Method for large scale spheroid and extracellular vesicle production

Abstract

The present invention provides a method for producing spheroids and cellular by-products of spheroids, such as extracellular vesicles. The method comprises providing a bioreactor having a cavity for culturing cells; inserting a cell into the cavity to form a spheroid; and perfusing a cell culture medium through the cavity to culture the spheroid. A porous scaffold may be soluble or insoluble, and the cells are inoculated into pores of the porous scaffold, where the cells aggregate in the pores of the porous scaffold to form spheroids. The bioreactor is continuously perfused for large scale spheroid production and enables collection of spheroids and / or extracellular vesicles or other components from cell secretory groups.

Description

[0001] Cross - reference to related applications

[0002] This application claims the priority of U.S. Provisional Application No. 63 / 426,908, filed on November 21, 2022, under 35 U.S.C.§119, the content of which is hereby incorporated by reference in its entirety and for all purposes. Technical field

[0003] The present disclosure generally relates to methods for generating spheroids and / or by - products (including extracellular vesicles) of cells. More specifically, the present disclosure relates to methods for large - scale generation of spheroids and / or extracellular vesicles in a porous scaffold. Background art

[0004] In vitro models of cell function have been developed in various fields, such as cancer and stem cell research, drug discovery and drug screening, and regenerative medicine. The goal of these models is to understand how these cells function and interact at the molecular and cellular levels. These in vitro models are generated using two - dimensional (“2D”) systems. Although these 2D systems have made significant discoveries in these fields, there are still many deficiencies. In the case of 2D systems, cells adhere to a rigid surface and are geometrically constrained, resulting in a flat morphology, which alters cytoskeleton regulation, which plays an important role in intracellular signal transduction, and may thus affect cell growth, migration, and apoptosis. These limitations of 2D culture often lead to a significant difference between in vitro biological responses and those observed in vivo. This is because these 2D models cannot fully capture in vivo tissue complexity and the role of the cellular and acellular microenvironment.

[0005] Currently, in drug discovery, the standard procedure for screening compounds starts with tests based on 2D cell culture, followed by animal model tests, and then clinical trials. Only about 10% of compounds successfully progress through clinical development. Many drugs fail during clinical trials (especially during Phase III, which is the most costly phase of clinical development), mainly due to insufficient clinical efficacy and / or unacceptable toxicity. Some of these failures are attributed to data collected from 2D culture tests, in which the response of cells to drugs is altered due to their non - native microenvironment. Due to the high costs associated with drug discovery, there is an increasing need to be able to eliminate ineffective and / or toxic compounds with unacceptable toxicity as early as possible in the drug discovery process. There is a need for in vitro cell - based systems that can more realistically mimic in vivo cell behavior and provide more predictable results for in vivo testing.

[0006] Particularly of interest are three-dimensional (“3D”) systems. Compared to 2D cell culture monolayers, 3D cell culture provides enhanced cell-to-cell interactions and more realistically mimics the native microenvironment of tissues. Over the past decade, 3D cell culture has 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 have shown that 3D cell culture more accurately presents the environment that cells experience in vivo, and that the cellular responses in 3D culture are more similar to in vivo behavior than those in 2D culture. It is thought that the additional dimension of 3D culture causes differences in cellular responses because it not only affects the spatial organization of cell surface receptors involved in interacting with surrounding cells, but also induces physical constraints on the cells. These spatial and physical aspects in 3D culture are thought to affect signal transduction from the outside to the inside of the cell and ultimately affect gene expression and cell behavior.

[0007] There is also interest in cell culture systems due to components produced by cells and secreted into their environment. One component of particular interest (especially with respect to regenerative medicine and other therapies) is extracellular vesicles. Extracellular vesicles (“EVs”) are nanoparticles produced by most cell types and have strong therapeutic potential. Current methods for EV production use adherent cells that adhere to 2D surfaces that require large culture spaces to meet the concentrations needed for therapy, or 3D surfaces (such as microcarriers) or other fixed-bed materials under perfusion in a bioreactor. However, despite the need for large-scale production of this important cellular byproduct, a method for large-scale and efficient EV production has not yet been achieved.

[0008] Cells aggregated into 3D structures are called spheroids, and spheroids better mimic in vivo tissues than 2D-grown cells. For a variety of reasons, it is advantageous to produce large numbers of spheroids, including for drug testing and as cell factories for producing cellular byproducts such as EVs and exosomes. Since EVs are cellular byproducts, they can be collected from the fluid surrounding cells and cell aggregates such as spheroids. However, in cases where drug testing is needed for a cell type (or when other applications are required, such as research on cell function or regenerative medicine), it is also advantageous to capture large numbers of spheroids themselves. Currently, spheroids are grown on 2D platforms such as microtiter plates and flasks. However, the 2D nature of these platforms limits the number of spheroids produced. In addition, since EVs are produced by the cells in the spheroids, the limited number of spheroids produced by 2D systems also limits the amount of EVs that can be produced.

[0009] Capturing (collecting) spheroids from 2D systems is labor-intensive and inefficient because each plate must be processed individually to collect spheroids from the microwells, and growing spheroids in 2D systems typically requires changing the liquid, making it difficult to culture spheroids without disturbing them. Similarly, capturing EVs and other cellular by-products produced by spheroids is also labor-intensive and inefficient because the liquid must be collected and separated from the spheroids.

[0010] Accordingly, there is a continuing need for 3D matrices that can perform 3D cell culture to produce spheroids and / or cellular by-products (e.g., extracellular vesicles) on a large scale. More preferably, nutrients can be adequately supplied to the cultured cells, and ultimately the cultured cells and / or cellular by-products can be collected or recovered. The present disclosure provides 3D porous scaffolds for culturing spheroids and cellular by-products produced by spheroids, which solve the above problems encountered in 2D culture. SUMMARY OF THE INVENTION

[0011] In accordance with aspects of the present disclosure, a method for cell spheroid production is provided herein. The method includes providing a bioreactor having a cavity for culturing cells, inserting cells into the cavity to form spheroids, and perfusing a cell culture medium through the cavity to culture the spheroids. Aspects of the embodiments include a bioreactor that includes a porous scaffold in the cavity. The porous scaffold is made of an interconnected scaffold material that defines pores. In certain aspects, the porous scaffold is a foam scaffold, which is soluble in some embodiments and insoluble in other embodiments. The method may further include inoculating the pores of the porous scaffold with cells by inserting the cells into the cavity. In some embodiments, the cells aggregate in the pores of the foam scaffold to form spheroids. The pores may be sized to confine at least some of the spheroids within the pores. As an aspect of the embodiments, the cells do not adhere to the porous scaffold. In the embodiments, the porous scaffold does not have a cell adhesion coating. The method may further include collecting at least one spheroid or secreted material from the spheroids, where the secreted material may include extracellular vesicles, proteins, or other components of the cell secretome. The method may include digesting any soluble foam scaffold by exposing the soluble foam scaffold to an enzyme and / or a chelating agent. In some embodiments, perfusing the cell culture medium includes continuously passing the cell culture medium over the porous scaffold.

[0012] According to aspects of the present disclosure, provided herein are soluble foam scaffolds for cell culture. In some embodiments, the soluble foam scaffold comprises: an ionically crosslinked polygalacturonic acid compound selected from at least one of the following: pectic acid, partially esterified pectic acid, partially amidated pectic acid, and salts thereof; and a water-soluble polymer having surface activity and a hydrophilic-lipophilic balance value (HLB) greater than about 10, and preferably greater than about 20. Alternatively, the soluble foam scaffold may alternatively comprise alginic acid as the ionically crosslinked polymer. In some embodiments, the HLB of the water-soluble polymer is greater than or equal to about 22. In some embodiments, the soluble foam scaffold does not contain glycerol, sorbitol, ethylene glycol, propylene glycol, polyethylene glycol. In some embodiments, the soluble foam scaffold comprises glycerol, or one of sorbitol, ethylene glycol, propylene glycol, and polyethylene glycol. According to aspects of the embodiments, the soluble foam scaffold comprises only a single water-soluble polymer having surface activity and may not comprise any polymer without surface activity. In some aspects of the present disclosure, provided herein are insoluble foam scaffolds for cell culture. Cells can form spheroids in the soluble foam scaffold or the insoluble foam scaffold.

[0013] According to embodiments of the present disclosure, provided herein is a method for forming a soluble foam scaffold. The method includes forming a first aqueous mixture by adding alginic acid or a polygalacturonic acid compound selected from at least one of the following to an aqueous solution: pectic acid, partially esterified pectic acid, partially amidated pectic acid, and salts thereof; 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 gelling inducer to the combined aqueous mixture to form a foaming solution; and introducing gas bubbles into the foaming solution to form a foam scaffold. The hydrophilic-lipophilic balance value (HLB) of the water-soluble polymer is greater than about 10, or greater than about 20. In an embodiment, the HLB of the water-soluble polymer is greater than or equal to about 22. In aspects of the embodiment, forming the second aqueous mixture includes adding a water-soluble plasticizer to the second aqueous mixture and may not include adding a second polymer without surface activity. According to additional 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 the embodiment, the method includes adding a first amount of the gelling inducer to the aqueous mixture before foaming, and adding a second amount of the gelling inducer to the aqueous mixture during or after foaming. The ratio of the second amount of the gelling inducer to the first amount of the gelling inducer is greater than about 2, or about 7 or greater.

[0014] According to embodiments of the present disclosure, provided herein is a method for culturing cells in a soluble foam scaffold. The method includes: inoculating cells in the soluble foam scaffold such that the cells enter the pores of the soluble foam scaffold, the soluble scaffold comprising: ionically crosslinked alginic acid and its salts, or an ionically crosslinked polygalacturonic acid compound selected from at least one of the following: pectic acid, partially esterified pectic acid, partially amidated pectic acid, and their salts; and a water-soluble polymer having surface activity and a hydrophilic-lipophilic balance value (HLB) greater than about 20; and contacting the soluble foam scaffold with a cell culture medium. According to various aspects of the embodiments, the soluble foam scaffold has 0 wt% water-soluble plasticizer and does not include a non-surface-active polymer. According to other aspects of the embodiments, the soluble foam scaffold may contain a plasticizer, including a water-soluble plasticizer.

[0015] According to an aspect of the present disclosure, provided is a method for producing spheroids or by-products of spheroids, which includes: (a) providing a bioreactor comprising a cavity for culturing cells, an inlet and an outlet of the cavity, and a porous scaffold, (b) inserting cells of a certain cell type into the porous scaffold to form spheroids, and (c) perfusing a cell culture medium through the cavity to culture the spheroids. The porous scaffold comprises pores and an interconnected structure (channels) between the pores. In some embodiments, the step of inserting cells of a certain cell type into the porous scaffold comprises inoculating the cells into the porous scaffold. The cells aggregate in large numbers in the pores of the porous scaffold to form spheroids. In some embodiments, at least 80% of the cells in the pores of the porous scaffold aggregate to form spheroids. In some embodiments, the channels between the pores are sized to confine at least some of the spheroids within the porous scaffold. In some embodiments, the method comprises the step of collecting at least one spheroid or at least one by-product of the spheroids. In some embodiments, the by-product of the collected spheroids is extracellular vesicles. In some embodiments, the step of perfusing the cell culture medium comprises continuously passing the cell culture medium over the porous scaffold.

[0016] In some embodiments, the porous scaffold is made of a material that is non-adhesive to cells, or the porous scaffold material is treated to be non-adhesive to cells, or both of the above are satisfied. In some embodiments, when perfused at a linear velocity (Darcy velocity) of about 1.0×10 -5 m / s to about 5.0×10 -4 m / s, the porous scaffold is non-adhesive to cells. In some embodiments, the porous scaffold is non-adhesive to at least 80% of the cells in the bioreactor.

[0017] The porous scaffold comprises: pores, an interconnection structure (channels) between the pores that permits fluid passage between the pores, and the number of interconnection structures per pore. In some embodiments, when measured in the dry scaffold, at least 70% of the pores have a pore diameter of from about 200 μm to about 1000 μm. In some embodiments, when measured in the dry scaffold, at least 80% of the pores have a pore diameter of from about 400 μm to about 800 μm. In some embodiments, when measured in the dry scaffold, at least 70% of the channels have a maximum channel width of from about 30 μm to about 500 μm. In some embodiments, when measured in the dry scaffold, at least 80% of the channels have a maximum channel width of from about 60 μm to about 400 μm. In some embodiments, at least 70% of the pores have 5 to 18 channels / pore. In some embodiments, at least 80% of the pores have from about 6 to about 14 channels / pore.

[0018] In some embodiments, the porous scaffold comprises ionically crosslinked alginic acid or a salt thereof, or an ionically crosslinked polygalacturonic acid compound selected from at least one of the following: pectic acid, partially esterified pectic acid, partially amidated pectic acid, and salts thereof. In some embodiments, the porous scaffold is soluble. In some embodiments having a soluble scaffold, the method further comprises the step of digesting the soluble scaffold by exposing the soluble scaffold to an enzyme. In some embodiments, this exposure step can include exposing the scaffold to an enzyme between about 1 U and about 200 U. In some embodiments, the enzyme used to dissolve the scaffold comprises a non-proteolytic enzyme. In some embodiments, the non-proteolytic enzyme is selected from the group consisting of pectin hydrolase, pectinase, and alginate lyase. In some embodiments having a soluble scaffold, the method further comprises the step of exposing the soluble porous scaffold to a chelating agent. In some embodiments, this exposure step can include exposing the scaffold to a chelating agent between about 1 mM and about 200 mM. In some embodiments, the digestion of the soluble porous scaffold is completed in less than about 1 hour.

[0019] According to aspects of the present disclosure, a bioreactor is provided that includes a cavity for culturing cells, an inlet and an outlet of the cavity, and a porous scaffold in the cavity. The porous scaffold includes pores and channels between the pores, is non-adhesive to cells, and is configured to accommodate spheroids. In some embodiments, at least 75% of the pores have a pore diameter of from about 200 μm to about 1000 μm when measured in the dry scaffold. In some embodiments, at least 75% of the channels have a maximum channel width of from about 30 μm to about 500 μm when measured in the dry scaffold. In some embodiments, at least 75% of the pores have 5 to 18 channels per pore. In some embodiments, the porous scaffold includes an ionically crosslinked polysaccharide selected from alginic acid and its salts, pectic acid and its salts, partially esterified pectic acid and its salts, partially amidated pectic acid and its salts, or combinations thereof. In a particular embodiment, the ionically crosslinked polysaccharide is polygalacturonic acid. In some embodiments, the porous scaffold of the bioreactor is soluble. 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.

[0020] According to aspects of the present disclosure, a porous scaffold for culturing spheroids or by-products from spheroids is provided that includes pores, channels between the pores, and a number of channels per pore. This porous scaffold has: at least 75% of the pores with a pore diameter of from about 200 μm to about 1000 μm when measured in the dry scaffold, at least 75% of the channels with a maximum channel width of from about 30 μm to about 500 μm when measured in the dry scaffold, and at least 75% of the pores having a number of channels per pore of 5 to 18. This porous scaffold is also non-adhesive to cells and is configured to grow spheroids from cells of a certain cell type in the pores. In some embodiments of this porous scaffold, the cells of a certain cell type are selected from primary cell lines and immortalized cell lines. In some embodiments, the primary cell line is human mesenchymal stem cells derived from bone marrow and the immortalized cell line is HEK293T cells.

[0021] According to aspects of the present disclosure, a perfusion bioreactor is provided that includes a cavity for culturing cells, an inlet and an outlet of the cavity, and a porous scaffold within the cavity. The porous scaffold of this perfusion bioreactor is non-adhesive to cells and includes pores and channels between the pores. The porous scaffold further has: pores with at least 75% of the pore diameters being from about 200 μm to about 1000 μm when measured in the dry scaffold, channels with at least 75% of the maximum widths being from about 30 μm to about 500 μm when measured in the dry scaffold, at least 75% of the pores having 5 to 18 channels per pore, and the porous scaffold is suitable for growing spheroids or by-products of spheroids. 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 suitable for producing extracellular vesicles as by-products of spheroids. In some embodiments, the extracellular vesicles produced by the perfusion bioreactor are produced in an amount between about 1×10 3 EVs / cell and about 1×10 7 EVs / cell. In some embodiments, these extracellular vesicles are produced by perfusion with a linear velocity (Darcy velocity) between about 1.0×10 - 5 m / s and about 5.0×10 -4 m / s. In some embodiments, increasing the perfusion flow rate increases the number of extracellular vesicles produced per cell. In some embodiments, the number of extracellular vesicles produced per cell with the perfusion bioreactor is greater than the number of extracellular vesicles produced with a static 2D microplate.

[0022] In some embodiments, as measured by the wound healing assay of HT-1080 cells, at 6 hours post-injury, the wound healing by the extracellular vesicles produced by the perfusion bioreactor is greater than the wound healing by the extracellular vesicles produced in a 2D flask for 3D cultures. In some embodiments, as measured by the wound healing assay of HT-1080 cells, at 6 hours post-injury, compared to the wound healing by 2×10 9 extracellular vesicles produced in a 2D flask for 3D cultures, the wound healing by about 2×10 9 extracellular vesicles produced by the perfusion bioreactor has at least 5% better wound closure.

[0023] According to aspects of the present disclosure, a foamed scaffold product is provided herein. The foamed scaffold product is formed from a composition comprising: ionically crosslinked alginic acid and its salts, or an ionically crosslinked polygalacturonic acid compound selected from at least one of the following: pectic acid, partially esterified pectic acid, partially amidated pectic acid, and their salts; and at least one first water-soluble polymer having surface activity and a hydrophilic-lipophilic balance value (HLB) greater than about 20; and 0 wt% water-soluble plasticizer. In other embodiments, the water-soluble plasticizer may be present at a value greater than 0 wt%.

[0024] Additional features and advantages will be set forth in the detailed description which follows, and in part will be obvious from the description, or may be learned by practice of the embodiments described herein, which embodiments include the detailed description which follows, the claims, as well as the drawings.

[0025] It is to be understood that both the foregoing general description and the following detailed description are exemplary and intended to provide an overview or framework for understanding the nature and character of the claims. 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 operations of the various embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] The present disclosure will be more clearly understood from the following description and drawings, which are provided only as non-limiting examples, wherein:

[0027] Figure 1 is a perspective view of a soluble foam scaffold according to the present disclosure;

[0028] Figure 2 shows an SEM image of the foam scaffold prepared in Example 1;

[0029] Figure 3 shows a phase contrast image of spheroids formed by Vero cells in the pores of the foam scaffold prepared in Example 1 according to the embodiment;

[0030] Figure 4 shows an SEM image of the foam scaffold prepared in Example 3 according to the embodiment;

[0031] Figure 5A is an illustration of a foam scaffold for spheroid production in a culture plate according to the embodiment;

[0032] Figure 5B is an illustration of a foam scaffold for spheroid or cell product production in a perfusion bioreactor according to the embodiment;

[0033] Figure 6A Photograph of an uncoated foam scaffold made from composition XP49, according to an embodiment;

[0034] Figure 6B Photograph of an uncoated foam scaffold made from composition XP64, according to an embodiment;

[0035] Figure 6C Photograph of an uncoated foam scaffold made from composition XP76, according to an embodiment;

[0036] Figure 6D Photograph of an uncoated foam scaffold made from composition FMXP005, according to an embodiment;

[0037] Figure 6E Graph showing the measured scaffold pore sizes in different scaffolds, according to an embodiment;

[0038] Figure 6F Graph showing the measured scaffold interconnect structure sizes in different scaffolds, according to an embodiment;

[0039] Figure 6G Graph showing the number of interconnect structures per pore in different scaffolds, according to an embodiment;

[0040] Figure 7A Fluorescence microscopy images of HEK293 spheroids 1 day and 5 days after inoculating scaffolds with HEK293 cells. The left column depicts spheroids in a foam scaffold using composition XP64. The right column depicts spheroids in a foam scaffold using composition XP76.

[0041] Figure 7B Fluorescence microscopy images of hMSC spheroids 1 day and 7 days after inoculating scaffolds with hMSC cells. The left column depicts spheroids in a foam scaffold using composition XP64. The right column depicts spheroids in a foam scaffold using composition XP76.

[0042] Figure 8 Bar graph showing the increase in HEK293T 1 day and 5 days after inoculating cells in Figure 7A and the increase in hMSC cells 1 day and 7 days after inoculating cells in Figure 7B , according to an embodiment;

[0043] Figure 9A Schematic of cell seeding data in a perfusion bioreactor, according to an embodiment;

[0044] Figure 9B Bar graph showing the percentage of recovered cells from scaffolds 1 day after inoculation, according to an embodiment;

[0045] Figure 10 Showing fluorescence microscopy images from two perfusion bioreactors one day after inoculating HEK293T cells into the foam scaffolds, according to an embodiment;

[0046] Figure 11 Showing fluorescence microscopy images of different foam scaffold compositions and spheroids after 48 hours of perfusion at perfusion flow rates of 1 mL / min and 10 mL / min, according to an embodiment;

[0047] Figure 12 To show, according to an embodiment, the bar graph of the cell count of cells recovered from the Figure 11 scaffolds at 48 hours after the start of perfusion;

[0048] Figure 13A Fluorescence microscopy image of hMSC spheroids in the foam scaffold after 48 hours of perfusion at 10 mL / min, according to an embodiment;

[0049] Figure 13B Fluorescence microscopy image of hMSC spheroids in the foam scaffold after 48 hours of perfusion at 2 mL / min, according to an embodiment;

[0050] Figure 13C Bright-field phase-contrast microscopy image of 2D adherent hMSCs on a 2D static surface after 48 hours, according to an embodiment;

[0051] Figure 14A Graph showing the size distribution of EV / particles produced by hMSCs in a 3D foam scaffold using a perfusion flow rate of 10 mL / min, based on MADLS analysis, according to an embodiment;

[0052] Figure 14B Graph showing the size distribution of EV / particles produced by hMSCs in a 3D foam scaffold using a perfusion flow rate of 2 mL / min, based on MADLS analysis, according to an embodiment;

[0053] Figure 14C Graph showing the size distribution of EV / particles produced by hMSCs in a 2D static environment, based on MADLS analysis, according to an embodiment;

[0054] Figure 15A Graph showing the concentration of EV / particles produced by hMSCs in a 3D foam scaffold using a perfusion flow rate of 10 mL / min, based on MADLS analysis, according to an embodiment;

[0055] Figure 15BGraph of the concentration of EVs / particles produced by hMSCs in a 3D foam scaffold with a perfusion flow rate of 2 mL / min, based on MADLS analysis, according to an embodiment;

[0056] Figure 15C Graph of the concentration of EVs / particles produced by hMSCs in a 2D static environment, based on MADLS analysis, according to an embodiment;

[0057] Figure 16 Bar graph of the number of EVs per million cells from each of three culture conditions, based on MADLS analysis, according to an embodiment; Figures 15A to 15C from each of three culture conditions, according to an embodiment;

[0058] Figure 17 Bar graph of the number of EVs per million cells from each of three culture conditions, based on ELISA analysis for CD63-positive EVs, according to an embodiment;

[0059] Figure 18 Image of Western blot analysis for CD81 and TSG101 of EV isolation samples generated from 3D foam culture with 10 ml / min perfusion and static 2D culture, according to an embodiment;

[0060] Figure 19 Showing bright-field phase-contrast microscopic images of the HT1080 wound healing assay at 0 and 8 hours after creating a wound and treating the cells with 2×10 9 CD63+ EVs or no treatment, according to an embodiment;

[0061] Figure 20 Graph of EVs produced by hMSCs under different conditions analyzed using an in vitro wound healing assay, according to an embodiment;

[0062] Figure 21A Showing fluorescence microscopic images of hMSC spheroids observed after 48 hours of exposure to EV collection medium by perfusion at 10 mL / min. The spheroids were stained with calcein AM before taking the pictures, according to an embodiment;

[0063] Figure 21B Showing bright-field phase-contrast microscopic images of hMSC spheroids in a microchamber vessel observed after 48 hours of exposure to EV collection medium under stirring at 35 rpm, according to an embodiment;

[0064] Figure 21C Showing bright-field phase-contrast microscopic images of 2D adherent hMSCs in a T-75 flask observed after 48 hours of exposure to EV collection medium under static conditions, according to an embodiment;

[0065] Figure 22A For an example, the size distribution pattern of EVs produced by hMSC spheroids according to the MADLS analysis after exposure to EV collection medium by perfusion at 10 mL / min for 48 hours;

[0066] Figure 22B For an example, the size distribution pattern of EVs produced by hMSC spheroids according to the MADLS analysis after exposure to EV collection medium under stirring at 35 rpm for 48 hours;

[0067] Figure 22C For an example, the size distribution pattern of EVs produced by 2D adherent hMSCs according to the MADLS analysis after exposure to EV collection medium under static conditions for 48 hours;

[0068] Figure 22D A bar graph of the amount of particles and EVs produced after exposure to EV collection medium for 48 hours according to an example;

[0069] Figure 23A A bar graph of the number of EVs produced by hMSC spheroids and 2D adherent hMSCs under different conditions after exposure to EV collection medium for 48 hours according to an example, as analyzed by ELISA for CD63;

[0070] Figure 23B Shows Western blot analysis of EVs produced by hMSC spheroids and 2D adherent hMSCs under different conditions after exposure to EV collection medium for 48 hours according to an example, using CD81 and TSG101 markers;

[0071] Figure 24 For an example, a graph showing the results of EV function analysis using the HT-1080 cell wound healing assay with control and EVs produced by hMSCs under different conditions after exposure to EV collection medium for 48 hours;

[0072] Figure 25 Shows fluorescence micrographs of hMSCs and 2D adherent hMSCs after exposure to EV collection medium by perfusion at 20, 10, 2, and 0.5 mL / min for 48 hours according to an example. The spheroids were stained with calcein AM before taking the pictures;

[0073] Figure 26A Shows a graph of the average diameter of EVs obtained in clarified EV conditioned perfusion media under the indicated conditions using MADLS analysis. The EVs were produced by hMSCs from a bioreactor;

[0074] Figure 26B Figure showing total EVs obtained in clarified EV-conditioned perfusion medium under the indicated conditions using MADLS analysis, according to an embodiment. EVs are produced by hMSCs from a bioreactor;

[0075] Figure 27A Figure showing the size distribution pattern of EVs obtained from a purified EV sample under the indicated conditions using MADLS, according to an embodiment. EVs are produced by hMSCs from a bioreactor;

[0076] Figure 27B Bar graph showing total EVs obtained from a purified EV sample under the indicated conditions using MADLS, according to an embodiment. EVs are produced by hMSCs from a bioreactor;

[0077] Figure 27C Bar graph showing EVs produced per million hMSC cells calculated from the total EVs in a purified EV sample and the total number of cells in the bioreactor, according to an embodiment;

[0078] Figure 28A Bar graph showing the total number of EVs obtained from a purified EV sample under the indicated conditions using ELISA CD63 analysis, according to an embodiment;

[0079] Figure 28B Bar graph showing EVs produced per million hMSC cells calculated from the total EVs in a purified EV sample and the total number of cells in the bioreactor, according to an embodiment;

[0080] Figure 29 Bar graph showing the wound healing % level of EVs obtained from a purified EV-conditioned perfusion medium under the indicated conditions using the HT-1080 wound healing assay, as observed 6 hours after wounding. DETAILED DESCRIPTION

[0081] Reference will now be made in detail to one or more embodiments of the invention, one or more examples 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.

[0082] Unless the context clearly indicates otherwise, the singular forms "a", "an" and "the" include plural referents. All ranges of values of the same feature may be combined independently and include the recited end values. All references are incorporated herein by reference.

[0083] As used herein, when used in a list of two or more items, the term "and / or" means that any one of the listed items can be taken alone, or any combination of two or more of the listed items can be taken. For example, if a composition is described as containing components A, B, and / or C, the composition can contain only A; only B; only C; a combination of A and B; a combination of A and C; a combination of B and C; or a combination of A, B, and C.

[0084] Unless otherwise indicated, the use of individual numerical values is stated as an approximation, as if the value were preceded by the word "about" or "approximately". Similarly, unless otherwise explicitly stated, the numerical values within various ranges specified in this application are stated as approximations, as if the words "about" or "approximately" preceded both the minimum and maximum values within the stated range. In this way, variations above and below the stated range can be used to achieve substantially the same results as the values within the range. As used herein, when referring to numerical values, the terms "about" and "approximately" should have their ordinary and general meaning to a person of ordinary skill in the art in the field most closely related to the disclosed subject matter or in the field related to the range or element being discussed. The amount of broadening from the strict numerical boundaries depends on many factors. For example, some factors that can be considered include the criticality of the element and / or the effect of a given amount of variation on the performance of the claimed subject matter, as well as other considerations known to those skilled in the art. As used herein, using different numbers of significant figures for different numerical values does not mean a limitation on the way the terms "about" or "approximately" are used to broaden a particular numerical value or range. Thus, as a general rule, "about" or "approximately" broadens a numerical value. In addition, the disclosure of a range is intended as a continuous range, including every value between the minimum and maximum values plus the broadening of the range provided by using the terms "about" or "approximately". Thus, the recitation of a range of values herein is merely intended as a shorthand method for individually referring to each separate value falling within the range, and each separate value is incorporated into this specification as if it were individually recited herein.

[0085] As used herein, "have", "having", "include", "including", "comprise", "comprising", etc. are used in their open-ended sense and generally mean "including (but not limited to)".

[0086] "Optional" or "optionally" means that the subsequently described element, component, or circumstance may or may not occur, such that the description includes the case where the element, component, or circumstance occurs and the case where it does not occur.

[0087] As used herein, "shear stress" or "wall shear stress" are interchangeable and refer to the tangential force per unit area exerted on spheroids cultured within the pores of a 3D scaffold by a fluid flowing through those pores.

[0088] Unless otherwise noted, all scientific and technical terms used herein have the meanings commonly used in the art. The definitions provided herein are intended to facilitate understanding of certain terms frequently used herein and are not intended to limit the scope of the present disclosure.

[0089] The present disclosure will first be described generally below and then in detail based on several exemplary embodiments. Features shown in combination with one another in individual exemplary embodiments do not all have to be implemented. Rather, individual features may also be omitted or combined in some other way with other features shown in the same exemplary embodiment or other exemplary embodiments.

[0090] Embodiments of the present disclosure relate to methods for generating large quantities of spheroids from isolated cells and producing high concentrations of extracellular vesicles and other cellular by-products secreted by these spheroids. Embodiments herein provide for the first time methods for generating spheroids by perfusion of a cell suspension inside a bioreactor having a scaffold, as well as methods for generating EVs and other secreted by-products from cells within spheroids under perfusion inside the bioreactor. Embodiments include forming spheroids in a soluble foam scaffold, after which the spheroids can be recovered from the scaffold and used for other applications or directly inside the scaffold for a specific application, such as EV production. In other embodiments, an insoluble scaffold enables the formation of spheroids within the insoluble scaffold, after which the spheroids can be directly used inside the scaffold for a specific application, such as EV production. According to embodiments, the soluble and insoluble foam scaffolds of the present disclosure for growing and culturing spheroids can be used without an external cell adhesion coating on the scaffold, thereby preventing cell adhesion to the scaffold. After inoculating the cells in the scaffold, the cells will 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 the case of soluble scaffolds, the spheroids can be recovered after formation and used for other applications, including, for example, toxicity analysis or other applications that would be understood by one of ordinary skill in the art.

[0091] Existing methods for generating spheroids typically involve growing spheroids in plates having a plurality of cavities that have been treated to prevent cell adhesion. The limited number of cavities on these plates results in a limited number of spheroids. Additionally, the large footprint of these plates results in inefficient use of space compared to the 3D scaffold structures disclosed herein. The high density of pores inside the scaffold results in a much higher spheroid density than any current method for generating spheroids. Other methods for generating spheroids do not allow for easy recovery of these spheroids, especially in large quantities.

[0092] Cells cultured in spheroids can produce useful by-products (secretome) in the extracellular space surrounding the cells. The secretome is a collection of proteins and other biological components that are expressed by the cells and secreted into the extracellular space. One component of the secretome is EV. According to embodiments of the present disclosure, methods are provided that allow for the large-scale production of functional EVs by generating spheroids within a porous material and exposing these spheroids to perfusion of a culture medium in a bioreactor.

[0093] Advantages of embodiments of the present disclosure include large-scale spheroid formation, and the spheroids can be recovered if a soluble scaffold is used. For spheroids whose growth depends in part on the pore size, controlling the spheroid size by the engineered pore size of the scaffold material can help provide a uniform population of spheroids, which is important for some downstream applications. Additionally, aspects of the embodiments herein provide the advantage of large-scale production of EVs and production of other cell secretomes or particles (e.g., exosomes) from spheroids under perfusion in the scaffold. The amount of EVs, particles, and / or secretome produced by the spheroids can also be controlled by the dependence of these amounts on the perfusion flow rate. Furthermore, embodiments of the present disclosure are scalable such that it is possible to produce spheroids, EVs, and other cell by-products at various scales, including large-scale production. For example, by increasing the diameter of the scaffold, spheroid and EV / secretome production increases.

[0094] Embodiments of the present disclosure relate to soluble and insoluble foam scaffolds for cell culture and methods of making soluble foam scaffolds. Embodiments of the present disclosure further relate to methods of culturing adherent cells, cell aggregates, or spheroids in soluble and insoluble foam scaffolds. Additionally, embodiments of the present disclosure relate to bioreactors and bioreactor systems comprising soluble and insoluble foam scaffolds. As will become clearer in the discussion below, in some embodiments, the foam scaffolds disclosed herein are described as soluble and insoluble. As used herein, the term "insoluble" is used to refer to a material or combination of materials that is insoluble and remains crosslinked under conventional cell culture conditions, including, for example, cell culture media. Additionally, as used herein, the term "soluble" is used to refer to a material or combination of materials that is digested when exposed to an enzyme that digests or degrades the material or combination of materials at an appropriate concentration. The soluble and insoluble foam scaffolds described herein are porous scaffolds having an open pore architecture and highly interconnected pores. The pores of the scaffold provide a protected environment for cell culture, where cell-cell interactions and the formation of the extracellular matrix are assisted in a 3D manner. The soluble foam scaffold can be completely digested, which allows for the collection of cells without damaging the cells using protease treatment and / or mechanical collection techniques.

[0095] Figure 1FIG. 0 is a perspective view of a soluble foam (porous) scaffold 10 according to the present disclosure. As will be described in further detail below and as will become more apparent from other figures of the present disclosure, the soluble foam scaffold 10 is a porous foam including an open pore architecture. The soluble foam scaffold 10 has a porosity of about 85% to about 96% and an average pore diameter between about 50 μm and about 500 μm. The soluble foam scaffold 10 provides a protected environment for culturing spheroids within the pores of the foam scaffold. Additionally, the soluble foam scaffold 10 is also soluble when exposed to an enzyme of an appropriate digestive or degrading material, which facilitates the collection of spheroids cultured in the scaffold without damaging the cells.

[0096] The soluble foam scaffolds as described herein include at least one ionically crosslinked polysaccharide and can be used for culturing three-dimensional cell cultures (e.g., spheroids or organoids) and by-products of these cultures (e.g., extracellular vesicles and exosomes). Generally, polysaccharides have properties beneficial for cell culture applications. Polysaccharides are hydrophilic, non-cytotoxic, and stable in culture media. Examples include pectic acid, also known as polygalacturonic acid (PGA) or its salts, partially esterified pectic acid or its salts, or partially amidated pectic acid or its salts. Another example of an ionically crosslinked polysaccharide that can be used in the scaffolds of the present disclosure includes alginic acid or its salts. Pectic acid can be formed by hydrolysis of certain pectin esters. Pectin is a cell wall polysaccharide and has a structural role in plants in nature. The main sources of pectin include citrus peels (e.g., peels from lemons and limes) and apple peels. Pectin is a linear polymer mainly based on a 1,4-linked α-D-galacturonate backbone and randomly interspersed with 1,2-linked L-rhamnose. The average molecular weight ranges from about 50,000 to about 200,000 daltons.

[0097] The polygalacturonic acid chains of pectin can be partially esterified, e.g., with methyl groups, and the free acid groups can be partially or fully neutralized with monovalent ions such as sodium, potassium, or ammonium ions. Polygalacturonic acid partially esterified with methanol is called pectic acid, and its salts are called pectates. The degree of methylation (DM) of high-methoxyl (HM) pectin can be, for example, 60 to 75 mol%, and the degree of methylation of low-methoxyl (LM) pectin can be 1 to 40 mol%. The degree of esterification of the partially esterified polygalacturonic acid as 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 wishing to be bound by any particular theory, it is believed that a very small amount of free carboxylic acid groups (unesterified) contributes to a certain degree of ionic crosslinking, thereby allowing the formation of an insoluble soluble scaffold.

[0098] Alternatively, the polygalacturonic acid chains of pectin can be partially amidated. Partially amidated pectin of polygalacturonic acid can be produced, for example, by treatment with ammonia. Amidated pectin contains carboxyl groups (~COOH), methyl ester groups (~COOCH3), and amidated groups (-CONH2). The degree of amidation can vary and can be, for example, from about 10% to about 40% amidation.

[0099] According to embodiments of the present disclosure, the soluble foam scaffolds as described herein can include a mixture of pectic acid and partially esterified pectic acid. Blends with compatible polymers can also be used. For example, pectic acid and / or partially esterified pectic acid can be mixed with other polysaccharides such as dextran, substituted cellulose derivatives, alginic acid, starch, glycogen, arabinoxylan, agarose, etc. Glycosaminoglycans such as hyaluronic acid and chondroitin sulfate, or various proteins such as elastin, fibrin, fibroin, collagen, and their derivatives can also be used. Water-soluble synthetic polymers can also be blended with pectic acid and / or partially esterified pectic acid. Exemplary water-soluble synthetic polymers include (but are not limited to) polyalkylene glycols, poly((meth)acrylic acid hydroxyalkyl esters), poly((meth)acrylamides) and derivatives, poly(N-vinyl-2-pyrrolidone), and polyvinyl alcohol.

[0100] According to embodiments of the present disclosure, the soluble foam scaffold as described herein may further comprise at least one first polymer. The at least one first polymer is water-soluble, non-ion crosslinkable and has surface activity. As used herein, the term "surface activity" refers to the activity of a reagent to reduce or eliminate the surface tension (or interfacial tension) between two liquids, or 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 value (HLB) greater than about 8 or even greater than about 10. For example, the HLB of the at least one first polymer may be between about 8 and about 40 or between about 10 and about 40. The HLB of the at least one first polymer may be between about 8 and about 15, or even between about 10 and about 12. The HLB provides a reference for the degree of lipophilicity or hydrophilicity of the polymer. A larger HLB value indicates stronger hydrophilicity, while a smaller HLB value indicates stronger lipophilicity. Generally, the HLB value varies within the range of 1 to 40, and the hydrophilic-lipophilic transition is generally considered to be between about 8 and about 10. When the HLB value is less than the hydrophilic-lipophilic transition, the material is lipophilic, and when 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 between 10 and 40, between 15 and 40, between 20 and 40, or between 20 and 30.

[0101] Exemplary first polymers according to embodiments of the present disclosure may be any one 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) poloxamer available under the trade name (commercially available from Croda International, Snaith, United Kingdom), poloxamer available under the trade name (commercially available from BASF Corp., Parsippany, NJ), and poloxamer available under the trade name (commercially available from BASF Corp., Parsippany, NJ).

[0102] The soluble foam scaffolds as described herein may further comprise at least one second polymer. The at least one second polymer is water-soluble and non-surface active. Exemplary second polymers can be any of synthetic polymers, semi-synthetic polymers, natural polymers, and combinations thereof. Exemplary synthetic polymers include, but are not limited to, polyethylene glycol, polyvinylpyrrolidone, polyvinyl alcohol, carboxyvinyl polymer, polyacrylic acid, polyacrylamide, homopolymers and copolymers of N-(2-hydroxypropyl)methacrylamide, polyethylene methyl ether-maleic anhydride, and polyethylene oxide / polypropylene oxide block copolymers. Exemplary semi-synthetic polymers include, but are not limited to, dextran derivatives, carboxymethyl cellulose, hydroxyethyl cellulose and derivatives, methyl cellulose and derivatives, ethyl cellulose, ethyl hydroxyethyl cellulose, and hydroxypropyl cellulose. 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, alginate, and carrageenan), seed extracts (such as guar gum and derivatives and locust bean gum), hyaluronic acid, and chondroitin sulfate.

[0103] The soluble foam scaffolds as described herein can be crosslinked to increase their mechanical strength and prevent dissolution of the scaffolds upon contact with cell culture media. Crosslinking can be carried out by ion-promoted gelation as described below, wherein ion-promoted gelation is based on the ability of polyelectrolytes to crosslink in the presence of multivalent counterions to form crosslinked scaffolds. Without wishing to be bound by any particular theory, it is believed that the ion-promoted gelation of the polysaccharides of the soluble foam scaffolds is the result of strong interactions between divalent cations and the polysaccharides.

[0104] It should be understood that only in some embodiments, the scaffolds for forming and generating the spheroids and cell by-products of the present disclosure are soluble scaffolds. Insoluble 3D scaffolds as also described herein having pore sizes, interconnected structure (channel) sizes, and number of interconnected structures per pore similar to any soluble 3D scaffold can be used to form spheroids and cell by-products. The insoluble scaffolds can be made of, 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 insoluble scaffold material is cell-adhesive, the insoluble scaffold material is coated with a non-adhesive material, or made non-adhesive by chemical modification or other known methods that render the scaffold material non-adhesive.

[0105] According to embodiments of the present disclosure, the soluble and insoluble scaffolds described herein are porous foam scaffolds. The foam scaffolds described herein can have a porosity of from about 85% to about 96%. For example, the foam scaffolds described herein can have a porosity of from about 91% to about 95% or from about 94% to about 96%. As used herein, the term "porosity" refers to a measure of the open pore volume in the soluble scaffold and is expressed as porosity %, where porosity % is the percentage of voids in the total volume of the soluble foam scaffold.

[0106] When measured in the dry scaffold, the average pore diameter of the foam scaffolds described herein can be between about 200 μm and about 1000 μm. Drying of the foam (porous) scaffold can be carried out by any method used by a person of ordinary skill in the art, but is preferably carried out by freeze-drying. The diameter of the pore is the widest distance across the pore and is measured by scanning electron microscopy ("SEM"). In some embodiments, the average pore diameter in the dry scaffold can be between about 250 μm and about 650 μm, or between about 300 μm and about 600 μm, or even between about 350 μm and about 500 μm, and all values therebetween. In some embodiments, when measured in the dry scaffold, the average pore diameter can be between about 450 μm and about 850 μm, between about 500 μm and about 700 μm, or between about 550 μm and about 600 μm. In some embodiments, when measured in the dry scaffold, the average pore diameter can be between about 300 μm and about 650 μm, between about 350 μm and about 550 μm, or between about 400 μm and about 525 μm. In some embodiments, when measured in the dry scaffold, the average pore diameter can be between about 350 μm and about 1000 μm, between about 450 μm and about 850 μm, or between about 550 μm and about 650 μm. The average pore diameter is the average of the pore diameters of the pores in the scaffold.

[0107] In some embodiments, when measured in the dry scaffold, at least 50% of the pores have a pore diameter of from about 200 μm to about 1000 μm. In another embodiment, when measured in the dry scaffold, 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 diameter of from about 200 μm to about 1000 μm. In a particular embodiment, when measured in the dry scaffold, between 65% and 85% of the pores have a pore diameter of from about 200 μm to about 1000 μm. In another particular embodiment, when measured in the dry scaffold, at least 70% of the pores have a pore diameter of from about 200 μm to about 1000 μm. In some embodiments, when measured in the dry scaffold, at least 50% of the pores have a pore diameter of from about 400 μm to about 800 μm. In another embodiment, when measured in the dry scaffold, 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 diameter of from about 400 μm to about 800 μm. In a particular embodiment, when measured in the dry scaffold, between 70% and 90% of the pores have a pore diameter of from about 400 μm to about 800 μm. In another particular embodiment, when measured in the dry scaffold, at least 80% of the pores have a pore diameter of from about 400 μm to about 800 μm.

[0108] The scaffolds described herein may have a wet density of less than about 0.40 g / cc. By way of 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 between about 0.16 g / cc and about 0.40 g / cc, or between about 0.16 g / cc and about 0.35 g / cc, or between about 0.16 g / cc and about 0.30 g / cc, or even between about 0.16 g / cc and 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. By way of 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 between about 0.02 g / cc and about 0.20 g / cc, or between about 0.02 g / cc and about 0.15 g / cc, or between about 0.02 g / cc and about 0.10 g / cc, or even between about 0.02 g / cc and about 0.05 g / cc, and all values therebetween.

[0109] There may be several types of pores in a foam (porous) scaffold. Open pores allow cells to enter both sides of the scaffold and permit fluid flow and nutrient transport through the soluble scaffold. Partially open pores allow cells to enter one side of the scaffold, but mass transport of nutrients and waste is limited to diffusion. Closed pores have no openings and cannot be utilized by cells or for mass transport of nutrients and waste. Both soluble and insoluble foam scaffolds as described herein have an open pore architecture and highly interconnected pores. Generally, the open pore architecture and highly interconnected pores enable cells to migrate into the pores of the soluble foam scaffold and also facilitate enhanced mass transport of nutrients, oxygen, and waste. The open pore architecture also affects cell adhesion and cell migration by providing a high surface area for cell-cell interaction and space for extracellular matrix (“ECM”) regeneration.

[0110] The porous scaffolds (soluble and insoluble) of the present disclosure have channels (interconnecting structures) between the pores that permit fluid to flow through the scaffold. In some embodiments, when measured in a dry scaffold, at least 50% of the interconnecting structures (channels) have a maximum channel width of from about 30 μm to about 500 μm. As used herein, the measured value of the channel width is the value measured in the dry scaffold. In another embodiment, when measured in a dry scaffold, at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more (or any value between 50% and 100%) of the interconnecting structures (channels) have a maximum channel width of from about 30 μm to about 500 μm. In a particular embodiment, when measured in a dry scaffold, between 65% and 85% of the channels have a maximum channel width of from about 30 μm to about 500 μm. In another particular embodiment, when measured in a dry scaffold, at least 70% of the channels have a maximum channel width of from about 30 μm to about 500 μm. In some embodiments, when measured in a dry scaffold, at least 50% of the channels have a maximum channel width of from about 60 μm to about 400 μm. In another embodiment, when measured in a dry scaffold, at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more (or any value between 50% and 100%) of the channels have a maximum channel width of from about 60 μm to about 400 μm. In a particular embodiment, when measured in a dry scaffold, between 70% and 90% of the channels have a maximum channel width of from about 60 μm to about 400 μm. In another particular embodiment, when measured in a dry scaffold, at least 80% of the channels have a maximum channel width of from about 60 μm to about 400 μm. The maximum channel width is the widest part of the cross-sectional area of the channel through the dry scaffold measured by scanning electron microscopy.

[0111] In some embodiments, when measured in the dry scaffold, the average interconnect structure diameter (i.e., the average maximum distance across the cross-sectional area of the channel space connecting the pores) is between about 150 μm and about 500 μm, between about 250 μm and about 400 μm, or between about 275 μm and about 375 μm. In some embodiments, when measured in the dry scaffold, the average interconnect structure diameter is between about 30 μm and about 300 μm, between about 80 μm and about 250 μm, or between about 130 μm and about 200 μm. In some embodiments, when measured in the dry scaffold, the average interconnect structure diameter is between about 70 μm and about 400 μm, between about 120 μm and about 350 μm, or between about 170 μm and about 300 μm.

[0112] The porous scaffolds (soluble and insoluble) of the present disclosure also have the number of interconnect structures (channels) present per pore. This number can generally range from 1 interconnect structure per pore to 40 interconnect structures per pore, or more. In some embodiments, at least 50% of the pores have 5 to 18 channels / pore. 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 pores have 5 to 18 channels / pore. In one particular embodiment, between 65% and 85% of the pores have 5 to 18 channels / pore. In another particular embodiment, at least 70% of the pores have 5 to 18 channels / pore. In some embodiments, at least 50% of the pores have a per-pore channel number of about 6 to about 14 channels / pore. 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 pores have a per-pore channel number of about 6 to about 14 channels / pore. In one particular embodiment, between 70% and 90% of the pores have a per-pore channel number of about 6 to about 14 channels / pore. In another particular embodiment, at least 80% of the pores have a per-pore channel number of about 6 to about 14 channels / pore. The number of interconnect structures (channels) per pore can be measured by scanning electron microscopy.

[0113] In some embodiments, the average number of interconnect structures (channels) per pore is between 6 and 20, between 8 and 18, or between 10 and 14. In some embodiments, the average number of interconnect structures (channels) per pore is between 4 and 16, between 6 and 16, or between 7 and 11. In some embodiments, the average number of interconnect structures (channels) per pore is between 6 and 12, or between 7 and 10. In some embodiments, the average number of interconnect structures (channels) per pore is between 12 and 18, between 13 and 17, or between 14 and 16.

[0114] For embodiments using a soluble foam scaffold, the scaffold may dissolve in certain circumstances. The soluble foam scaffolds as described herein are digested when exposed to appropriate enzymes that digest or degrade materials. Non-proteolytic enzymes suitable for digesting the foam scaffold, collecting cells, or both include pectin hydrolases or pectinases, which are a heterogeneous group of related enzymes that hydrolyze pectin substances. Pectinase (polygalacturonase) is an enzyme that breaks down complex pectin molecules into shorter galacturonic acid molecules. Commercially available sources of pectinase are typically multi-enzymatic, such as Pectinex TM ULTRA SP-L (commercially available from Novozyme North American, Inc., Franklinton, NC), a pectin hydrolase preparation produced by a selected strain of Aspergillus aculeatus. Pectinex TM ULTRA SP-L mainly contains polygalacturonase (EC 3.2.1.15), pectin transeliminase (EC 4.2.2.2), and pectin esterase (EC 3.1.1.11). The EC designation is based on the Enzyme Commission enzyme classification scheme for the chemical reactions catalyzed by the enzyme. Non-proteolytic enzymes suitable for digesting the foam scaffold, collecting cells, or both also include alginate lyase (EC 4.2.2.3). Alginate lyase is an enzyme that breaks down alginic acid into shorter molecules. Commercially available sources of alginate lyase can be used, such as those from Sigma those.

[0115] According to some embodiments of the present disclosure, digestion of the soluble 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), ethylene glycol tetraacetic acid (EGTA), citric acid, and tartaric acid.

[0116] The time to completely digest a soluble foam scaffold as described herein can be less than about 1 hour. For example, the time to completely digest the foam scaffold 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 between about 1 minute and about 25 minutes, or between about 3 minutes and about 20 minutes, or even between about 5 minutes and about 15 minutes.

[0117] According to embodiments of the present disclosure, methods for forming a soluble foam scaffold as described herein are also disclosed. The methods as described herein may include forming a first aqueous mixture that includes dissolving a polysaccharide in an aqueous solution. The polysaccharide may be those as described above, such as pectic acid or its salts, partially esterified pectic acid or its salts, or partially amidated pectic acid or its salts, and blends of the polysaccharides.

[0118] The method for forming a soluble foam scaffold as described herein may further include forming a second aqueous mixture that includes a water-insoluble divalent metal salt in an aqueous solution. The metal in the divalent metal salt may include (but is not limited to) cations such as magnesium, calcium, zinc, strontium, barium, etc. and combinations thereof. The anions in the divalent metal salt may include (but is not limited to) organic and inorganic anions such as oxalate, tartrate, phosphate, carbonate, citrate, etc. and combinations thereof.

[0119] According to embodiments of the present disclosure, forming the second aqueous mixture may further include adding at least one first polymer as described above to the second aqueous mixture. Optionally, the methods as described herein may further include adding at least one second polymer as 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 to the second aqueous mixture separately or may be added to the second aqueous mixture together. When added in a mixture, the mixture may include about 50% of the at least one first polymer and about 50% of the at least one second polymer. By way of example, the mixture may include between about 35% and about 65% (and all values therebetween) of the at least one first polymer and between about 35% and about 65% (and all values therebetween) of the at least one second polymer.

[0120] According to embodiments of the present disclosure, forming the second aqueous mixture may further comprise adding at least one water-soluble plasticizer to the second aqueous mixture. The plasticizers as described herein are non-toxic and do not affect the solubility of the polysaccharides of the soluble foam scaffold. The plasticizer provides flexibility and softness to the resulting foam, such that the resulting foam is soft and flexible. The plasticizers as described herein may include (but are not limited to) polyols such as glycerol, sorbitol, ethylene glycol, propylene glycol, polyethylene glycol, and combinations thereof. The water-soluble plasticizer added to the second aqueous mixture may be less than about 55 wt% of all solid additives added to form the second aqueous mixture. For example, the water-soluble plasticizer added to the second aqueous mixture may be less than about 50 wt% of all solid additives added to form the second aqueous mixture, or less than about 40 wt%, or less than about 30 wt%, or less than about 25 wt%, or between about 15 wt% and about 55 wt%, or between about 15 wt% and about 50 wt%, or between about 15 wt% and about 40 wt%, or between about 15 wt% and about 30 wt%, or between about 15 wt% and about 25 wt%, and all values therebetween. As used herein, the term "all solid additives added to form the second aqueous mixture" refers to all components of the aqueous mixture other than water.

[0121] According to embodiments of the present disclosure, forming the second aqueous mixture may further comprise adding at least one emulsifier to the second aqueous mixture. The emulsifiers as described herein may include (but are not limited to) sodium dodecyl sulfate (SDS) and polysorbates such as polyethylene glycol sorbitan monolaurate ( 20) and polyethylene oxide sorbitan monooleate ( 80) (each available from Croda International PLC of Snaith, UK).

[0122] According to embodiments of the present disclosure, forming the second aqueous mixture may further comprise adding at least one leachable solid to the second aqueous mixture. The leachable solids as described herein include materials that strengthen or create pores during the formation of the foam scaffold. The leachable solids may be (but are not limited to) non-toxic leachable materials such as 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, etc. 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.

[0123] Each of the materials described above with respect to the second aqueous mixture can optionally be added to the second aqueous mixture, and can be added to the second aqueous mixture in any order, and it is possible to add two or more materials to the second aqueous mixture simultaneously. In one exemplary method, forming the second aqueous mixture includes adding leachable solids to an aqueous solution comprising a divalent metal salt, and mixing the aqueous mixture to facilitate dissolution of the leachable solids in the aqueous mixture. Subsequently, at least one first polymer, at least one second polymer, and / or a water-soluble plasticizer are added to the second aqueous mixture.

[0124] The method of forming a soluble foam scaffold as 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. The foam can be formed from the combined aqueous mixture by introducing air bubbles into the aqueous mixture by mixing, slapping, stirring, inflating, whipping, injecting, or other mechanical actions. The gas can be, for example (but not limited to), air, nitrogen, helium, hydrogen, argon, carbon dioxide, or other inert gases. Introducing air bubbles into the combined aqueous mixture can then be carried out for a period of less than about 30 minutes, such as between about 1 minute and about 30 minutes, or between about 3 minutes and about 25 minutes, or even between about 5 minutes and about 20 minutes. While introducing air bubbles into the combined aqueous mixture, the method of forming a soluble foam scaffold may further include adding a gelling inducer to the combined aqueous mixture. The gelling inducer can be an acid that provides a buffering action and / or a material that slowly generates an acid. Exemplary acids include (but are not limited to) lactide, glycolide, glucono-δ-lactone, and acid anhydrides.

[0125] The method of forming a soluble foam scaffold as described herein can further include coating the soluble foam scaffold with an adhesive polymer coating. Coating the soluble foam scaffold can include exposing the scaffold to an aqueous solution having an adhesive polymer in the aqueous solution. As previously described, the adhesive polymer can include peptides. Exemplary peptides can include (but are not limited to) BSP, vitronectin, fibronectin, laminin, type I collagen and type IV collagen, denatured collagen (gelatin), and other peptides and mixtures thereof. Additionally, the peptide can be a peptide having an RGD sequence. The coating can be, for example II-SC (commercially available from Corning, Incorporated, Corning, NY).

[0126] According to an embodiment of the present disclosure, a method for culturing cells and spheroids in soluble and insoluble foam scaffolds as described herein is also disclosed. Any type of cells or spheroids can be cultured in the foam scaffolds, including (but not limited to) immortalized cells, primary cultured cells, cancer cells, stem cells (e.g., embryonic or induced pluripotent stem cells), etc. The cells can be mammalian cells, avian cells, fish cells, etc. The cells can be of any tissue type, including (but not limited to) kidney, fibroblast, mammary, skin, brain, ovary, lung, bone, nerve, muscle, heart, colorectal, pancreas, immune (e.g., B cells), blood, etc. The cells can be seeded into the foam scaffolds in any culture format, including dispersed (e.g., freshly seeded), confluent, 2D, 3D, spheroids, etc. Culturing cells or spheroids in the foam scaffolds can include seeding the cells in the foam scaffolds. Seeding the cells in the foam scaffolds can include contacting the scaffold with a solution containing the cells. During seeding the cells in the foam scaffolds, the cells enter the pores of the foam scaffolds.

[0127] Culturing cells and spheroids in the foam scaffolds can further include contacting the scaffold with a cell culture medium. Generally, contacting the scaffold with a cell culture medium includes placing the cells to be cultured in a scaffold in an environment having a culture medium in which the cells to be cultured are present. Contacting the scaffold with a cell culture medium can include pipetting the cell culture medium onto the scaffold, or immersing the scaffold in the cell culture medium, or passing the cell culture medium over the scaffold in a continuous manner. Generally, as used herein, the term "continuous" refers to culturing cells with a constant inflow and outflow of cell culture medium in the cell culture environment. Passing the cell culture medium over the scaffold in a continuous manner can include immersing the scaffold in the cell culture medium for a predetermined period of time, and then removing at least some of the cell culture medium and adding fresh cell culture medium after a predetermined period of time, such that the volume of the cell culture medium in contact with the soluble foam scaffold remains substantially constant. The cell culture medium can be removed and replaced according to any predetermined schedule. For example, at least some 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. The cells can be cultured for hours, days, or weeks. For example, the 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 only examples of the culture times, and any predetermined amount of time is acceptable.

[0128] Cell culture media can be, for example (but not limited to), sugars, salts, amino acids, serum (such as fetal bovine serum), antibiotics, growth factors, differentiation factors, coloring agents, or other desired factors. Exemplary cell culture media include Dulbecco's Modified Eagle Medium (DMEM), Ham's F12 Nutrient Mixture, Minimum Essential Media (MEM), RPMI medium, Iscove's Modified Dulbecco's Media (IMDM), Mesencult TM -XF medium, RoosterNourish-MSC, RoosterNourish-MSC XF, etc. Other exemplary cell culture media include those that are adapted to collect by-products of cell cultures, such as chemically defined low particulate media, including extracellular matrix collection media, such as RoosterCollect TM -EV medium, RoosterCollect TM -EV-CC medium, RoosterBio M2001 medium, etc.

[0129] According to embodiments of the present disclosure, methods for collecting cells (including three-dimensional cell structures) from a foam scaffold as described herein are also disclosed. In embodiments having a soluble foam scaffold, the methods for collecting cells as described herein may include digesting the soluble foam scaffold by exposing the soluble foam scaffold to an enzyme. As previously discussed, non-proteolytic enzymes suitable for digesting foam scaffolds, collecting cells, or both include pectin hydrolases or pectinases (which are a heterogeneous group of enzymes that hydrolyze pectin substances) and alginate lyases. Commercially available sources of pectinases are typically multi-enzymatic, such as Pectinex TM ULTRA SP-L (commercially available from Novozymes North America, Inc., Franklin County, North Carolina), a pectin hydrolase preparation produced by a selected strain of Aspergillus aculeatus. Pectinex TM ULTRA SP-L mainly contains polygalacturonase (EC 3.2.1.15), pectin trans-eliminase (EC 4.2.2.2), and pectin esterase (EC 3.1.1.11). Alginate lyase (EC 4.2.2.3) degrades alginate and alginic acid and is commercially available from Sigma- etc. The EC designation is based on the Enzyme Commission enzyme classification scheme for enzyme-catalyzed chemical reactions.

[0130] Exposing the soluble foam scaffold to an enzyme can include exposing the scaffold to an enzyme concentration between about 1 and about 200 U. For example, the method can include exposing the scaffold to an enzyme concentration between about 2 U and about 150 U, or between about 5 U and about 100 U, or even between about 10 U and about 75 U, and all values therebetween.

[0131] The method of collecting cells as described herein can 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 soluble foam scaffold to a chelating agent can include exposing the scaffold to a chelating agent concentration between about 1 mM and about 200 mM. For example, the method can include exposing the scaffold to a chelating agent concentration between about 10 mM and about 150 mM, or between about 20 mM and about 100 mM, or even between about 25 mM and about 50 mM, and all values therebetween.

[0132] In aspects according to embodiments of the present disclosure, the foam scaffolds (soluble or insoluble) of the present disclosure can be used in perfusion bioreactors for three-dimensional cell culture (e.g., spheroids and organoids), and for generating cell culture by-products such as extracellular vesicles and extracellular matrix proteins. The perfusion bioreactor of the present disclosure has an inlet that allows fluid to enter the bioreactor and an outlet that allows fluid to leave the bioreactor. The perfusion bioreactor of the present disclosure also has a substrate (i.e., a porous scaffold) within the bioreactor to assist in culturing cells. Prior to placing the foam scaffold in the bioreactor to culture cells, cells of the desired cell type can be directly added (referred to as "seeding") into the foam scaffold. Alternatively, cells of the desired cell type can be added through the inlet (the location on the bioreactor where fluid can enter the bioreactor) to reach the foam scaffold inside the bioreactor. Under either option, the added cells will enter the pores and the interconnected spaces (referred to as "interconnected structures") between the pores present in the foam scaffold. The pores and interconnected structures of the foam scaffold accommodate the cells and allow the formation of spheroids, organoids, or other three-dimensional cell types. In some embodiments, at least 70%, 80%, 90% or more of the cells in the pores of the porous scaffold aggregate to form spheroids. In one particular embodiment, at least 80% of the cells in the pores of the porous scaffold aggregate to form spheroids. During cell culture in the bioreactor, the formed spheroids, organoids, or other three-dimensional cell types remain accommodated by the pores and interconnected structures. For three-dimensional cell culture, the foam scaffold is preferably non-adhesive to the 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 are to adhere to each other to form a three-dimensional culture. Culturing three-dimensional cells can produce a variety of by-products, including extracellular vesicles, proteins, and other secreted materials. Any particular by-product mentioned (e.g., EV) is not intended to limit the scope of embodiments regarding that particular by-product, which is for illustrative purposes only. It should be understood that exosomes and other cell by-products secreted by spheroids can be cultured using the scaffolds of the present disclosure rather than the EVs already described herein.

[0133] Figures 5A to 5B Illustrative examples of foam (porous) scaffolds for three-dimensional cell cultures are shown. Figure 5A A porous scaffold fabricated from scaffold material 2500 is shown, where pores 2502 are formed in the scaffold material 2500. The scaffold is placed in a tissue culture plate 2506 and cells are seeded onto it. After the culture phase, the cells form spheroids 2504 in the pores 2502. Figure 5BA similar scaffold for growing spheroids is shown, but this time the scaffold material 2510 with pores 2512 is placed in the bioreactor 2516 of a perfusion bioreactor system. Cells are inoculated into the bioreactor 2516 through the inlet 2517 to form spheroids 2514 in the pores 2512. The bioreactor 2516 has an outlet 2518 through which waste fluid can leave the bioreactor. Figure 5B The bioreactor system shown in is a simplified depiction of such a system that has a media conditioning vessel 2520 and a fluid flow path 2522 that allows media perfusion and / or recirculation through the bioreactor 2516. The use of a porous scaffold inside a bioreactor with media perfusion can increase extracellular vesicle production. Figure 5B The system shown in is only an example and is not intended to limit the scope of the embodiments of the present disclosure. For example, cells can alternatively be added to the foam scaffold 2500 through the outlet 2518 of the bioreactor, and fluid can leave through the inlet 2517 of the bioreactor. As another example, a media feed container can be added to the perfusion bioreactor system that conditions the media conditioning vessel. As yet another example, a pump or a controller or both can be present in the pipeline between the bioreactor and the media conditioning vessel.

[0134] Aspects of embodiments of the present disclosure cultivate and collect two-dimensional and three-dimensional cell structures (spheroids, organoids, etc.) and by-products of these cells and structures using the foam scaffolds of the present disclosure. From the time of cell seeding to the time of cell collection, the increase in cells can be from about 0.2-fold to about 200-fold. In one embodiment, the increase in cells from a primary cell line (e.g., hMSC cells, etc.) is between about 0.2-fold and about 100-fold, between about 0.2-fold and about 50-fold, between about 0.2-fold and about 30-fold, between about 0.2-fold and about 10-fold, or between about 0.5-fold and about 5-fold. In another embodiment, the increase in cells from a non-primary cell line (e.g., HEK293T cells, etc.) is between about 5-fold and about 200-fold, between about 50-fold and about 150-fold, between about 50-fold and about 100-fold, between about 5-fold and about 50-fold, between about 7-fold and about 40-fold, or between about 8-fold and about 30-fold. In a particular embodiment, five days after seeding cells in the foam scaffold and culturing the cells in a perfusion bioreactor, the increase in cells from a primary cell line is between about 0.2-fold and about 5-fold. In another particular embodiment, seven days after seeding cells in the foam scaffold and culturing the cells in a perfusion bioreactor, the increase in cells from a non-primary cell line is between about 8-fold and about 30-fold. In another particular embodiment, one day after seeding cells in the foam scaffold and culturing the cells in a perfusion bioreactor, the increase in cells from a non-primary cell line is between about 50-fold and about 100-fold. The time after cell seeding and the fold increase after seeding cells with the foam scaffold are exemplary in nature, and other cell seeding times and fold increases are also contemplated.

[0135] When introducing cells into a foam (porous) scaffold in a bioreactor by perfusion, the capture of cells in the foam scaffold can be at least 1% or more of the added cells with each pass through the bioreactor and can accumulate with more passes through the bioreactor. For example, the cell capture with each pass through 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 added cells. In one embodiment, the cell capture in the foam scaffold is at least 5% of the added cells after a single pass through the bioreactor. In another embodiment, the cell capture in the foam scaffold is at least 10% of the added cells after a single pass through the bioreactor. Cells that exit the bioreactor during any pass through the bioreactor can be recycled back into the bioreactor for more passes. The cells that accumulate 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 added cells. In a particular embodiment, the cells that accumulate in the bioreactor at the end of all passes are at least 30%. In another particular embodiment, the cells that accumulate in the bioreactor at the end of all passes are at least 40%. In yet another particular embodiment, the cells that accumulate in the bioreactor at the end of all passes can be at least 50%.

[0136] In some embodiments, cells are added at the inlet of the bioreactor. In some embodiments, cells are added at a point prior to the inlet of the bioreactor. In one non-limiting example, cells are added to a fluid line that feeds a culture medium into the bioreactor through a port in the fluid line. In another non-limiting example, cells are added to a container containing a culture medium and then perfused into the bioreactor at a set flow rate (such as 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, cells are perfused through the bioreactor at between about 0.1 mL / min and about 20 mL / min. In some embodiments, cells are perfused through the bioreactor at a linear velocity (Darcy velocity) between about 2.3×10 -6 m / s and about 1.2×10 -3 m / s. As used herein, the term "linear velocity" refers to the Darcy velocity. In some embodiments, cells are at about 1.0×10 -5The linear velocity (Darcy velocity) between m / s and approximately 5.0×10 -4 m / s is perfused through the bioreactor. In some embodiments, the cells are at approximately 1.0×10 -5 m / s and approximately 4.8×10 -5 m / s, between approximately 1.0×10 -5 m / s and approximately 2.4×10 -4 m / s, or between approximately 1.0×10 - 5 m / s and approximately 3.6×10 -4 m / s is perfused through the bioreactor. In some embodiments, any cells exiting the bioreactor are recirculated back through the inlet of the bioreactor in an attempt to capture the cells during another pass through the bioreactor.

[0137] It should be understood that the perfusion rate does not need to be the same throughout the process. For example, when the cells are in the process of inoculating the foam (porous) scaffold present in the bioreactor, a lower or higher flow rate can 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 lower linear velocity (Darcy velocity) can be used during cell seeding, such as (but not limited to) between approximately 1.0×10 -5 m / s and approximately 4.8×10 -5 m / s, between approximately 1.0×10 -5 m / s and approximately 2.4×10 -4 m / s, or greater than 0 m / s and less than 2.4×10 -4 m / s. After the foam scaffold has been inoculated, a higher or lower flow rate than that used for loading can be used, 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 is performed at a perfusion flow rate of approximately 0.1 mL / min to approximately 5 mL / min. In another preferred embodiment, cell culture is performed at a perfusion flow rate of approximately 5 mL / min to approximately 15 mL / min. In some embodiments, the perfusion flow rate of the medium during cell seeding is approximately 2.3×10 -6m / s to about 1.2×10 -3 m / s of linear velocity (Darcy velocity). In some embodiments, the cells are perfused through the bioreactor at a linear velocity (Darcy velocity) between about 1.0×10 -5 m / s and about 5.0×10 -4 m / s. In some embodiments, the cells are perfused through the bioreactor at a linear velocity (Darcy velocity) between about 1.0×10 -5 m / s and about 4.8×10 -5 m / s, between about 1.0×10 -5 m / s and about 2.4×10 - 4 m / s, or between about 1.0×10 -5 m / s and about 3.6×10 -4 m / s of linear velocity (Darcy velocity).

[0138] According to some embodiments, during cell culture, the flow rate (Darcy velocity) of the fluid flowing through the scaffold is in the range of about 2.3×10 -6 m / s and about 1.2×10 -3 m / s. In some embodiments, the flow rate (Darcy velocity) of the fluid flowing through the scaffold is between 2.3×10 -6 m / s and about 1.2×10 -5 m / s, between about 1.2×10 -5 m / s and about 5.0×10 -4 m / s, or between about 5.0×10 -4 m / s and about 1.2×10 -3 m / s. In some embodiments, the linear velocity (Darcy velocity) of the fluid flowing through the scaffold is between about 1.0×10 -5 m / s and about 5.0×10 -4 m / s. In some embodiments, the cells are perfused through the bioreactor at a linear velocity (Darcy velocity) between about 1.0×10 -5 m / s and about 4.8×10 -5 m / s, between about 1.0×10 -5 m / s and about 2.4×10 -4 m / s, or between about 1.0×10 -5 m / s and about 3.6×10 -4 m / s of linear velocity (Darcy velocity). The flow rate of the culture medium through the scaffold depends on the size of the scaffold and the flow rate of the culture medium.

[0139] A fluid with a certain velocity can exert wall shear stress on cells cultured in a porous scaffold. For a cell culture medium flowing through a porous scaffold with spheroids or other cells, the flow is typically 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 the culture medium moving through at least one porous scaffold containing the spheroids. In some embodiments, a pump is used to generate shear stress on the spheroids or cells. In some embodiments, shear stress on the spheroids or cells is generated by any other method that causes the culture medium within the scaffold to move, including (but not limited to) agitation, vibration, rotation, undulation, or tilting.

[0140] The value of the shear stress can be determined by computer fluid dynamics simulation using computer modeling of the scaffold. This is done for the scaffolds of the present disclosure. A representative computer-aided drawing geometry of the scaffold is created by MATLAB coding and using the pore size, interconnect structure size, and number of interconnect structures per pore measured on the scaffold, thereby creating a computer grid. This computer grid is then imported into ANSY Fluent for computer fluid dynamics simulation. The calculated Reynolds number is less than 1, which means the flow in the porous scaffold is laminar. The shear stress provided to the cells and spheroids of the present disclosure is determined to be between 0 mPa and about 300 mPa. However, shear stress values greater than 300 mPa can be provided, such as 400 mPa, 500 mPa, 600 mPa, 700 mPa, 800 mPa, 900 mPa, or 1000 mPa or greater, and the spheroids or cells can still maintain their aggregation and / or still produce by-products of interest.

[0141] According to some embodiments, the shear stress provided to the spheroids housed 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 housed within the pores of the scaffold is greater than 300 mPa. In some embodiments, the shear stress provided to the spheroids housed within the pores of the scaffold is greater than 0 mPa. In some embodiments, the shear stress provided to the spheroids housed 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 housed 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 and about 150 mPa, or between about 150 mPa and 300 mPa, or any value within these ranges.

[0142] According to some embodiments of the present disclosure, by-products are collected from cultured cells or three-dimensional cell structures 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 in 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, ectosomes, and other nanoparticles, proteins, polypeptides, peptides, amino acids, lipids, polynucleotide sequences, and hormones. The by-products can be genetically engineered or naturally occurring. The collection of the by-products can be carried out by any one of a number of methods known to those skilled in the art.

[0143] Compared to conventional 3D culture methods and devices, perfusion using the foam (porous) scaffolds of the present disclosure produces an increased number of by-products. For vesicular particles, after culturing the cells for about 48 hours, perfusion of spheroids cultured in the foam scaffolds of the present disclosure produces about 1×10 9 between about 1×10 13 EVs per million cells (i.e., about 1×10 3 to about 1×10 7 EVs per cell). In one embodiment, after a culture time of about 48 hours at a perfusion rate with a linear velocity (Darcy velocity) of about 2.4×10 -4 m / s, for every 1×10 6 cells of a certain cell type, at least 1×10 11 EVs are produced (i.e., at least 1×10 5 EVs per cell). In another embodiment, after a culture time of about 48 hours at a perfusion rate with a linear velocity (Darcy velocity) of about 2.4×10 -4 m / s, for every 1×10 6 cells of a certain cell type, at least 1×10 12 EVs are produced (i.e., at least 1×10 6 EVs per cell). In another embodiment, after a culture time of about 48 hours at a perfusion rate with a linear velocity (Darcy velocity) of about 2.4×10 -4 m / s, for every 1×10 6 cells of a certain cell type, at least 1×10 13 EVs are produced (i.e., at least 1×10 7 EVs per cell). In yet another embodiment, after a culture time of about 48 hours at a perfusion rate with a linear velocity (Darcy velocity) of about 2.4×10 -4 m / s, for every 1×10 6 cells of a certain cell type, about 1×10 11between about 1×10 13 EVs per cell of a particular cell type (i.e., between about 1×10 5 and about 1×10 7 EVs). As used herein, the term “extracellular vesicle” or “EV” refers to membrane-bound vesicles that are secreted from cells and can be generated in endosomal compartments. EVs contain various molecular components from the cell. These components are referred to as “cargo” and can have biological functions. The cargo can contain some or all of the following: proteins, lipids, mitochondrial components, and genetic material (RNA and / or DNA).

[0144] EVs generated by culturing cells with the foam (porous) scaffolds of the present disclosure each have a size referred to as the vesicle diameter, and collectively the EVs have an average vesicle diameter. The size of the EVs can be determined by multi-angle dynamic light scattering (MADLS). In one embodiment, when measured by MADLS, the diameter of EVs collected from cells cultured in the foam scaffolds of the present disclosure is between about 40 nm and about 200 nm, between about 50 nm and about 150 nm, or between about 60 nm and about 140 nm, between about 70 nm and about 130 nm, between about 80 nm and about 120 nm, or between about 90 nm and about 110 nm. In another embodiment, when measured by MADLS, the average diameter of EVs collected from cells cultured in the foam scaffolds of the present disclosure is between about 80 nm and about 120 nm, or between about 90 nm and about 110 nm, or between about 95 nm and about 105 nm. In a particular embodiment, the average diameter of the EVs is about 100 nm to about 110 nm.

[0145] EVs produced by cells cultured in the foam scaffolds of the present disclosure also have a level of functionality associated therewith. This functionality of the EVs can be measured by a wound healing assay using HT-1080 cells (epithelial cells derived from connective tissue, ATCC, CCL-121) to determine directed cell migration in vitro. The wound healing assay of the present disclosure comprises generating a cell monolayer from HT-1080 cells, creating a wound in the cell monolayer, imaging the wound after wound formation, treating the wound with 2×10 9 EVs collected from the cell culture, and then periodically imaging the treated wound during cell migration to close the wound. The same procedure can be done for a control (no EVs added). A plot of the percentage of wound healing over time for each wound analyzed can then be generated to compare the functionality of different EV batches or processes.

[0146] According to some embodiments, EVs collected from cell cultures in the 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, when measured by a wound healing assay, after 6 hours of creating a wound in monolayer HT-1080 cells, the improvement in EV functionality is such that wound healing by perfusion-based culture is about 2% to 50% faster than static culture (i.e., in the micropores of a culture plate or flask). In a specific embodiment, when measured by a wound healing assay, as measured 6 hours after creating a wound in monolayer HT-1080 cells, the improvement in EV functionality is such that EVs produced by perfusion-based culture are about 2% to 10%, about 10% to 20%, about 20% to 30%, about 30% to 40%, or about 40% to 50% faster than EVs produced by static culture. In some embodiments, when measured by a wound healing assay, after 6 hours of creating a wound in monolayer HT-1080 cells, the improvement in EV functionality is such that wound healing is about 2% to 30% faster than EVs produced by non-perfusion-based cell culture methods. In a specific embodiment, when measured by a wound healing assay, after 6 hours of creating a wound in monolayer HT-1080 cells, the improvement in EV functionality 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.

[0147] Example

[0148] Certain exemplary and specific embodiments of the present disclosure are further described below. These embodiments are illustrative only and are not intended to be limiting.

[0149] Example 1

[0150]

[0151] A first aqueous mixture containing 2.0 wt% polygalacturonic acid (PGA) was prepared by dissolving approximately 162 grams of sodium polygalacturonate 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 it in an 800 W microwave for approximately 30 seconds. Approximately 1.06 grams of CaCO3 and approximately 0.125 grams 20 was added to the second aqueous mixture. Subsequently, the second aqueous mixture was sonicated for about 1 minute and then transferred to the bowl of a KitchenAid mixer equipped with a wire loop whisk. Then, about 17.5 g of sucrose and about 1.94 g of Methocel HPMC Culminal 724 were added to the bowl of the KitchenAid mixer, and the aqueous mixture was stirred for about 5 minutes. The first aqueous mixture containing 2.0 wt% PGA was added to the mixing bowl to form a combined aqueous mixture, which was mixed at a certain stirring speed (Speed 1 of the KitchenAid mixer) for about 3 minutes. Then, the combined aqueous mixture was whipped at a high whipping speed (Speed 10 of the KitchenAid mixer) for about 20 minutes to introduce air into the combined aqueous mixture. While continuing to whip the combined aqueous mixture, a solution of about 3.77 g of glucono-delta-lactone (GDL) in about 30 mL of water was added to the mixing bowl and whipping was continued for about 1 minute.

[0152] Following the method discussed above, an opaque white foam was obtained. At room temperature, the foam was left open in the mixing bowl for about 1 hour to allow time for crosslinking to occur within the foam. Then, the foam was exposed to a temperature of about -80 °C for about 16 hours to freeze the foam, and then it was 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 the foam was observed to be porous with highly interconnected pores. Figure 2 SEM pictures of the foam prepared in Example 1 are shown.

[0153] Example 2

[0154] Vero cells were studied in the foam formed according to the method of Example 4. Vero cells ( CCL-81, commercially available from ATCC, Manassas, VA) were cultured on a cell culture plate in IMDM medium supplemented with 10% fetal bovine serum (FBS). The foam was cut into sections with a thickness of about 2 to 3 mm and a diameter of about 22 mm. The foam sections were sterilized in an aqueous solution of 70% ethanol for about 5.0 minutes and then placed into the corresponding wells of a 6-well ultra-low attachment cell culture plate. The foam sections were washed twice in ultrapure water and once in IMDM medium. Before inoculation, the excess medium was removed from the wells.

[0155] Vero cells were collected from a cell culture plate using trypsin, resuspended in IMDM medium, and 150 μL containing approximately 100,000 cells was inoculated into each foam portion placed in the wells of 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 portions were observed using phase-contrast microscopy. Figure 3 The image obtained by phase-contrast microscopy is depicted in Figure 3 , which shows that the cells did not adhere to the uncoated foam portion but formed spheroids in the pores of the foam portion. Thus, it was determined that the soluble foam scaffolds of the present disclosure can be used for culturing spheroids or non-adherent cells.

[0156] Example 3

[0157] Some of the soluble foams disclosed herein use various components (e.g., sucrose, glycerol, dextran, etc.), which are added to facilitate the foaming process and constitute a large portion (e.g., by weight) of the solution used in the foaming process. For example, the components can account for up to 90% of the final weight of the foam. Most of these components do not covalently associate with the foam structure and will dissociate from the foam during coating or may need to be removed prior to cell culture. In cell culture, if these components are not removed, these materials can potentially alter the cell culture medium composition, block cell-binding epitopes, and reduce cell attachment. After being released into the cell culture medium, these materials can significantly change the medium osmotic pressure. During the foaming process, surface-active molecules or foaming agents, such as surfactants, are required. For example, P123 can be used during the foaming process, as disclosed in some of the following examples. However, the above examples also use other components, such as sucrose, dextran, glycerol, and 20 for foaming. To simplify the process, reduce production costs, and enable the direct use of soluble foams in cell culture without removing additional components, it would be beneficial to eliminate these additional materials in the foam, as discussed in the following paragraphs.

[0158] In a foam, as in any porous material, the pore size is important for hydrodynamics and the total surface area. It has been found that by using smaller-sized calcium carbonate particles as the source of gelling, much smaller pore sizes are observed. It is believed that the smaller-sized calcium carbonate particles can cause better distribution and easier release of calcium during mixing before the addition of acid, which increases the viscosity of the PGA solution. This makes it difficult for the foam pores to merge together to form large pores. However, the faster release of calcium during foaming can also cause the PGA to become over-crosslinked before complete mixing and can damage the foam structure. It is desirable to be able to control the pore size by controlling the viscosity of the PGA solution during foaming and at the same time prevent uncontrolled over-crosslinking.​

[0159] In this Example 3, by using a high HLB surfactant polymer (such as poloxamer 407 or F127) instead of the two surfactants used in the previous examples (such as a surfactant polymer, such as P123, and an emulsifier, such as 20), a plasticizer (such as glycerol), a foam enhancing agent or a sugar (such as sucrose) and a foam enhancing agent in the form of a non-surfactant polymer (such as dextran) are no longer used. This provides a much simpler formulation for the soluble foam and eliminates components that require further downstream processing or may have a potential negative impact on cell culture. In addition, it is possible to control the viscosity of the simplified formulation without adding any new components. This simplified formulation has several advantages. For example, the new formulation removes non-critical materials that may be undesirable in the finished soluble foam product in some cases. The elimination of such materials also greatly simplifies the process and reduces costs. In addition, a large amount of leachable materials are removed from the foam structure, which otherwise may potentially change the osmotic pressure of the cell culture medium and block cell-binding epitopes during application. This will enable the subsequent separate coating step to be eliminated and enable the foam to be used directly in cell culture. In addition, the simplified formulation provides adjustable viscosity without adding additional components. This makes it easier and less costly to optimize the foaming process and control the foam structure in the future. Finally, by using the shear-thinning properties of the material, the simplified formulation can be beneficial for a continuous foaming process.

[0160] The foaming formulation according to an exemplary embodiment from some of the above examples (which will be referred to as the comparative example for the remainder of this Example 3) includes a relatively long list of materials as shown in Table 1 below. In contrast, the simplified formulation of this Example 3 omits a plasticizer (such as glycerol), an emulsifier or a second surfactant polymer (such as 20) and other foam enhancing agents or non-surfactant polymers (such as sucrose and dextran). The simplified formulation in Table 1 also uses a poloxamer (such as, F127) having a higher HLB than the HLB of P123 used in the comparative example ( The HLB of P123 is 8, in contrast, the HLB of F127 is 22). Most of the additional components in the comparative example are used to provide physical properties to enable the foaming process, as highlighted in Table 1. These additional components ( 20. Glycerol, sucrose, and dextran) account for approximately 80% of the total weight (excluding water) of the formulation in the comparative example. Table 2 below compares the weight percentages of the components in the comparative examples of Table 1 and the simplified formulation. These materials may have no value for the final product and may need to be removed during subsequent coating processes. If foam products are used for cell culture, these materials need to be removed to prevent significant changes in osmotic pressure or blockage of cell-binding epitopes.

[0161]

[0162] Table 1.

[0163]

[0164] Table 2.

[0165] In this Example 3, F127 can replace P123 and the mixed surfactant of 20. It also enables good foaming without adding any sucrose, glycerol, or dextran. The measurement results show that the amount of the required porosity in the foam can be comparable to the porosity of the examples (including comparative examples) previously disclosed herein. Figure 4 Show the SEM image of the foam formed from the simplified formulation of Example 3, which has a pore structure comparable to that of the foams manufactured by other examples using more complex formulations herein.

[0166] Replacing P123 with F127 is also beneficial to the process. P123 is a pasty material that is difficult to aliquot. It is also difficult to dissolve in water, and the dissolution process may take overnight. F127 is a powder that is easy to weigh during formulation, and the dissolution can be completed within one hour. These characteristics can be beneficial to the manufacturing process.

[0167] By adding a small amount of calcium ions to the PGA solution, the PGA molecules can be partially crosslinked and the viscosity of the solution increases. This enables the production of shear-thinning and soluble materials for 3D bioprinting. When a certain amount of calcium ions is added to bind approximately 10% of the carboxyl groups in the PGA molecules, the viscosity of the solution increases 1000 times at a shear rate of 1 / s, which is approximately 10 times that of glycerol (1.4×10 3 mPa*s). When exposed to a shear rate of 1000 1 / s, the viscosity drops to 1 / 100 and becomes 1 / 10 that of glycerol. After removing the shear force, the decrease in viscosity can be quickly restored. By adjusting the amount of calcium, it is possible to further adjust the range of viscosity and the shear-thinning reaction.

[0168] Thus, 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 the viscosity and provide shear-thinning properties. Under the shear of the mixing blades, the solution viscosity decreases, which helps incorporate air bubbles into the foam. When mixing stops, the solution viscosity rapidly increases, which helps stabilize the foam and prevent liquid drainage. This can further eliminate the need for foaming stabilizer materials such as glycerol, sucrose, and dextran. By controlling the solution viscosity, the rate of bubble incorporation can be increased or decreased, and the pore size in the foam can be altered. The shear-thinning property is also beneficial for the continuous foaming process. After the foam is extruded from the continuous foaming mixer, it can rapidly stabilize the pore structure. For partial crosslinking, instead of adding all of the GDL at the end of the foaming step, a portion of the GDL (e.g., 0.5 g in Table 1) can be introduced before foaming, which causes partial release of calcium from calcium carbonate and partial crosslinking of PGA. At the end of the foaming step, the remaining GDL (e.g., 3.5 g in Table 1) can be added, or acid molecules such as acetic acid can be introduced in the form of vapor to complete the gelling process.

[0169] ***

[0170] According to aspects of embodiments of the present disclosure, spheroids can be formed from cells entering a scaffold by directly inoculating cells on top of the scaffold in a tissue culture plate ( Figure 5A ) or by perfusion in a bioreactor ( Figure 5B ). The cells will enter the porous material and be "trapped" or confined within the pores. Since the cells cannot adhere to the material, they will 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. Any particular by-product mentioned (e.g., EVs) is not intended to limit the scope of embodiments regarding that particular by-product, and is only for illustrative purposes. Figure 5A Shows a porous scaffold fabricated from scaffold material 2500, where pores 2502 are formed in the scaffold material 2500. The scaffold is placed in a tissue culture plate 2506 and cells are inoculated thereon. After the culturing phase, the cells form spheroids 2504 within the pores 2502. Figure 5B Shows a similar scaffold for growing spheroids, but this time the scaffold material 2510 having pores 2512 is placed in a bioreactor 2516 of a perfusion bioreactor system. Cells are inoculated into the bioreactor 2516 to form spheroids 2514 within the pores 2512. Figure 5BThe bioreactor system shown is a simplified depiction of such a system having a media conditioning vessel 2520 and a fluid flow path 2522 that permits media perfusion and / or recirculation through the bioreactor 2516. Use of a porous scaffold inside a bioreactor with media perfusion can increase extracellular vesicle production. The rate of media perfusion through the bioreactor will vary depending on the application, cell type, and other factors. Some examples of embodiments may use a media perfusion flow rate of from about 0.5 mL / min to about 50 mL / min. Figure 5B The system shown is only an example and is not intended to limit the scope of embodiments of the present disclosure. Cells will enter the porous material and be “trapped” or confined within the pores. Since the cells cannot adhere to the material, they will adhere to each other to form spheroids.

[0171] According to an embodiment, the scaffold is soluble and thus the spheroids can be easily recovered from the scaffold and used for other applications such as toxicity analysis or other applications known to those skilled in the art. To achieve spheroid formation and EV production, any type of scaffold material made of a biocompatible material with a controlled porosity that does not permit cell adhesion can be used. To recover the spheroids, the scaffold material is made of a biocompatible digestible material with a controlled porosity that does not permit cell adhesion. The porosity and structure of the porous scaffold can be controlled. In some preferred embodiments, the porous scaffold comprises a scaffold having a high porosity and large pores but with a smaller interconnecting structure of the scaffold material.

[0172] Example 4

[0173] This example compares different scaffold characteristics based on the composition. These data look at the effect of certain reagents on the size or number of pores or interconnecting structures and how these specific characteristics then affect cell proliferation, spheroid maintenance in foams, or cell production, as well as other parameters.

[0174] Four different compositions based on 2.0 wt% PGA were evaluated: XP49, XP64, XP76, and FMXP005. XP49 ( Figure 6A ) uses 20 as the sole emulsifier, P123 as the surface-active polymer, and dextran as the non-surface-active polymer (the compositions are in Table 2).

[0175]

[0176] Table 2.

[0177] XP64 ( Figure 6B ) uses both 20 and SDS as emulsifiers, P123 is used as a surface-active polymer, and dextran is used as a non-surface-active polymer (the composition is shown in Table 3).

[0178]

[0179] Table 3.

[0180] 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 polymer (the composition is shown in Table 4).

[0181]

[0182] Table 4.

[0183] FMXP005 ( Figure 6D ) uses SDS as the sole emulsifier and dextran as the non-surface-active polymer. It does not contain any surface-active polymer (the composition is shown in Table 5).

[0184]

[0185] Table 5.

[0186] Scaffolds producing each of the XP49, XP64, XP76, and FMXP005 compositions are dried and sliced for microscopic analysis. Three parameters are quantified by scanning electron microscopy: pore size, interconnect structure (channel) size, and the number of interconnect structures (channels) per pore.

[0187] For all compositions, as Figure 6E shown, the pore size distribution (i.e., the maximum distance across the cross-sectional area of the pore) of each composition is relatively wide. However, the pore sizes of XP49 and XP64 are mainly between 450 μm and 850 μm in diameter. The XP76 scaffold produces the smallest pore sizes, where the pore sizes are mainly between 300 μm and 650 μm in diameter. The FMXP005 scaffold produces the widest pore size distribution, where the pore sizes are mainly between 350 μm and 1000 μm. The pore size distributions of all four foam compositions are mainly between 300 μm and 1000 μm.

[0188] The distribution of the interconnect structure diameter (i.e., the maximum distance across the cross-sectional area of the channel space connecting the pores) of the four compositions is similar to the pore size distribution ( Figure 6F)。The XP49 and XP64 interconnect structure sizes are mainly between 150 μm and 500 μm in diameter. XP76 has the smallest interconnect structure diameter in the composition, where the diameter is mainly between 30 μm and 300 μm. Although the FMXP005 composition has the largest pore size, it does not have the largest interconnect structure diameter, where the diameter is mainly between 70 μm and 400 μm.

[0189] The number of interconnect structures (channels) per pore ( Figure 6G ) was also studied. Compared with the XP49 scaffold, adding SDS as an emulsifier in the XP64 scaffold seems to affect the number of interconnect structures per pore. For XP64, the number of interconnect structures per pore is mainly between 6 and 20 (median 12) interconnect structures / pore, while for XP49 it is mainly between 4 and 16 (median 8) interconnect structures / pore. For XP76, the number of interconnect structures per pore is mainly between 6 and 12 (median 8) interconnect structures / pore. For FMXP005, the number of interconnect structures per pore is larger, resulting in between 12 and 18 (median 15) interconnect structures / pore.

[0190] In addition, the density of the dried scaffolds was measured. The XP49 scaffold exhibited a density of approximately 0.038 to 0.046 g / cm 3 , the XP64 exhibited a density of approximately 0.029 to 0.030 g / cm 3 , the XP76 exhibited a density of approximately 0.071 to 0.09 g / cm 3 , and the FMXP005 exhibited a density of approximately 0.048 to 0.057 g / cm 3 .

[0191] In summary, these data indicate that the scaffold composition can affect the pore size, interconnect structure diameter, and the number of interconnect structures per pore, as shown above.

[0192] Example 5

[0193] In this example, the use of an immortalized cell line (HEK293T cells) or a primary cell line (human mesenchymal stem cells derived from bone marrow, hMSC) in scaffolds in a tissue culture plate demonstrated spheroid formation. 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) were tested, which produced different pore sizes and interconnect structure sizes as described in Example 4.

[0194] Figure 6B and 6CExemplary photographs showing uncoated foam scaffolds are presented. After preparing the foam, 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 attachment 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. The cells were dissociated from their culture flasks using Tryspin (HEK293T) or TrypLE (hMSC), and the concentration was adjusted to 1×10 6 cells / mL. To seed the foam scaffolds, the medium was first removed from the wells, and 150 μL of the cell suspension was added to the top of the foam, for a total of 1.5×10 5 cells / scaffold. The plate was transferred back to the cell culture incubator and kept for 2 hours, then 3 mL of the appropriate medium was added to each well without disturbing the scaffolds. One day after seeding, initial spheroid formation was evaluated by staining the live cells with calcein AM. Spheroids were observed under all conditions and for both cell types ( Figure 7A (HEK293T cells) and 7B (hMSC), top row). Interestingly, more spheroids were present in the XP76 scaffolds compared to the XP64 scaffolds, indicating better cell maintenance in the XP76 scaffolds. This may be due to smaller interconnecting structures and / or fewer numbers of interconnecting structures per pore in XP76 compared to the XP64 scaffolds, as described in Example 4. Seven days after seeding, spheroid growth was evaluated, as shown in the photographs in the bottom row for Figure 7A (HEK293T cells) and 7B (hMSC). Under all conditions, the spheroids were larger on day 7 than on day 1, especially for HEK293T cells. Cell counts after scaffold digestion and spheroid dissociation showed similar results.

[0195] Figure 8 is a bar graph showing the cell counts after scaffold digestion and spheroid dissociation in terms of the fold increase in cells after seeding for two experiments involving HEK293T cells and hMSC cells. The data show cell growth five days after seeding HEK293T cells or seven days after seeding hMSC cells. The data clearly indicate spheroid and / or cell growth in the foam scaffolds. The increase in cell mass for HEK29T cells was from about 9-fold to about 27-fold, while the increase for hMSC cells was about 2 to 3-fold. Regarding HEK293T cells, the lower number of cell growth in the XP64 scaffolds seems to be mainly due to cell loss during seeding, as the XP64 scaffolds have larger interconnecting structures and / or more interconnecting structures per pore compared to the XP76 scaffolds. The growth of hMSC cells thus remained limited in both scaffolds, as expected for these cells.

[0196] Example 6

[0197] As described above, cells can also be seeded into a porous (foam) scaffold within a bioreactor under perfusion conditions, as Figure 5B shown. In this example, an immortalized cell line (HEK293T cells) was seeded into two such perfusion bioreactors containing uncoated soluble 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 cartridges. The scaffolds were then washed with sterile filtered demineralized water and sterilized within the bioreactor by perfusion. Then an appropriate cell culture medium was added to the medium reservoir bottle and perfused through the scaffolds. The bioreactors were transferred to a cell culture incubator set at 37 °C, 5% CO2 for 1 hour to equilibrate. The cells were dissociated from their culture flasks by Tryspin 0.25% and their concentration was adjusted to 1×10 6 cells / mL. To seed the scaffolds, 10 mL of the cell suspension was added to the medium reservoir bottle, a total of 10×10 6 cells / scaffold. The perfusion flow rate was adjusted to 10 mL / min for cell seeding and the cell seeding was monitored for 3 hours, as Figure 9A shown, and then the medium flow rate was reduced to 1 mL / min. Figure 9A A graph showing the number of cells counted in the recycled medium over time, with the downward slope indicating the decrease in HEK293T cells in the medium over time, indicating that the cells were successfully seeded into the foam scaffolds. After overnight perfusion in the bioreactor, the bioreactor cartridges were opened to collect the scaffolds and the spheroid formation was evaluated by staining the live cells with calcein AM. The top and bottom of the scaffolds were observed, as Figure 10 shown. Figure 10 The resulting spheroids formed one day after seeding at the top and bottom of the two bioreactors are shown. Spheroids were present in both scaffolds and throughout each scaffold from the bottom to the top. As Figure 9B shown, cell counts after scaffold digestion and spheroid dissociation showed a slight cell loss compared to the initial cells seeded into the medium reservoir bottle, with a cell recovery of 90% in reactor 1 and 60% in reactor 2. These data indicate that the scaffolds can be colonized by cells directly by perfusing a medium carrying suspended cells, and within the scaffolds, these cells form spheroids under perfusion.

[0198] Example 7

[0199] In this experiment, the hMSC spheroid formation and maintenance at a medium perfusion flow rate of up to 10 mL / min for 48 hours were evaluated for the optimal scaffold composition (pore and interconnect structure dimensions). Figures 6A to 6D An uncoated foam scaffold composition (FMXP005( Figure 6D ), XP64(Figure 6B ), XP76( Figure 6C ) and XP49( Figure 6A )) and inoculate hMSC cells in a perfusion bioreactor within the foam scaffold composition.

[0200] Inoculate cells as described for hMSC in Example 5, but inoculate 75 μL of a cell inoculation solution containing 2 × 10 6 cells / mL. After spheroid formation overnight, transfer the scaffold to the bioreactor cartridge. To test the spheroid maintenance dependence on possible perfusion flow rates, test each foam scaffold at perfusion flow rates of 1 mL / min and 10 mL / min. Figure 11 The first row in Figure 11 shows the resulting spheroids present after 48 hours in the bioreactor perfused at 1 mL / min. Figure 6D and 11 ), some hMSC adhesion to the scaffold was observed, especially at the perfusion flow rate of 10 mL / min. This may be due to the specific composition of this scaffold, or due to the smaller interconnect structure or the number of interconnect structures per pore, thus increasing the shear force / turbulence in this scaffold at this higher flow rate. Therefore, at least when using hMSC, other compositions may be preferred for spheroid formation and EV production. In addition, both the XP64 and XP49 foam scaffolds induced good spheroid formation at a perfusion flow rate of 1 mL / min, indicating that both scaffold compositions are suitable for spheroid formation. However, as Figure 12 shows, at a perfusion flow rate of 10 mL / min, more obvious spheroid 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 most likely due to the higher number of interconnect structures per pore in the XP64 scaffold. These data may indicate that for this scaffold composition, a higher perfusion flow rate may not be the optimal condition for stimulating EV and other particle production through spheroids. In addition, these data show that for certain applications, a specific scaffold composition with certain pore and interconnect structure dimensions may be preferred for maintaining spheroids within the scaffold at higher perfusion flow rates. Accordingly, embodiments of the present disclosure include adjusting the pore and / or interconnect structure dimensions according to the cell type or desired cell by-products.

[0201] Example 8

[0202] In this experiment, it was demonstrated that foam scaffolds according to embodiments of the present disclosure can be used to significantly increase extracellular vesicle (EV) and particle production from spheroids. For this example, only XP49 scaffold composition was used. Cell seeding (with hMSC) was performed as in Example 6. After spheroid formation overnight, the scaffolds were inoculated with RoosterCollect from RoosterBio, Inc. TM -EV medium (defined low particle medium) to remove any remaining EVs and other particles present in the medium. The scaffolds were then added to the bioreactor cartridge. TM -EV medium was added to the medium reserve bottle and the perfusion flow rate was adjusted to 10 mL / min or 2 mL / min. For 2D static conditions, use RoosterCollect TM - Wash the cells with EV medium and then in RoosterCollect under static conditions TM -EV medium for 48 hours. After 48 hours, the bioreactor was opened to collect the scaffolds and stained with calcein AM for spheroids or cells. In this example, the scaffolds were cultured at 10 mL / min ( Figure 13A ) and 2mL / min( Figure 13B ) flow rate perfused scaffolds. Cells showed normal shape under 2D static conditions ( Figure 13C ).

[0203] Collect conditional RoosterCollect TM -EV medium was processed to remove cell debris and large particles by centrifugation at 300 × g for 15 min and filtration through a 200 nm filter. This clarified medium was then concentrated using a 100 kDa ultrafiltration membrane and purified by filtration in an ExoQuick-TC TM EVs were isolated by overnight precipitation in PBS (System Biosciences) and centrifugation at 1500 × g for 30 min. 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 was performed by multi-angle dynamic light scattering (MADLS) and the results are shown in Figures 14A to 14C , 15A to 15C and 16. Figures 14A to 14C The size distribution of detected particles or EVs is shown based on the number (which is proportional to the abundance of particles in the sample). Figures 15A to 15CShows the concentration of particles / EVs for different images of the detected particles. MALDS combines the scattering angle information from Mie theory and the particle size distribution analysis from dynamic light scattering measurements in an integrated method. The lower noise and thus reduced smoothing enable a reliable and accurate representation of the particle size distribution and a good characterization of the individual components in a multi-component sample. Using the MADLS method, a population of extracellular vesicles with an average diameter of approximately 100 nm was identified, as Figures 14A to 14C shown in 15A to 15C. The abundance of this population in the perfused sample at 10 mL / min ( Figure 14A and 15A ) was significantly higher than that in the perfused sample at 2 mL / min ( Figure 14B and 15B ) or in the samples from static culture conditions ( Figure 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 Figure 16 shown, compared to the 2D growth condition, the perfused 3D scaffolds produced a higher EV count per million cells after 48 hours.

[0204] Similar results were observed by quantifying CD63-positive EVs (a specific EV population) by enzyme-linked immunosorbent assay (ELISA), as Figure 17 shown. Compared to the 2D environment, the perfused foam scaffolds showed a higher number of CD63-positive EVs per million cells, with the higher perfusion rate of 10 mL / min having a significantly higher number of EVs per million cells. The presence of EVs in these samples was also confirmed by Western blot analysis against the CD81 and TSG101 markers (two other markers of the EV population), as Figure 18 shown. Figure 18 The Western blot analysis in shows the results of CD81 and TSG101 for the foams used in the 10 mL / min perfused reactor and the static 2D culture plates.

[0205] Next, the functionality of these EVs was evaluated by the HT-1080 cell wound healing assay. The wound healing assay is a method for studying directed 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 at set time points or at regular intervals at the start and during cell migration to close the wound, and comparing the images to quantify the migration rate of the cells. After creating a wound on a tissue culture plate, 2 × 10 from the 10 mL / min perfused sample (using the XP49 foam scaffold) or from the 2D sample 9EV treatment wells, or treated only with phosphate buffered saline (PBS) as a control. Figure 19 Shows bright field phase contrast micrographs of cells immediately after creating the wound (0 hours) for both PBS-treated samples and EV-treated samples, followed by photographs of each sample 8 hours later. A significant increase in wound healing rate was observed when treating with EVs from a 10 mL / min perfused sample compared to PBS-treated wells, as Figure 20 shown.

[0206] In Figure 20 the wound healing percentages over time of PBS-treated samples, samples treated with EVs from hMSC spheroids in the foam scaffolds in the perfusion bioreactor (XP49, 10 mL / min flow rate), and samples treated with EVs from adherent hMSC spheroids on a 2D surface were plotted. Compared to PBS-treated samples, samples treated with EVs showed higher wound healing percentages, and samples treated with EVs from the perfusion bioreactor showed the best wound healing with a significant advantage. Thus, as Figure 20 shown, EVs from perfused samples are shown to be functional and even more effective than EVs from 2D samples. These data indicate that with a low-nutrient medium determined by perfusion components (such as RoosterCollect TM -EV medium), hMSC spheroids can be maintained in the scaffolds. Additionally, increasing the perfusion flow rate results in increased production of particles and EVs from these spheroids. The presence of EVs and their quality and quantity were confirmed.

[0207] Example 9

[0208] This example compares EV production from hMSC spheroids in different culture vessels that generate EVs through the scaffolds of the present disclosure. It determines the effect of shear stress on the ability of hMSC spheroids to produce large amounts of functional EVs when using perfusion of the scaffolds of the present disclosure, agitation, or classical 2D culture conditions (i.e., static conditions).

[0209] More specifically, the scaffold composition XP49 was used to evaluate EV production from hMSC spheroids under perfusion of the scaffolds of the present disclosure. It was then compared to EV production from hMSC spheroids in a container with microcavities under agitation (35 rpm) and in a T-75 flask (used as classical 2D static conditions).

[0210] hMSC cell seeding on the scaffolds of the present disclosure was performed as in Examples 6 and 7. For the container with microcavities with agitation, cell seeding was performed according to the manufacturer's recommendations. For the T-75 Flasks were inoculated with approximately 9×10 3 cells / cm 2 . All vessels and scaffolds were inoculated simultaneously. After allowing the vessels to stand overnight to form spheroids, the scaffolds were washed with RoosterCollect TM -EV medium (a defined low-particle medium, RoosterBio M2001) to remove EVs and other particles present in the medium.

[0211] For experiments using scaffolds, the scaffolds were then added to the bioreactor device as shown in the Figure 5B example. RoosterCollect TM -EV medium was added to the medium reservoir bottle, and the perfusion flow rate was adjusted to 10 mL / min. RoosterCollect TM -EV medium was also added to the vessel with microcavities with stirring and to a T-75 flask. For the vessel with microcavities with stirring and the T-75 flask, the cells were washed with RoosterCollect TM -EV medium and then incubated in RoosterCollect -EV medium for 48 hours under stirring at 35 rpm (microcavity vessel) or static conditions (T-75 TM flask).

[0212] After 48 hours under perfusion, stirring, or static conditions, the bioreactor was opened to collect the scaffolds and spheroids, and the cells were stained with calcein AM (results shown in Figures 21A to 21C ). Figure 21A Spheroids in the scaffold after perfusion conditions are shown. Figure 21B Spheroids in the microcavity vessel after stirring conditions are shown. Figure 21C Spheroids in the T-75 flask after static conditions are shown. Spheroids were observed in the scaffolds under 10 mL / min perfusion flow rate conditions, in the microcavity vessel with stirring conditions, and in 2D static conditions.

[0213] Conditioned RoosterCollect TM -EV medium was collected and processed similar to Example 8 to remove cell debris and large particles, then concentrated, EVs were isolated and resuspended in sterile Dulbecco's phosphate-buffered saline (dPBS). Each EV sample was then analyzed using the same technique as in Example 8 to evaluate particle size, concentration, EV quality, and functionality.

[0214] The MADLS method identified a population with a diameter of approximately 100 nm (average) under all conditions ( Figures 22A to 22C ). Figures 22A to 22C Triplicate measurements of the same samples for each of the perfusion, agitation, and static condition experiments are shown. This particle population was significantly more abundant in the perfusion samples at 10 mL / min than in the agitated microchamber vessels or 2D static conditions ( Figure 22D ). ELISA analysis quantifying CD63-positive EVs in these samples indicated no significant difference between the perfusion samples and the agitated microchamber samples. However, significantly more CD63-positive EVs were detected in the perfusion samples compared to the 2D static samples, and significantly more CD63-positive EVs were detected in the agitated microchamber samples compared to the 2D static samples ( Figure 23A ). The presence of EVs in these samples was also confirmed by Western blot analysis against CD81 and TSG101 ( Figure 23B ). Figures 23A to 23B Data from ELISA CD63 and Western blot (using CD81 and TSG-101 markers) experiments are shown, where the data are from four independent experiments and each point represents a sample. Points of the same color belong to the same experiment.

[0215] The functionality of these EVs was evaluated by the HT-1080 cell wound healing assay performed as described in Example 8, and after creating the wound, each well was treated with 2 × 10 9 EVs from the perfusion samples at 10 mL / min, the agitated microchamber samples, or the static condition samples. Controls were also performed where the wounds were treated with only phosphate buffered saline (without EVs) as a negative control.

[0216] A significant increase in the wound healing rate was observed for EVs from hMSC spheroids. EVs from the perfusion samples at 10 mL / min and the agitated or static condition microchamber samples caused faster wound healing compared to the PBS-treated wells (Figure 38). EVs from the perfusion samples were slightly more effective than EVs from the static condition samples ( Figure 24 ).

[0217] The results indicate that hMSC spheroids produce significantly more EVs per cell under a certain shear stress than classical 2D cultures. Additionally, according to the ELISA data, hMSC spheroids under perfusion produce significantly more particles than hMSC spheroids under agitation and 2D static conditions, but do not produce significantly more CD63-positive EVs compared to the microchamber condition. Furthermore, EVs from hMSC spheroids under perfusion or agitation conditions appear to be more effective in the wound healing assay than EVs from 2D static conditions.

[0218] Example 10

[0219] This example compared the EV production of hMSCs in the scaffold at perfusion flow rates higher and lower than those previously evaluated. The results provided the lower and upper limits of the perfusion flow rate that provides a higher EV yield per cell in EV production.

[0220] In this experiment, only the scaffold composition XP49 was used to evaluate the EV production from hMSC spheroids under perfusion. Similar to Examples 7 and 8, this scaffold was selected because it showed the best spheroid maintenance at high perfusion flow rates compared to other scaffolds previously tested. As a control, EVs were also produced from hMSCs adhered to a T-75 flask under 2D static conditions.

[0221] The scaffolds were seeded with hMSCs as in Examples 7 and 8. For the T-75 flask, approximately 9×10 3 cells / cm 2 were seeded. All vessels and scaffolds were seeded simultaneously. After overnight spheroid formation, the scaffolds were washed with RoosterCollect TM -EV medium (a defined low particle medium, RoosterBio M2001) to remove all remaining EVs and other particles present in the medium. The scaffolds were then added to the bioreactor cartridge. RoosterCollect TM -EV medium was added to the medium reservoir bottle, and the perfusion flow rate was adjusted to the different flow rates tested. Here, EV production was evaluated at 20, 10, 2, and 0.5 mL / min. After washing twice with RoosterCollect TM -EV medium, EV collection medium was also added to the T-75 flask.

[0222] After 48 hours of perfusion, EV production was evaluated. The bioreactor was opened to collect the scaffolds, and the spheroids were stained with calcein AM. For the 2D condition, the cells were observed without further staining.

[0223] As previously observed, no strong differences in spheroid shape or density in the scaffold were observed between the 10 mL / min and 2 mL / min perfusion conditions ( Figure 25 ). Similarly, no differences were observed under the 0.5 mL / min perfusion condition. However, under the 20 mL / min perfusion condition, almost half of the spheroids seemed to have lost their morphology and seemed to adhere to or contact the scaffold ( Figure 25 ). As observed in the previous examples, hMSCs under 2D conditions showed the expected spindle-like morphology ( Figure 25 ).

[0224] In addition to spheroid and cell morphology observations, conditioned RoosterCollect TM -EV medium was collected. To clarify the medium, the collected conditioned medium was centrifuged at 300 × 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 large particles. For this experiment, the clarified medium was also evaluated by MADLS to determine the size and concentration of EVs produced by hSMCs. For all conditions, the average diameter of the EVs was approximately 100 nm ( Figure 26A ). Regarding the particle concentration, a strong difference was observed between different conditions, up to approximately 2 × 10 11 particles / mL under the 20 mL / min perfusion condition, and approximately 1.23 × 10 11 particles / mL under the 10 mL / min perfusion condition ( Figure 26B ). These numbers decreased under other conditions, only approximately 3.28 × 10 10 particles / mL under the 2 mL / min perfusion condition, and 5.38 × 10 9 particles / mL under the 0.5 mL / min perfusion condition. However, the concentration could not be calculated for the 2D condition because the concentration of these particles was below the threshold of the equipment.

[0225] This clarified medium was then ultrafiltered to concentrate the EVs, and then the EVs were precipitated overnight in as described previously. The next day, all these samples were centrifuged at 1500 × g for 30 minutes, and the EVs were resuspended in sterile PBS.

[0226] These purified EVs were analyzed again by MADLS to determine their size and concentration. As in the clarified medium, the EVs had an average diameter of approximately 100 nm ( Figure 27A ). Regarding the amount of EVs produced, no strong difference was observed between the 20 mL / min and 10 mL / min perfusion conditions ( Figure 27B ). However, when compared with other conditions, under the 2 mL / min perfusion condition, there were approximately 7-fold fewer EVs than under the 10 or 20 mL / min perfusion conditions, and under the 0.5 mL / min perfusion condition, there were more than 10-fold fewer EVs compared with these higher flow rate conditions ( Figure 27B ). More interestingly, approximately 100-fold fewer EVs were produced under the 2D condition compared with these higher flow rate conditions ( Figure 27B ).

[0227] For the amount of EVs produced per cell number under each condition ( Figure 27C), similar results were observed, where the number of EVs produced was highest under perfusion conditions of 20 and 10 mL / min, and lowest under 2D conditions, but the EVs per million cells were 1000-fold less under 2D conditions compared to the 10 mL / min perfusion condition.

[0228] Next, these samples were further analyzed to determine the amount of CD63-positive EVs by ELISA. Regarding the total CD63-positive EVs ( Figure 28A ), the highest content of CD63-positive EVs was observed under perfusion conditions of 20 mL / min and 10 mL / min. No strong difference in CD63-positive EVs was observed between the lowest perfusion flow rates at 2 and 0.5 mL / min and the 2D condition ( Figure 28A ). However, compared to the 2D condition, the total CD63-positive EVs per million cells ( Figure 28B ) showed a slightly increased ~2-fold more CD63-positive EVs at these lowest perfusion flow rates.

[0229] The functionality of these EVs was also evaluated by the HT-1080 cell wound healing assay ( Figure 29 ). Here, this analysis was performed slightly differently than before. Wounds were created with a P1000 pipette tip, and wound healing was only evaluated 6 hours after injury. Each wound (except the control) was treated with 2×10 9 EVs from each condition. The control was treated with PBS only. Images were taken from each wound at 0 and 6 hours after injury, and the distance between the edges was measured using software (Olympus LS). At 6 hours, the distance between the two edges was compared to the distance at 0 hours to calculate the percentage of wound closure or wound healing. All perfusion conditions showed faster wound healing than the mock control ( Figure 29 ). Additionally, it appears here that increasing the perfusion flow rate and also compared to the 2D condition caused faster wound healing, indicating that other EVs besides CD63-positive EVs may play a role in the wound healing rate.

[0230] In summary, the data indicate that increasing the flow rate in perfused samples results in higher EV production. However, the upper limit appears to be approximately 10 to 20 mL / min, and the lower limit is even less than approximately 0.5 mL / min, as EV production increases even at this low flow rate compared to the 2D condition. Additionally, EVs produced by perfusion are functional and improve the wound healing rate even at higher flow rates, such as 20 mL / min, compared to the control mock treatment condition.

[0231] Although this disclosure includes a limited number of embodiments, those skilled in the art will appreciate that other embodiments can be designed that do not depart from the scope of this disclosure.

Claims

1. A method for generating spheroids or by-products of spheroids, comprising: Providing a bioreactor comprising a cavity for culturing cells, an inlet and an outlet of the cavity, and a porous scaffold; Inserting cells of a certain cell type into the porous scaffold to form spheroids; and perfusion of a cell culture medium through the cavity to culture the spheroids; wherein the porous scaffold comprises pores and channels between the pores.

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

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

4. The method according to any one of claims 1 to 3, wherein inserting the cells of the certain cell type into the porous scaffold comprises inoculating the cells into the porous scaffold.

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

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

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

8. The method according to claim 7, wherein the by-product of the collected 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 porous scaffold material is treated to be non-adhesive to cells, or both of the above are satisfied.

10. The method according to any one of claims 1 to 9, wherein the porous scaffold comprises ionically crosslinked alginic acid or its salt, or an ionically crosslinked polygalacturonic acid compound selected from at least one of the following: pectic acid, partially esterified pectic acid, partially amidated pectic acid, and their salts.

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

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

13. The method according to any one of claims 1 to 11, wherein when measured in the dry scaffold, at least 70% of the channels have a maximum channel width of from about 30 μm to about 500 μm.

14. The method according to any one of claims 1 to 11, wherein when measured in the dry scaffold, at least 80% of the channels have a maximum channel width of from about 60 μm to about 400 μm.

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

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

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

18. The method according to any one of the preceding claims, wherein the porous scaffold is soluble.

19. The method according to claim 18, further comprising digesting the soluble porous scaffold by exposing the soluble porous scaffold to an enzyme.

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

21. The method according to claim 19 or claim 20, wherein digestion of the soluble porous scaffold is completed in less than about 1 hour.

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

23. The method according to claim 22, wherein the non-proteolytic enzyme is selected from the group consisting of pectin hydrolase, pectinase, and alginate lyase.

24. The method according to any one of claims 19 to 23, wherein digesting the soluble porous scaffold comprises exposing the soluble foam scaffold to the enzyme in an amount between about 1 U and about 200 U.

25. The method according to any one of claims 20 to 24, comprising exposing the soluble porous scaffold to the chelating agent in an amount between about 1 mM and about 200 mM.

26. A bioreactor, comprising: A cavity for culturing cells; An inlet and an outlet of the cavity; and The porous scaffold in the cavity; wherein the porous scaffold comprises pores and channels between the pores; wherein the porous scaffold is non - adhesive to cells; and wherein the porous scaffold is configured to accommodate spheroids.

27. The bioreactor according to claim 26, wherein when measured in the dry scaffold, at least 75% of the pores have a pore diameter of about 200 μm to about 1000 μm.

28. The bioreactor according to claim 26, wherein when measured in the dry scaffold, at least 75% of the channels have a maximum channel width of about 30 μm to about 500 μm.

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

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

31. The bioreactor according to claim 30, wherein the soluble porous scaffold is a foam scaffold.

32. The bioreactor according to claim 31, wherein the soluble foam scaffold is dissolved by pectinase or alginate lyase.

33. The bioreactor according to any one of claims 26 to 32, wherein the porous scaffold has a composition comprising: Ion - crosslinked polysaccharides selected from alginic acid and its salts, pectic acid and its salts, partially esterified pectic acid and its salts, partially amidated pectic acid and its salts, or combinations thereof.

34. The bioreactor according to claim 33, wherein the ion - crosslinked polysaccharide is polygalacturonic acid.

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

36. The porous scaffold according to claim 35, wherein the cells of a certain cell type are selected from primary cell lines and immortalized cell lines.

37. The porous scaffold according to claim 36, wherein the primary cell line is human mesenchymal stem cells derived from bone marrow and the immortalized cell line is HEK293T cells.

38. A perfusion bioreactor, comprising: A cavity for culturing cells; An inlet and an outlet of the cavity; and A porous scaffold in the cavity, wherein the porous scaffold is non-adhesive to cells; Wherein the porous scaffold comprises pores and channels between the pores, Wherein when measured in the dry scaffold, at least 75% of the pores have a pore diameter of about 200 μm to about 1000 μm, Wherein when measured in the dry scaffold, at least 75% of the channels have a maximum width of about 30 μm to about 500 μm, Wherein at least 75% of the pores have 5 to 18 channels / pore; and Wherein the porous scaffold is suitable for growing spheroids or by-products of spheroids.

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

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

41. The perfusion bioreactor according to claim 35, wherein the bioreactor is suitable for producing extracellular vesicles as by-products of the spheroids.

42. The perfusion bioreactor according to claim 41, wherein when measured by the wound healing assay of HT-1080 cells, at 6 hours after injury, the wound healing by the extracellular vesicles produced by the perfusion bioreactor is greater than the wound healing by the extracellular vesicles produced in a 2D flask for 3D cultures.

43. The perfusion bioreactor according to claim 41, wherein when measured by the wound healing assay of HT-1080 cells, at 6 hours after injury, the wound closure achieved by the wound healing of about 2×10 9 extracellular vesicles produced by the perfusion bioreactor is at least 5% better than the wound healing of 2×10 9 extracellular vesicles produced in a 2D flask for 3D cultures.

44. The perfusion bioreactor according to claim 41, wherein the extracellular vesicles produced by the perfusion bioreactor are produced in an amount between about 1×10 3 EVs / cell and about 1×10 7 EVs / cell.

45. The perfusion bioreactor according to claim 44, wherein the extracellular vesicles are produced by perfusion having a linear velocity between about 1.1×10 - 5 m / s and about 5.0×10 -4 m / s.

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

47. The perfusion bioreactor according to claim 44, wherein the number of extracellular vesicles per cell produced with the perfusion bioreactor is greater than the number of extracellular vesicles produced with a static 2D microplate.

48. The method according to claim 1, wherein the porous scaffold is non-adhesive to the cells when perfused at a linear velocity between about 2.3×10 -6 m / s and about 1.2×10 -3 m / s.

49. The perfusion bioreactor according to claim 44, wherein the extracellular vesicles are produced by perfusion having a linear velocity between about 2.3×10 - 6 m / s and about 1.2×10 -3 m / s.

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