold, about 5-fold to about 50-fold, about 7-fold to about 40-fold, or about 8-fold to about 30-fold. In one particular embodiment, the expansion of cells from a primary cell line is about 0.2-fold to about 5-fold after 5 days of seeding the cells into a foam scaffold and culturing the cells in a perfusion bioreactor. In another particular embodiment, the expansion of cells from a non-primary cell line is about 8-fold to about 30-fold after 7 days of seeding the cells into a foam scaffold and culturing the cells in a perfusion bioreactor. In another particular embodiment, the expansion of cells from a non-primary cell line is about 50-fold to about 100-fold after 1 day of seeding the cells into a foam scaffold and culturing the cells in a perfusion bioreactor. The times after cell seeding and the fold expansion after cell seeding using a foam scaffold are exemplary in nature, and other cell seeding times and fold expansions are contemplated.

[0081] When cells are introduced into a foam (porous) scaffold within a bioreactor by perfusion, cell entrapment within the foam scaffold can be at least 1% or more of the cells added per pass through the bioreactor and can accumulate with further passes through the bioreactor. For example, cell entrapment per pass within the foam scaffold can be at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more (or any value therebetween) of the cells added. In one embodiment, cell entrapment within the foam scaffold is at least 5% of the cells added after one pass through the bioreactor. In another embodiment, cell entrapment within the foam scaffold is at least 10% of the cells added after one pass through the bioreactor. Cells exiting the bioreactor during any one pass through the bioreactor can be recycled through the bioreactor for further passages. The cells accumulated in the bioreactor at the end of all passes can be at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more (or any value therebetween) of the cells added. In one particular embodiment, the cells accumulated in the bioreactor at the end of all passes are at least 30%. In another particular embodiment, the cells accumulated in the bioreactor at the end of all passes are at least 40%. In yet another particular embodiment, the cells accumulated in the bioreactor at the end of all passes can be at least 50%.

[0082] In some embodiments, the addition of cells occurs at the inlet of the bioreactor. In some embodiments, the addition of cells occurs at a point prior to the inlet of the bioreactor. In one non-limiting example, the cells are added to a fluid line supplying medium to the bioreactor via a port on the fluid line. In another non-limiting example, the cells are added to a vessel containing medium, and then the cells are perfused into the bioreactor at a set flow rate (e.g., 500 mL / min, 200 mL / min, 50 mL / min, 20 mL / min, 10 mL / min, 5 mL / min, 1 mL / min, or 0.5 mL / min). In one embodiment, the cells are perfused through the bioreactor at about 0.1 mL / min to about 20 mL / min. In some embodiments, the cells are perfused at a rate of about 2.3 x 10 -6 m / s~approx. 1.2×10 -3 The cells are perfused through the bioreactor at a linear velocity (Darcy flow rate) of about 1.0 x 10 m / s. As used herein, the term "linear velocity" refers to the Darcy flow rate. In some embodiments, the cells are perfused at a linear velocity of about 1.0 x 10 m / s. -5 m / s~approx. 5.0×10 -4 In some embodiments, the cells are perfused through the bioreactor at a linear velocity of about 1.0 x 10 m / s (Darcy flow rate). -5 m / s~approx. 4.8×10 -5 m / s, approximately 1.0×10 -5 m / s~approx. 2.4×10 -4 m / s, or approximately 1.0 x 10 -5 m / s~approx. 3.6×10 -4 The cells are perfused through the bioreactor at a linear velocity (Darcy flow rate) of 1000 m / s. In some embodiments, any cells exiting the bioreactor are recycled back through the inlet of the bioreactor to attempt to capture the cells on another pass through the bioreactor.

[0083] It should be understood that the perfusion rate need not be the same throughout the entire process. For example, while cells are in the process of seeding cells into a foam (porous) scaffold present in a bioreactor, slower or faster flow rates may be used, such as 0.1 mL / min, 0.5 mL / min, 1 mL / min, 2 mL / min, 3 mL / min, 4 mL / min, or 5 mL / min, 10 mL / min, 15 mL / min, 20 mL / min, 50 mL / min, 100 mL / min, or some value between 0.1 mL / min and 100 mL / min. Similarly, a perfusion rate of about 1.0×10 -5 m / s~approx. 4.8×10 -5 m / s, approximately 1.0×10 -5 m / s~approx. 2.4×10 -4 m / s, or greater than 0 m / s and 2.4 x 10 -4 Slower linear velocities (Darcy flow rates), such as, but not limited to, less than m / s, may be used during cell seeding. After the foam scaffold is seeded, faster or slower flow rates than those used for loading may be used for culturing, such as 2 mL / min, 5 mL / min, 10 mL / min, 15 mL / min, 20 mL / min, 50 mL / min, 100 mL / min, 200 mL / min, 300 mL / min, 400 mL / min, 500 mL / min, or some value between 2 mL / min and 500 mL / min. In a preferred embodiment, cell seeding occurs at a perfusion rate of about 0.1 mL / min to about 5 mL / min. In another preferred embodiment, cell culture occurs at a perfusion rate of about 5 mL / min to about 15 mL / min. In some embodiments, the medium perfusion rate during cell seeding is about 2.3 x 10 -6 m / s~approx. 1.2×10 -3 In some embodiments, the cells are at a linear velocity of about 1.0 x 10 m / s (Darcy velocity). -5 m / s~approx. 5.0×10 -4 In some embodiments, the cells are perfused through the bioreactor at a linear velocity of about 1.0 x 10 m / s (Darcy flow rate). -5 m / s~approx. 4.8×10 -5 m / s, approximately 1.0×10 -5 m / s~approx. 2.4×10 -4 m / s, or approximately 1.0 x 10 -5 m / s~approx. 3.6×10 -4 The perfusion is carried out through the bioreactor at a linear velocity of 1000 s / s (Darcy flow rate).

[0084] According to some embodiments, the fluid flow through the scaffold during cell culture is about 2.3×10 -6 m / s~approx. 1.2×10 -3 In some embodiments, the fluid has a flow velocity in the range of 2.3×10 -6 m / s~approx. 1.2×10 -5 m / s, approx. 1.2×10 -5 m / s~approx. 5.0×10 -4 m / s, or approximately 5.0 x 10 -4 m / s~approx. 1.2×10 -3 In some embodiments, the fluid flow through the scaffold is about 1.0×10 -5 m / s~approx. 5.0×10 -4 In some embodiments, the cells are at a linear velocity of about 1.0 x 10 m / s (Darcy velocity). -5 m / s~approx. 4.8×10 -5 m / s, approximately 1.0×10 -5 m / s~approx. 2.4×10 -4 m / s, or approximately 1.0 x 10 -5 m / s~approx. 3.6×10 -4 The medium is perfused through the bioreactor at a linear velocity (Darcy flow rate) of 1000 m / s. The flow rate of the medium through the scaffold depends on the dimensions of the scaffold and the medium flow rate.

[0085] Fluids at a certain velocity can exert wall shear stress on cells cultured within a porous scaffold. For cell culture medium flowing within a porous scaffold containing spheroids or other cells, the flow is generally laminar, and the cell culture medium is a Newtonian fluid. According to some embodiments, shear stress stimulation on the spheroids and / or cells is generated by movement of the medium through at least one porous scaffold containing the spheroids. In some embodiments, shear stress on the spheroids or cells is generated using a pump. In some embodiments, shear stress on the spheroids or cells is generated by any other method that results in movement of the medium within the scaffold, including, but not limited to, stirring, shaking, rotation, vibration, or tilting.

[0086] Shear stress values ​​can be determined by in silico modeling of the scaffold using computational fluid dynamics simulations. This was performed for the scaffolds of the present disclosure. A representative computer-aided geometric drawing of the scaffold was created using MATLAB® coding and the pore size, interconnect size, and number of interconnects per pore measured on the scaffold to create a computational mesh. This computational mesh was then imported into ANSY Fluent for computational fluid dynamics simulations. The calculated Reynolds number was less than 1, indicating that the flow within the porous scaffold was laminar. The shear stress provided to the cells and spheroids of the present disclosure was determined to be between 0 mPa and approximately 300 mPa. However, shear stress values ​​greater than 300 mPa, e.g., 400 mPa, 500 mPa, 600 mPa, 700 mPa, 800 mPa, 900 mPa, or 1000 mPa or more, may be provided and the spheroids or cells may still retain their aggregation and / or still produce the desired by-product.

[0087] According to some embodiments, the shear stress provided to the spheroids contained within the pores of the scaffold is between 0 mPa and about 300 mPa, or any value therebetween. In some embodiments, the shear stress provided to the spheroids contained within the pores of the scaffold is greater than 300 mPa. In some embodiments, the shear stress provided to the spheroids contained within the pores of the scaffold is greater than 0 mPa. In some embodiments, the shear stress provided to the spheroids contained within the pores of the scaffold is between 0.001 mPa and about 300 mPa, or any value therebetween. In still other embodiments, the shear stress provided to the spheroids contained within the pores of the scaffold is between about 0.001 mPa and about 1 mPa, between about 1 mPa and about 50 mPa, between about 50 mPa and about 150 mPa, or between about 150 mPa and 300 mPa, or any value therebetween.

[0088] According to some embodiments of the present disclosure, by-products are collected from cells or three-dimensional cellular structures cultured using the foam (porous) scaffolds of the present disclosure, which can then be used for downstream applications. One such by-product is extracellular vesicles (EVs). EVs are produced by cells cultured within the foam scaffolds of the present disclosure. Other by-products that can be produced by perfusion using the foam scaffolds or methods described herein include, but are not limited to, microvesicles, exosomes, other exosome-like particles, exomers and other nanoparticles, proteins, polypeptides, peptides, amino acids, lipids, polynucleotide sequences, and hormones. By-products can be genetically engineered or naturally occurring. Collection of by-products can be accomplished by any of a number of methods known to those skilled in the art.

[0089] Perfusion using the disclosed foam (porous) scaffold produces an increased number of by-products compared to conventional 3D culture methods and devices. In the case of vesicle particles, culturing spheroids by perfusion within the disclosed foam scaffold produces approximately 1 x 10 vesicle particles per million cells after culturing the cells for approximately 48 hours. 9 ~Approx. 1×10 13 EVs (i.e., approximately 1 x 10 per cell) 3 ~Approx. 1×10 7 In one embodiment, approximately 2.4 x 10 EVs are generated. -4 After approximately 48 hours of culture at a perfusion rate with a linear velocity (Darcy flow rate) of 1 × 10 of a given cell type 6 At least 1 x 10 cells per 11 EVs (i.e., at least 1 x 10 per cell) 5 In another embodiment, about 2.4×10 EVs are generated. -4 After approximately 48 hours of culture at a perfusion rate with a linear velocity (Darcy flow rate) of 1 × 10 of a given cell type 6 At least 1 x 10 cells per 12 EVs (i.e., at least 1 x 10 per cell) 6 In another embodiment, about 2.4×10 EVs are generated. -4After approximately 48 hours of culture at a perfusion rate with a linear velocity (Darcy flow rate) of 1 × 10 of a given cell type 6 At least 1 x 10 cells per 13 EVs (i.e., at least 1 x 10 per cell) 7 In yet another embodiment, about 2.4×10 EVs are generated. -4 After approximately 48 hours of culture at a perfusion rate with a linear velocity (Darcy flow rate) of 1 × 10 of a given cell type 6 1 x 10 cells per 11 EV ~ approx. 1 x 10 13 EVs (i.e., approximately 1 x 10 per cell of a given cell type) 5 ~Approx. 1×10 7 As used herein, the term "extracellular vesicles" or "EVs" refers to membrane-bound vesicles that are secreted from cells and may be generated within endosomal compartments. EVs contain various molecular components from cells. These components are called "cargo" and may have biological functions. These cargoes may include some or all of the following: proteins, lipids, mitochondrial components, and genetic material (RNA and / or DNA).

[0090] EVs produced by cell culture using the foam (porous) scaffolds of the present disclosure each have a size referred to as the vesicle diameter; collectively, the EVs have a mean vesicle diameter. EV size can be determined by multi-angle dynamic light scattering (MADLS). In one embodiment, EVs collected from cells cultured in a foam scaffold of the present disclosure have a diameter of about 40 nm to about 200 nm, about 50 nm to about 150 nm, about 60 nm to about 140 nm, about 70 nm to about 130 nm, about 80 nm to about 120 nm, or about 90 nm to about 110 nm, as measured by MADLS. In another embodiment, EVs collected from cells cultured in a foam scaffold of the present disclosure have a mean diameter of about 80 nm to about 120 nm, about 90 nm to about 110 nm, or about 95 nm to about 105 nm, as measured by MADLS. In one specific embodiment, the EVs have a mean diameter of about 100 nm to about 110 nm.

[0091] EVs generated from cells cultured within the foam scaffolds of the present disclosure also have a level of functionality associated with them. Such functionality of EVs can be measured by a wound healing assay using HT-1080 cells (epithelial cells derived from connective tissue, ATCC, CCL-121) to determine directional cell migration in vitro. The wound healing assay of the present disclosure involves creating a cell monolayer from HT-1080 cells, creating a wound within the cell monolayer, imaging the wound after it has formed, and imaging 2 x 10 EVs collected from the cell culture. 9 This involves treating the wound with EVs and then imaging the treated wound at regular intervals during cell migration to close the wound. The same process can be performed on a control (no EVs added). Plots of percent wound healing over time for each wound assay can then be generated to compare the functionality of different EV batches or processes.

[0092] According to some embodiments, EVs collected from cell cultures within foam scaffolds of the present disclosure have similar or improved wound healing functionality compared to EVs collected from non-perfusion-based cell culture methods. In some embodiments, improved EV functionality is measured by a wound healing assay, where wound healing is about 2% to 50% faster in perfusion-based culture than in static culture (i.e., in microwells of a culture plate or culture flask) 6 hours after wound formation in a monolayer of HT-1080 cells. In one specific embodiment, improved EV functionality is measured by a wound healing assay, where wound healing is about 2% to 10%, about 10% to 20%, about 20% to 30%, about 30% to 40%, or about 40% to 50% faster for EVs generated by perfusion-based culture than EVs generated by static culture 6 hours after wound formation in a monolayer of HT-1080 cells. In some embodiments, improved EV functionality, as measured by a wound healing assay, results in wound healing that is about 2% to 30% faster than EVs produced by non-perfusion-based cell culture methods 6 hours after forming a wound in a monolayer of HT-1080 cells. In one particular embodiment, improved EV functionality, as measured by a wound healing assay, results in wound healing that is about 2% to 5%, about 5% to 10%, about 10% to 15%, about 15% to 20%, about 20% to 25%, or about 25% to 30% faster than EVs produced by non-perfusion-based cell culture methods 6 hours after forming a wound in a monolayer of HT-1080 cells. [Example]

[0093] Embodiments of the present disclosure are further described below with reference to certain exemplary and specific embodiments thereof, which are merely illustrative and not intended to be limiting. [Table 1]

[0094] A first aqueous mixture containing 2.0 wt% polygalacturonic acid (PGA) was prepared by dissolving approximately 162 grams of polygalacturonic acid sodium salt in demineralized water in an oil bath set at 104°C. The aqueous mixture was cooled to room temperature. A second aqueous mixture was prepared by adding approximately 7.5 grams of glycerol to ultrapure water and heating in a microwave at 800W for approximately 30 seconds. Approximately 1.06 grams of CaCO3 and approximately 0.125 grams of TWEEN® 20 were added to the second aqueous mixture. The second aqueous mixture was then sonicated for approximately 1 minute and then transferred to the bowl of a KitchenAid mixer equipped with a wire loop whisk. Approximately 17.5 grams of sucrose and approximately 1.94 grams of Methocel HPMC Culminal 724 were then added to the bowl of the KitchenAid mixer, and the aqueous mixture was stirred for approximately 5 minutes. The first aqueous mixture containing 2.0 wt. % PGA was added to form a combined aqueous mixture in a mixing bowl and mixed at an agitation speed (speed 1 on the KitchenAid mixer) for approximately 3 minutes. The combined aqueous mixture was then whipped at a fast whipping speed (speed 10 on the KitchenAid mixer) for approximately 20 minutes to introduce air into the combined aqueous mixture. While continuing to whip the combined aqueous mixture, an aqueous solution of approximately 3.77 grams of gluconolactone (GDL) in approximately 30 mL of water was added to the mixing bowl and whipping continued for approximately 1 minute.

[0095] An opaque white foam was obtained according to the above process. The foam was left uncovered in the mixing bowl at room temperature for about 1 hour to allow time for crosslinking to occur within the foam. The foam was then exposed to a temperature of about -80°C for about 16 hours to freeze the foam, and then exposed to a temperature of -86°C and a pressure of 0.11 mbar for about 72 hours. The resulting foam was observed to have a wet foam density of about 0.21 g / cc and a dry foam density of about 0.06 g / cc, and was observed to be porous with highly interconnected pores. Figure 2 shows an SEM image of the foam prepared in this Example 1.

[0096] Example 2 The culture of Vero cells in foams formed according to the process of Example 4 was investigated. Vero cells (ATCC® CCL-81, commercially available from ATCC, Manassas, VA, USA) were cultured on cell culture plates in IMDM medium supplemented with 10% fetal bovine serum (FBS). The foams were cut into sections approximately 2-3 mm thick and approximately 22 mm in diameter. The foam sections were disinfected with 70% aqueous ethanol for approximately 5.0 minutes and then placed into separate wells of a 6-well ultra-low attachment cell culture plate. The foam sections were washed twice with ultrapure water and once with IMDM medium. Excess medium was removed from the wells prior to seeding.

[0097] Vero cells were harvested from the cell culture plate using trypsin and resuspended in IMDM medium. Approximately 100,000 cells were seeded in 150 μL of IMDM medium into each foam section placed in a 6-well cell culture plate. The 6-well cell culture plate was placed in a cell culture incubator, and after approximately 2.0 hours, approximately 3.0 mL of IMDM medium was added to each well. After approximately 18 hours in the cell culture incubator, the foam sections were visualized using a phase-contrast microscope. Images obtained from the phase-contrast microscope are shown in Figure 3, demonstrating that the cells did not adhere to the uncoated foam sections but instead formed spheroids in the pores of the foam sections. Therefore, it was demonstrated that the dissolvable foam scaffold of the present disclosure can be used to culture spheroids or non-adherent cells.

[0098] Example 3 Some of the dissolvable foams disclosed herein use various ingredients (e.g., sucrose, glycerol, dextran, and others) that are added to facilitate the foaming process and account for a significant amount (e.g., by weight) of the solution used in the foaming process. For example, these ingredients can account for up to 90% of the final foam weight. Most of these ingredients are not covalently bound to the foam structure and may dissociate from the foam during coating or may need to be removed prior to cell culture. If not removed during cell culture, these materials could potentially alter the cell medium composition, block cell-binding epitopes, and reduce cell binding. After being released into the cell culture medium, these materials could significantly change the medium osmolality. The foaming process requires surface-active molecules, such as surfactants, or foaming agents. For example, as disclosed in some of the examples below, Pluronic® P123 may be used in the foaming process. However, the above examples still use other ingredients, such as sucrose, dextran, glycerol, and Tween® 20, for foaming. To simplify the process, reduce production costs, and allow for direct use of the dissolvable foam in cell culture without removing excess components, it would be beneficial to remove those excess materials in the foam, as discussed in the examples below.

[0099] In foams, as with any porous material, pore size is important for flow dynamics and total surface area. It has been observed that by using smaller calcium carbonate particles as a gelling source, much smaller pore sizes are observed. It is believed that smaller calcium carbonate particles lead to better distribution and easier release of calcium during mixing before adding acid, increasing the viscosity of the PGA solution. This makes it more difficult for foam cells to coalesce together to form larger pores. However, the more rapid release of calcium during foaming also leads to excessive crosslinking of the PGA before it is fully mixed, potentially damaging the foam structure. It is desirable to be able to control pore size by controlling the viscosity of the PGA solution during foaming, which also prevents uncontrolled excessive crosslinking.

[0100] In this Example 3, the use of plasticizers (e.g., glycerol), foam enhancers or sugars (e.g., sucrose), and foam enhancers in the form of non-surface-active polymers (e.g., dextran) is eliminated by substituting a high HLB surface-active polymer (e.g., Poloxamer 407 or Pluronic® F127) for the two surfactants used in the previous examples (e.g., a surface-active polymer such as P123 and an emulsifier such as Tween® 20). This provides a much simpler formulation for the dissolvable foam and eliminates components that may require further downstream processing or potentially adversely affect cell culture. Additionally, it is possible to control the viscosity of the simplified formulation without adding new components. This simplified formulation has several advantages. For example, this new formulation removes non-critical materials that, in some cases, may not be desirable in the finished dissolvable foam product. This elimination of materials also significantly simplifies the process and reduces costs. Additionally, the foam structure material eliminates a large amount of leachable material that could potentially alter the cell medium osmolality and block cell-binding epitopes during application. This allows for the elimination of a separate coating step in the future, allowing for direct use of the foam for cell culture. Furthermore, the simplified formulation provides tunable viscosity without the addition of additional components. This makes future optimization of the foaming process and control of the foam structure easier and less expensive. Finally, the simplified formulation can benefit the continuous foaming process by utilizing the shear-thinning properties of the material.

[0101] A foaming formulation according to an example embodiment from several of the above examples (referred to as a Comparative Example in the remainder of this Example 3) contained a relatively long list of ingredients, as shown in Table 1 below. In contrast, the simplified formulation of this Example 3 excludes plasticizers (e.g., glycerol), surface-active emulsifiers or second polymers (e.g., Tween® 20), and other foam-enhancing agents or polymers that are not surface-active (e.g., sucrose and dextran). The simplified formulation of Table 1 also uses a poloxamer (e.g., F127) with a higher HLB than the P123 used in the Comparative Example (Pluronic® P123 has an HLB of 8, while Pluronic® F127 has an HLB of 22). As highlighted in Table 1, most of the additional ingredients in the Comparative Example were used to provide physical properties to enable the foaming process. These additional ingredients (Tween® 20, glycerol, sucrose, and dextran) comprise approximately 80% of the total weight (excluding water) of the comparative formulation. Table 2 below compares the weight percentages of the ingredients in the comparative and simplified formulations of Table 1. These materials have no value to the final product and may need to be removed during the subsequent coating process. If the foam article is to be used for cell culture, these materials need to be removed to prevent significant changes in osmotic pressure or interference with cell-binding epitopes. [Table 2] [Table 3]

[0102] In this Example 3, Pluronic® F127 can be replaced with a mixed surfactant of Pluronic® P123 and Tween® 20. This also allows for good foaming without the addition of sucrose, glycerol, or dextran. Measurements indicated that the desired porosity in the foam can be comparable to that of previous examples (including comparative examples) disclosed herein. Figure 4 shows an SEM image of a foam formed from the simplified formulation of Example 3, which has a pore structure comparable to foams produced from other examples herein using more complex formulations.

[0103] Replacing P123 with F127 also benefits the process. P123 is a paste material that is difficult to divide into equal portions. It is also difficult to dissolve in water, and the dissolution process can take overnight. F127 is a powder that can be easily weighed during formulation, and dissolution can be completed in one hour. These properties can benefit the manufacturing process.

[0104] Adding small amounts of calcium ions to a PGA solution can partially crosslink the PGA molecules and increase the viscosity of the solution. This allows for the creation of shear-thinning, dissolvable materials for 3D bioprinting. Adding enough calcium ions to bind to approximately 10% of the carboxyl groups in the PGA molecules increased the viscosity of the solution by 1000-fold at a shear rate of 1 / s, which was approximately 10 times that of glycerol (1.4 × 10 3 mPa*s). When exposed to a shear rate of 1000 1 / s, the viscosity decreased 100-fold, to 1 / 10 of that of glycerol. The viscosity decrease can be quickly recovered after the shear force is removed. By adjusting the amount of calcium, the viscosity range and shear-thinning response can be further tuned.

[0105] Therefore, according to an embodiment of the simplified formulation, a small amount of calcium can be added to increase the viscosity of the PGA solution before foaming begins. This can help increase viscosity and provide shear-thinning properties. Under the shear of the mixing blade, the solution reduces viscosity, which helps incorporate gas bubbles into the form. When mixing is stopped, the solution viscosity rapidly increases, which helps stabilize the foam and prevent liquid drainage. This can further eliminate the need for foam stabilizer materials such as glycerol, sucrose, and dextran. By controlling the solution viscosity, the rate of gas bubble coalescence can be increased or decreased, which can change the pore size of the foam. Shear-thinning properties can also benefit the continuous foaming process. After the foam is extruded from the continuous foam mixer, the foam can quickly stabilize its pore structure. To achieve partial crosslinking, instead of adding all of the GDL at the end of the foaming step, a portion of the GDL can be introduced before foaming (e.g., 0.5 g in Table 1), which results in partial release of calcium from the calcium carbonate and partial crosslinking of the PGA. At the end of the foaming step, the remainder of the GDL can be added (eg, 3.5 g in Table 1) or an acid molecule such as acetic acid can be introduced in vapor form to complete the gelation process.

[0106] According to aspects of an embodiment of the present disclosure, spheroids can be formed by cells entering a scaffold, either by seeding cells directly on top of the scaffold in a tissue culture plate (FIG. 5A) or by perfusion in a bioreactor (FIG. 5B). The cells enter the porous material and become "trapped," or trapped, within the pores. Because the cells cannot adhere to the material, they adhere to each other to form spheroids. The cells or spheroids can produce a variety of by-products, including EVs, proteins, and other secreted materials. Reference to any particular by-product (e.g., EVs) is not intended to limit the scope of the embodiments to that particular by-product, which is used for illustrative purposes only. FIG. 5A shows a porous scaffold fabricated from scaffold material 2500, in which pores 2502 are formed within scaffold material 2500. The scaffold is placed in a tissue culture plate 2506, and cells are seeded thereon. After a culture period, the cells form spheroids 2504 within the pores 2502. FIG. 5B shows a similar scaffold for growing spheroids, but this time, a scaffold material 2510 with pores 2512 is placed within a bioreactor 2516 of a perfusion bioreactor system. Cells are seeded within the bioreactor 2516 to form spheroids 2514 within the pores 2512. The bioreactor system shown in FIG. 5B is a simplified depiction of a system with a medium conditioning vessel 2520 and fluid flow paths 2522 that allow for perfusion and / or recirculation of medium through the bioreactor 2516. The use of a porous scaffold within the bioreactor with medium perfusion can increase extracellular vesicle production. The medium perfusion rate through the bioreactor varies based on the application, cell type, and other factors. Some example embodiments may use medium perfusion with a flow rate of about 0.5 mL / min to about 50 mL / min. The system shown in FIG. 5B is merely an example and is not intended to limit the scope of the embodiments of the present disclosure. The cells enter the porous material and become "trapped" or trapped within the pores. Because the cells cannot adhere to the material, they adhere to each other to form spheroids.

[0107] According to embodiments, the scaffold is dissolvable, and therefore, spheroids can be easily recovered from the scaffold and used for other applications, such as toxicity assays or other applications that would be understood by those skilled in the art. To enable spheroid formation and EV generation, any type of scaffold material can be used that is made from a biocompatible material with controlled porosity and does not allow cell adhesion. To recover spheroids, the scaffold material is made from a biocompatible, digestible material with controlled porosity and does not allow cell adhesion. The porosity and structure of the porous scaffold can be controlled. In some preferred embodiments, the porous scaffold includes a scaffold with high porosity and large pores, but with smaller interconnections of the scaffold material.

[0108] Example 4 This example compares different scaffold properties based on their composition. These data examine the effect of certain agents on pore size or interconnect size or number of interconnects, and how these specific properties affect cell proliferation, maintenance of spheroids within the foam, or cell generation, among other parameters.

[0109] Four different compositions based on 2.0 wt% PGA were evaluated: XP49, XP64, XP76, and FMXP005. XP49 (Figure 6A) uses Tween® 20 as the sole emulsifier, Pluronic® P123 as the surface-active polymer, and dextran as the non-surface-active polymer (composition in Table 2). [Table 4]

[0110] XP64 (Figure 6B) uses both Tween® 20 and SDS as emulsifiers, Pluronic® P123 as a surface-active polymer, and dextran as a non-surface-active polymer (composition in Table 3). [Table 5]

[0111] XP76 (Figure 6C) uses soy lecithin as the sole emulsifier and Methocel HPMC Culminal 724 as the surface-active polymer. It does not contain any non-surface-active polymers (composition in Table 4). [Table 6]

[0112] FMXP005 (Figure 6D) uses SDS as the only emulsifier and dextran as the non-surface-active polymer, and does not contain any surface-active polymers (composition in Table 5). [Table 7]

[0113] Scaffolds of each of the XP49, XP64, XP76, and FMXP005 compositions were grown, dried, and sliced ​​for microscopic analysis. Three parameters, pore size, interconnect (passage) size, and number of interconnects (passages) per pore, were quantified by scanning electron microscopy.

[0114] For all compositions, the pore size distribution (i.e., the maximum distance across the cross-sectional area of ​​the pores) for each composition was relatively broad, as shown in Figure 6E. However, the pore sizes of XP49 and XP64 were primarily between 450 μm and 850 μm in diameter. The XP76 scaffold yielded the smallest pore size, with pore sizes primarily between 300 μm and 650 μm in diameter. The FMXP005 scaffold yielded the largest pore size distribution, with pore sizes primarily between 350 μm and 1000 μm. The pore size distribution across all four foam compositions was primarily between 300 μm and 1000 μm.

[0115] The distribution of interconnect diameters (i.e., the maximum distance across the cross-sectional area of ​​the passage spaces connecting the pores together) was similar to the distribution of pore sizes across the four compositions (Figure 6F). The interconnect sizes of both XP49 and XP64 were primarily between 150 μm and 500 μm in diameter. XP76 had the smallest interconnect diameters of the compositions, primarily between 30 μm and 300 μm in diameter. The FMXP005 composition had the largest pore diameters, but not the largest interconnect diameters, primarily between 70 μm and 400 μm in diameter.

[0116] The number of interconnections (passageways) per pore was also investigated (Figure 6G). Adding SDS as an emulsifier to XP64 scaffolds appears to affect the number of interconnections per pore compared to XP49 scaffolds. The number of interconnections per pore for XP64 was primarily 6–20 (median 12) interconnections per pore, while XP49 was primarily 4–16 (median 8) interconnections per pore. For XP76, the number of interconnections per pore was primarily 6–12 (median 8) interconnections per pore. For FMXP005, the number of interconnections per pore was higher, at 12–18 (median 15) interconnections per pore.

[0117] In addition, the density of the dry scaffolds was also measured. XP49 scaffolds had a density of approximately 0.038–0.046 g / cm 3 The density of XP64 is approximately 0.029-0.030 g / cm 3 The density of XP76 is approximately 0.071 to 0.09 g / cm 3 The density of FMXP005 is approximately 0.048-0.057g / cm 3 The density of was presented.

[0118] Taken together, these data indicate that scaffold composition can influence pore size, interconnect diameter, and number of interconnects per pore, as shown above.

[0119] Example 5 In this example, spheroid formation was demonstrated within scaffolds in tissue culture plates using either an immortalized cell line (HEK293T cells) or a primary cell line (bone marrow-derived human mesenchymal stem cells (hMSCs)). The foam scaffolds had a cylindrical shape with a diameter of 1 cm and a thickness of 0.4 cm. Two different compositions (XP64 and XP76) that result in different pore and interconnect sizes were tested as described in Example 4.

[0120] Figures 6B and 6C show exemplary photographs of uncoated foam scaffolds. After foam preparation, the scaffolds were washed with sterile-filtered demineralized water and sterilized with 70% ethanol. The scaffolds were added to the wells of an ultra-low adhesion 6-well plate, and then the appropriate cell culture medium was added to the wells. The plate was transferred to a cell culture incubator set at 37°C and 5% CO2 for equilibration. Cells were dissociated from the culture flasks with either Tryspin (HEK293T) or TrypLE (hMSC) and adjusted to a concentration of 1 x 10 6 To seed the foam scaffolds, the medium was first removed from the wells and 150 μL of cell suspension was added to the top of the foam for a total of 1.5 × 10 cells / mL. 5The plates were transferred back to the cell culture incubator for 2 hours, after which 3 mL of the appropriate medium was added to each well without disturbing the scaffolds. One day after seeding, initial spheroid formation was assessed by staining live cells with calcein AM. Spheroids were observed under all conditions and for both cell types (Figure 7A (HEK293T cells) and Figure 7B (hMSCs), first column). Interestingly, there were more spheroids in the XP76 scaffold compared to the XP64 scaffold, indicating better cell retention within the XP76 scaffold. This may be due to smaller interconnections per pore and / or fewer interconnections in XP76 compared to the XP64 scaffold, as described in Example 4. Seven days after seeding, spheroid growth was assessed, as shown in the photographs in the bottom row of Figure 7A (HEK293T cells) and Figure 7B (hMSCs). Under all conditions, spheroids, especially for HEK293T cells, were larger on day 7 than on day 1. Cell numbers after scaffold digestion and spheroid dissociation showed similar results.

[0121] Figure 8 is a bar graph showing cell numbers after scaffold digestion and spheroid dissociation in terms of fold increase in cells after seeding for both experiments involving HEK293T cells and hMSC cells. Data show cell growth either 5 days after seeding of HEK293T cells or 7 days after seeding of hMSC cells. The data clearly demonstrate spheroid and / or cell growth within the foam scaffolds. The increase in cell mass for HEK29T cells was approximately 9-fold to approximately 27-fold, while the increase for hMSC cells was approximately 2-3-fold. For HEK293T cells, the lower cell growth numbers within the XP64 scaffold appear to be primarily due to cell loss during seeding due to the higher number of interconnections and / or more interconnections per pore compared to the XP76 scaffold. Therefore, hMSC cell growth remained limited within both scaffolds, as expected for these cells.

[0122] Example 6 As described above, cells can be seeded into porous (foam) scaffolds in bioreactors under perfusion conditions, as shown in Figure 5B. In this example, an immortalized cell line (HEK293T cells) was seeded into two perfusion bioreactors containing uncoated dissolvable foam scaffolds. The foam scaffolds had a cylindrical shape with a diameter of 3 cm and a thickness of 1 cm. After preparation, the dry scaffolds were placed into the bioreactor cartridge. The scaffolds were then washed with sterile filtered demineralized water and sterilized inside the bioreactor by perfusion. The appropriate cell culture medium was then added to the medium stock bottle and perfused through the scaffolds. The bioreactors were then transferred to a cell culture incubator set at 37°C and 5% CO2 for 1 hour for equilibration. Cells were dissociated from their culture flasks with 0.25% Tryspin, and their concentration was adjusted to 1 x 10 6 To seed the scaffolds, 10 mL of cell suspension was added to the medium stock bottle for a total of 10 × 10 cells / mL. 6The cells / scaffolds were then seeded. As shown in Figure 9A, the perfusion rate was adjusted to 10 mL / min for cell seeding. After monitoring cell seeding for 3 hours, the medium flow rate was reduced to 1 mL / min. Figure 9A shows a plot of the number of cells counted in the recirculating medium over time. The downward sloping line represents the decrease in HEK293T cells in the medium over time, indicating that the cells were successfully seeded within the foam scaffolds. After perfusing the bioreactor overnight, the bioreactor cartridge was opened to collect the scaffolds. Spheroid formation was assessed by staining live cells with calcein AM. Both the top and bottom of the scaffolds were observed, as shown in Figure 10. Figure 10 shows the resulting spheroids formed 1 day after seeding in both the top and bottom of both bioreactors. Spheroids were present throughout both scaffolds and each scaffold, from bottom to top. As shown in Figure 9B, cell counts after scaffold digestion and spheroid dissociation showed little cell loss compared to initial cell seeding in the medium stock bottle, with 90% cell recovery in reactor 1 and 60% cell recovery in reactor 2. These data indicate that scaffolds can be directly colonized by cells by perfusion of medium carrying cells in suspension, and that within the scaffolds, these cells form spheroids under prefusion.

[0123] Example 7 In this experiment, optimal scaffold compositions (pore size and interconnect size) were evaluated for the formation and maintenance of hMSC spheroids under perfusion for 48 hours at medium flow rates up to 10 mL / min. Figures 6A-6D show the performance of uncoated foam scaffold compositions (FMXP005 (Figure 6D), XP64 (Figure 6B), XP76 (Figure 6C), and XP49 (Figure 6A)) seeded with hMSC cells inside a perfusion bioreactor.

[0124] Cell seeding was performed as described in Example 5 for hMSCs, but with 2×10 6The scaffolds were seeded with 75 μL of cell seeding solution containing 1000 cells / mL. After overnight spheroid formation, the scaffolds were transferred to a bioreactor cartridge. To test the perfusion rate, which may be dependent on spheroid maintenance, each foam scaffold was tested at perfusion rates of 1 mL / min and 10 mL / min. The first column in Figure 11 shows the resulting spheroids after 48 hours in the 1 mL / min perfusion bioreactor. The second column in Figure 11 shows the resulting spheroids after 48 hours in the 10 mL / min perfusion bioreactor. After 48 hours of perfusion in the bioreactor, the bioreactor was opened, the scaffolds were collected, and the spheroids or cells were observed after calcein AM staining. Interestingly, even in the absence of a cell adhesive coating on the FMXP005 foam scaffold (Figures 6D and 11), some hMSC adhesion was observed on the scaffold, especially at a perfusion rate of 10 mL / min. This could be due to the specific composition of this scaffold or to increased shear forces / turbulence in this scaffold at this faster flow rate due to smaller interconnects or fewer interconnects per pore. Therefore, at least when using hMSCs, other compositions may be preferable for spheroid formation and EV generation. Additionally, both XP64 and XP49 foam scaffolds resulted in good spheroid formation at a perfusion rate of 1 mL / min, indicating that both scaffold compositions may be suitable for spheroid formation. However, as shown in Figure 12, at a perfusion rate of 10 mL / min, more significant spheroid loss and total cell loss were observed in the XP64 foam scaffold (compared to the XP49 foam scaffold). This spheroid loss at 10 mL / min in the XP64 scaffold compared to the XP49 scaffold is likely due to the greater number of interconnects per pore in the XP64 scaffold. These data may suggest that for this scaffold composition, faster perfusion rates may not be optimal for spheroid-stimulated EV and other particle generation. Additionally, these data indicate that certain scaffold compositions with pore and interconnect sizes may be preferable for maintaining spheroids within the scaffold at faster perfusion rates for certain applications.Thus, embodiments of the present disclosure include tailoring pore size and / or interconnect size depending on the cell type or desired cell by-product.

[0125] Example 8 This experiment demonstrates that foam scaffolds according to embodiments of the present disclosure can be used to enhance extracellular vesicle (EV) and particle generation from spheroids. In this example, only the XP49 scaffold composition was used. Cell seeding (with hMSCs) was performed as in Example 6. After overnight spheroid formation, the scaffolds were washed with RoosterCollect™-EV medium (a defined, low-particle medium) from RoosterBio, Inc. to remove any remaining EVs and other particles present in the culture medium. The scaffolds were then added to the cartridge of the bioreactor. RoosterCollect™-EV medium was added to the medium stock bottle, and the perfusion rate was adjusted to either 10 mL / min or 2 mL / min. For 2D static conditions, cells were washed with RoosterCollect™-EV medium and then incubated in RoosterCollect™-EV medium under static conditions for 48 hours. After 48 hours, the bioreactor was opened to collect the scaffolds, and the spheroids or cells were stained with calcein AM. In this example, similar numbers of spheroids were observed in scaffolds perfused at flow rates of 10 mL / min (FIG. 13A) and 2 mL / min (FIG. 13B). Cells displayed normal morphology under 2D static conditions (FIG. 13C).

[0126] Conditioned RoosterCollect™-EV media was collected and processed by centrifugation at 300 x g for 15 minutes and filtration through a 200 nm filter to remove cellular debris and large particles. The clarified media was then concentrated using a 100 kDa ultrafiltration membrane, and EVs were isolated by overnight precipitation in ExoQuick-TC™ (System Biosciences) and centrifugation at 1500 x g for 30 minutes. EVs were then resuspended in sterile dPBS. Each EV sample was then analyzed to assess particle size, concentration, and EV quality and functionality. Particle analysis by multi-angle dynamic light scattering (MADLS) was performed, and the results are shown in Figures 14A-14C, 15A-15C, and 16. Figures 14A-14C show the size distribution of detected particles or EVs by number (proportional to particle abundance in the sample). Figures 15A-15C show the particle / EV concentration for different images of detected particles. MALDS combines scattering angle information from Mie theory with particle size distribution analysis from dynamic light scattering measurements in an integrated manner. Lower noise, and therefore reduced smoothing, allows for reliable and accurate presentation of particle size distributions with better characterization of individual components of multicomponent samples. Using the MADLS method, we identified a population of extracellular vesicles with an average diameter of approximately 100 nm, as shown in Figures 14A-14C and 15A-15C. This population was significantly more abundant in samples perfused at 10 mL / min (Figures 14A and 15A) than in samples perfused at 2 mL / min (Figures 14B and 15B) or under static culture conditions (Figures 14C and 15C). Figure 16 shows the number of EVs per million cells for each condition: 10 mL / min perfusion, 2 mL / min perfusion, and static 2D culture. As shown in Figure 16, perfused 3D scaffolds generated a higher number of EVs per million cells compared to 2D growth conditions after 48 hours.

[0127] Similar results were observed by quantifying CD63-positive EVs (specific EV populations) by enzyme-linked immunosorbent assay (ELISA), as shown in Figure 17. Perfused foam scaffolds exhibited higher numbers of CD63-positive EVs per million cells compared to 2D environments, with the faster perfusion rate of 10 mL / min having significantly higher numbers of EVs per million cells. The presence of EVs in these samples was also confirmed by Western blot analysis for two other markers of EV populations, CD81 and TSG101, as shown in Figure 18. The blot analysis in Figure 18 shows CD81 and TSG101 results for foams used in the 10 mL / min perfusion reactor and static 2D culture plates.

[0128] The functionality of these EVs was then evaluated by a wound-healing assay using HT-1080 cells. The wound-healing assay is a method for studying directional cell migration in vitro. This method mimics cell migration during wound healing in vivo. The basic steps involve creating a "wound" in a cell monolayer, capturing images either initially and at set time points or at regular intervals during cell migration to close the wound, and comparing the images to quantify the cell migration rate. After wounding on tissue culture plates, wells were perfused with 10 mL / min of perfusion samples (using XP49 foam scaffolds) or 2 × 10 cells from 2D samples. 9 Cells were treated with either EVs from the 10 mL / min perfusion well or with phosphate-buffered saline (PBS) alone as a control. Figure 19 shows bright-field contrast phase microscopy images of cells immediately after wounding (0 h) for both PBS- and EV-treated samples, followed by photographs of each sample 8 h later. As shown in Figure 20, a significant increase in the rate of wound healing was observed when treated with EVs from the 10 mL / min perfusion well compared to the PBS-treated wells.

[0129] Figure 20 plots the percent wound healing over time for PBS-treated samples, samples treated with EVs from hMSC spheroids in foam scaffolds in a perfusion bioreactor (XP49, 10 mL / min flow rate), and samples treated with EVs from adherent hMSC spheroids on a 2D surface. The EV-treated samples exhibited higher Wood's would healing rates compared to the PBS-treated samples, with the EV-treated samples from the perfusion bioreactor exhibiting the best wound healing by a significant margin. Thus, as shown in Figure 20, EVs from perfused samples are functional and even more efficient than EVs from 2D samples. These data demonstrate that hMSC spheroids can be maintained within the scaffold by perfusion with a defined, low-nutrient medium such as RoosterCollect™-EV medium. Additionally, increasing the perfusion rate resulted in increased particle and EV production from these spheroids. The presence, quality, and quantity of EVs were confirmed.

[0130] Example 9 This example compares EV generation from hMSC spheroids in different culture vessels with EV generation from the scaffolds of the present disclosure to determine the effect of shear stress on the ability of hMSC spheroids to generate functional EV mass using perfusion, agitation, or classical 2D culture conditions (i.e., static conditions) with the scaffolds of the present disclosure.

[0131] More specifically, scaffold composition XP49 was used to evaluate EV generation from hMSC spheroids using the disclosed scaffolds under perfusion, which was then compared to EV generation from hMSC spheroids in a microcavity-containing vessel and a T-75 CellBIND® flask (Corning®) under stirring (35 rpm), which were used as classical 2D static conditions.

[0132] Cell seeding with hMSCs was performed on scaffolds of the present disclosure as in Examples 6 and 7. For stirred microcavity-containing vessels, cell seeding was performed according to the manufacturer's recommendations. For T-75 CellBIND® flasks, approximately 9 x 10 3 cells / cm 2 All vessels and scaffolds were seeded simultaneously. After leaving the vessels overnight for spheroid formation, the scaffolds were washed with RoosterCollect™-EV medium (defined low-particle medium, RoosterBio M2001) to remove EVs and other particles present in the culture medium.

[0133] For experiments using scaffolds, the scaffolds were then added to a bioreactor set up as in the embodiment shown in FIG. 5B. RoosterCollect™-EV medium was added to the medium stock bottle, and the perfusion rate was adjusted to 10 mL / min. RoosterCollect™-EV medium was also added to the stirred microcavity-containing vessels and T-75 CellBIND® flasks. For the stirred microcavity-containing vessels and T-75 CellBIND® flasks, the cells were washed with RoosterCollect™-EV medium and then incubated in RoosterCollect™-EV medium for 48 hours either under stirring at 35 rpm (microcavity vessels) or under static conditions (T-75 CellBIND® flasks).

[0134] After 48 hours of perfusion, stirring, or static conditions, the bioreactor was opened, the scaffolds and spheroids were collected, and the cells were stained with calcein AM (results shown in Figures 21A-21C). Figure 21A shows spheroids in the scaffold after perfusion. Figure 21B shows spheroids in the microcavity vessel after stirring. Figure 21C shows spheroids in a T-75 CellBIND® flask after static conditions. Spheroids were observed in the scaffold at a perfusion rate of 10 mL / min, in the microcavity vessel with stirring, and under 2D static conditions.

[0135] Conditioned RoosterCollect™-EV media was collected and processed as in Example 8 to remove cellular debris and large particles, then concentrated, and EVs were isolated and resuspended in sterile Dulbecco's phosphate-buffered saline (dPBS). Each EV sample was then analyzed using the same techniques as in Example 8 to assess particle size, concentration, EV quality, and functionality.

[0136] The MADLS method identified a population with an average size diameter of approximately 100 nm for all conditions (Figures 22A-C). Figures 22A-C show triplicate measurements of the same sample for each of the perfusion, stirring, and static experiments. This particle population was significantly more abundant in the 10 mL / min perfusion sample than in the stirred microcavity vessel or 2D static conditions (Figure 22D). ELISA assays quantifying CD63-positive EVs in these samples showed no significant difference between the perfusion and stirred microcavity samples. However, significantly more CD63-positive EVs were detected in the perfusion sample compared to the 2D static sample, and in the stirred microcavity sample compared to the 2D static sample (Figure 23A). The presence of EVs in these samples was also confirmed by Western blot for CD81 and TSG101 (Figure 23B). Figures 23A-B show data from ELISA CD63 and Western blot (using CD81 and TSG-101 markers) experiments with data from four independent experiments, where each dot represents one sample. Dots of the same color belong to the same experiment.

[0137] The functionality of these EVs was assessed by a wound healing assay using HT-1080 cells performed as described in Example 8. After wound creation, each well was filled with 2–10 ml of EVs from the 10 mL / min perfusion sample, the stirred microcavity sample, or the static condition sample. 9 The wounds were treated with EVs. A negative control (no EVs) was also prepared in which the wounds were treated with phosphate-buffered saline only.

[0138] A significant increase in the rate of wound healing was observed with EVs from hMSC spheroids. EVs from the 10 mL / min perfusion sample and the stirred or static microcavity samples resulted in faster wound healing compared to wells treated with PBS (Figure 38). EVs from the perfusion sample were slightly more efficient than EVs from the static sample (Figure 24).

[0139] The results show that hMSC spheroids under shear stress generate significantly more EVs per cell than classical 2D cultures. Additionally, perfused hMSC spheroids generate significantly more particles than spheroids under agitation and 2D static conditions, but ELISA data indicate that they do not generate significantly more CD63-positive EVs compared to microcavity spheroids. Furthermore, EVs from hMSC spheroids under perfused or agitated conditions appear to be more efficient in wound healing assays than EVs from 2D static conditions.

[0140] Example 10 This example compares EV generation from hMSCs within scaffolds at perfusion rates faster and slower than those previously evaluated. The results provide lower and upper limits for perfusion rates that provide higher EV yields per cell during EV generation.

[0141] In this experiment, EV generation from hMSC spheroids under perfusion was evaluated using only scaffold composition XP49. Similar to Examples 7 and 8, this scaffold was chosen because it offered the best spheroid maintenance at high perfusion rates compared to other scaffolds previously tested. As a control, EVs were also generated from hMSCs adhered to T-75 CellBIND® flasks under 2D static conditions.

[0142] Cell seeding using hMSCs was performed on the scaffolds as in Examples 7 and 8. For T-75 CellBIND® flasks, approximately 9 x 10 3 cells / cm 2All vessels and scaffolds were seeded simultaneously. After overnight spheroid formation, the scaffolds were washed with RoosterCollect™-EV medium (defined low-particle medium, RoosterBio M2001) to remove any remaining EVs and other particles present in the culture medium. The scaffolds were then added to the bioreactor cartridge. RoosterCollect™-EV medium was added to the medium stock bottle, and the perfusion rate was adjusted to the different flow rates tested. EV generation was evaluated at 20, 10, 2, and 0.5 mL / min. After washing twice with RoosterCollect™-EV medium, the EV collection medium was also added to the T-75 CellBIND® flask.

[0143] After 48 hours of perfusion, EV production was assessed. The bioreactor was opened, the scaffolds were collected, and the spheroids were stained with calcein AM. In the 2D condition, cells were observed without further staining.

[0144] As observed previously, no significant differences in spheroid shape or density within the scaffold were observed between the 10 mL / min and 2 mL / min perfusion conditions (Figure 25). Similarly, no differences were observed with the 0.5 mL / min perfusion condition. However, under the 20 mL / min perfusion condition, nearly half of the spheroids appeared to have lost their shape and were either attached to or in contact with the scaffold (Figure 25). As observed in the previous example, hMSCs under 2D conditions displayed the expected spindle-shaped morphology (Figure 25).

[0145] Apart from spheroid and cell morphology observations, the conditioned RoosterCollect™-EV medium was collected. To clarify the medium, the collected conditioned medium was centrifuged at 300 x g for 15 minutes to remove remaining cells and debris, and then filtered through a 200 nm filter to further remove smaller cell debris and larger particles. For this experiment, the clarified medium was also evaluated by MADLS to determine the size and concentration of EVs generated by hSMCs. For all conditions, EVs had an average diameter of approximately 100 nm (Figure 26A). Regarding particle concentration, large differences were observed between different conditions, with a maximum of approximately 2 x 10 under 20 mL / min perfusion conditions. 11 particles / mL, which is approximately 1.23 x 10 under a perfusion condition of 10 mL / min. 11 These figures were approximately 3.28 × 10 particles / mL under the 2 mL / min perfusion condition, which was significantly lower than the other conditions (Figure 26B). 10 / mL, and under a perfusion condition of 0.5 mL / min, the 9 particles / mL only. However, because the concentrations of these particles were below the instrument threshold, it was not possible to calculate the concentration for the 2D condition.

[0146] The clarified medium was then ultrafiltered to concentrate EVs, as previously described, and the EVs were then precipitated overnight in ExoQuick®. The following day, all samples were centrifuged at 1500 x g for 30 minutes, and the EVs were resuspended in sterile PBS.

[0147] These purified EVs were again analyzed by MADLS to determine their size and concentration. Similar to the clarified medium, the EVs had an average diameter of approximately 100 nm (Figure 27A). No significant difference was observed between the 20 mL / min and 10 mL / min perfusion conditions in terms of the amount of EVs generated (Figure 27B). However, when compared with other conditions, the 2 mL / min perfusion condition produced approximately 7-fold fewer EVs than the 10 mL / min or 20 mL / min perfusion conditions, and the 0.5 mL / min perfusion condition produced over 10-fold fewer EVs than these faster flow rate conditions (Figure 27B). More interestingly, approximately 100-fold fewer EVs were generated under the 2D condition compared with these faster flow rate conditions (Figure 27B).

[0148] Regarding the amount of EVs generated per cell number for each condition (Figure 27C), similar results were observed, with the highest number of EVs generated under 20 mL / min and 10 mL / min perfusion conditions and the lowest number of EVs generated under 2D conditions, although 1000-fold lower EVs per million cells under 2D conditions compared to 10 mL / min perfusion conditions.

[0149] These samples were then further analyzed to determine the amount of CD63-positive EVs by ELISA. Regarding total CD63-positive EVs (Figure 28A), the highest levels of CD63-positive EVs were observed at perfusion rates of 20 mL / min and 10 mL / min. No significant differences in CD63-positive EVs were observed between the lowest perfusion rates of 2 mL / min and 0.5 mL / min and the 2D condition (Figure 28A). However, total CD63-positive EVs per million cells for each condition (Figure 28B) show a slight increase of approximately twofold in CD63-positive EVs at these slowest perfusion rates compared to the 2D condition.

[0150] The functionality of these EVs was also assessed by a wound healing assay using HT-1080 cells (Figure 29). Here, the assay was performed slightly differently than before. Wounds were created with a P1000 pipette tip, and wound healing was assessed only at 6 hours post-wounding. Each wound (except for the control) was treated with 2x10 EVs from each condition. 9The wounds were treated with 100 EVs. Controls were treated with PBS only. Images were taken from each wound at time 0 and 6 hours after injury, and the distance between the wound edges was measured using OlyVIA® software (Olympus LS). At 6 hours, the distance between the two wound edges was compared to the distance at time 0 to calculate the wound closure or wound healing rate. All perfusion conditions exhibited faster wound healing than the mock control (Figure 29). In addition, increasing the perfusion rate here appeared to accelerate wound healing, even compared to the 2D condition, suggesting that EVs other than CD63-positive EVs may play a role in wound healing rate.

[0151] Taken together, the data indicate that increasing the flow rate in perfused samples leads to higher EV generation. However, even at this slower flow rate, increased EV generation is observed compared to 2D conditions, with an upper limit of approximately 10-20 mL / min and a lower limit of approximately 0.5 mL / min. Additionally, EVs generated by perfusion, even at faster flow rates such as 20 mL / min, are functional and improve wound healing rates compared to control mock-treated conditions.

[0152] While the present disclosure includes a limited number of embodiments, those skilled in the art, having the benefit of this disclosure, will recognize that other embodiments may be devised that do not depart from the scope of the present disclosure.

Claims

1. 1. A method for producing spheroids or spheroid by-products, comprising: providing a bioreactor comprising a cavity for culturing cells, an inlet and an outlet to said cavity, and a porous scaffold; inserting cells of a certain cell type into the porous scaffold to form spheroids; and culturing the spheroids by perfusing a cell culture medium through the cavity; A method wherein the porous scaffold comprises pores and passageways between the pores.

2. Approximately 1.1×10 －５ m / s ~ approx. 5.0×10 －４ The method of claim 1, wherein the porous scaffold is non-adhesive to the cells when perfused at a linear velocity of 1000 rpm.

3. 3. The method of claim 2, wherein the porous scaffold is non-adherent to at least 80% of the cells in the bioreactor.

4. 4. The method of claim 1, wherein inserting cells of a cell type into the porous scaffold comprises seeding the cells into the porous scaffold.

5. The method of any one of claims 2 to 4, wherein at least 80% of the cells within the pores of the porous scaffold aggregate to form spheroids.

6. 6. The method of any one of claims 2 to 5, wherein the pores and the passages between the pores are sized to trap at least some of the spheroids within the porous scaffold.

7. 7. The method of any one of claims 1 to 6, further comprising recovering at least one of the spheroids or at least one by-product of the spheroids.

8. 8. The method of claim 7, wherein the by-product of the harvested spheroids is extracellular vesicles.

9. The method according to any one of claims 1 to 8, wherein the porous scaffold is made of a material that is non-adhesive to cells, or the material of the porous scaffold has been treated to be non-adhesive to cells, or a combination thereof.

10. 10. The method of any one of claims 1 to 9, wherein the porous scaffold comprises an ionotropically crosslinked polygalacturonic acid compound selected from at least one of ionotropically crosslinked alginic acid or a salt thereof, or pectinic acid; partially esterified pectinic acid, partially amidated pectinic acid, and salts thereof.

11. 10. The method of any one of the preceding claims, wherein at least 70% of the pores have a pore diameter of from about 200 μm to about 1000 μm, as measured in the dry scaffold.

12. 10. The method of any one of the preceding claims, wherein at least 80% of the pores have a pore diameter of from about 400 μm to about 800 μm, as measured in the dry scaffold.

13. 12. The method of any one of claims 1 to 11, wherein at least 70% of the passageways have a maximum passageway width, measured within the dry scaffold, of from about 30 μm to about 500 μm.

14. 12. The method of any one of claims 1 to 11, wherein at least 80% of the passageways have a maximum passageway width, measured within the dry scaffold, of from about 60 μm to about 400 μm.

15. 12. The method of any one of claims 1 to 11, wherein at least 70% of the pores have 5 to 18 passages per pore.

16. The method of any one of claims 1 to 11, wherein at least 80% of the pores have a number of passages per pore of from about 6 to about 14.

17. 10. The method of any one of the preceding claims, wherein perfusing the cell culture medium comprises continuously passing cell culture medium over the porous scaffold.

18. 10. The method of any one of the preceding claims, wherein the porous scaffold is dissolvable.

19. 20. The method of claim 18, further comprising digesting the dissolvable porous scaffold by exposing the dissolvable porous scaffold to an enzyme.

20. 20. The method of claim 19, further comprising exposing the dissolvable porous scaffold to a chelating agent.

21. 21. The method of claim 19 or 20, wherein digestion of the dissolvable porous scaffold is complete in less than about 1 hour.

22. 22. The method of any one of claims 19 to 21, wherein the enzyme comprises a non-proteolytic enzyme.

23. 23. The method of claim 22, wherein the non-proteolytic enzyme is selected from the group consisting of pectin-degrading enzymes, pectinases, and alginate lyases.

24. 24. The method of any one of claims 19 to 23, wherein digesting the dissolvable porous scaffold comprises exposing the dissolvable foam scaffold to about 1 U to about 200 U of the enzyme.

25. 25. The method of any one of claims 20 to 24, comprising exposing the dissolvable porous scaffold to about 1 mM to about 200 mM of the chelating agent.

26. 1. A bioreactor comprising: a cavity for culturing cells; an inlet and an outlet to the cavity; a porous scaffold within the cavity; the porous scaffold comprises pores and passageways between the pores; the porous scaffold is non-adhesive to cells; A bioreactor wherein the porous scaffold is configured to contain spheroids.

27. 27. The bioreactor of claim 26, wherein at least 75% of the pores have a pore size of about 200 μm to about 1000 μm as measured within the dry scaffold.

28. 27. The bioreactor of claim 26, wherein at least 75% of the passages have a maximum passage width, as measured within the dry scaffold, of about 30 μm to about 500 μm.

29. 27. The bioreactor of claim 26, wherein at least 75% of the pores have 5 to 18 passages per pore.

30. The bioreactor of any one of claims 26 to 29, wherein the porous scaffold is dissolvable.

31. 31. The bioreactor of claim 30, wherein the dissolvable porous scaffold is a foam scaffold.

32. 32. The bioreactor of claim 31 , wherein the dissolvable foam scaffold is dissolved by pectinase or alginate lyase.

33. The porous scaffold comprises:

33. The bioreactor of any one of claims 26 to 32, having a composition comprising an ionotropically crosslinked polysaccharide, wherein the ionotropically crosslinked polysaccharide is selected from alginic acid and its salts, pectinic acid and its salts, partially esterified pectinic acid and its salts, partially amidated pectinic acid and its salts, or combinations thereof.

34. 34. The bioreactor of claim 33, wherein the ionotropically cross-linked polysaccharide is polygalacturonic acid.

35. A porous scaffold for culturing spheroids or by-products from spheroids, comprising: pores, at least 75% of said pores having a pore diameter of about 200 μm to about 1000 μm, as measured in the dry scaffold; passages between the pores, at least 75% of the passages having a maximum width of the passage, as measured in the dry scaffold, of about 30 μm to about 500 μm; a number of passages per pore, wherein at least 75% of the pores have a number of passages per pore of between 5 and 18; the porous scaffold is non-adhesive to cells; A porous scaffold configured to grow spheroids within the pores from cells of a cell type.

36. 36. The porous scaffold of claim 35, wherein the cells of a cell type are selected from a primary cell line and an immortalized cell line.

37. 37. The porous scaffold of claim 36, wherein said primary cell line is a bone marrow-derived human mesenchymal stem cell and said immortalized cell line is a HEK293T cell.

38. 1. A perfusion bioreactor comprising: a cavity for culturing cells; an inlet and an outlet to the cavity; a porous scaffold within the cavity, wherein the porous scaffold is non-adhesive to cells; the porous scaffold comprises pores and passageways between the pores; at least 75% of the pores have a pore size, as measured in the dry scaffold, of about 200 μm to about 1000 μm; at least 75% of the channels have a maximum width, as measured in the dry scaffold, of between about 30 μm and about 500 μm; at least 75% of the pores have 5 to 18 passages per pore; A perfusion bioreactor, wherein the porous scaffold is adapted to grow spheroids or by-products of spheroids.

39. 40. The perfusion bioreactor of claim 38, wherein the bioreactor is configured to retain at least 20% of any cells added to the cavity through the inlet.

40. 40. The perfusion bioreactor of claim 38, wherein the bioreactor is configured to retain at least 60% of any cells added to the cavity through the inlet.

41. 36. The perfusion bioreactor of claim 35, wherein the bioreactor is adapted to produce extracellular vesicles as the by-product of spheroids.

42. 42. The perfusion bioreactor of claim 41, wherein wound healing by the extracellular vesicles produced by the perfusion bioreactor exceeds wound healing by extracellular vesicles produced in 2D flasks for 3D culture at 6 hours post-injury as measured by a wound healing assay using HT-1080 cells.

43. Approximately 2 x 10 ９ Wound healing using 2 x 10 extracellular vesicles generated in 2D flasks for 3D culture was measured by a wound healing assay using HT-1080 cells at 6 hours after injury. ９ 42. The perfusion bioreactor of claim 41, having a wound closure that is at least 5% better than wound healing with extracellular vesicles.

44. The extracellular vesicles produced by the perfusion bioreactor were approximately 1 x 10 per cell. ３ EVs ~ approximately 1 x 10 per cell ７ 42. The perfusion bioreactor of claim 41, wherein the amount of EVs produced is

45. The extracellular vesicles are about 1.1 x 10 －５ m / s ~ approx. 5.0×10 －４ 45. The perfusion bioreactor of claim 44, wherein the perfusion bioreactor is produced from perfusion at a linear velocity of 1000 rpm.

46. 45. The perfusion bioreactor of claim 44, wherein increasing the perfusion rate increases the number of extracellular vesicles produced per cell.

47. 45. The perfusion bioreactor of claim 44, wherein the number of extracellular vesicles per cell produced using the perfusion bioreactor exceeds the number of extracellular vesicles produced using a static 2D microwell plate.

48. Approximately 2.3 x 10 －６ m / s ~ approx. 1.2×10 －３ The method of claim 1, wherein the porous scaffold is non-adhesive to the cells when perfused at a linear velocity of 1000 rpm.

49. The extracellular vesicles are about 2.3 x 10 －６ m / s ~ approx. 1.2×10 －３ 45. The perfusion bioreactor of claim 44, wherein the perfusion bioreactor is produced from perfusion at a linear velocity of 1000 rpm.

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