Dissolvable foam scaffold for cell culture and method of manufacturing same

Abstract

Provided herein is a dissolvable foam scaffold for cell culture, which comprises an ionotropically crosslinked polygalacturonic acid compound selected from at least one of pectinic acid, partially esterified pectinic acid, partially amidated pectinic acid, and salts thereof, and a water-soluble polymer that is surface-active and has a hydrophilic-lipophilic balance (HLB) greater than about 20.

Description

[Technical Field]

[0001] Cross-reference to related art This application claims the benefit of priority under 35 U.S.C. § 119 to U.S. Provisional Patent Application No. 63 / 425,127, filed November 14, 2022, the contents of which are relied upon and incorporated by reference herein in its entirety.

[0002] The present disclosure relates generally to dissolvable cell culture materials. Specifically, the present disclosure relates to dissolvable foam scaffolds for cell culture and methods for making such dissolvable foam scaffolds. [Background technology]

[0003] In vitro research is important for the drug discovery process and the search for potential new therapeutics. However, traditional in vitro cell culture on two-dimensional (2D) culture substrates fails to simulate the in vivo environment. Because nearly all cells in an in vivo environment are surrounded by other cells and extracellular matrix (ECM) in a three-dimensional (3D) manner, 2D cell culture does not adequately simulate a cell's natural 3D environment. Cells in 2D culture are forced to adhere to a rigid surface, are geometrically constrained, and adopt a flat morphology. This alters cytoskeletal regulation, which is important for intracellular signaling, and can consequently affect cell growth, migration, and apoptosis. Furthermore, the organization of the ECM, which is important for cell differentiation, proliferation, and gene expression, is absent in most 2D cells. These limitations of 2D culture often result in in vitro biological responses that differ significantly from those observed in vivo.

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

[0005] Recent studies suggest that 3D cell culture, in contrast to 2D culture, more accurately represents the environment experienced by cells in vivo, demonstrating that cellular responses in 3D culture are more similar to in vivo behavior than cellular responses in 2D culture. The additional dimensionality of 3D culture is thought to lead to differences in cellular responses because it not only affects the spatial organization of cell surface receptors involved in interactions with surrounding cells, but also imposes physical constraints on cells. These spatial and physical aspects of 3D culture are thought to affect signaling from the outside to the inside of cells, ultimately affecting gene expression and cellular behavior.

[0006] The benefits of 3D culture have inspired the development of cell culture technologies that simulate the cells' natural 3D environment. Some bioreactors contain carriers in the form of fixed packing material, forming a fixed or packed bed to promote cell attachment and growth. The packing arrangement of the fixed bed influences localized fluid, heat, and mass transfer and is typically very dense to maximize cell culture in a given space. Yet another 3D cell culture technology is the creation of porous 3D matrices or scaffolds that promote the growth and proliferation of cultured cells within the pores and other interior spaces of the matrix.

[0007] In each of the above techniques, protease treatment can be used to harvest cells. However, commonly used harvesting procedures, such as protease treatment, expose cells to harsh conditions that can damage cellular structure and function. In addition, protease treatment alone often results in only a limited amount of cell detachment. In the case of fixed-bed materials, the problem is due, in part, to the densely packed nature of the fixed-bed material, which makes it more difficult to circulate the protease agent throughout the bed to increase the yield of harvested cells. Similarly, it can be difficult to circulate the protease agent throughout the interior space of the 3D matrix, which in turn makes it difficult to remove cells during the harvesting process. This difficulty is exacerbated by the presence of extracellular macromolecules secreted by cultured cells that serve to attach cells to the surface of the fixed-bed material or matrix.

[0008] Alternatively, or in combination with protease treatment, methods and systems for cell harvesting have been developed that apply mechanical force to release cultured cells from a fixed bed material or 3D matrix. For example, the fixed bed material or 3D matrix, or a larger system containing the fixed bed material or 3D matrix, can be shaken or vibrated to release the cultured cells. The application of mechanical force can physically damage the cultured cells, which can in turn reduce cell culture yield. Summary of the Invention

[0009] According to embodiments of the present disclosure, a dissolvable foam scaffold for cell culture is provided herein. The dissolvable foam scaffold comprises an ionotropically crosslinked polygalacturonic acid compound selected from at least one of pectinic acid; partially esterified pectinic acid, partially amidated pectinic acid, and salts thereof; and a water-soluble polymer having surface activity and a hydrophilic-lipophilic balance (HLB) greater than about 20. In embodiments, the HLB of the water-soluble polymer is about 22 or greater. In embodiments, the dissolvable foam scaffold is produced from a simplified formulation containing 0% by weight of a water-soluble plasticizer. The dissolvable foam scaffold does not contain glycerol, sorbitol, ethylene glycol, propylene glycol, or polyethylene glycol. According to aspects of the embodiment, the dissolvable foam scaffold may contain only a single water-soluble polymer having surface activity and may not contain any polymers that do not have surface activity.

[0010] According to embodiments of the present disclosure, a method for forming a dissolvable foam scaffold is provided herein. The method includes: adding a polygalacturonic acid compound selected from pectinic acid; partially esterified pectinic acid, partially amidated pectinic acid, and at least one of their salts to an aqueous solution to form a first aqueous mixture; adding a surface-active water-soluble polymer and a divalent metal salt to the aqueous solution to form a second aqueous mixture; combining the first aqueous mixture with the second aqueous mixture to form a combined aqueous mixture; adding a gel-inducing agent to the combined aqueous mixture to form a foamed solution; and introducing gas bubbles into the foamed solution to form a foamed scaffold. The water-soluble polymer has a hydrophilic-lipophilic balance (HLB) greater than about 20. In embodiments, the HLB of the water-soluble polymer is about 22 or greater. In some embodiments, forming the second aqueous mixture does not include adding a water-soluble plasticizer to the second aqueous mixture, and may not include adding a non-surface-active second polymer. According to further aspects of the embodiment, the method does not include adding an emulsifier to any of the first aqueous mixture, the second aqueous mixture, the combined aqueous mixture, and the foaming solution. According to embodiments, the method includes adding a first amount of gel-inducing agent to the aqueous mixture before foaming and adding a second amount of gel-inducing agent to the aqueous mixture during or after foaming. The ratio of the second amount of gel-inducing agent to the first amount of gel-inducing agent is greater than about 2, or about 7 or greater.

[0011] According to embodiments of the present disclosure, a method for culturing cells on a dissolvable foam scaffold is provided herein. The method includes seeding cells onto a dissolvable foam scaffold such that the cells enter the pores of the dissolvable foam scaffold, the dissolvable scaffold comprising an ionotropically crosslinked polygalacturonic acid compound selected from at least one of pectinic acid; partially esterified pectinic acid, partially amidated pectinic acid, and salts thereof, and a water-soluble polymer that is surface-active and has a hydrophilic-lipophilic balance (HLB) greater than about 20; and contacting the dissolvable foam scaffold with a cell culture medium. According to various aspects of the embodiments, the dissolvable foam scaffold has 0% by weight of a water-soluble plasticizer and is free of a non-surface-active polymer.

[0012] According to embodiments of the present disclosure, there is provided herein a foamed scaffold product formed from a composition comprising: an ionotropically crosslinked polygalacturonic acid compound selected from at least one of pectinic acid, partially esterified pectinic acid, partially amidated pectinic acid, and salts thereof; at least one first water-soluble polymer having surface activity and a hydrophilic-lipophilic balance (HLB) greater than about 20; and 0% by weight of a water-soluble plasticizer.

[0013] Additional features and advantages will be set forth in the following detailed description, and in part will be readily apparent to those skilled in the art from that description, or may be learned by practicing the embodiments described herein, including the following detailed description, claims, and accompanying drawings.

[0014] It is to be understood that both the foregoing general description and the following detailed description are merely exemplary and are intended to provide an overview or framework for understanding the nature and character of the claims. The accompanying drawings are included to provide a further understanding, and are incorporated into and constitute a part of this specification. The drawings illustrate one or more embodiments, and together with the description, serve to explain the principles and operation of the various embodiments.

[0015] The present disclosure will be more clearly understood from the following description and the accompanying drawings, given purely by way of non-limiting example. [Brief explanation of the drawings]

[0016] [Figure 1] FIG. 1 is a perspective view of a dissolvable foam scaffold according to the present disclosure. [Figure 2] 1 shows an SEM photograph of the foam scaffold prepared in Example 1. [Figure 3] 1 shows an SEM photograph of the foam scaffold prepared in Example 2. [Figure 4] 1 shows an SEM photograph of the foam scaffold prepared in Example 3. [Figure 5] 1 shows an SEM photograph of the foam scaffold prepared in Example 4. [Figure 6] 1 shows an SEM photograph of the foam scaffold prepared in Example 5. [Figure 7] 1 shows an SEM photograph of the foam scaffold prepared in Example 6. [Figure 8] 1 shows an SEM photograph of the foam scaffold prepared in Example 7. [Figure 9] 1 shows an SEM photograph of the foam scaffold prepared in Example 8. [Figure 10] 1 shows an SEM photograph of the foam scaffold prepared in Example 9. [Figure 11] 1 shows an SEM photograph of the foam scaffold prepared in Example 10. [Figure 12] 1 shows an SEM photograph of the foam scaffold prepared in Example 11. [Figure 13] 1 shows four foams prepared in Example 12 with varying amounts of plasticizer added to form four foam scaffolds. [Figure 14] 1 shows an SEM photograph of the foam scaffold prepared in Example 13. [Figure 15] 1 shows spheroids formed within the pores of the foam scaffold prepared in Example 4. [Figure 16] 1 shows cells attached to the foam scaffold prepared in Example 14. [Figure 17]1 shows cells attached to the foam scaffold prepared in Example 15. [Figure 18] 1 shows cells attached to a foam scaffold prepared in Example 15 after 6 days of growth. [Figure 19] 1 is a bar graph showing the GFP-positive rate of transfected HEK cells for each of the culture conditions in Example 24. [Figure 20] 10 is a bar graph showing the GFP-positive rate of transfected cells per foam scaffold set of Example 25. [Figure 21] 1 is a bar graph showing the number of viral particles (vp) per foam scaffold set for Example 25. [Figure 22] 10 is a bar graph showing the number of virus particles per cell obtained per foam scaffold set of Example 25. [Figure 23] 10 is a bar graph showing the percentage of cells showing GFP expression for each of the foam scaffold sets of Example 25 following infection. [Figure 24] 1 shows an SEM photograph of the foam scaffold prepared in Example 26. DETAILED DESCRIPTION OF THE INVENTION

[0017] Reference will now be made in detail to the present embodiment(s), example(s) of which are illustrated in the accompanying drawings. Wherever possible, the same reference numbers will be used throughout the drawings to refer to the same or like parts.

[0018] The singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. The endpoints of all ranges reciting the same characteristic are independently combinable and inclusive of the recited endpoint. All references are incorporated herein by reference.

[0019] As used herein, the words "have," "having," "include," "including," "comprise," "comprising," and the like are used in an open-ended sense and generally mean "including, but not limited to."

[0020] All scientific and technical terms used herein have meanings commonly used in the art unless otherwise specified. The definitions provided herein are intended to facilitate understanding of certain terms used frequently herein and are not intended to limit the scope of the present disclosure.

[0021] The present disclosure will be described below generally at first and then in detail based on several exemplary embodiments. The features shown in combination with each other in each exemplary embodiment do not necessarily have to be realized. In particular, each feature may be omitted or may be combined in some other way with other features shown in the same exemplary embodiment or other exemplary embodiments.

[0022] Embodiments of the present disclosure relate to dissolvable foam scaffolds for cell culture and methods for manufacturing such dissolvable foam scaffolds. Embodiments of the present disclosure further relate to cell culture methods for adhering cells, cell aggregates, or spheroids on dissolvable foam scaffolds. Furthermore, embodiments of the present disclosure relate to bioreactor systems including dissolvable foam scaffolds. As will become apparent from the following discussion, the foam scaffolds disclosed herein are described as both dissolvable and insoluble. As used herein, the term "insoluble" refers to a material or combination of materials that is not soluble and remains crosslinked under conventional cell culture conditions, including, for example, cell culture medium. Also, as used herein, the term "dissolvable" refers to a material or combination of materials that is digested when exposed to an appropriate concentration of enzyme that digests or degrades the material or combination of materials. The dissolvable foam scaffolds described herein are porous scaffolds with an open pore structure and highly interconnected pores. The pores of the scaffold provide a protected environment for cell culture, supporting cell-cell interactions and the formation of extracellular matrix (ECM) in a 3D format. The dissolvable foam scaffold is completely digested and cells can be harvested using protease treatment and / or mechanical harvesting techniques without damaging the cells.

[0023] FIG. 1 is a perspective view of a dissolvable foam scaffold 10 according to the present disclosure. As will be described in more detail below and become more apparent from other figures of the present disclosure, the dissolvable foam scaffold 10 is a porous foam comprising an open pore structure. The dissolvable foam scaffold 10 has a porosity of about 85% to about 96% and an average pore size of about 50 μm to about 500 μm. The dissolvable foam scaffold 10 provides a protected environment within the pores of the foam scaffold for the cultivation of cells. Additionally, the dissolvable foam scaffold 10 is also dissolvable when exposed to appropriate enzymes that digest or degrade the material, facilitating the recovery of cells cultured in the scaffold without damaging the cells.

[0024] The dissolvable foam scaffolds described herein contain at least one ionotropically cross-linked polysaccharide. Generally, polysaccharides have beneficial properties 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 pectinic acid or its salts, or partially amidated pectinic acid or its salts. Pectin can be formed by hydrolysis of certain pectin esters. Pectin is a cell wall polysaccharide that naturally plays a structural role in plants. Major sources of pectin include citrus peels (e.g., lemon and lime peels) and apple peels. Pectin is a primarily linear polymer based on a 1,4-linked alpha-D-galacturonate backbone randomly interrupted by 1,2-linked L-rhamnose. Its average molecular weight ranges from about 50,000 to about 200,000 daltons.

[0025] The polygalacturonic acid chains of pectin may be partially esterified, e.g., with methyl groups, and the free acid groups may be partially or completely neutralized with monovalent ions such as sodium, potassium, or ammonium ions. Polygalacturonic acid partially esterified with methanol is called pectinic acid, and its salts are called pectinates. The degree of methylation (DM) of high-methoxyl (HM) pectin can be, for example, 60-75 mol%, while the degree of methylation of low-methoxyl (LM) pectin can be 1-40 mol%. The degree of esterification of the partially esterified polygalacturonic acid described herein can be less than about 70 mol%, or less than about 60 mol%, or less than 50 mol%, or even less than about 40 mol%, and all values ​​therebetween. Without being bound by theory, it is believed that a minimum amount of free carboxylic acid groups (unesterified) promotes some degree of ionotropic crosslinking, allowing for the formation of an insoluble, dissolvable scaffold.

[0026] Alternatively, the polygalacturonic acid chains of pectin may be partially amidated. Pectin with partially amidated polygalacturonic acid can be produced, for example, by treatment with ammonia. Amidated pectin contains carboxyl groups (-COOH), methyl ester groups (-COOCH), and amidated groups (-CONH). The degree of amidation may vary, for example, from about 10% to about 40%.

[0027] According to embodiments of the present disclosure, the dissolvable foam scaffolds described herein may comprise a mixture of pectinic acid and partially esterified pectinic acid. Blends with compatible polymers can also be used. For example, pectinic acid and / or partially esterified pectinic acid can be mixed with other polysaccharides, such as dextran, substituted cellulose derivatives, alginic acid, starch, glycogen, arabinoxylan, and agarose. Glycosaminoglycans, such as hyaluronic acid and chondroitin sulfate, or various proteins, such as elastin, fibrin, silk fibroin, collagen, and their derivatives, can also be used. Water-soluble synthetic polymers can also be blended with pectinic acid and / or partially esterified pectinic acid. Exemplary water-soluble synthetic polymers include, but are not limited to, polyalkylene glycols, poly(hydroxyalkyl(meth)acrylates), poly(meth)acrylamides and derivatives, poly(N-vinyl-2-pyrrolidone), and polyvinyl alcohol.

[0028] According to embodiments of the present disclosure, the dissolvable foam scaffolds described herein may further comprise at least one first polymer. The at least one first polymer is water-soluble, nonionotropically crosslinkable, and surface-active. As used herein, the term "surface-active" refers to the activity of an agent to reduce or eliminate surface tension (or interfacial tension) between two liquids, between a liquid and a solid, or between a gas and a liquid. The at least one first polymer may have a hydrophilic-lipophilic balance (HLB) greater than about 8, or even greater than about 10. For example, the at least one first polymer may have an HLB of about 8 to about 40, or about 10 to about 40. The at least one first polymer may have an HLB of about 8 to about 15, or even about 10 to about 12. HLB provides a measure of the degree of lipophilicity or hydrophilicity of a polymer. A higher HLB value indicates greater hydrophilicity, and a lower HLB value indicates greater lipophilicity. Generally, HLB values ​​range from 1 to 40, with the hydrophilic-lipophilic transition often considered to be about 8 to about 10. If the HLB value is less than the hydrophilic-lipophilic transition, the material is lipophilic; if the HLB value is greater than the hydrophilic-lipophilic transition, the material is hydrophilic.

[0029] Exemplary first polymers according to embodiments of the present disclosure may be any of cellulose derivatives, proteins, synthetic amphiphilic polymers, and combinations thereof. Exemplary cellulose derivatives include, but are not limited to, hydroxyethyl cellulose (HEC), hydroxypropyl cellulose (HPC), methyl cellulose (MC), hydroxyethyl methyl cellulose (HEMC), and hydroxypropyl methyl cellulose (HPMC). Exemplary proteins include, but are not limited to, bovine serum albumin (BSA), gelatin, casein, and hydrophobin. Exemplary synthetic amphiphilic polymers include, but are not limited to, poloxamers available under the tradename Synperonics® (commercially available from Croda International, Snaith, UK), poloxamers available under the tradename Pluronics® (commercially available from BASF Corp., Parsippany, New Jersey, USA), and poloxamers available under the tradename Kolliphor® (commercially available from BASF Corp., Parsippany, New Jersey, USA).

[0030] The dissolvable foam scaffolds described herein may further comprise at least one second polymer. The at least one second polymer is water-soluble and not surface-active. Exemplary second polymers may be synthetic polymers, semi-synthetic polymers, natural polymers, or combinations thereof. Exemplary synthetic polymers include, but are not limited to, polyethylene glycol, polyvinylpyrrolidone, polyvinyl alcohol, carboxyvinyl polymers, polyacrylic acid, polyacrylamide, N-(2-hydroxypropyl)methacrylamide homopolymers and copolymers, polyvinyl methyl ether-maleic anhydride, and polyethylene oxide / polypropylene oxide block copolymers. Exemplary semi-synthetic polymers include, but are not limited to, dextran derivatives, carboxymethylcellulose, hydroxyethylcellulose and derivatives, methylcellulose and derivatives, ethylcellulose, ethylhydroxyethylcellulose, and hydroxypropylcellulose. Exemplary natural polymers include, but are not limited to, starch and starch derivatives, polymers obtained by microbial fermentation such as curdlan, pullulan, and gellan gum, xanthan gum, dextran, proteins such as albumin, casein, and caseinates, gelatin, seaweed extracts such as agar, alginates, and carrageenan, seed extracts such as guar gum and derivatives and locust bean gum, hyaluronic acid, and chondroitin sulfate.

[0031] The dissolvable foam scaffolds described herein can be crosslinked to increase their mechanical strength and prevent dissolution of the scaffolds when placed in contact with cell culture medium. Crosslinking can be achieved by ionotropic gelation, as described below, which is based on the ability of polyelectrolytes to crosslink to form crosslinked scaffolds in the presence of multivalent counterions. Without being bound by theory, it is believed that the ionotropic gelation of the polysaccharides of the dissolvable foam scaffolds is the result of strong interactions between divalent cations and the polysaccharides.

[0032] According to embodiments of the present disclosure, the scaffolds described herein are porous foam scaffolds. The foam scaffolds described herein may have a porosity of about 85% to about 96%. For example, the foam scaffolds described herein may have a porosity of about 91% to about 95%, or about 94% to about 96%. As used herein, the term "porosity" refers to a measure of the open pore volume of a dissolvable scaffold and is referred to in terms of % porosity, where % porosity is the percentage of voids in the total volume of the dissolvable foam scaffold. The foam scaffolds described herein may have an average pore size of about 50 μm to about 500 μm. For example, the average pore size may be about 75 μm to about 450 μm, or about 100 μm to about 400 μm, or even 150 μm to about 350 μm, and all values ​​therebetween.

[0033] The scaffolds described herein may have a wet density of less than about 0.40 g / cc. For example, the scaffolds described herein may have a wet density of less than about 0.35 g / cc, or less than about 0.30 g / cc, or less than about 0.25 g / cc. The scaffolds described herein may have a wet density of about 0.16 g / cc to about 0.40 g / cc, or about 0.16 g / cc to about 0.35 g / cc, or about 0.16 g / cc to about 0.30 g / cc, or even about 0.16 g / cc to about 0.25 g / cc, and all values ​​therebetween. The scaffolds described herein may have a dry density of less than about 0.20 g / cc. For example, the scaffolds described herein may have a dry density of less than about 0.15 g / cc, or less than about 0.10 g / cc, or less than about 0.05 g / cc. The scaffolds described herein may have a dry density of from about 0.02 g / cc to about 0.20 g / cc, or from about 0.02 g / cc to about 0.15 g / cc, or from about 0.02 g / cc to about 0.10 g / cc, or even from about 0.02 g / cc to about 0.05 g / cc, and all values ​​therebetween.

[0034] Several pore types are possible in scaffolds. Open pores allow cell access on both sides of the scaffold and allow fluid flow and nutrient transport through the dissolvable scaffold. Partially open pores allow cell access on one side of the scaffold, but nutrient and waste mass transport is limited to diffusion. Closed pores have no openings and do not allow access by cells or nutrient and waste mass transport. The dissolvable foam scaffolds described herein have an open pore structure and highly interconnected pores. Generally, the open pore structure and highly interconnected pores allow cells to migrate into the pores of the dissolvable foam scaffold and also facilitate the mass transport of nutrients, oxygen, and waste products. The open pore structure also influences cell adhesion and migration by providing a large surface area for cell-cell interactions and space for ECM regeneration.

[0035] The dissolvable foam scaffolds described herein are digested when exposed to appropriate enzymes that digest or degrade materials. Non-proteolytic enzymes suitable for digesting foam scaffolds, recovering cells, or both include pectinolytic enzymes, or pectinases, a heterogeneous group of related enzymes that hydrolyze pectinic substances. Pectinases (polygalacturonases) are enzymes that break down complex pectin molecules into shorter molecules of galacturonic acid. Commercially available sources of pectinases are generally multienzymes, such as Pectinex™ ULTRA SP-L (commercially available from Novozyme North American, Inc., Franklinton, North Carolina, USA), a pectin-degrading enzyme preparation produced from selected strains of Aspergillus aculeatus. Pectinex™ ULTRA SP-L contains primarily polygalacturonase (EC 3.2.1.15), pectin transeliminase (EC 4.2.2.2), and pectinesterase (EC:3.1.1.11). The EC designation is the Enzyme Commission's classification scheme for enzymes based on the chemical reaction they catalyze.

[0036] According to embodiments of the present disclosure, digestion of the dissolvable foam scaffold also includes exposing the scaffold to a divalent cation chelator. Exemplary chelators include, but are not limited to, ethylenediaminetetraacetic acid (EDTA), cyclohexanediaminetetraacetic acid (CDTA), ethyleneglycoltetraacetic acid (EGTA), citric acid, and tartaric acid.

[0037] The time to complete digestion of the dissolvable foam scaffolds described herein can be less than about 1 hour. For example, the time to complete digestion of the foam scaffolds can be less than about 45 minutes, or less than about 30 minutes, or less than about 15 minutes, or from about 1 minute to about 25 minutes, or from about 3 minutes to about 20 minutes, or even from about 5 minutes to about 15 minutes.

[0038] According to embodiments of the present disclosure, the scaffolds described herein may further comprise an adhesive polymer coating. The adhesive polymer may comprise a peptide. Exemplary peptides may include, but are not limited to, BSP, vitronectin, fibronectin, laminin, type I and type IV collagen, denatured collagen (gelatin), and similar peptides, as well as mixtures thereof. Additionally, the peptide may have an RGD sequence. The coating may be, for example, Synthemax® II-SC (commercially available from Corning, Incorporated, Corning, NY, USA). Optionally, the adhesive polymer may comprise an extracellular matrix. The coating may be, for example, Matrigel® (commercially available from Corning, Incorporated, Corning, NY, USA).

[0039] Also disclosed are methods for forming the dissolvable foam scaffolds described herein, according to embodiments of the present disclosure. The methods described herein may include forming a first aqueous mixture, which includes dissolving a polysaccharide in an aqueous solution. The polysaccharide may be one of those described above, such as pectinic acid or a salt thereof, partially esterified pectinic acid or a salt thereof, or partially amidated pectinic acid or a salt thereof, or blends of these polysaccharides.

[0040] The method for forming a dissolvable foam scaffold described herein may further include forming a second aqueous mixture containing a water-insoluble divalent metal salt in an aqueous solution. The metal of the divalent metal salt may include, but is not limited to, magnesium, calcium, zinc, strontium, barium, and similar cations, and combinations thereof. The anion of the divalent metal salt may include, but is not limited to, oxalate, tartrate, phosphate, carbonate, citrate, and similar organic and inorganic anions, and combinations thereof.

[0041] According to embodiments of the present disclosure, forming the second aqueous mixture may further include adding at least one first polymer described above to the second aqueous mixture. Optionally, the methods described herein may further include adding at least one second polymer described above to the second aqueous mixture. According to embodiments of the present disclosure, the at least one first polymer and the at least one second polymer may be added separately to the second aqueous mixture, or may be added together to the second aqueous mixture. When added as a mixture, the mixture may contain about 50% of the at least one first polymer and about 50% of the at least one second polymer. For example, the mixture may contain about 35% to about 65% (and all values ​​therebetween) of the at least one first polymer and about 35% to about 65% (and all values ​​therebetween) of the at least one second polymer.

[0042] According to embodiments of the present disclosure, forming the second aqueous mixture may further include adding a water-soluble plasticizer to the second aqueous mixture. The plasticizers described herein are non-toxic and do not affect the solubility of the polysaccharides of the dissolvable foam scaffold. The plasticizer provides flexibility and softness to the resulting foam, making it soft and pliable. Plasticizers described herein may include, but are not limited to, polyhydric alcohols such as glycerol, sorbitol, ethylene glycol, propylene glycol, polyethylene glycol, and combinations thereof. Adding the water-soluble plasticizer to the second aqueous mixture may include adding less than about 55% by weight of the total solid additives added to form the second aqueous mixture. For example, adding the water-soluble plasticizer to the second aqueous mixture can include adding less than about 50% by weight, or less than about 40% by weight, or less than about 30% by weight, or less than about 25% by weight, or about 15% to about 55% by weight, or about 15% to about 50% by weight, or about 15% to about 40% by weight, or about 15% to about 30% by weight, or about 15% to about 25% by weight, and all values ​​therebetween. As used herein, the term "total solid additives added to form the second aqueous mixture" refers to all of the components of the aqueous mixture except water.

[0043] According to embodiments of the present disclosure, forming the second aqueous mixture may further include adding an emulsifier to the second aqueous mixture. Emulsifiers described herein may include, but are not limited to, Tween® 20, Tween® 80 (each commercially available from Croda International, Snaith, UK).

[0044] According to embodiments of the present disclosure, forming the second aqueous mixture may further include adding at least one leachable solid to the second aqueous mixture. Leachable solids described herein include materials that reinforce or generate pores during the formation of the foamed scaffold. The leachable solid may be a non-toxic leachable material, such as, but not limited to, salts, biocompatible monosaccharides and disaccharides, and water-soluble proteins. Exemplary salts include, but are not limited to, sodium chloride, potassium chloride, calcium chloride, sodium tartrate, sodium citrate, and the like. Exemplary biocompatible monosaccharides and disaccharides include, but are not limited to, glucose, fructose, dextrose, maltose, lactose, and sucrose. Exemplary water-soluble proteins include, but are not limited to, gelatin and agarose.

[0045] Each of the above-mentioned materials associated with the second aqueous mixture may be optionally added to the second aqueous mixture, and may be added to the second aqueous mixture in any order, with the possibility that two or more materials may be added to the second aqueous mixture. In one exemplary method, forming the second aqueous mixture includes adding a leachable solid to an aqueous solution containing a divalent metal salt and mixing the aqueous mixture to promote dissolution of the leachable solid in the aqueous mixture. Thereafter, at least one first polymer, at least one second polymer, and / or a water-soluble plasticizer are added to the second aqueous mixture.

[0046] The methods for forming a dissolvable foam scaffold described herein may further include, after forming the first and second aqueous mixtures, combining the second aqueous mixture with the first aqueous mixture to form a combined aqueous mixture. A foam can be formed from the combined aqueous mixture by introducing gas bubbles into the aqueous mixture by mixing, pulsating, stirring, aerating, whipping, injecting, or other mechanical action. The gas can be, for example, but not limited to, air, nitrogen, helium, hydrogen, argon, carbon dioxide, or other inert gas. The subsequent introduction of gas bubbles into the combined aqueous mixture can be carried out for less than about 30 minutes, for example, from about 1 minute to about 30 minutes, or from about 3 minutes to about 25 minutes, or even from about 5 minutes to about 20 minutes. While introducing gas bubbles into the combined aqueous mixture, the methods for forming a dissolvable foam scaffold may further include adding a gel-inducing agent to the combined aqueous mixture. The gel-inducing agent can be an acid that provides buffering and / or a substance that slowly generates acid. Exemplary acids include, but are not limited to, lactic acid lactone, glycolic acid lactone, glucono-delta-lactone, and acid anhydrides.

[0047] The methods for forming a dissolvable foam scaffold described herein may further include coating the dissolvable foam scaffold with an adhesive polymer coating. Coating the dissolvable foam scaffold may include exposing the scaffold to an aqueous solution having an adhesive polymer in the aqueous solution. As described above, the adhesive polymer may include a peptide. Exemplary peptides may include, but are not limited to, BSP, vitronectin, fibronectin, laminin, type I and type IV collagen, denatured collagen (gelatin), and similar peptides, as well as mixtures thereof. Additionally, the peptide may have an RGD sequence. The coating may be, for example, Synthemax® II-SC (commercially available from Corning, Incorporated, Corning, NY, USA).

[0048] Also disclosed according to embodiments of the present disclosure are methods for culturing cells on the dissolvable foam scaffold described herein. Any cell type can be cultured on the dissolvable foam scaffold, including, but not limited to, immortalized cells, primary cells, cancer cells, stem cells (e.g., embryonic or induced pluripotent), 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, breast, skin, brain, ovarian, lung, bone, nerve, muscle, cardiac, colorectal, pancreatic, immune (e.g., B cells), blood, etc. The cells can be in any cultured form within the bag, including dispersed (e.g., freshly seeded), confluent, two-dimensional, three-dimensional, spheroid, etc. Culturing cells on the dissolvable foam scaffold can include seeding the cells onto the dissolvable foam scaffold. Seeding the cells onto the dissolvable foam scaffold can include contacting the scaffold with a solution containing the cells. During seeding of the dissolvable foam scaffold with cells, the cells enter the pores of the dissolvable foam scaffold. If the dissolvable foam scaffold includes an adhesive polymer coating, the cells can enter the pores of the dissolvable foam scaffold and attach to the scaffold material.

[0049] Culturing cells on the dissolvable foam scaffold may further include contacting the scaffold with cell culture medium. Generally, contacting the scaffold with cell culture medium includes placing the cells to be cultured on the scaffold in an environment having a medium in which the cells are cultured. Contacting the scaffold with cell culture medium may include pipetting the cell culture medium onto the scaffold, submerging the scaffold in cell culture medium, or continuously passing the cell culture medium over the scaffold. Generally, as used herein, the term "continuous" refers to culturing cells with a consistent flow of cell culture medium into and out of the cell culture environment. Such continuous passing of cell culture medium over the scaffold may include submerging the scaffold in cell culture medium for a predetermined period of time, then removing at least a portion of the cell culture medium after the predetermined period of time, and adding fresh cell culture medium so that the volume of cell culture medium in contact with the dissolvable foam scaffold remains substantially constant. The cell culture medium may be removed and replaced according to a predetermined schedule. For example, at least a portion of the cell culture medium can be removed and replaced every hour, or every 12 hours, or every 24 hours, or every 2 days, or every 3 days, or every 4 days, or every 5 days.

[0050] The cell culture medium may contain, for example, but is not limited to, sugars, salts, amino acids, serum (e.g., fetal bovine serum), antibiotics, growth factors, differentiation factors, coloring agents, or other desired factors. Exemplary cell culture media include Dulbecco's Modified Eagle's Medium (DMEM), Ham's F12 Nutrient Mixture, Minimum Essential Medium (MEM), RPMI medium, Iscove's Modified Dulbecco's Medium (IMDM), Mesencult™-XF medium, and the like.

[0051] Also disclosed according to embodiments of the present disclosure are methods for recovering cells from the dissolvable foam scaffolds described herein. The methods for recovering cells described herein may include digesting the dissolvable foam scaffold by exposing the scaffold to an enzyme. As previously mentioned, non-proteolytic enzymes suitable for digesting the foam scaffold, recovering cells, or both, include pectinolytic enzymes or pectinases, a heterogeneous group of related enzymes that hydrolyze pectinic substances. Commercially available sources of pectinases are generally multienzymes, such as Pectinex™ ULTRA SP-L (commercially available from Novozyme North American, Inc., Franklinton, North Carolina, USA), a pectin-degrading enzyme preparation produced from selected strains of Aspergillus aculeatus. Pectinex™ ULTRA SP-L contains primarily polygalacturonase (EC 3.2.1.15), pectin transeliminase (EC 4.2.2.2), and pectinesterase (EC:3.1.1.11). The EC designation is the Enzyme Commission's classification scheme for enzymes based on the chemical reaction they catalyze.

[0052] Exposing the dissolvable foam scaffold to an enzyme can include exposing the scaffold to an enzyme concentration of about 1 to about 200 U. For example, the method can include exposing the scaffold to an enzyme concentration of about 2 U to about 150 U, or about 5 U to about 100 U, or even about 10 U to about 75 U, and all values ​​therebetween.

[0053] The methods for recovering cells described herein may further include exposing the material to a chelating agent. Exemplary chelating agents include, but are not limited to, ethylenediaminetetraacetic acid (EDTA), cyclohexanediaminetetraacetic acid (CDTA), ethylene glycol tetraacetic acid (EGTA), citric acid, and tartaric acid. Exposing the dissolvable foam scaffold to a chelating agent may include exposing the scaffold to a chelating agent concentration of about 1 mM to about 200 mM. For example, the method may include exposing the scaffold to a chelating agent concentration of about 10 mM to about 150 mM, or about 20 mM to about 100 mM, or even about 25 mM to about 50 mM, and all values ​​therebetween. [Example]

[0054] Embodiments of the present disclosure are further described below with reference to certain exemplary and specific embodiments thereof, which are merely illustrative and not intended to be limiting. Example 1 [Table 1]

[0055] A first aqueous mixture containing 2.0 wt% polygalacturonic acid (PGA) was prepared by dissolving approximately 162 grams of polygalacturonic acid sodium salt in demineralized water in an oil bath set at a temperature of 104°C. The aqueous mixture was cooled to room temperature. A second aqueous mixture was prepared by adding approximately 1.06 grams of CaCO3 to approximately 24.52 grams of ultrapure water in the bowl of a KitchenAid mixer equipped with a wire loop whisk. Approximately 0.125 grams of TWEEN® 20 (commercially available from Sigma-Aldrich, St. Louis, Missouri, USA) was also added to the bowl of the KitchenAid mixer. Approximately 17.5 grams of sucrose was then added to the mixer bowl and mixed to promote dissolution of the sucrose in the second aqueous mixture. Approximately 7.5 grams of glycerol, approximately 1.94 grams of Methocel HPMC Culminal 724, and the first aqueous mixture were added to form a combined aqueous mixture in a mixing bowl and mixed at an agitation speed (speed 1 on a KitchenAid mixer) for approximately 5 minutes. The combined aqueous mixture was then whipped at a high whisking speed (speed 10 on a KitchenAid mixer) for approximately 20 minutes to introduce air into the combined aqueous mixture. While continuing to whip the combined aqueous mixture, an aqueous solution of approximately 3.77 grams of gluconolactone (GDL) in approximately 30 mL of water was added to the mixing bowl and whipping continued for approximately 1 minute.

[0056] Following the process described above, an opaque white foam with a wet foam density of approximately 0.25 g / cc was obtained. The foam was left uncovered in the mixing bowl at room temperature for approximately 1 hour to allow time for crosslinking to occur within the foam. The foam was then exposed to a temperature of approximately -80°C for approximately 16 hours to freeze the foam, and then exposed to a temperature of -86°C and a pressure of 0.11 mbar for approximately 72 hours. The resulting foam was observed to have a dry foam density of approximately 0.04 to approximately 0.045 g / cc and was observed to be porous with highly interconnected pores. Figure 2 shows an SEM image of the foam prepared in this Example 1. The composition of the resulting foam was determined to include approximately 1.3 wt% PGA, approximately 0.78 wt% HPM, and approximately 2.08 wt% total solids. Example 2 [Table 2]

[0057] The process described in Example 1 was repeated, except that a second aqueous mixture was prepared by adding about 0.53 grams of cellulose acetate. The resulting foam was observed to be porous with highly interconnected pores. Figure 3 shows an SEM photograph of the foam prepared in this Example 2. Example 3 [Table 3]

[0058] A first aqueous mixture containing 2.0 wt% polygalacturonic acid (PGA) was prepared by dissolving approximately 162 grams of polygalacturonic acid sodium salt in demineralized water in an oil bath set at 104°C. The aqueous mixture was cooled to room temperature. A second aqueous mixture was prepared by adding approximately 7.5 grams of glycerol to ultrapure water and heating in a microwave at 800W for approximately 30 seconds. Approximately 0.97 grams of bovine gelatin was cold swollen in approximately 5.8 mL of ultrapure water and then added to the second aqueous mixture and stirred until dissolution was observed. Approximately 1.06 grams of CaCO3 and approximately 0.125 grams of TWEEN® 20 were added to the second aqueous mixture. The second aqueous mixture was then sonicated for approximately 1 minute and then transferred to the bowl of a KitchenAid mixer equipped with a wire loop whisk. Then, about 17.5 grams of sucrose and about 0.97 grams of Methocel HPMC Culminal 724 were added to the bowl of a KitchenAid mixer, and the aqueous mixture was stirred for about 5 minutes. A first aqueous mixture containing 2.0 wt. % PGA was added to form a combined aqueous mixture in the mixing bowl and mixed at a stirring speed (speed 1 on the KitchenAid mixer) for about 3 minutes. The combined aqueous mixture was then whipped at a high whisking speed (speed 10 on the KitchenAid mixer) for about 20 minutes to introduce air into the combined aqueous mixture. While continuing to whip the combined aqueous mixture, an aqueous solution of about 3.77 grams of gluconolactone (GDL) in about 30 mL of water was added to the mixing bowl, and whipping continued for about 1 minute.

[0059] An opaque white foam was obtained according to the process described above. The foam was left uncovered in the mixing bowl at room temperature for approximately 1 hour to allow time for crosslinking to occur within the foam. The foam was then exposed to a temperature of approximately -80°C for approximately 16 hours to freeze the foam, and then exposed to a temperature of -86°C and a pressure of 0.11 mbar for approximately 72 hours. The resulting foam was observed to have a dry foam density of approximately 0.04 g / cc and was observed to be porous with highly interconnected pores. Figure 4 shows an SEM image of the foam prepared in this Example 3. Example 4 [Table 4]

[0060] The process described in Example 3 was repeated, except that instead of bovine gelatin, about 0.97 grams of porcine gelatin was cold swollen in about 5.8 mL of ultrapure water and then added to the second aqueous mixture and stirred until dissolution was observed. The resulting foam was observed to have a wet foam density of about 0.21 g / cc and a dry foam density of about 0.06 g / cc, and was observed to be porous with highly interconnected pores. Figure 5 shows an SEM photograph of the foam prepared in this Example 4. Example 5 [Table 5]

[0061] The process described in Example 1 was repeated, except that a first aqueous mixture was prepared by dissolving approximately 172 grams of 3.0 wt. % polygalacturonic acid sodium salt in demineralized water in an oil bath set at a temperature of 104°C. Additionally, Methocel HPMC Culminal 724 was omitted from the second aqueous mixture. The resulting foam was observed to have a wet foam density of approximately 0.40 g / cc and a dry foam density of approximately 0.11 g / cc. The foam was observed to be less porous than the foam formed in Example 1, and the pores were less interconnected than those of the foam formed in Example 1. It is believed that the omission of Methocel HPMC Culminal 724, or any surface-active polymer, reduced the porosity and level of interconnectivity of the foam. Figure 6 shows an SEM photograph of the foam prepared in this Example 5. It was demonstrated that foams having a wet density greater than about 0.40 g / cc and a dry density greater than about 0.11 g / cc can support the cultivation of cells, while foams having a wet density less than about 0.40 g / cc and a dry density less than about 0.11 g / cc exhibited improved cell culture conditions compared to foams having wet and dry densities such as those of the foam formed in this Example 5. Example 6 [Table 6]

[0062] The process described in Example 1 was repeated, except that a first aqueous mixture was prepared by dissolving approximately 162 grams of 1.59 wt. % polygalacturonic acid sodium salt in demineralized water in an oil bath set at a temperature of 104°C. In addition, a second aqueous mixture was prepared by adding 0.53 grams of CaCO to approximately 24.52 grams of ultrapure water, and approximately 2.58 grams of Methocel HPMC Culminal 724 was added to the second aqueous mixture. The resulting foam was observed to have a wet foam density of approximately 0.36 g / cc and a dry foam density of approximately 0.09 g / cc. Figure 7 shows an SEM photograph of the foam prepared in this Example 6. Example 7 [Table 7]

[0063] The process described in Example 1 was repeated, except that no sucrose was added to the second aqueous mixture. The resulting foam was observed to have a wet foam density of about 0.23 g / cc and a dry foam density of about 0.03 g / cc. Figure 8 shows an SEM photograph of the foam prepared in this Example 7. Example 8 [Table 8]

[0064] The process described in Example 1 was repeated, except that a first aqueous mixture was prepared by dissolving approximately 162 grams of 2.59 wt. % polygalacturonic acid sodium salt in demineralized water in an oil bath set at a temperature of 104°C. In addition, a second aqueous mixture was prepared by adding 0.97 grams of Methocel HPMC Culminal 724 to the second aqueous mixture. The ratio of PGA to Methocel HPMC Culminal 724 in the combined aqueous mixture was controlled to be 81 / 19. The resulting foam was observed to have a wet foam density of approximately 0.18 g / cc and a dry foam density of approximately 0.057 g / cc. Figure 9 shows an SEM photograph of the foam prepared in this Example 8. Example 9 [Table 9]

[0065] A second aqueous mixture was prepared by adding 1.94 grams of dextran to the second aqueous mixture, and the process described in Example 1 was repeated, except that Methocel HPMC Culminal 724 was omitted from the aqueous mixture. The resulting foam was observed to have a wet foam density of approximately 0.34 g / cc and a dry foam density of approximately 0.095 g / cc. The foam was observed to be less porous than the foam formed in Example 1, and the pores were less interconnected than those of the foam formed in Example 1. The omission of Methocel HPMC Culminal 724, or any surface-active polymer, is believed to have reduced the level of interconnectivity between the pores in the foam. Furthermore, the addition of a non-surface-active polymer did not contribute to the formation of pores in the foam. Figure 10 shows an SEM photograph of the foam prepared in this Example 9. Example 10 [Table 10]

[0066] The process described in Example 1 was repeated, except that a second aqueous mixture was prepared by adding 1.94 grams of Pluronic® P123 to the second aqueous mixture instead of Methocel HPMC Culminal 724. The ratio of PGA to Pluronic P123 in the combined aqueous mixture was controlled to be 62.5 / 37.5. The resulting foam was observed to have a wet foam density of about 0.18 g / cc and a dry foam density of about 0.03 g / cc, and was observed to have a higher porosity than the foam formed in Example 1. Figure 11 shows an SEM photograph of the foam prepared in this Example 10. Example 11 [Table 11]

[0067] The process described in Example 1 was repeated, except that a second aqueous mixture was prepared by adding a 50:50 weight ratio blend of Pluronic P123 and dextran instead of Methocel HPMC Culminal 724. The resulting foam was observed to have a wet foam density of about 0.20 g / cc and a dry foam density of about 0.038 g / cc, and was observed to have a higher porosity than the foam formed in Example 1. Figure 12 shows an SEM photograph of the foam prepared in this Example 11. Example 12 [Table 12]

[0068] The process described in Example 11 was repeated to form multiple foams with varying amounts of glycerol added to the second aqueous mixture. In forming the first foam in this example, 6.5 grams of glycerol were added to the second aqueous mixture. The glycerol constituted 19% by weight of the total solid additives added to form the combined aqueous mixture. The resulting foam was observed to have a wet foam density of approximately 0.19 g / cc and a dry foam density of approximately 0.055 g / cc. In forming the second foam in this example, 7.5 grams of glycerol were added to the second aqueous mixture. The glycerol constituted 22% by weight of the total solid additives added to form the combined aqueous mixture. The resulting foam was observed to have a wet foam density of approximately 0.21 g / cc and a dry foam density of approximately 0.048 g / cc. In forming the third foam in this example, 19.44 grams of glycerol were added to the second aqueous mixture. The glycerol constituted 42% by weight of the total solid additives added to form the combined aqueous mixture. The resulting foam was observed to have a wet foam density of about 0.23 g / cc and a dry foam density of about 0.23 g / cc. In forming the fourth foam of this example, 29.16 grams of glycerol was added to the second aqueous mixture. The glycerol constituted 52% by weight of the total solid additives added to form the combined aqueous mixture. The resulting foam was observed to have a wet foam density of about 0.26 g / cc and a dry foam density of about 0.35 g / cc.

[0069] The four foams in Example 12 demonstrate the effect of the amount of plasticizer added during foam formation on density and porosity. Increasing the plasticizer content was observed to result in slower drying, higher density, and lower porosity. Also, as shown in Figure 13, when the amount of glycerol was increased beyond about 20-25 wt.% of the total solid additives added to form the second aqueous mixture, the foam lost its approximately cylindrical shape. The foam's pore porosity and interconnectivity were observed to be greatest when the amount of plasticizer added to the second aqueous mixture was less than about 52 wt.% of the total solid additives added to form the second aqueous mixture. Example 13 [Table 13]

[0070] The process described in Example 1 was repeated, except that a second aqueous mixture was prepared by adding a 50:50 weight ratio blend of Pluronic P127 and dextran. The resulting foam was observed to have similar porosity and pore interconnectivity to the foam prepared in Example 11. Figure 14 shows an SEM photograph of the foam prepared in this Example 13.

[0071] Example 14 Foams formed according to the processes of Examples 1-12 were coated with Synthemax® II-SC. A 250 μg / ml Synthemax® II-SC aqueous ethanol solution was prepared by adding approximately 5.0 mg of Corning® Synthemax® II-SC powder to approximately 20 mL of an ethanol-to-water solution with a 70:30 ethanol:water ratio. Each foam, approximately 2-3 mm thick and approximately 22 mm in diameter, was placed in a separate well of a polystyrene 6-well cell culture plate. Approximately 4.0 mL of the 250 μg / ml Synthemax® II-SC aqueous ethanol solution was added to each well, and the plate was then allowed to stand at room temperature for approximately 1.5 hours. Excess solution was removed from the wells, and the foams were washed once with approximately 5.0 mL of 70% ethanol. The Synthemax® II-SC coating was then crosslinked by adding approximately 4.0 mL of 0.05% v / v glutaraldehyde in 70% aqueous ethanol (prepared by mixing 40 μl of a 25% glutaraldehyde solution in 6.0 mL of water and 14 mL of ethanol). The plates were allowed to stand at room temperature for approximately 1.5 hours to allow time for crosslinking to occur. The foam was then rinsed three times with ultrapure water.

[0072] Example 15 Foams formed according to the processes of Examples 1-12 were coated with gelatin. A 0.1 wt% gelatin solution was prepared by swelling approximately 500 mg of gelatin powder (derived from pig skin) in ultrapure water, followed by dissolving and homogenizing in 20 mL of ultrapure water. Each foam, approximately 2-3 mm thick and approximately 22 mm in diameter, was placed in a separate well of a polystyrene 6-well cell culture plate. Approximately 4.0 mL of gelatin solution was added to each well, and the plate was then allowed to stand at room temperature for approximately 1.5 hours. Excess solution was removed from the wells, and the foams were washed once with approximately 5.0 mL of 70% aqueous ethanol. The gelatin coating was then crosslinked by adding approximately 4.0 mL of 0.05% v / v glutaraldehyde in 70% aqueous ethanol (prepared by mixing 50 μl of 25% glutaraldehyde solution in 25 mL of ultrapure water). The plate was left at room temperature for approximately 1.5 hours to allow time for cross-linking to occur, after which the foam was rinsed three times with ultrapure water.

[0073] Example 16 The culture of Vero cells on foams formed according to the process of Example 4 was investigated. Vero cells (ATCC® CCL-81, commercially available from ATCC, Manassas, Virginia, USA) were cultured on cell culture plates in IMDM medium supplemented with 10% fetal bovine serum (FBS). The foams were cut into sections approximately 2-3 mm thick and approximately 22 mm in diameter. The foam sections were disinfected with 70% aqueous ethanol for approximately 5.0 minutes and then placed into separate wells of a 6-well ultra-low attachment cell culture plate. The foam sections were washed twice with ultrapure water and once with IMDM medium. Excess medium was removed from the wells prior to seeding.

[0074] Vero cells were harvested from the cell culture plate using trypsin and resuspended in IMDM medium. Approximately 100,000 cells were seeded in 150 μL of IMDM medium into each foam section placed in a well 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 sections were visualized using a phase-contrast microscope. Images obtained from the phase-contrast microscope are shown in Figure 15, demonstrating that the cells did not adhere to the uncoated foam sections but instead formed spheroids in the pores of the foam sections. Therefore, it was demonstrated that the dissolvable foam scaffold of the present disclosure can be used to culture spheroids or non-adherent cells.

[0075] Example 17 The culture of human mesenchymal stem cells (hMSCs) on foams formed according to the process of Example 4 and coated according to the process of Example 14 was investigated. hMSCs, passage 2 (commercially available from RoosterBio Inc., Frederick, MD, USA) were cultured on cell culture plates in Mesencult™-XF medium (a serum-free medium commercially available from STEMCELL Technologies, Vancouver, British Columbia, Canada). The foams were cut into sections approximately 2-3 mm thick and approximately 22 mm in diameter. The foam sections were sanitized with 70% aqueous ethanol for approximately 5.0 minutes and then placed into separate wells of a 6-well ultra-low attachment cell culture plate. The foam sections were washed twice with ultrapure water and once with Mesencult™-XF medium. Excess medium was removed from the wells prior to seeding.

[0076] hMSCs were harvested from cell culture plates using trypsin and resuspended in Mesencult™-XF medium. Approximately 100,000 cells were seeded in 150 μL of Mesencult™-XF medium into each foam section placed in a well 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 Mesencult™-XF medium was added to each well. After approximately 18 hours in the cell culture incubator, the cells were stained with 1 μg / mL of Calcein-AM and visualized using a fluorescent microscope. Images obtained from the fluorescent microscope are shown in Figure 16, demonstrating that the cells were able to adhere to the foam sections and spread within the pores of the foam sections. Therefore, the Corning® Synthemax® II-SC-coated dissolvable foam scaffold of the present disclosure can be used to culture adherent cells in addition to spheroids or non-adherent cells, and the scaffold was found to support cell culture in serum-free medium.

[0077] Example 18 The culture of human mesenchymal stem cells (hMSCs) on foams formed according to the process of Example 4 and coated according to the process of Example 15 was investigated. hMSCs, passage 2, used in Example 17, were cultured on cell culture plates in IMDM medium supplemented with 10% fetal bovine serum (FBS). The foams were cut into sections approximately 2-3 mm thick and approximately 22 mm in diameter. The foam sections were disinfected with 70% aqueous ethanol for approximately 5.0 minutes and then placed into separate wells of a 6-well ultra-low attachment cell culture plate. The foam sections were washed twice with ultrapure water and once with Mesencult™-XF medium. Excess medium was removed from the wells prior to seeding.

[0078] hMSCs were harvested from the cell culture plate using trypsin and resuspended in IMDM medium. Approximately 100,000 cells were seeded in 150 μL of the solution into each foam section placed in a well 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 cells were stained with 1 μg / mL Calcein-AM and visualized using a fluorescent microscope. Images obtained from the fluorescent microscope, showing that the cells were able to adhere to the foam section and spread within the pores of the foam section, are shown in Figure 17. hMSC cell growth was allowed to proceed on the foam for 6 days and again visualized using a fluorescent microscope. Images obtained from the fluorescent microscope after 6 days, showing that the cells had adhered to the foam section and spread within the pores of the foam section, are shown in Figure 18. Thus, it has been found that the gelatin-coated dissolvable foam scaffolds of the present disclosure can be utilized for culturing adherent cells in addition to spheroids or non-adherent cells, and that the scaffolds support cell culture in serum-containing media.

[0079] Example 19 Vero cell growth on foams formed according to the processes of Examples 2 and 4 and coated according to the process of Example 15 was investigated. Vero cells used in Example 16 were cultured on tissue culture-treated (TCT) plates in IMDM medium supplemented with 10% fetal bovine serum (FBS). The foams were cut into sections approximately 2-3 mm thick and approximately 22 mm in diameter. The foam sections were sanitized with 70% aqueous ethanol for approximately 5.0 minutes and then placed into separate wells of a polystyrene 6-well cell culture plate. The foam sections were washed twice with ultrapure water and once with IMDM medium. Excess medium was removed from the wells prior to seeding.

[0080] Vero cells were harvested from the TCT plates using trypsin and resuspended in IMDM medium. Some foam sections were seeded with 150 μL containing approximately 25,000 cells, and other foam sections were seeded with 150 μL containing approximately 50,000 cells. The foam sections were then placed into wells of a 6-well cell culture plate. The 6-well cell culture plate was placed in a cell culture incubator for approximately 6 days. After approximately 6 days, the medium was removed, and the foam was dissolved by adding approximately 2.0 mL of a digestion solution containing approximately 50 U / mL pectinase and approximately 5.0 mM EDTA to each well. The foam was observed to dissolve within 5.0 minutes. After dissolution of the foam, cells were counted using the trypan blue exclusion protocol as detailed in “Protocol for Performing a Trypan Blue Viability Test: Technical Reference Guide.” Lonza Cologne GmbH, September 2012, retrieved from http: / / bio.lonza.com / uploads / tx_mwaxmarketingmaterial / Lonza_BenchGuides_Protocol_for_Performing_a_Trypan_Blue_Viability_Test__Technical_Reference_Guide.pdf.

[0081] It was observed that cells colonized all pore surfaces of the foam portion. Approximately 70-90 fold proliferation was observed for the coated foam formed according to the process of Example 4, and approximately 40-70 fold proliferation was observed for the coated foam formed according to the process of Example 2. Therefore, it was found that the gelatin-coated dissolvable foam scaffolds of the present disclosure can be used for the proliferation of Vero cells.

[0082] Example 20 The differentiation of human mesenchymal stem cells (hMSCs) was investigated on foams formed according to the process of Example 4 and coated according to the process of Example 15. hMSCs, passage 2, used in Example 17, were cultured on cell culture plates in Mesencult™-XF medium. The foams were cut into sections approximately 2-3 mm thick and approximately 22 mm in diameter. The foam sections were disinfected with 70% aqueous ethanol for approximately 5.0 minutes and then placed into separate wells of a polystyrene 6-well cell culture plate. The foam sections were washed twice with ultrapure water and once with Mesencult™-XF medium. Excess medium was removed from the wells prior to seeding.

[0083] hMSCs were harvested from cell culture plates using trypsin and resuspended in Mesencult™-XF medium. Approximately 100,000 cells were seeded in 150 μL of Mesencult™-XF medium into each foam well of a 6-well cell culture plate. The 6-well cell culture plate was placed in a cell culture incubator for approximately 3 days. After approximately 3 days, the Mesencult™-XF medium was removed, and osteogenic differentiation medium, chondrogenic differentiation medium, and adipogenic differentiation medium (commercially available from Thermo Fisher Scientific, Waltham, Massachusetts, USA, under the trade names StemPro™ Osteogenic Differentiation Kit, StemPro™ Chondrogenic Differentiation Kit, and StemPro™ Adipogenic Differentiation Kit) were added to the wells (each type of differentiation medium was added to a different foam portion of the well from the other types of differentiation medium). After 3 weeks, the foam sections exposed to the osteocyte differentiation medium were stained with Alizarin Red, the foam sections exposed to the chondrocyte differentiation medium were stained with Alcian Blue, and the foam sections exposed to the adipocyte differentiation medium were stained with Oil Red O. After staining, it was observed that hSMCs cultured on the gelatin-coated dissolvable foam scaffold of the present disclosure maintained chondrogenic, osteogenic, and adipogenic differentiation. Thus, it was found that cells cultured on the dissolvable foam scaffold of the present disclosure exhibit biological responses similar to those of cells in vivo.

[0084] Example 21 The proliferation of human mesenchymal stem cells (hMSCs) was investigated on foams formed according to the processes of Examples 2, 11, and 13 and coated according to the process of Example 14. hMSCs, passage 2, used in Example 17, were cultured on cell culture plates in Mesencult™-XF medium. The foams were cut into sections approximately 2-3 mm thick and approximately 22 mm in diameter. The foam sections were disinfected with 70% aqueous ethanol for approximately 5.0 minutes and then placed into separate wells of a 6-well ultra-low attachment cell culture plate. The foam sections were washed twice with ultrapure water and once with Mesencult™-XF medium. Excess medium was removed from the wells prior to seeding.

[0085] hMSCs were harvested from the cell culture plate using trypsin and resuspended in Mesencult™-XF medium. Approximately 100,000 cells were seeded in 150 μL of Mesencult™-XF medium into each foam section placed in a well of a 6-well cell culture plate. The 6-well cell culture plate was placed in a cell culture incubator for approximately 7 days. On day 4, the medium was removed and replaced with fresh medium. After approximately 7 days, the medium was removed and the foam was dissolved by adding approximately 2.0 mL of a digestion solution containing approximately 50 U / mL pectinase and approximately 5.0 mM EDTA to each well. The foam was observed to dissolve within 5.0 minutes. After foam dissolution, cells were counted using the trypan blue exclusion protocol described above. Cells colonized all pore surfaces of the foam section, demonstrating successful hSMC proliferation on the Corning® Synthemax® II-SC-coated dissolvable foam scaffold of the present disclosure.

[0086] Example 22 A comparison of the culture of human mesenchymal stem cells (hMSCs) under static and dynamic conditions on foams formed according to the processes of Examples 2 and 11 and coated according to the process of Example 14 was investigated. hMSCs passage 2 were cultured as in Example 17. hMSCs were harvested from cell culture plates using trypsin, resuspended in Mesencult™-XF medium, and 150 μL containing approximately 100,000 cells was seeded onto 20 foam scaffolds formed in Example 2 and 20 foam scaffolds formed in Example 11. The scaffolds were placed into wells of a 6-well cell culture plate, which was then placed in a cell culture incubator for approximately 2 hours. After approximately 2 hours, the 6-well cell culture plate containing the scaffolds formed according to Example 2 and the 6-well cell culture plate containing the scaffolds formed according to the Examples were removed from the incubator, and the scaffolds were transferred to separate bioreactors. The other 6-well cell culture plate remained in the incubator. All scaffolds were maintained under the respective conditions for approximately 6 days and then dissolved by adding a digestion solution containing approximately 50 U / mL pectinase and approximately 5.0 mM EDTA. After foam dissolution, cells were counted using the trypan blue exclusion protocol described above. Cell proliferation on the Corning® Synthemax® II-SC-coated dissolvable foam scaffolds of the present disclosure was observed to be similar under static and dynamic conditions.

[0087] Example 23 Multi-passage culture of human mesenchymal stem cells (hMSCs) was investigated. As described herein, the passage number of a cell culture is a record of the number of times the culture has been subcultured, i.e., harvested and reseeded. Therefore, in the context of this disclosure, multi-passage culture describes the process in which cells are harvested from one dissolvable foam scaffold and reseeded onto a different dissolvable foam scaffold. hMSCs were harvested from cell culture plates using trypsin, resuspended in Mesencult™-XF medium, and 150 μL containing approximately 100,000 cells was seeded onto the first set of foam scaffolds formed in Example 2 and coated in Example 14. The scaffolds were placed in Mesencult™-XF medium and placed in a cell culture incubator for approximately 6 days. After approximately 6 days, the scaffolds were dissolved by adding a digestion solution containing approximately 50 U / mL pectinase and approximately 5.0 mM EDTA. After dissolution of the foam, cells were counted using the trypan blue exclusion protocol described above. A solution containing approximately 100,000 cells recovered from the first set of scaffolds was then used to seed a second set of foam scaffolds formed in Example 2 and coated in Example 14. The scaffolds were placed in Mesencult™-XF medium and placed in a cell culture incubator for approximately 7 days. After approximately 7 days, the scaffolds were dissolved by adding a digestion solution containing approximately 50 U / mL pectinase and approximately 5.0 mM EDTA. After dissolution of the foam, cells were counted using the trypan blue exclusion protocol. A solution containing approximately 100,000 cells recovered from the second set of scaffolds was then used to seed a third set of foam scaffolds formed in Example 2 and coated in Example 14. The scaffolds were placed in Mesencult™-XF medium and placed in a cell culture incubator for approximately 7 days. After about 7 days, the scaffolds were dissolved by adding a digestion solution containing about 50 U / mL pectinase and about 5.0 mM EDTA. After foam dissolution, cells were counted using a trypan blue exclusion protocol and then seeded onto cell culture plates and exposed to the differentiation medium, chondrocyte differentiation medium, and adipocyte differentiation medium used in Example 20.After 3 weeks, cells exposed to osteogenic differentiation medium were stained with Alizarin Red, cells exposed to chondrogenic differentiation medium were stained with Alcian Blue, and cells exposed to adipogenic differentiation medium were stained with Oil Red O. After staining, it was observed that hSMCs maintained chondrogenic, osteogenic, and adipogenic differentiation after multiple passages on the Corning® Synthemax® II-SC-coated dissolvable foam scaffolds of the present disclosure.

[0088] Example 24 Transfection of human embryonic kidney (HEK) cells on foam scaffolds formed according to the process of Example 11 and coated according to the process of Example 14 was compared with transfection of HEK cells on Corning® Synthemax® II-SC dissolvable microcarriers (commercially available from Corning Incorporated, Corning, NY, USA). HEK cells were seeded on the dissolvable microcarriers. A first set of dissolvable microcarriers was placed in a 6-well cell culture plate and exposed to IMDM medium supplemented with 10% fetal bovine serum (FBS). The 6-well cell culture plate was placed in a cell incubator and grown under static conditions for approximately 3 days. A second set of dissolvable microcarriers was placed in a disposable spinner flask and suspended in IMDM medium supplemented with 10% fetal bovine serum (FBS) for approximately 3 days with intermittent agitation (15 minutes of agitation every 2.0 hours). After approximately 3 days, approximately 150 μL of transfection reagent was added to the dissolvable microcarriers in the wells of a 6-well cell culture plate, and approximately 1.0 mL of transfection reagent was added to the spinner flask with intermittent agitation.

[0089] HEK cells were seeded onto some of the foam. Some foam sections were placed in a 6-well cell culture plate and exposed to IMDM medium supplemented with 10% fetal bovine serum (FBS). The 6-well culture plate was then placed in a cell incubator for approximately 3 days. Other foam sections were placed in the chamber of a radial flow perfusion cartridge device for approximately 3 days. The perfusion cartridge device allowed for the continuous removal of spent IMDM medium supplemented with 10% fetal bovine serum (FBS) and the addition of fresh IMDM medium supplemented with 10% fetal bovine serum (FBS) from the chamber. After approximately 3 days, approximately 150 μL of transfection reagent was added to the foam sections within the wells of the 6-well cell culture plate, and approximately 1.0 mL of transfection reagent was added to the foam sections within the perfusion cartridge device.

[0090] After adding the transfection reagent, cells were collected from the dissolvable microcarriers and foam portions, and reporter gene expression levels were detected by measuring the response of green fluorescent protein (GFP) as a reporter gene. The GFP-positive rate of transfected HEK cells, which can be used as an indicator of transfection efficiency, was measured using flow cytometry. As used herein, the term "transfection efficiency" refers to the percentage of cells that have a given nucleic acid or biologically active molecule present within the cells after exposure to a transfection reagent. Figure 19 is a bar graph showing the GFP-positive rate of transfected HEK cells for each culture condition. As shown, bar 1610 represents the GFP-positive rate of transfected HEK cells cultured on dissolvable foam portions in a 6-well cell culture plate; bar 1620 represents the GFP-positive rate of transfected HEK cells cultured on dissolvable foam portions in a perfusion cartridge device; bar 1630 represents the GFP-positive rate of transfected HEK cells cultured on dissolvable microcarriers in a 6-well cell culture plate; and bar 1640 represents the GFP-positive rate of transfected HEK cells cultured on dissolvable microcarriers in a spinner flask. Cells cultured on Corning® Synthemax® II-SC-coated dissolvable foam scaffolds of the present disclosure were observed to exhibit greater transfection efficiency than cells cultured on microcarriers. Without being bound by theory, it is believed that cells within the pores of the foam scaffold experienced greater exposure to the transfection reagent than cells attached to the surface of the microcarriers.

[0091] Example 25 The production of adeno-associated virus (AAV) vectors was demonstrated in foam scaffolds formed according to the process of Example 11 and coated according to the process of Example 14. A first set of foam scaffolds and a second set of foam scaffolds were seeded with 1 million 293aav cells and transferred to the chamber of a radial flow perfusion cartridge device where they were exposed to IMDM media supplemented with 10% fetal bovine serum (FBS).

[0092] Approximately 24 hours later, cells from the first set of foam scaffolds were transfected with an AAV-2 helper-free packaging system (commercially available from Cell Biolabs, Inc., San Diego, CA, USA) using the calcium phosphate transfection method. Also approximately 24 hours later, cells from the second set of foam scaffolds were transfected with an AAV-2 helper-free packaging system using the PEI method. For the calcium phosphate method, cells were incubated for approximately 18 hours with calcium phosphate / DNA complexes prepared using a plasmid concentration of 6.4 μg / mL and a 1:1:1 molar ratio of plasmid. For the PEI method, cells on the foam scaffolds were transfected with 2 μg of plasmid / mL. Each PEI method used a 2:1 PEI / DNA ratio and a 1:1:1 molar ratio of plasmid.

[0093] The radial flow perfusion cartridge device was operated at a perfusion rate of 20 mL / min for approximately 2 hours, followed by 5 mL / min for approximately 16 hours. For all cell sets, the medium was removed after approximately 18 hours and replaced with fresh medium. The cells were then further incubated and harvested 72 hours after transfection. The foam scaffolds were collected and lysed by adding a digestion solution containing approximately 50 U / mL pectinase and approximately 5.0 mM EDTA. After dissolution of the foam scaffolds, the cells were collected using centrifugation and washed with dPBS. A portion of the cells was retained for flow cytometry analysis, while the remaining cells were lysed using 0.1% sodium deoxycholate buffer supplemented with 20 mM Tris pH 8, 150 mM NaCl, and 50 U / mL benzonase.

[0094] After incubation at 37°C for approximately 30 minutes, the viral extract was clarified by centrifugation at 14,000 rpm for approximately 5 minutes. Viral titers were measured using an AAV-2 ELISA assay (commercially available from PROGEN Biotechnik GmbH, Heidelberg, Germany).

[0095] Transfection efficiency was analyzed using flow cytometry, and good transfection efficiency was observed. Figure 20 is a bar graph showing the GFP-positive rate of transfected cells for each foam scaffold set. As shown, bar 1710 represents the GFP-positive rate of cells transfected using the calcium phosphate transfection method, and the transfection efficiency was measured to be approximately 86.4%, while bar 1720 represents the GFP-positive rate of cells transfected using the PEI transfection method, and the transfection efficiency was measured to be approximately 73.8%.

[0096] Viral titers were also measured using an ELISA assay. Figure 21 is a bar graph showing the number of viral particles (vp) per foam scaffold set. As shown, bar 1810 represents the viral particles of cells transfected using the calcium phosphate transfection method, which was 8.7 x 10 11 5.7 x 10 viral particles were measured, bar 1,820 represents viral particles in cells transfected using the PEI transfection method. 11 Virus particles were measured. Figure 22 is a bar graph showing the number of virus particles per cell per foam scaffold set. As shown, bar 1910 represents virus particles / cell for cells transfected using the calcium phosphate transfection method, which was 1.17 x 10 5 6.8 x 10 viral particles / cell were measured, bar 1920 represents viral particles / cell for cells transfected using the PEI transfection method. 4 virus particles / cell were measured.

[0097] Finally, the functionality of the viral vectors was assessed by testing the ability of the extracts to induce GFP expression in infected HEK293 cells. Viral vector infectivity was assessed using 10 5 The virus was evaluated by infecting HEK293 cells with extracts prepared using 100 viral particles / cell. Approximately 72 hours after infection, the GFP-positive rate of infected HEK cells was measured by flow cytometry. Infection of cells derived from each foam scaffold set was observed. GFP expression was used to determine the efficiency of gene transfer, which in this example ranged from approximately 10% to approximately 26%. Therefore, it was concluded that functional AAV vectors can be produced using the foam scaffolds disclosed herein. Figure 23 is a bar graph showing the percentage of cells expressing GFP for each foam scaffold set after infection. As shown, bar 2010 represents the percentage of cells expressing GFP for cells transfected using the calcium phosphate transfection method, with approximately 26% of cells expressing GFP, and bar 2020 represents the percentage of cells expressing GFP for cells transfected using the PEI transfection method, with approximately 10% of cells expressing GFP.

[0098] Example 26 Some of the dissolvable foams disclosed herein use various ingredients (e.g., sucrose, glycerol, dextran, and others) that are added to facilitate the foaming process and account for a significant amount (e.g., by weight) of the solution used in the foaming process. For example, these ingredients can account for up to 90% of the final foam weight. Most of these ingredients are not covalently bound to the foam structure and may dissociate from the foam during coating or may need to be removed prior to cell culture. If not removed during cell culture, these materials could potentially alter the cell medium composition, block cell-binding epitopes, and reduce cell binding. After being released into the cell culture medium, these materials could significantly change the medium osmolality. The foaming process requires surface-active molecules, such as surfactants, or foaming agents. For example, as disclosed in some of the examples above, Pluronic® P123 may be used in the foaming process. However, the examples above still use other ingredients, such as sucrose, dextran, glycerol, and Tween® 20, for foaming. To simplify the process, reduce production costs, and allow for direct use of the dissolvable foam in cell culture without removing the excess components, it would be beneficial to remove these excess materials in the foam, as discussed in Example 26 below.

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

[0100] In this Example 26, the use of a plasticizer (e.g., glycerol), a foam enhancer or sugar (e.g., sucrose), and a foam enhancer in the form of a non-surface-active polymer (e.g., dextran) is eliminated by substituting a high HLB surface-active polymer (e.g., Poloxamer 407 or Pluronic® F127) for the two surfactants used in the previous example (e.g., a surface-active polymer such as P123 and an emulsifier such as Tween® 20). This provides a much simpler formulation for the dissolvable foam, eliminating components that may require further downstream processing or potentially adversely affect cell culture. Additionally, it is possible to control the viscosity of the simplified formulation without adding new components. This simplified formulation has several advantages. For example, this new formulation removes non-critical materials that, in some cases, may not be desirable in the finished dissolvable foam product. This elimination of materials also significantly simplifies the process and reduces costs. Additionally, the foam structure material eliminates a large amount of leachable material that could potentially alter the cell medium osmolality and block cell-binding epitopes during application. This allows for the elimination of a separate coating step in the future, allowing for direct use of the foam for cell culture. Furthermore, the simplified formulation provides tunable viscosity without the addition of additional components. This makes future optimization of the foaming process and control of the foam structure easier and less expensive. Finally, the simplified formulation can benefit the continuous foaming process by utilizing the shear-thinning properties of the material.

[0101] A foaming formulation according to an example embodiment from several of the above examples (referred to as a Comparative Example in the remainder of this Example 26) contained a relatively long list of ingredients, as shown in Table 1 below. In contrast, the simplified formulation of this Example 26 excludes plasticizers (e.g., glycerol), surface-active emulsifiers or second polymers (e.g., Tween® 20), and other foam-enhancing agents or polymers that are not surface-active (e.g., sucrose and dextran). The simplified formulation of Table 1 also uses a poloxamer (e.g., F127) with a higher HLB than the P123 used in the Comparative Example (Pluronic® P123 has an HLB of 8, while Pluronic® F127 has an HLB of 22). As highlighted in Table 1, most of the additional ingredients in the Comparative Example were used to provide physical properties to enable the foaming process. These additional ingredients (Tween® 20, glycerol, sucrose, and dextran) comprise approximately 80% of the total weight (excluding water) of the comparative formulation. Table 2 below compares the weight percentages of the ingredients in the comparative and simplified formulations of Table 1. These materials have no value to the final product and may need to be removed during the subsequent coating process. If the foam article is to be used for cell culture, these materials need to be removed to prevent significant changes in osmotic pressure or interference with cell-binding epitopes. [Table 14] [Table 15]

[0102] In this Example 26, Pluronic® F127 can be replaced with a mixed surfactant of Pluronic® P123 and Tween® 20. This also allows for good foaming without the addition of sucrose, glycerol, or dextran. Measurements indicated that the desired porosity in the foam can be comparable to that of previous examples (including comparative examples) disclosed herein. Figure 24 shows an SEM image of a foam formed from the simplified formulation of Example 26, which has a pore structure comparable to foams produced from other examples herein using more complex formulations.

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

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

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

[0106] Exemplary Implementations Below are descriptions of various aspects of embodiments of the disclosed subject matter. Each aspect may include one or more of various features, characteristics, or advantages of the disclosed subject matter. These embodiments are intended to illustrate a few aspects of the disclosed subject matter and should not be considered a comprehensive or exhaustive description of all possible embodiments.

[0107] Aspect 1 relates to a dissolvable foam scaffold for cell culture, comprising an ionotropically crosslinked polygalacturonic acid compound selected from at least one of pectinic acid; partially esterified pectinic acid, partially amidated pectinic acid, and salts thereof, and a water-soluble polymer that is surface-active and has a hydrophilic-lipophilic balance (HLB) greater than about 20.

[0108] Aspect 2 relates to the dissolvable foam scaffold of aspect 1, wherein the water-soluble polymer has an HLB of about 22 or greater.

[0109] Aspect 3 relates to the dissolvable foam scaffold of aspect 1 or 2, wherein the dissolvable foam scaffold comprises 0 wt. % water soluble plasticizer.

[0110] Aspect 4 relates to a dissolvable foam scaffold according to any one of aspects 1 to 3, wherein the dissolvable foam scaffold does not comprise glycerol, sorbitol, ethylene glycol, propylene glycol, polyethylene glycol.

[0111] Aspect 5 relates to a dissolvable foam scaffold according to aspects 1-4, further comprising an adhesive polymer coating.

[0112] Aspect 6 relates to a dissolvable foam scaffold according to aspect 5, wherein the adhesive polymer coating comprises a peptide.

[0113] Aspect 7 relates to a dissolvable foam scaffold according to aspect 5, wherein the adhesive polymer coating comprises a peptide selected from the group consisting of BSP, vitronectin, fibronectin, laminin, type I collagen, type IV collagen, denatured collagen, and mixtures thereof.

[0114] Example 8 relates to a dissolvable foam scaffold according to Example 5, wherein the adhesive polymer coating comprises Synthemax® II-SC.

[0115] Embodiment 9 relates to a dissolvable foam scaffold according to any of embodiments 1 to 8, wherein the at least one first polymer comprises a protein.

[0116] Example 10 relates to a dissolvable foam scaffold according to any of Examples 1 to 8, wherein the at least one first polymer comprises a synthetic amphiphilic polymer.

[0117] Example 11 relates to a dissolvable foam scaffold according to any of Examples 1 to 10, wherein the dissolvable foam scaffold comprises only a single water-soluble polymer that is surface active.

[0118] Aspect 12 relates to a dissolvable foam scaffold according to any of aspects 1 to 11, wherein the dissolvable foam scaffold does not comprise a non-surface active polymer.

[0119] Aspect 13 relates to a dissolvable foam scaffold according to any of aspects 1 to 12, wherein the dissolvable foam scaffold does not comprise dextran.

[0120] Aspect 14 relates to a dissolvable foam scaffold according to any one of aspects 1 to 13, wherein the dissolvable foam scaffold consists of an ionotropically crosslinked polygalacturonic acid compound and a surface-active water-soluble polymer.

[0121] Example 15 relates to a dissolvable foam scaffold according to any one of Examples 1 to 13, wherein the dissolvable foam scaffold consists of an ionotropically crosslinked polygalacturonic acid compound, a surface-active water-soluble polymer, and an adhesive polymer coating.

[0122] A sixteenth embodiment relates to a dissolvable foam scaffold according to any one of the first to fifth embodiments, comprising an average pore size of about 50 μm to about 500 μm.

[0123] Example 17 relates to a dissolvable foam scaffold according to any of Examples 1 to 16, comprising an open pore structure.

[0124] Example 18 relates to a dissolvable foam scaffold according to any of Examples 1 to 17, wherein digestion of the dissolvable foam scaffold is complete in less than about 1 hour.

[0125] Aspect 19 relates to a method for forming a dissolvable foamed scaffold, the method comprising: adding a polygalacturonic acid compound selected from at least one of pectinic acid; partially esterified pectinic acid, partially amidated pectinic acid, and salts thereof to an aqueous solution to form a first aqueous mixture; adding a surface-active water-soluble polymer and a divalent metal salt to the aqueous solution to form a second aqueous mixture; combining the first aqueous mixture with the second aqueous mixture to form a combined aqueous mixture; adding a gel-inducing agent to the combined aqueous mixture to form a foamed solution; and introducing gas bubbles into the foamed solution to form the foamed scaffold, wherein the water-soluble polymer has a hydrophilic-lipophilic balance (HLB) of greater than about 20.

[0126] Example 20 relates to the method of example 19, wherein the water-soluble polymer has an HLB of about 22 or greater.

[0127] Example 21 relates to the method of example 19 or example 20, wherein forming the second aqueous mixture does not include adding a water-soluble plasticizer to the second aqueous mixture.

[0128] Example 22 relates to the method of any of Examples 19-21, wherein forming the second aqueous mixture does not include adding a second polymer that is not surface active.

[0129] Example 23 relates to a method according to any one of Examples 19 to 22, wherein forming the second aqueous mixture comprises adding only a single water-soluble polymer that is surface active.

[0130] Example 24 relates to the method of any one of Examples 19 to 23, wherein the divalent metal salt comprises a cation selected from the group consisting of magnesium, calcium, zinc, strontium, barium, and combinations thereof, and an anion selected from the group consisting of oxalate, tartrate, phosphate, carbonate, citrate, and combinations thereof.

[0131] Example 25 relates to the method of any of Examples 19 to 24, wherein 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.

[0132] Example 26 relates to the method of any of Examples 19-25, wherein forming the second aqueous mixture further comprises adding at least one leachable solid to the second aqueous mixture.

[0133] Example 27 relates to the method of example 26, wherein the at least one leachable solid is selected from the group consisting of salts and water-soluble proteins.

[0134] Aspect 28 relates to a method according to any one of aspects 19 to 27, wherein the gel-inducing agent is selected from the group consisting of lactic acid lactone, glycolic acid lactone, glucono-delta-lactone, and acid anhydrides.

[0135] Example 29 relates to the method of any of Examples 19-28, wherein adding a gel-inducing agent to the combined aqueous mixture comprises adding a first amount of gel-inducing agent to the aqueous mixture before foaming, and adding a second amount of gel-inducing agent to the aqueous mixture during or after foaming.

[0136] Example 30 relates to the method of example 29, wherein the ratio of the second amount of gel-inducing agent to the first amount of gel-inducing agent is greater than about 2.

[0137] Example 31 relates to the method of example 30, wherein the ratio is about 7 or greater.

[0138] Example 32 relates to the method of Examples 19-31, further comprising coating the dissolvable foam scaffold with an adhesive polymer coating.

[0139] Example 33 relates to a method according to example 32, wherein the adhesive polymer coating comprises a peptide.

[0140] Example 34 relates to the method of example 32, wherein the adhesive polymer coating comprises a peptide selected from the group consisting of BSP, vitronectin, fibronectin, laminin, type I collagen, type IV collagen, denatured collagen, and mixtures thereof.

[0141] Example 35 relates to the method of example 32, wherein the adhesive polymer coating comprises Synthemax® II-SC.

[0142] Example 36 relates to the method of any one of Examples 19 to 35, wherein the foaming solution comprises more than 10% by weight of the polygalacturonic acid compound.

[0143] Example 37 relates to the method of example 36, wherein the foaming solution comprises more than 30% by weight of the polygalacturonic acid compound.

[0144] Example 38 relates to the method of any of Examples 19 to 37, wherein the foaming solution comprises more than 3 wt. % of a water-soluble polymer having a surface activity.

[0145] Example 39 relates to the method of example 38, wherein the foaming solution comprises greater than about 10% by weight of a water-soluble polymer having a surface activity.

[0146] Example 40 relates to the method of any one of Examples 19 to 39, wherein the foaming solution comprises more than 2 wt. % of a divalent metal salt.

[0147] Example 41 relates to the method of example 40, wherein the foaming solution comprises greater than about 5% by weight of a divalent metal salt.

[0148] Example 42 relates to the method of any of Examples 19 to 41, wherein the foaming solution comprises more than 10 wt% of a gel-inducing agent.

[0149] Example 43 relates to the method of example 42, wherein the foaming solution comprises more than 40 wt. % of the gel-inducing agent.

[0150] Aspect 44 relates to a method for culturing cells on a dissolvable foam scaffold, the method comprising: seeding cells onto a dissolvable foam scaffold such that the cells enter the pores of the dissolvable foam scaffold, wherein the dissolvable scaffold comprises an ionotropically crosslinked polygalacturonic acid compound selected from at least one of pectinic acid; partially esterified pectinic acid, partially amidated pectinic acid, and salts thereof, and a water-soluble polymer that is surface-active and has a hydrophilic-lipophilic balance (HLB) of greater than about 20; and contacting the dissolvable foam scaffold with cell culture medium.

[0151] Example 45 relates to the method of example 44, wherein the water-soluble polymer has an HLB of about 22 or greater.

[0152] Example 46 relates to the method of example 44 or 45, wherein the dissolvable foam scaffold further comprises 0 wt% water soluble plasticizer.

[0153] Example 47 relates to a method according to any one of Examples 44 to 46, wherein the dissolvable foam scaffold does not comprise glycerol, sorbitol, ethylene glycol, propylene glycol, or polyethylene glycol.

[0154] Example 48 relates to a method according to any of Examples 44 to 47, wherein the dissolvable foam scaffold does not comprise a polymer that is not surface active.

[0155] Example 49 relates to a method according to any one of Examples 44 to 48, wherein the dissolvable foam scaffold consists of an ionotropically crosslinked polygalacturonic acid compound and a surface-active water-soluble polymer.

[0156] Embodiment 50 relates to a method according to any of embodiments 44 to 48, wherein the cells aggregate within the pores of the dissolvable foam scaffold to form spheroids.

[0157] Example 51 relates to a method according to any of Examples 44 to 48 and 50, wherein the dissolvable foam scaffold comprises an adhesive polymer coating, and seeding the dissolvable foam scaffold with cells comprises adhering the cells to the surface of the dissolvable foam scaffold.

[0158] Example 52 relates to the method of example 51, wherein the dissolvable foam scaffold consists of an ionotropically crosslinked polygalacturonic acid compound, a surface-active water-soluble polymer, and an adhesive polymer coating.

[0159] Example 53 relates to the method of any of Examples 44 to 48 and 50 to 52, wherein contacting the dissolvable foam scaffold with cell culture medium comprises submerging the dissolvable foam scaffold in the cell culture medium.

[0160] Example 54 relates to the method of any of Examples 44 to 53, wherein contacting the dissolvable foam scaffold with cell culture medium comprises continuously passing cell culture medium over the dissolvable foam scaffold.

[0161] Example 55 relates to the method of example 54, wherein continuously passing cell culture medium over the dissolvable foam scaffold comprises removing at least a portion of the cell culture medium from contact with the dissolvable foam scaffold and contacting the dissolvable foam scaffold with fresh cell culture medium such that a volume of cell culture medium in contact with the dissolvable foam scaffold remains substantially constant.

[0162] Example 56 relates to the method of any of Examples 44 to 55, further comprising digesting the dissolvable foam scaffold by exposing the dissolvable foam scaffold to an enzyme; and exposing the dissolvable foam scaffold to a chelating agent.

[0163] Embodiment 57 relates to the method according to embodiment 56, wherein the enzyme comprises a non-proteolytic enzyme.

[0164] Aspect 58 relates to the method of aspect 57, wherein the non-proteolytic enzyme is selected from the group consisting of pectin-degrading enzymes and pectinases.

[0165] Example 59 relates to the method of any of Examples 56 to 58, wherein digesting the dissolvable foam scaffold comprises exposing the dissolvable foam scaffold to between about 1 U and about 200 U of the enzyme.

[0166] Example 60 relates to a method according to any of Examples 56 to 59, comprising exposing the dissolvable foam scaffold to between about 1 mM and about 200 mM of a chelating agent.

[0167] Example 61 relates to a foamed scaffold product formed from a composition comprising: an ionotropically crosslinked polygalacturonic acid compound selected from at least one of pectinic acid; partially esterified pectinic acid, partially amidated pectinic acid, and salts thereof; at least one first water-soluble polymer that is surface-active and has a hydrophilic-lipophilic balance (HLB) greater than about 20; and 0 wt. % of a water-soluble plasticizer.

[0168] Example 62 relates to the foamed scaffold product of example 61, further comprising an adhesive polymer coating.

[0169] Example 63 relates to a foam scaffold product according to example 62, wherein the adhesive polymer coating comprises a peptide.

[0170] Example 64 relates to a foam scaffold product of example 62, wherein the adhesive polymer coating comprises a peptide selected from the group consisting of BSP, vitronectin, fibronectin, laminin, type I collagen, type IV collagen, denatured collagen, and mixtures thereof.

[0171] Example 65 relates to a foam scaffold product of example 62, wherein the adhesive polymer coating comprises Synthemax® II-SC.

[0172] Example 66 relates to an expanded scaffold product of any of Examples 61-65, wherein the at least one first polymer comprises a cellulose derivative.

[0173] Example 67 relates to a foamed scaffold product of any of Examples 61 to 65, wherein the at least one first polymer comprises a protein.

[0174] Example 68 relates to an expanded scaffold product of any of Examples 61-65, wherein the at least one first polymer comprises a synthetic amphiphilic polymer.

[0175] Example 69 relates to a foamed scaffold product of any of Examples 61 to 68, wherein the dissolvable foamed scaffold comprises only a single water-soluble polymer that is surface active.

[0176] Example 70 relates to a foam scaffold product of any of Examples 61-69, wherein the dissolvable foam scaffold does not comprise a non-surface active polymer.

[0177] Example 71 relates to a foamed scaffold product of any of Examples 61 to 70, comprising a porosity of about 85% to about 96%.

[0178] Example 72 relates to a foamed scaffold product of any of Examples 61 to 71, comprising an average pore size of from about 50 μm to about 500 μm.

[0179] Example 73 relates to a foamed scaffold product according to any of Examples 61-72, comprising an open pore structure.

[0180] Example 74 relates to a foamed scaffold product according to any of Examples 61 to 73, wherein the composition does not comprise an emulsifier.

[0181] Example 75 relates to a foamed scaffold product according to any of Examples 61 to 74, wherein the composition does not comprise glycerol, sorbitol, ethylene glycol, propylene glycol, polyethylene glycol, dextran, or sucrose.

[0182] Example 76 relates to a foam scaffold product of any of Examples 61 to 75, wherein the composition comprises greater than 10 wt.% of a polygalacturonic acid compound.

[0183] Example 77 relates to a foam scaffold product according to example 76, wherein the composition comprises more than 30% by weight of a polygalacturonic acid compound.

[0184] Example 78 relates to a foamed scaffold product of any of Examples 61 to 77, wherein the composition comprises greater than 3 wt.% of a water-soluble polymer having a surface activity.

[0185] Example 79 relates to the foamed scaffold product of example 78, wherein the composition comprises greater than about 10% by weight of a water-soluble polymer having a surface activity.

[0186] Example 80 relates to a foam scaffold product of any of Examples 61-79, wherein the composition comprises greater than 2 wt. % of a divalent metal salt.

[0187] Example 81 relates to a foam scaffold product of example 80, wherein the composition comprises greater than about 5% by weight of a divalent metal salt.

[0188] Example 82 relates to a foam scaffold product of any of Examples 61 to 81, wherein the composition comprises greater than 10 wt% of a gel-inducing agent.

[0189] Example 83 relates to a foam scaffold product of example 82, wherein the composition comprises more than 40 wt% of a gel-inducing agent.

[0190] While the present disclosure includes a limited number of embodiments, those skilled in the art, having the benefit of this disclosure, will recognize that other embodiments may be devised that do not depart from the scope of the present disclosure.

Claims

1. 1. A dissolvable foam scaffold for cell culture comprising: pectic acid; an ionotropically crosslinked polygalacturonic acid compound selected from at least one of partially esterified pectinic acid, partially amidated pectinic acid, and salts thereof; a water-soluble polymer that is surface-active and has a hydrophilic-lipophilic balance (HLB) of greater than about 20.

2. 10. The dissolvable foam scaffold of claim 1, wherein the HLB of the water-soluble polymer is about 22 or greater.

3. 3. The dissolvable foam scaffold of claim 1 or 2, wherein the dissolvable foam scaffold comprises 0 wt% water soluble plasticizer.

4. 10. The dissolvable foam scaffold of any one of the preceding claims, wherein the dissolvable foam scaffold does not comprise glycerol, sorbitol, ethylene glycol, propylene glycol, polyethylene glycol.

5. 10. The dissolvable foam scaffold according to any one of the preceding claims, further comprising an adhesive polymer coating.

6. The dissolvable foam scaffold of claim 5 , wherein the adhesive polymer coating comprises a peptide.

7. 6. The dissolvable foam scaffold of claim 5, wherein the adhesive polymer coating comprises a peptide selected from the group consisting of BSP, vitronectin, fibronectin, laminin, type I collagen, type IV collagen, denatured collagen, and mixtures thereof.

8. The dissolvable foam scaffold of claim 5, wherein the adhesive polymer coating comprises Synthemax® II-SC.

9. The dissolvable foam scaffold according to any one of claims 1 to 8, wherein the at least one first polymer comprises a protein.

10. The dissolvable foam scaffold according to any one of claims 1 to 8, wherein the at least one first polymer comprises a synthetic amphiphilic polymer.

11. 10. The dissolvable foam scaffold according to any one of the preceding claims, wherein the dissolvable foam scaffold comprises only a single water-soluble polymer that has surface activity.

12. 10. The dissolvable foam scaffold of any one of the preceding claims, wherein the dissolvable foam scaffold does not comprise a polymer that is not surface active.

13. 10. The dissolvable foam scaffold of any one of the preceding claims, wherein the dissolvable foam scaffold does not comprise dextran.

14. the dissolvable foam scaffold comprising: the ionotropically crosslinked polygalacturonic acid compound, and 10. A dissolvable foam scaffold according to any one of the preceding claims, consisting of said surface-active water-soluble polymer.

15. the dissolvable foam scaffold comprising: the ionotropically crosslinked polygalacturonic acid compound, the surface-active water-soluble polymer, and A dissolvable foam scaffold according to any one of claims 1 to 13, consisting of said adhesive polymer coating.

16. 10. The dissolvable foam scaffold according to any one of the preceding claims, comprising an average pore size of from about 50 μm to about 500 μm.

17. 10. A dissolvable foam scaffold according to any one of the preceding claims, comprising an open pore structure.

18. 10. The dissolvable foam scaffold of any one of the preceding claims, wherein digestion of the dissolvable foam scaffold is complete in less than about 1 hour.

19. 1. A method for forming a dissolvable foam scaffold, said method comprising: forming a first aqueous mixture by adding a polygalacturonic acid compound selected from pectinic acid; at least one of partially esterified pectinic acid, partially amidated pectinic acid, and salts thereof to an aqueous solution; forming a second aqueous mixture by adding a surface-active water-soluble polymer and a divalent metal salt to an aqueous solution; combining the first aqueous mixture with a second first aqueous mixture to form a combined aqueous mixture; adding a gel-inducing agent to the combined aqueous mixture to form a foaming solution; introducing air bubbles into the foam solution to form a foam scaffold; The method wherein the water-soluble polymer has a hydrophilic-lipophilic balance (HLB) of greater than about 20.

20. 20. The method of claim 19, wherein the HLB of the water-soluble polymer is about 22 or greater.

21. 21. The method of claim 19 or 20, wherein forming the second aqueous mixture does not include adding a water-soluble plasticizer to the second aqueous mixture.

22. 22. The method of any one of claims 19 to 21, wherein forming the second aqueous mixture does not include adding a second polymer that is not surface active.

23. 23. The method of any one of claims 19 to 22, wherein forming the second aqueous mixture comprises adding only a single water-soluble polymer that is surface active.

24. The divalent metal salt is a cation selected from the group consisting of magnesium, calcium, zinc, strontium, barium, and combinations thereof; and an anion selected from the group consisting of oxalate, tartrate, phosphate, carbonate, citrate, and combinations thereof.

25. 25. The method of any one of claims 19 to 24, wherein 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.

26. 26. The method of any one of claims 19 to 25, wherein forming a second aqueous mixture further comprises adding at least one leachable solid to the second aqueous mixture.

27. 27. The method of claim 26, wherein the at least one leachable solid is selected from the group consisting of salts and water-soluble proteins.

28. 28. The method of any one of claims 19 to 27, wherein the gel-inducing agent is selected from the group consisting of lactic acid lactone, glycolic acid lactone, glucono-delta-lactone, and acid anhydrides.

29. 29. The method of any one of claims 19-28, wherein adding the gel-inducing agent to the combined aqueous mixture comprises adding a first amount of the gel-inducing agent to the aqueous mixture before foaming, and adding a second amount of the gel-inducing agent to the aqueous mixture during or after foaming.

30. 30. The method of claim 29, wherein the ratio of the second amount of gel-inducing agent to the first amount of gel-inducing agent is greater than about 2.

31. 31. The method of claim 30, wherein the ratio is about 7 or greater.

32. 32. The method of any one of claims 19 to 31, further comprising coating the dissolvable foam scaffold with an adhesive polymer coating.

33. 33. The method of claim 32, wherein the adhesive polymer coating comprises a peptide.

34. 33. The method of claim 32, wherein the adhesive polymer coating comprises a peptide selected from the group consisting of BSP, vitronectin, fibronectin, laminin, type I collagen, type IV collagen, denatured collagen, and mixtures thereof.

35. The method of claim 32, wherein the adhesive polymer coating comprises Synthemax® II-SC.

36. 36. The method of any one of claims 19 to 35, wherein the foaming solution comprises more than 10% by weight of the polygalacturonic acid compound.

37. 37. The method of claim 36, wherein the foaming solution comprises greater than 30% by weight of the polygalacturonic acid compound.

38. 38. The method of any one of claims 19 to 37, wherein the foaming solution comprises more than 3% by weight of the surface-active water-soluble polymer.

39. 39. The method of claim 38, wherein the foaming solution comprises greater than about 10% by weight of the surface-active water-soluble polymer.

40. 40. The method of any one of claims 19 to 39, wherein the foaming solution comprises more than 2% by weight of the divalent metal salt.

41. 41. The method of claim 40, wherein the foaming solution comprises greater than about 5% by weight of the divalent metal salt.

42. 42. The method of any one of claims 19 to 41, wherein the foaming solution comprises more than 10% by weight of the gel-inducing agent.

43. 43. The method of claim 42, wherein the foaming solution comprises greater than 40% by weight of the gel-inducing agent.

44. 1. A method for culturing cells on a dissolvable foam scaffold, said method comprising: Seeding the cells into a dissolvable foam scaffold such that the cells are contained within the pores of the dissolvable foam scaffold, wherein the dissolvable scaffold comprises: pectic acid; an ionotropically crosslinked polygalacturonic acid compound selected from at least one of partially esterified pectinic acid, partially amidated pectinic acid, and salts thereof; a water-soluble polymer that is surface active and has a hydrophilic-lipophilic balance (HLB) greater than about 20; contacting the dissolvable foam scaffold with cell culture medium.

45. 45. The method of claim 44, wherein the HLB of the water-soluble polymer is about 22 or greater.

46. 46. ​​The method of claim 44 or 45, wherein the dissolvable foam scaffold further comprises 0 wt% water soluble plasticizer.

47. 47. The method of any one of claims 44 to 46, wherein the dissolvable foam scaffold does not comprise glycerol, sorbitol, ethylene glycol, propylene glycol, or polyethylene glycol.

48. 48. The method of any one of claims 44 to 47, wherein the dissolvable foam scaffold does not comprise a non-surface active polymer.

49. the dissolvable foam scaffold comprising: the ionotropically crosslinked polygalacturonic acid compound, and The method according to any one of claims 44 to 48, comprising the surface-active water-soluble polymer.

50. 49. The method of any one of claims 44 to 48, wherein cells aggregate within the pores of the dissolvable foam scaffold to form spheroids.

51. 51. The method of any one of claims 44-48 and 50, wherein the dissolvable foam scaffold comprises an adhesive polymer coating, and seeding the dissolvable foam scaffold with cells comprises adhering cells to a surface of the dissolvable foam scaffold.

52. the dissolvable foam scaffold comprising: the ionotropically crosslinked polygalacturonic acid compound, the surface-active water-soluble polymer, and 52. The method of claim 51, comprising the adhesive polymer coating.

53. 53. The method of any one of claims 44-48 and 50-52, wherein contacting the dissolvable foam scaffold with cell culture medium comprises submerging the dissolvable foam scaffold in cell culture medium.

54. 54. The method of any one of claims 44 to 53, wherein contacting the dissolvable foam scaffold with cell culture medium comprises continuously passing cell culture medium over the dissolvable foam scaffold.

55. 55. The method of claim 54, wherein continuously passing cell culture medium over the dissolvable foam scaffold comprises removing at least a portion of the cell culture medium from contact with the dissolvable foam scaffold and contacting the dissolvable foam scaffold with fresh cell culture medium such that a volume of cell culture medium in contact with the dissolvable foam scaffold remains substantially constant.

56. digesting the dissolvable foam scaffold by exposing the dissolvable foam scaffold to an enzyme; 56. The method of any one of claims 44-55, further comprising exposing the dissolvable foam scaffold to a chelating agent.

57. 57. The method of claim 56, wherein the enzyme comprises a non-proteolytic enzyme.

58. 58. The method of claim 57, wherein the non-proteolytic enzyme is selected from the group consisting of pectin-degrading enzymes and pectinases.

59. 59. The method of any one of claims 56-58, wherein digesting the dissolvable foam scaffold comprises exposing the dissolvable foam scaffold to about 1 U to about 200 U of the enzyme.

60. 60. The method of any one of claims 56-59, comprising exposing the dissolvable foam scaffold to about 1 mM to about 200 mM of the chelating agent.

61. 1. A foamed scaffold product formed from a composition, said composition comprising: pectic acid; an ionotropically crosslinked polygalacturonic acid compound selected from at least one of partially esterified pectinic acid, partially amidated pectinic acid, and salts thereof; at least one first water-soluble polymer that is surface active and has a hydrophilic-lipophilic balance (HLB) greater than about 20; 0% by weight of a water-soluble plasticizer.

62. 62. The foam scaffold product of claim 61, further comprising an adhesive polymer coating.

63. 63. The foam scaffold product of claim 62, wherein the adhesive polymer coating comprises a peptide.

64. 63. The foam scaffold product of claim 62, wherein said adhesive polymer coating comprises a peptide selected from the group consisting of BSP, vitronectin, fibronectin, laminin, type I collagen, type IV collagen, denatured collagen, and mixtures thereof.

65. 63. The foam scaffold product of claim 62, wherein the adhesive polymer coating comprises Synthemax® II-SC.

66. 66. The expanded scaffold product of any one of claims 61 to 65, wherein the at least one first polymer comprises a cellulose derivative.

67. 66. The foam scaffold product of any one of claims 61 to 65, wherein the at least one first polymer comprises a protein.

68. 66. The foam scaffold product of any one of claims 61 to 65, wherein the at least one first polymer comprises a synthetic amphiphilic polymer.

69. 69. A foam scaffold product according to any one of claims 61 to 68, wherein the dissolvable foam scaffold comprises only a single water-soluble polymer that has surface activity.

70. 70. A foam scaffold product according to any one of claims 61 to 69, wherein the dissolvable foam scaffold does not comprise a non-surface active polymer.

71. 71. The foam scaffold product of any one of claims 61 to 70, comprising a porosity of about 85% to about 96%.

72. 72. The foam scaffold product of any one of claims 61 to 71, comprising an average pore size of from about 50 μm to about 500 μm.

73. 73. The foam scaffold product of any one of claims 61 to 72, comprising an open pore structure.

74. 74. The foam scaffold product of any one of claims 61 to 73, wherein the composition is emulsifier-free.

75. 75. The foam scaffold product of any one of claims 61 to 74, wherein the composition does not comprise glycerol, sorbitol, ethylene glycol, propylene glycol, polyethylene glycol, dextran, or sucrose.

76. 76. The foam scaffold product of any one of claims 61 to 75, wherein the composition comprises more than 10% by weight of the polygalacturonic acid compound.

77. 77. The foam scaffold product of claim 76, wherein said composition comprises greater than 30% by weight of said polygalacturonic acid compound.

78. 78. The expanded scaffold product of any one of claims 61 to 77, wherein the composition comprises more than 3 wt% of said surface-active water-soluble polymer.

79. 79. The foam scaffold product of claim 78, wherein said composition comprises greater than about 10% by weight of said surface-active water-soluble polymer.

80. 80. The foam scaffold product of any one of claims 61 to 79, wherein the composition comprises more than 2% by weight of said divalent metal salt.

81. 81. The foam scaffold product of claim 80, wherein the composition comprises greater than about 5% by weight of the divalent metal salt.

82. 82. The foam scaffold product of any one of claims 61 to 81, wherein the composition comprises more than 10% by weight of the gel-inducing agent.

83. 83. The foam scaffold product of claim 82, wherein the composition comprises greater than 40% by weight of the gel-inducing agent.

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