Dissolvable foamed scaffold for cell culture and method for producing the same

Abstract

Dissolvable foam scaffolds for cell culture are provided herein, which comprise an ionotropic cross-linked polygalacturonic acid compound selected from at least one of pectinic acid, partially esterified pectinic acid, partially amidated pectinic acid, and salts thereof, and at least one first water-soluble polymer having surface activity.

Description

Cross - reference to related applications

[0001] This application claims the benefit of priority of U.S. Provisional Patent Application No. 62 / 589,238, filed Nov. 21, 2017, and U.S. Provisional Patent Application No. 62 / 632,178, filed Feb. 19, 2018, the contents of which are relied upon and incorporated herein by reference in their entirety as if fully set forth below.

Technical Field

[0002] The present disclosure generally relates to dissolvable cell culture materials. In particular, the present disclosure relates to dissolvable foamed scaffolds for cell culture and methods of manufacturing such dissolvable foamed scaffolds.

Background Art

[0003] In vitro studies are important for drug discovery processes and the search for potential new therapies. However, conventional in vitro cell culture on two - dimensional (2D) culture substrates cannot simulate the in vivo environment. Almost all cells in the in vivo environment are surrounded in a three - dimensional (3D) manner by other cells and the extracellular matrix (ECM), so 2D cell culture does not appropriately simulate the natural 3D environment of cells. Cells in 2D culture are forced to adhere to a hard surface, are geometrically constrained, and adopt a flat morphology, which can alter cytoskeletal regulation important for intracellular signaling and, as a result, can affect cell growth, migration, and apoptosis. Furthermore, the ECM organization, 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 are significantly different from those observed in vivo.

[0004] Currently, in drug discovery, the standard procedure for screening compounds starts with tests based on 2D cell culture, followed by tests in animal models, and then clinical trials. According to publicly available data, only about 10% of compounds successfully progress through clinical development. Many drugs fail during clinical trials (especially during Phase III, which is the most costly phase of clinical development), mainly due to lack of clinical efficacy and / or unacceptable toxicity. Some of these failures are due to data collected from 2D culture tests where the cellular response to the (one or more) drugs changes due to an unnatural microenvironment. Due to the high costs associated with drug discovery, there is an increasing need for the ability to eliminate ineffective and / or unacceptable toxicity compounds as early as possible in the drug discovery process. In vitro cell-based systems that can more realistically mimic the behavior of cells in vivo and provide more predictable results in in vivo tests are currently being investigated.

[0005] Recent studies have suggested that, in contrast to 2D culture, 3D cell culture more accurately represents the environment that cells experience in vivo, and it has been demonstrated that the cellular response in 3D culture is more similar to in vivo behavior than the cellular response in 2D culture. The additional dimension 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 causes physical constraints on the cells. These spatial and physical aspects in 3D culture are thought to affect signal transduction from the outside to the inside of the cells and ultimately gene expression and cellular behavior.

[0006] The benefits of 3D culture have influenced the development of cell culture technologies that simulate the natural 3D environment of cells. Some bioreactors include carriers in the form of fixed packing materials that form a fixed bed or packed bed to promote cell adhesion and growth. The composition of the fixed bed packing material affects local fluid, heat, and mass transfer and is usually very dense to maximize cell culture in a given space. Yet another 3D cell culture technology is a porous 3D matrix or scaffold that promotes the growth and proliferation of cultured cells within the pores and other internal spaces of the matrix.

[0007] In each of the above techniques, protease treatment can be used for cell retrieval. However, commonly used retrieval procedures, such as protease treatment, expose cells to harsh conditions that can damage their structure and function. In addition, protease treatment alone often results in only a limited amount of cell detachment. With fixed-bed materials, the problem lies partly in the dense nature of the fixed-bed material, which makes it even more difficult to circulate the protease agent throughout the bed to increase the yield of retrieved cells. Similarly, circulating the protease agent within the internal space of a 3D matrix may be difficult, making it difficult to remove cells during the retrieval process. This difficulty is exacerbated by the presence of extracellular macromolecules secreted by cultured cells that help cells adhere to the surface of the fixed-bed material or matrix.

[0008] Methods and systems have been developed for cell recovery that use mechanical force to release cultured cells from fixed bed material or 3D matrix, either as an alternative or in combination with protease treatment. For example, cultured cells can be released by shaking or vibrating the fixed bed material or 3D matrix, or a larger system containing the fixed bed material or 3D matrix. Applying mechanical force can cause physical damage to the cultured cells, which may reduce cell culture yield. [Overview of the Initiative]

[0009] Embodiments of the present disclosure provide a soluble foam scaffold for cell culture. The soluble foam scaffold comprises pectic acid; an ionotropic crosslinked polygalacturonic acid compound selected from at least one of partially esterified pectic acid, partially amidated pectic acid, and salts thereof; and at least one first water-soluble polymer having surface activity.

[0010] Embodiments of the present disclosure provide a method for forming a soluble foamed scaffold. The method includes forming a first aqueous mixture by adding pectic acid; a polygalacturonic acid compound selected from at least one of partially esterified pectic acid, partially amidated pectic acid, and salts thereof to an aqueous solution; forming a second aqueous mixture by adding at least one first water-soluble polymer having surface activity and a divalent metal salt to an aqueous solution; combining the first aqueous mixture and the second aqueous mixture to form a combined aqueous mixture; adding a gel inducer to the combined aqueous mixture; and introducing bubbles into the combined aqueous mixture to form a foamed scaffold.

[0011] Embodiments of the present disclosure provide a method for culturing cells on a soluble foam scaffold. The method comprises seeding cells onto the soluble foam scaffold so that the cells enter the pores of the soluble foam scaffold, and contacting the soluble foam scaffold with a cell culture medium. The soluble scaffold comprises pectic acid; an ionotropic crosslinked polygalacturonic acid compound selected from at least one of partially esterified pectic acid, partially amidated pectic acid, and salts thereof; and at least one first water-soluble polymer having surface activity.

[0012] Embodiments of the present disclosure provide a method for recovering cells from a soluble foam scaffold. The method comprises digesting the soluble foam scaffold by exposing it to an enzyme, and exposing the soluble foam scaffold to a chelating agent. The soluble scaffold comprises pectic acid; an ionotropic cross-linked polygalacturonic acid compound selected from at least one of partially esterified pectic acid, partially amidated pectic acid, and salts thereof; and at least one first water-soluble polymer having surface activity. Embodiments of the present disclosure provide a foam scaffold product formed from a composition. The composition comprises pectic acid; an ionotropic cross-linked polygalacturonic acid compound selected from at least one of partially esterified pectic acid, partially amidated pectic acid, and salts thereof; at least one first water-soluble polymer having surface activity; and less than about 55% by mass of a water-soluble plasticizer.

[0013] Additional features and advantages are described below in detail and some will be readily apparent to those skilled in the art from that description, or will be recognized by carrying out the embodiments described herein, including the following detailed description, claims, and accompanying drawings.

[0014] It should be understood that both the above summary and the detailed description below are merely illustrative and intended to provide an overview or framework for understanding the nature and features of the claims. The accompanying drawings are included to provide further understanding and are incorporated herein and constitute part thereof. The drawings illustrate one or more embodiments and, together with their descriptions, serve to illustrate the principles and operation of the various embodiments.

[0015] This disclosure will be better understood from the following description and accompanying drawings, which are given purely as non-limiting examples. [Brief explanation of the drawing]

[0016] [Figure 1]Perspective view of the dissolvable foam scaffolding according to this disclosure [Figure 2] SEM image of the foamed scaffolding prepared in Example 1 [Figure 3] SEM image of the foam scaffolding prepared in Example 2 [Figure 4] SEM image of the foamed scaffolding prepared in Example 3 [Figure 5] SEM image of the foamed scaffolding prepared in Example 4 [Figure 6] SEM image of the foamed scaffolding prepared in Example 5 [Figure 7] SEM image of the foamed scaffolding prepared in Example 6 [Figure 8] SEM image of the foamed scaffolding prepared in Example 7 [Figure 9] SEM image of the foam scaffolding prepared in Example 8 [Figure 10] SEM image of the foamed scaffolding prepared in Example 9 [Figure 11] SEM image of the foamed scaffolding prepared in Example 10 [Figure 12] SEM image of the foamed scaffolding prepared in Example 11 [Figure 13] Photographs of the four foams prepared in Example 12, to which varying amounts of plasticizer were added to form four foam scaffolds. [Figure 14] SEM image of the foamed scaffolding prepared in Example 13 [Figure 15] Photograph showing spheroids formed in the pores of the foamed scaffold prepared in Example 4. [Figure 16] Photograph showing cells attached to the foam scaffold prepared in Example 14. [Figure 17] Photograph showing cells attached to the foam scaffold prepared in Example 15. [Figure 18] Photograph showing cells after 6 days of proliferation attached to the foam scaffold prepared in Example 15. [Figure 19] Bar graph showing the GFP positivity rate of transfected HEK cells for each of the culture conditions in Example 24. [Figure 20]Bar graph showing the GFP positivity rate of transfected cells per set of foam scaffolds in Example 25. [Figure 21] Bar graph showing the number of virus particles (VP) per set of foam scaffolding in Example 25. [Figure 22] Bar graph showing the number of virus particles per cell obtained per set of foam scaffolds in Example 25. [Figure 23] Bar graph showing the percentage of cells exhibiting GFP expression in each of the 25 foam scaffold sets after infection. [Modes for carrying out the invention]

[0017] Hereafter, we will refer in detail to this embodiment, an example of which is shown in the attached drawings. Wherever possible, the same reference numerals will be used throughout the drawings to refer to the same or similar parts.

[0018] The singular forms "a," "an," and "the" refer to multiple objects unless the context clearly indicates otherwise. All endpoints of ranges enumerating the same characteristic are independently combinable and include the enumerated endpoints. All references are incorporated herein by reference.

[0019] As used herein, "have," "possess," "include," "include," "equip," and "equip" are used in an open-ended sense and generally mean "include but not limited to."

[0020] All scientific and technical terms used herein have their common meanings in the art unless otherwise specified. The definitions provided herein are for the purpose of facilitating the understanding of certain terms that are frequently used herein and are not intended to limit the scope of this disclosure.

[0021] This disclosure is described in detail below, first in general terms, and then based on several exemplary embodiments. Features shown in combination with each other in individual exemplary embodiments do not necessarily have to be realized. In particular, individual features may be omitted or combined in several other ways with other features shown in the same exemplary embodiment or other exemplary embodiments.

[0022] Embodiments of this disclosure relate to soluble foam scaffolds for cell culture and methods for manufacturing such soluble foam scaffolds. Embodiments of this disclosure further relate to methods for cell culture of adherent cells, cell aggregates, or spheroids on soluble foam scaffolds. Furthermore, embodiments of this disclosure relate to bioreactor systems comprising soluble foam scaffolds. As will become apparent in the following discussion, the foam scaffolds disclosed herein are described as both soluble and insoluble. As used herein, the term “insoluble” is used to refer to a material or combination of materials that does not dissolve and remains crosslinked under conventional cell culture conditions, such as cell culture media. Also, as used herein, the term “soluble” is used to refer to a material or combination of materials that is digested when exposed to an appropriate concentration of enzymes that digest or decompose the material or combination of materials. The soluble foam scaffolds described herein are porous scaffolds having open structures and highly interconnected pores. The pores of the scaffold provide a protected environment for cell culture that facilitates cell-cell interactions and the formation of an ECM in 3D form. The soluble foam scaffold is completely digested, and cells can be recovered without damaging them using protease treatment and / or mechanical recovery techniques.

[0023] Figure 1 is a perspective view of the soluble foam scaffold 10 according to the present disclosure. As will be described in more detail below and will be more apparent from other figures of the present disclosure, the soluble foam scaffold 10 is a porous foam containing an open structure. The soluble foam scaffold 10 has a porosity of about 85% to about 96% and an average pore diameter of about 50 μm to about 500 μm. The soluble foam scaffold 10 provides a protected environment within the pores of the foam scaffold for cell culture. In addition, the soluble foam scaffold 10 is also soluble when exposed to appropriate enzymes that digest or decompose the material, facilitating the recovery of cells cultured on the scaffold without damaging the cells.

[0024] The soluble foam scaffolds described herein contain at least one ionotropically crosslinked polysaccharide. Generally, polysaccharides possess attributes beneficial to cell culture applications. Polysaccharides are hydrophilic, non-cytotoxic, and stable in culture media. Examples include pectic acid or salts thereof, also known as polygalacturonic acid (PGA), partially esterified pectic acid or salts thereof, or partially amidated pectic acid or salts thereof. Pectic acid can be formed by the hydrolysis of certain pectin esters. Pectin is a cell wall polysaccharide and plays a structural role in plants in nature. Major sources of pectin include citrus peels (e.g., lemon and lime peels) and apple peels. Pectin is a predominantly linear polymer based on a 1,4-linked alpha-D-galacturonate skeleton randomly interrupted by 1,2-linked L-rhamnose. The average molecular weight ranges from approximately 50,000 to approximately 200,000 daltons.

[0025] The polygalacturonic acid chains of pectin may be partially esterified, for example, 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 pectic acid, and its salts are called pectinates. The degree of methylation (DM) of high methoxyl (HM) pectin may be, for example, 60-75 mol%, and the degree of methylation of low methoxyl (LM) pectin may be 1-40 mol%. The degree of esterification of partially esterified polygalacturonic acid described herein may 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 ​​in between. Although not bound by any particular theory, it is thought that a minimum amount of free carboxylic acid groups (unesterified) promotes some degree of ionotropic crosslinking, enabling the formation of an insoluble, soluble scaffold.

[0026] Alternatively, the polygalacturonic acid chains of pectin may be partially amidated. Partially amidated pectin can be produced, for example, by treatment with ammonia. Amidated pectin contains a carboxyl group (~COOH), a methyl ester group (~COOCH3), and an amidating group (-CONH2). The degree of amidation may vary, for example, about 10% to about 40% may be amidated.

[0027] According to embodiments of the present disclosure, the soluble 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 alcohols.

[0028] According to embodiments of the present disclosure, the soluble foam scaffolds described herein may further comprise at least one first polymer. The at least one first polymer is water-soluble, non-ionotropically crosslinkable, and surface-active. As used herein, the term “surface-active” refers to the activity of an agent that reduces or eliminates surface tension (or interfacial tension) between two liquids, or between a liquid and a solid, or between a gas and a liquid. The at least one first polymer may have a hydrophilic-lipophilic balance (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 greater than about 10 to about 12. HLB provides a criterion for the degree of lipophilicity or hydrophilicity of a polymer. A higher HLB value indicates stronger hydrophilicity, and a lower HLB value indicates stronger lipophilicity. Generally, HLB values ​​vary in the range of 1 to 40, and the hydrophilic-lipophilic transition is often considered to be between approximately 8 and 10. If the HLB value is less than the hydrophilic-lipophilic transition, the material is lipophilic, and if the HLB value is greater than the hydrophilic-lipophilic transition, the material is hydrophilic.

[0029] The exemplary first polymers according to embodiments of the present disclosure may be cellulose derivatives, proteins, synthetic amphiphilic polymers, or combinations thereof. Exemplary cellulose derivatives include, but are not limited to, hydroxyethylcellulose (HEC), hydroxypropylcellulose (HPC), methylcellulose (MC), hydroxyethylmethylcellulose (HEMC), and hydroxypropylmethylcellulose (HPMC). Exemplary proteins include, but are not limited to, bovine serum albumin (BSA), gelatin, casein, and hydrophobin. Exemplary synthetic amphiphilic polymers include, but are not limited to, poloxamer available under the trade name Synperonics® (commercially available from Croda International in Snice, UK), poloxamer available under the trade name Pluronics® (commercially available from BASF Corp. in Parsippany, New Jersey, USA), and poloxamer available under the trade name Kolliphor® (commercially available from BASF Corp. in Parsippany, New Jersey, USA).

[0030] The soluble foam scaffolds described herein may further comprise at least one second polymer. At least one second polymer is water-soluble and non-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; homopolymers and copolymers of (2-hydroxypropyl)methacrylamide; 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, ethylcellulosecellulose, ethylhydroxyethylcellulose, and hydroxypropylcellulose. Exemplary natural polymers include, but are not limited to, polymers obtained by microbial fermentation such as starch and starch derivatives, curdlan, pullulan and gellan gum, xanthan gum, and dextran; proteins such as albumin, casein, and casein salts; 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 soluble foam scaffolds described herein may be crosslinked to increase their mechanical strength and to prevent dissolution of the scaffold when placed in contact with cell culture media. Crosslinking can be carried out by ionotropic gelation, as described below, which is based on the ability of polyelectrolytes to crosslink and form crosslinked scaffolds in the presence of polyvalent counterions. Although not bound by any particular theory, the ionotropic gelation of polysaccharides in soluble foam scaffolds is thought to be the result of a strong interaction between divalent cations and polysaccharides.

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

[0033] The scaffolds described herein can have a wet density of less than about 0.40 g / cm 3 [[ID=⑥]]For example, the scaffolds described herein can have a wet density of less than about 0.35 g / cm 3 [[ID=⑧]]or less than about 0.30 g / cm 3 [[ID=⑩]]or less than about 0.25 g / cm 3 The scaffolds described herein can have a wet density of from about 0.16 g / cm 3 to about 0.40 g / cm 3 or from about 0.16 g / cm 3 to about 0.35 g / cm 3 or from about 0.16 g / cm 3 to about 0.30 g / cm 3 or even from about 0.16 g / cm 3 to about 0.25 g / cm 3 and can have a wet density of all values in between. The scaffolds described herein can have a dry density of less than about 0.20 g / cm 3 For example, the scaffolds described herein can have a dry density of less than about 0.15 g / cm 3 or less than about 0.10 g / cm 3 or less than about 0.05 g / cm 3 The scaffolds described herein can have a dry density of from about 0.02 g / cm 3 to about 0.20 g / cm 3 or from about 0.02 g / cm3 ~Approx. 0.15g / cm 3 , or approximately 0.02 g / cm³ 3 ~Approx. 0.10g / cm 3 , or even approximately 0.02 g / cm³ 3 ~about 0.05g / cm 3 It can have dry densities of all values, and all values ​​in between.

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

[0035] The soluble foam scaffolds described herein are digested when exposed to suitable enzymes that digest or break down the material. Non-proteolytic enzymes suitable for digesting foam scaffolds, retrieving cells, or both include pectinases, which are a heterogeneous group of related enzymes that hydrolyze pectin. Pectinases (polygalacturonases) are enzymes that break down complex pectin molecules into shorter molecules of galacturonic acid. Commercially available sources of pectinases are generally polyenzymes such as Pectinex® ULTRA SP-L (commercially available from Novozyme North American, Inc., Franklington, North Carolina, USA), which are pectinase preparations produced from selected strains of Aspergillus aculeatus. Pectinex ULTRA SP-L primarily contains polygalacturonase (EC 3.2.1.15), pectin trans-eliminase (EC 4.2.2.2), and pectin esterase (EC 3.1.1.11). EC designation is the Enzyme Committee's classification scheme for enzymes based on the chemical reactions they catalyze.

[0036] According to embodiments of the present disclosure, the digestion of a soluble foam scaffold also includes exposing the scaffold to a divalent cation chelating agent. Exemplary chelating agents include, but are not limited to, ethylenediaminetetraacetic acid (EDTA), cyclohexanediaminetetraacetic acid (CDTA), ethylene glycoltetraacetic acid (EGTA), citric acid, and tartaric acid.

[0037] The time required to extinguish the dissolvable foam scaffolding described herein may be less than approximately one hour. For example, the time required to extinguish the foam scaffolding may be less than approximately 45 minutes, or less than approximately 30 minutes, or less than approximately 15 minutes, or approximately 1 to approximately 25 minutes, or approximately 3 to approximately 20 minutes, or even approximately 5 to approximately 15 minutes.

[0038] According to embodiments of this disclosure, the scaffold described herein may further include an adhesive polymer coating. The adhesive polymer may include peptides. 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. In addition, the peptides may have an RGD sequence. The coating may be, for example, Synthemax® II-SC (commercially available from Corning, Incorporated, Corning, New York, USA). Optionally, the adhesive polymer may include an extracellular matrix. The coating may be, for example, Matrigel® (commercially available from Corning, Incorporated, Corning, New York, USA).

[0039] Embodiments of this disclosure also disclose a method for forming a soluble foam scaffold as described herein. The method described herein may include forming a first aqueous mixture, which comprises dissolving a polysaccharide in an aqueous solution. The polysaccharide may be any of the above, such as pectinic acid or salts thereof, partially esterified pectinic acid or salts thereof, or partially amidated pectinic acid or salts thereof, and blends thereof.

[0040] A method for forming a soluble 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 compatible cations, as well as combinations thereof. The anion of the divalent metal salt may include, but is not limited to, oxalates, tartrates, phosphates, carbonates, citrates, and similar organic and inorganic anions, as well as combinations thereof.

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

[0042] According to embodiments of this disclosure, forming a 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 polysaccharides in the soluble foam scaffold. The plasticizers provide flexibility and softness to the resulting foam so that the resulting foam is soft and pliable. The 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 a water-soluble plasticizer to the second aqueous mixture may include adding less than about 55% by mass of the total solid additives added to form the second aqueous mixture. For example, adding a water-soluble plasticizer to a second aqueous mixture may include adding less than about 50% by mass, or less than about 40% by mass, or less than about 30% by mass, or less than about 25% by mass, or about 15% to about 55% by mass, or about 15% to about 50% by mass, or about 15% to about 40% by mass, or about 15% to about 30% by mass, or about 15% to about 25% by mass, and all values ​​in between. As used herein, the term “total solid additives added to form a second aqueous mixture” refers to all components of the aqueous mixture except water.

[0043] According to embodiments of this disclosure, forming a second aqueous mixture may further include adding an emulsifier to the second aqueous mixture. The emulsifiers described herein may include, but are not limited to, Tween® 20 and “Tween” 80 (each commercially available from Croda International, Snays, UK).

[0044] According to embodiments of the present disclosure, forming a second aqueous mixture may further include adding at least one leaching solid to the second aqueous mixture. Leaching solids described herein include materials that reinforce or generate pores during the formation of a foam scaffold. Leaching solids may be, but are not limited to, salts, biocompatible monosaccharides and disaccharides, and non-toxic leaching materials such as 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 relating to the second aqueous mixture may be optionally added to the second aqueous mixture, may be added to the second aqueous mixture in any order, and it is possible to add two or more materials to the second aqueous mixture. In one exemplary method, forming the second aqueous mixture involves adding a leaching solid to an aqueous solution containing a divalent metal salt, and mixing the aqueous mixture to facilitate the dissolution of the leaching solid that dissolves 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] A method for forming a soluble foam scaffold described herein may further include, after the formation of 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 bubbles into the aqueous mixture by mixing, pulsating, stirring, aeration, foaming, injection, or other mechanical action. The gas may be, for example, but not limited to, air, nitrogen, helium, hydrogen, argon, carbon dioxide, or other inert gas. The introduction of bubbles into the combined aqueous mixture can be carried out in a time of less than about 30 minutes, for example, about 1 minute to about 30 minutes, or about 3 minutes to about 25 minutes, or even about 5 minutes to about 20 minutes. A method for forming a soluble foam scaffold while introducing bubbles into the combined aqueous mixture may further include adding a gel inducer to the combined aqueous mixture. The gel inducer may be an acid that provides a buffering effect, and / or a substance that slowly produces an acid. Examples of acids include, but are not limited to, lactones, lactones, glucono delta-lactones, and acid anhydrides.

[0047] A method for forming a soluble foam scaffold described herein may further include coating the soluble foam scaffold with an adhesive polymer coating. Coating the soluble foam scaffold may include exposing the scaffold to an aqueous solution, or an aqueous solution having an adhesive polymer. As previously stated, the adhesive polymer may include peptides. 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. In addition, the peptides may have an RGD sequence. The coating may be, for example, "Synthemax" II-SC (commercially available from Corning, Incorporated, Corning, New York, USA).

[0048] Embodiments of this disclosure also disclose methods for culturing cells on a soluble foam scaffold as described herein. Any type of cell, including but not limited to immortalized cells, primary cultured cells, cancer cells, and stem cells (e.g., embryonic or induced pluripotent), can be cultured on a soluble foam scaffold. The cells may be mammalian cells, avian cells, fish cells, etc. The cells may be of any tissue type, including but not limited to kidney, fibroblast, mammary gland, skin, brain, ovary, lung, bone, nerve, muscle, heart, colorectal gland, pancreas, immune cells (e.g., B cells), and blood. The cells may be cultured in any form within a bag, including dispersed (e.g., newly seeded), confluent, two-dimensional, three-dimensional, spheroid, etc. Culturing cells on a soluble foam scaffold may include seeding cells onto the soluble foam scaffold. Seeding cells onto a soluble foam scaffold may include bringing the scaffold into contact with a solution containing the cells. During the seeding of cells onto a soluble foam scaffold, the cells enter the pores of the soluble foam scaffold. If the soluble foam scaffold includes an adhesive polymer coating, the cells can enter the pores of the soluble foam scaffold and adhere to the scaffold material.

[0049] Culturing cells on a soluble foam scaffold may further include bringing the scaffold into contact with a cell culture medium. Generally, bringing a scaffold into contact with a cell culture medium includes placing the cells to be cultured on the scaffold within an environment having the medium on which the cells are cultured. Bringing a scaffold into contact with a cell culture medium may include pipetting the cell culture medium onto the scaffold, or dipping the scaffold into the cell culture medium, or continuously passing the cell culture medium over the scaffold. Generally, as used herein, the term “continuous” means culturing cells in a consistent flow of cell culture medium entering and leaving the cell culture environment. Such continuous passage of cell culture medium over the scaffold may include dipping the scaffold into the cell culture medium for a predetermined period, then removing at least a portion of the cell culture medium after the predetermined period, and adding fresh cell culture medium so that the volume of cell culture medium in contact with the soluble 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 may be removed and replaced every hour, every 12 hours, every 24 hours, every 2 days, every 3 days, every 4 days, or every 5 days.

[0050] Cell culture media may include, but are not limited to, sugars, salts, amino acids, serum (e.g., fetal bovine serum), antibiotics, growth factors, differentiation factors, colorants, or other desired factors. Exemplary cell culture media include Dulbecco's Modified Eagle Medium (DMEM), Ham's F12 Nutrient Mixture, Minimum Essential Medium (MEM), RPMI medium, Iskov Modified Dulbecco's Medium (IMDM), and Mesencult™-XF medium.

[0051] Embodiments of this disclosure also disclose a method for recovering cells from a soluble foam scaffold described herein. The method for recovering cells described herein may include digesting the soluble foam scaffold by exposing the soluble foam scaffold to an enzyme. As previously stated, non-proteolytic enzymes suitable for digesting foam scaffolds, recovering cells, or both include pectinases, which are a heterogeneous group of related enzymes that hydrolyze pectin. Commercially available sources of pectinases are generally polyenzymes such as "Pectinex" ULTRA SP-L (commercially available from Novozyme North American, Inc., Franklington, North Carolina, USA), a pectinase preparation produced from selected strains of Aspergillus aculeatus. Pectinex ULTRA SP-L primarily contains polygalacturonase (EC 3.2.1.15), pectin trans-eliminase (EC 4.2.2.2), and pectin esterase (EC 3.1.1.11). EC designation is the Enzyme Committee's classification scheme for enzymes based on the chemical reactions they catalyze.

[0052] Exposure of a soluble foam scaffold to an enzyme may include exposing the scaffold to enzyme concentrations of about 1 to about 200 U. For example, the method may include exposing the scaffold to enzyme concentrations 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 ​​in between.

[0053] The methods for recovering cells described herein may further include exposing the material to a chelating agent. Examples of chelating agents include, but are not limited to, ethylenediaminetetraacetic acid (EDTA), cyclohexanediaminetetraacetic acid (CDTA), ethylene glycoltetraacetic acid (EGTA), citric acid, and tartaric acid. Exposure of a soluble foam scaffold to a chelating agent may include exposing the scaffold to chelating agent concentrations of about 1 mM to about 200 mM. For example, the method may include exposing the scaffold to chelating agent concentrations 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 ​​in between. [Examples]

[0054] The embodiments described herein are merely illustrative and not intended to limit you; certain exemplary embodiments and specific embodiments are described further below.

[0055] Example 1

[0056] [Table 1]

[0057] A first aqueous mixture containing 2.0% by mass of polygalacturonic acid (PGA) was prepared by dissolving approximately 162 grams of sodium polygalacturonate in demineralized water in an oil bath set to 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. Next, approximately 17.5 grams of sucrose was added to the mixer bowl and mixed to facilitate the dissolution of 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 a mixing bowl to form a combined aqueous mixture, which was then mixed at a certain stirring speed (KitchenAid mixer speed 1) for approximately 5 minutes. Next, the combined aqueous mixture was foamed at a fast foaming speed (KitchenAid mixer speed 10) for approximately 20 minutes to introduce air into the combined aqueous mixture. While continuing to foam the combined aqueous mixture, approximately 30 mL of water and an aqueous solution of approximately 3.77 grams of gluconolactone (GDL) were added to the mixing bowl, and foaming was continued for approximately 1 minute.

[0058] Following the process described above, approximately 0.25 g / cm³ 3 An opaque white foam with a wet foam density was obtained. The foam was left uncovered in a mixing bowl at room temperature for about 1 hour to allow time for crosslinking to occur within the foam. Next, the foam was exposed to a temperature of about -80°C for about 16 hours to freeze it, and then exposed to a temperature of -86°C and a pressure of 0.11 mbar (about 11 Pa) for about 72 hours. The resulting foam had a density of about 0.04 to about 0.045 g / cm³. 3It was observed to have a dry foam density and 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 obtained foam was determined to contain approximately 1.3 mass% PGA, approximately 0.78 mass% HPM, and approximately 2.08 mass% total solids.

[0059] Example 2

[0060] [Table 2]

[0061] The process described in Example 1 was repeated, except that a second aqueous mixture was prepared by adding approximately 0.53 grams. The resulting foam was observed to be porous with highly interconnected pores. Figure 3 shows an SEM image of the foam prepared in this Example 2.

[0062] Example 3

[0063] [Table 3]

[0064] A first aqueous mixture containing 2.0% by mass of polygalacturonic acid (PGA) was prepared by dissolving approximately 162 grams of sodium polygalacturonate in demineralized water in an oil bath set to 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 under microwave at 800W for approximately 30 seconds. Approximately 0.97 grams of bovine gelatin was cooled and swollen in approximately 5.8 mL of ultrapure water, 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 a bowl of KitchenAid mixer equipped with a wire loop whisk. Next, approximately 17.5 grams of sucrose and approximately 0.97 grams of Methocel HPMC Culminal 724 were added to the bowl of the KitchenAid mixer, and the aqueous mixture was stirred for approximately 5 minutes. The first aqueous mixture containing 2.0% by mass of PGA was added to form a combined aqueous mixture in the mixing bowl, and it was mixed at a certain stirring speed (KitchenAid mixer speed 1) for approximately 3 minutes. Next, the combined aqueous mixture was foamed at a fast foaming speed (KitchenAid mixer speed 10) for approximately 20 minutes to introduce air into the combined aqueous mixture. While continuing to foam the combined aqueous mixture, approximately 30 mL of water and approximately 3.77 grams of gluconolactone (GDL) solution were added to the mixing bowl, and the mixture was continued to foam for approximately 1 minute.

[0065] An opaque white foam was obtained following the process described above. The foam was left uncovered in a mixing bowl at room temperature for about 1 hour to allow time for crosslinking to occur within the foam. Next, the foam was exposed to a temperature of about -80°C for about 16 hours to freeze it, and then exposed to a temperature of -86°C and a pressure of 0.11 mbar (about 11 Pa) for about 72 hours. The resulting foam had a viscosity of about 0.04 g / cm³. 3 It was observed to have a dry foam density and to be porous with highly interconnected pores. Figure 4 shows an SEM image of the foam prepared in this Example 3.

[0066] Example 4

[0067] [Table 4]

[0068] The process described in Example 3 was repeated, except that instead of bovine gelatin, approximately 0.97 grams of porcine gelatin was cold-swelled in approximately 5.8 mL of ultrapure water, then added to the second aqueous mixture, and stirred until dissolution was observed. The resulting foam was approximately 0.21 g / cm³. 3 The wet foam density is approximately 0.06 g / cm³. 3 It was observed to have a dry foam density and to be porous with highly interconnected pores. Figure 5 shows an SEM image of the foam prepared in this Example 4.

[0069] Example 5

[0070] [Table 5]

[0071] The process described in Example 1 was repeated, except that a first aqueous mixture was prepared by dissolving approximately 172 grams of 3.0% by mass sodium polygalacturonate in demineralized water in an oil bath set at 104°C. Furthermore, Methocel HPMC Culminal 724 was omitted from the second aqueous mixture. The resulting foam had a viscosity of approximately 0.40 g / cm³. 3 The wet foam density is approximately 0.11 g / cm³. 3It was observed that the foam had a dry foam density of approximately 0.40 g / cm³. The foam was observed to have lower porosity than the foam formed in Example 1, and the pores were less interconnected than those of the foam formed in Example 1. It is thought that the level of porosity and interpore interconnection of the foam decreased due to the omission of Methocel HPMC Culminal 724, or any polymer with surface activity. Figure 6 shows an SEM image of the foam prepared in this Example 5. 3 A wet density exceeding 0.11 g / cm³ 3 Foams with a dry density exceeding approximately 0.40 g / cm³ have been shown to support cell culture, although this is not the case for foams with a dry density of approximately 0.40 g / cm³. 3 Wet density less than 0.11 g / cm³ 3 Foams with a dry density of less than 3% exhibited improved cell culture conditions compared to foams with wet and dry densities, such as those formed in Example 5.

[0072] Example 6

[0073] [Table 6]

[0074] The process described in Example 1 was repeated, except that a first aqueous mixture was prepared by dissolving approximately 162 grams of 1.59% by mass sodium polygalacturonate in demineralized water in an oil bath set at 104°C. In addition, a second aqueous mixture was prepared by adding 0.53 grams of CaCO3 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 had a viscosity of approximately 0.36 g / cm³. 3 The wet foam density is approximately 0.09 g / cm³. 3 It was observed that it had a dry foam density of . Figure 7 shows an SEM image of the foam prepared in this Example 6.

[0075] Example 7

[0076] [Table 7]

[0077] The process described in Example 1 was repeated, except that sucrose was not added to the second aqueous mixture. The resulting foam was approximately 0.23 g / cm³. 3 The wet foam density is approximately 0.03 g / cm³. 3 It was observed that the foam had a dry foam density of . Figure 8 shows an SEM image of the foam prepared in this Example 7.

[0078] Example 8

[0079] [Table 8]

[0080] The process described in Example 1 was repeated, except that a first aqueous mixture was prepared by dissolving approximately 162 grams of 2.59% by mass sodium polygalacturonate in demineralized water in an oil bath set at 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 8 1 / 19. The resulting foam had a viscosity of approximately 0.18 g / cm³. 3 The wet foam density is approximately 0.057 g / cm³. 3 It was observed that it had a dry foam density of . Figure 9 shows an SEM image of the foam prepared in this Example 8.

[0081] Example 9

[0082] [Table 9]

[0083] 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 had a viscosity of approximately 0.34 g / cm³. 3 The wet foam density is approximately 0.095 g / cm³. 3 It was observed that the foam had a dry foam density of . The foam was observed to have lower porosity than the foam formed in Example 1, and the pores were less interconnected than the pores of the foam formed in Example 1. It is thought that the level of interconnectivity between pores in the foam decreased due to the omission of Methocel HPMC Culminal 724, or any polymer with surface activity. Furthermore, the addition of polymers without surface activity did not contribute to pore formation in the foam. Figure 10 shows an SEM image of the foam prepared in this Example 9.

[0084] Example 10

[0085] [Table 10]

[0086] 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 62.5 / 37.5. The resulting foam had a viscosity of approximately 0.18 g / cm³. 3 The wet foam density is approximately 0.03 g / cm³. 3 It was observed that it had a dry foam density and a higher porosity than the foam formed in Example 1. Figure 11 shows an SEM image of the foam prepared in Example 10.

[0087] Example 11

[0088] [Table 11]

[0089] The process described in Example 1 was repeated, except that a second aqueous mixture was prepared by adding a 50:50 mass ratio blend of Pluronic P123 and dextran instead of Methocel HPMC Culminal 724. The resulting foam was approximately 0.20 g / cm³. 3 The wet foam density is approximately 0.038 g / cm³. 3 It was observed that it had a dry foam density and a higher porosity than the foam formed in Example 1. Figure 12 shows an SEM image of the foam prepared in Example 11.

[0090] Example 12

[0091] [Table 12]

[0092] The process described in Example 11 was repeated to form several foams by adding varying amounts of glycerol to the second aqueous mixture. In the formation of the first foam in this example, 6.5 grams of glycerol were added to the second aqueous mixture. The glycerol constituted 19% by mass of the total solid additives added to form the combined aqueous mixture. The resulting foam had a viscosity of approximately 0.19 g / cm³. 3 The wet foam density is approximately 0.055 g / cm³. 3 It was observed that it had a dry foam density of . In the formation of the second foam in this example, 7.5 grams of glycerol were added to the second aqueous mixture. The glycerol constituted 22% by mass of the total solid additives added to form the combined aqueous mixture. The resulting foam had a density of approximately 0.21 g / cm³. 3 The wet foam density is approximately 0.048 g / cm³. 3It was observed that it had a dry foam density of . In the formation of the third foam in this example, 19.44 grams of glycerol were added to the second aqueous mixture. The glycerol constituted 42% by mass of the total solid additives added to form the combined aqueous mixture. The resulting foam had a density of approximately 0.23 g / cm³. 3 The wet foam density is approximately 0.23 g / cm³. 3 It was observed that it had a dry foam density of . In the formation of the fourth foam in this example, 29.16 grams of glycerol were added to the second aqueous mixture. The glycerol constituted 52% by mass of the total solid additives added to form the combined aqueous mixture. The resulting foam had a density of approximately 0.26 g / cm³. 3 The wet foam density is approximately 0.35 g / cm³. 3 It was observed that it had a dry foam density of [value missing].

[0093] The four foams of Example 12 demonstrate the effect of the amount of plasticizer added during foam formation on density and porosity. It was observed that increasing the plasticizer content resulted in slower drying, higher density, and lower porosity. Furthermore, as shown in Figure 13, when the amount of glycerol increased beyond approximately 20-25% by mass of the total solid additives added to form the second aqueous mixture, the nearly cylindrical shape of the foam was lost. The porosity and interconnectivity of the foam pores were observed to be maximized when the amount of plasticizer added to the second aqueous mixture was less than approximately 52% by mass of the total solid additives added to form the second aqueous mixture.

[0094] Example 13

[0095] [Table 13]

[0096] The process described in Example 1 was repeated, except that a second aqueous mixture was prepared by adding a 50:50 mass ratio blend of Pluronic P127 and dextran. The resulting foam was observed to have porosity and pore interconnectivity similar to the foam prepared in Example 11. Figure 14 shows an SEM image of the foam prepared in this Example 13.

[0097] Example 14 The foams formed according to the processes of each of Examples 1 to 12 were coated with "Synthemax" II-SC. A 250 μg / ml "Synthemax" II-SC ethanol aqueous solution was prepared by adding approximately 5.0 mg of Corning® "Synthemax" II-SC powder to approximately 20 mL of ethanol aqueous solution having an ethanol:water ratio of 70:30. Each foam, having a thickness of approximately 2-3 mm and a diameter of approximately 22 mm, was placed in separate wells of a 6-well polystyrene cell culture plate. Approximately 4.0 mL of the 250 μg / ml "Synthemax" II-SC ethanol aqueous solution was added to each well, and the plate was 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 aqueous solution. Next, the "Synthemax" II-SC coating was crosslinked by adding approximately 4.0 mL of 0.05% v / v glutaraldehyde (prepared by mixing 40 μl of 25% glutaraldehyde solution in 6.0 mL of water and 14 mL of ethanol) to a 70% ethanol aqueous solution. The plate was left 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.

[0098] Example 15 The foams formed according to the processes of each of Examples 1 to 12 were coated with gelatin. A 0.1% by mass gelatin solution was prepared by swelling approximately 500 mg of gelatin powder (derived from pig skin) with ultrapure water, followed by dissolution and homogenization with 20 mL of ultrapure water. Each foam, having a thickness of approximately 2-3 mm and a diameter of approximately 22 mm, was placed in a separate well of a 6-well polystyrene cell culture plate. Approximately 4.0 mL of gelatin solution was added to each well, and the plate was left 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 aqueous solution. Next, the gelatin coating was crosslinked by adding approximately 4.0 mL of 0.05% v / v glutaraldehyde (prepared by mixing 50 μl of 25% glutaraldehyde solution in 25 mL of ultrapure water) to the 70% ethanol aqueous solution. The plate was left to stand at room temperature for approximately 1.5 hours to allow time for crosslinking to occur. Next, the foam was rinsed three times with ultrapure water.

[0099] Example 16 The culture of Vero cells on foam formed according to the process of Example 4 was investigated. Vero cells (ATCC® CCL-81, commercially available from ATCC in Manassas, Virginia, USA) were cultured on cell culture plates in IMDM medium supplemented with 10% fetal bovine serum (FBS). The foam was cut into sections approximately 2-3 mm thick and 22 mm in diameter. The foam sections were disinfected with a 70% ethanol aqueous solution for approximately 5.0 minutes and then placed in separate wells of a 6-well ultra-low adhesion cell culture plate. The foam sections were washed twice with ultrapure water and once with IMDM medium. Excess medium was removed from the wells before seeding.

[0100] Vero cells were harvested from the cell culture plate using trypsin, resuspended in IMDM medium, and seeded into each of the foam portions placed in the wells of a 6-well cell culture plate with approximately 150 μL of cells. The 6-well cell culture plate was placed in a cell culture incubator, and after approximately 2.0 hours, approximately 3.0 mL of IMDM medium was added to each well. After approximately 18 hours in the cell culture incubator, the foam portions were visualized using a phase-contrast microscope. Figure 15 shows images obtained from the phase-contrast microscope, indicating that cells did not adhere to the uncoated foam portions, but instead formed spheroids in the pores of the foam portions. Thus, the soluble foam scaffold of this disclosure is found to be usable for the culture of spheroids or non-adherent cells.

[0101] Example 17 The culture of human mesenchymal stem cells (hMSCs) on foam formed according to the process of Example 4 and coated according to the process of Example 14 was investigated. hMSC passage 2 (commercially available from RoosterBio Inc., Frederick, Maryland, USA) was cultured on cell culture plates in Mesencult-XF medium (serum-free medium commercially available from STEMCELL Technologies, Vancouver, British Columbia, Canada). The foam was cut into sections approximately 2-3 mm thick and 22 mm in diameter. The foam sections were disinfected with a 70% ethanol aqueous solution for approximately 5.0 minutes and then placed in separate wells of a 6-well ultra-low adhesion 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 before seeding.

[0102] hMSCs were harvested from the cell culture plate using trypsin, resuspended in Mesencult-XF medium, and seeded at 150 μL containing approximately 100,000 cells into each of the foam portions placed in the wells of a 6-well cell culture plate. The 6-well cell culture plate was placed in a cell culture incubator, and after approximately 2.0 hours, approximately 3.0 mL of 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 Calcein-AM and visualized using a fluorescence microscope. Figure 16 shows fluorescence microscope images indicating that the cells were unable to adhere to the foam portions and instead spread within the pores of the foam portions. Thus, the soluble foam scaffold coated with Corning Synthemax II-SC as disclosed herein can be used for culturing adherent cells in addition to spheroids or non-adherent cells, and the scaffold was found to support cell culture in serum-free medium.

[0103] Example 18 The culture of human mesenchymal stem cells (hMSCs) on foam formed according to the process of Example 4 and coated according to the process of Example 15 was investigated. hMSC passage 2 used in Example 17 was cultured on a cell culture plate in IMDM medium supplemented with 10% fetal bovine serum (FBS). The foam was cut into sections approximately 2-3 mm thick and 22 mm in diameter. The foam sections were disinfected with 70% ethanol aqueous solution for approximately 5.0 minutes and then placed in separate wells of a 6-well ultra-low adhesion 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 before seeding.

[0104] hMSCs were harvested from the cell culture plate using trypsin, resuspended in IMDM medium, and seeded into each of the foam portions in the wells of a 6-well cell culture plate with approximately 150 μL of cells. 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 fluorescence microscope. Figure 17 shows fluorescence microscope images indicating that the cells were unable to adhere to the foam portion but spread within the pores of the foam portion. The hMSC cells were allowed to proliferate on the foam for 6 days and visualized again using a fluorescence microscope. Figure 18 shows fluorescence microscope images after 6 days, indicating that the cells had adhered to the foam portion and spread further within the pores of the foam portion. Thus, the gelatin-coated, soluble foam scaffold of this disclosure can be used for culturing adherent cells in addition to spheroids or non-adherent cells, and the scaffold has been found to support cell culture in serum-containing media.

[0105] Example 19 The proliferation of Vero cells on foams formed according to the processes of Examples 2 and 4 and coated according to the process of Example 15 was investigated. The 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 portions approximately 2-3 mm thick and 22 mm in diameter. The foam portions were disinfected with a 70% ethanol aqueous solution for approximately 5.0 minutes and then placed in separate wells of a 6-well polystyrene cell culture plate. The foam portions were washed twice with ultrapure water and once with IMDM medium. Excess medium was removed from the wells before seeding.

[0106] Vero cells were harvested from the TCT plate using trypsin and resuspended in IMDM medium. 150 μL containing approximately 25,000 cells was seeded into one portion of the foamy layer, and 150 μL containing approximately 50,000 cells was seeded into the other portion of the foamy layer, and the foamy layers were placed in the 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 foamy layers were dissolved by adding approximately 2.0 mL of digestion solution containing approximately 50 U / mL pectinase and approximately 5.0 mM EDTA to each well. The foamy layers were observed to dissolve within 5.0 minutes. After the foam dissolved, 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, obtained from http: / / bio.lonza.com / uploads / tx_mwaxmarketingmaterial / Lonza_BenchGuides_Protocol_for_Performing_a_Trypan_Blue_Viability_Test__Technical_Reference_Guide.pdf.

[0107] It was observed that cells formed colonies on all pore surfaces of the foam portion. Approximately 70-90 times proliferation was observed in the coated foam formed according to the process of Example 4, and approximately 40-70 times proliferation was observed in the coated foam formed according to the process of Example 2. Thus, it was found that the gelatin-coated, soluble foam scaffold of this disclosure can be used for the proliferation of Vero cells.

[0108] Example 20 The differentiation 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. hMSC passage 2 used in Example 17 was cultured on a cell culture plate in "Mesencult"-XF medium. The foam was cut into sections approximately 2-3 mm thick and 22 mm in diameter. The foam sections were disinfected with a 70% ethanol aqueous solution for approximately 5.0 minutes and then placed in separate wells of a 6-well polystyrene 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 before seeding.

[0109] hMSCs were harvested from the cell culture plate using trypsin, resuspended in Mesencult-XF medium, and seeded into each of the foamy portions in the wells of a 6-well cell culture plate with approximately 100,000 cells in 150 μL. 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 osteocyte differentiation medium, chondrocyte differentiation medium, and adipocyte differentiation medium (commercially available from Thermo Fisher Scientific in Waltham, Massachusetts, USA, under the trade names StemPro® Osteogenesis Differentiation Kit, StemPro® Chondrocyte Differentiation Kit, and StemPro® Adipogenesis Differentiation Kit, respectively) were added to the wells (each type of differentiation medium was added to a different foamy portion in a different well than the other types of differentiation medium). After three weeks, the foam portion exposed to osteocyte differentiation medium was stained with alizarin red, the foam portion exposed to chondrocyte differentiation medium was stained with Alcian blue, and the foam portion exposed to adipocyte differentiation medium was stained with oil red O. After staining, it was observed that hSMCs cultured on the soluble foam scaffold coated with the gelatin of this disclosure maintained chondrogenesis, osteogenesis, and adipogenesis differentiation. Thus, it was found that cells cultured on the soluble foam scaffold of this disclosure exhibited biological responses similar to those of in vivo cells.

[0110] Example 21 The proliferation of human mesenchymal stem cells (hMSCs) on foams formed according to the processes of Examples 2, 11, and 13 and coated according to the process of Example 14 was investigated. hMSC passage 2 used in Example 17 was cultured on a cell culture plate in "Mesencult"-XF medium. The foam was cut into sections approximately 2-3 mm thick and 22 mm in diameter. The foam sections were disinfected with a 70% ethanol aqueous solution for approximately 5.0 minutes and then placed in separate wells of a 6-well ultra-low adhesion 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 before seeding.

[0111] hMSCs were harvested from the cell culture plate using trypsin, resuspended in Mesencult-XF medium, and seeded at 150 μL containing approximately 100,000 cells into each of the foamy portions placed in the wells of a 6-well cell culture plate. The 6-well cell culture plate was placed in a cell culture incubator for approximately 7 days, and 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 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 the dissolution of the foam, cells were counted using the trypan blue exclusion protocol described above. Cells colonized on all pore surfaces of the foamy portions, and it was observed that the proliferation of hMSCs on the soluble foamy scaffold coated with Corning Synthemax II-SC of this disclosure was successfully achieved.

[0112] Example 22 The culture of human mesenchymal stem cells (hMSCs) under static and dynamic conditions was compared on foams formed according to the processes of Examples 2 and 11 and coated according to the process of Example 14. Subcult 2 of hMSCs was cultured as in Example 17. The hMSCs were harvested from the cell culture plate 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 in the wells of a 6-well cell culture plate and placed in a cell culture incubator for approximately 2 hours. After approximately 2 hours, the 6-well cell culture plate containing the scaffold formed according to Example 2 and the 6-well cell culture plate containing the scaffold formed according to Example 11 were removed from the incubator, and the scaffolds were transferred to separate bioreactors. The other 6-well cell culture plate was left in the incubator. All scaffolds were held under their respective conditions for approximately 6 days, after which they were dissolved by adding a digestion solution containing approximately 50 U / mL of pectinase and approximately 5.0 mM EDTA. After dissolution of the foam, cells were counted using the trypan blue exclusion protocol described above. Cell proliferation on the soluble foam scaffolds coated with Corning Synthemax II-SC of this disclosure was observed to be similar under both static and dynamic conditions.

[0113] Example 23 We investigated the multi-passage culture of human mesenchymal stem cells (hMSCs). As described herein, the passage number of a cell culture is a record of the number of times the culture has been passaged, i.e., the number of times it has been harvested and reseeded. Therefore, the multi-passage culture as relating to this disclosure describes the process in which cells are harvested from one soluble foam scaffold and reseeded on a different soluble foam scaffold. Using trypsin, hMSCs were harvested from the cell culture plate, resuspended in Mesencult-XF medium, and 150 μL containing approximately 100,000 cells was seeded onto a first set of foam scaffolds formed in Example 2 and coated in Example 14. The scaffolds were placed in Mesencult-XF medium and in a cell culture incubator for approximately 6 days. After approximately 6 days, the scaffolds were lysed by adding a digestion solution containing approximately 50 U / mL of pectinase and approximately 5.0 mM of EDTA. After the foam was dissolved, the cells were counted using the trypan blue exclusion protocol described above. Next, a solution containing approximately 100,000 cells recovered from the first set of scaffolds was 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 left 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 of pectinase and approximately 5.0 mM of EDTA. After the foam was dissolved, the cells were counted using the trypan blue exclusion protocol. Next, a solution containing approximately 100,000 cells recovered from the second set of scaffolds was 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 left in a cell culture incubator for approximately 7 days. Approximately 7 days later, the scaffold was dissolved by adding a digestion solution containing approximately 50 U / mL of pectinase and approximately 5.0 mM EDTA. After the dissolution of the foam, the cells were counted using the trypan blue exclusion protocol, then seeded onto cell culture plates, and exposed to the differentiation medium, chondrocyte differentiation medium, and adipocyte differentiation medium used in Example 20.Three weeks later, cells exposed to osteocyte differentiation medium were stained with alizarin red, cells exposed to chondrocyte differentiation medium were stained with Alcian blue, and cells exposed to adipocyte differentiation medium were stained with oil red O. After staining, hSMCs that underwent multiple passages on a soluble foam scaffold coated with Corning Synthemax II-SC as disclosed herein were observed to maintain chondrogenesis, osteogenesis, and adipogenesis differentiation.

[0114] Example 24 The transfection of human embryonic kidney (HEK) cells on a foam scaffold formed according to the process of Example 11 and coated according to the process of Example 14 was compared with the transfection of HEK cells on Corning Synthemax II-SC lysatellite microcarriers (commercially available from Corning Incorporated, Corning, New York, USA). HEK cells were seeded onto the lysatellite microcarriers. A first set of lysatellite 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 lysatellite 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 agitation every 2.0 hours). Approximately 3 days later, about 150 μL of transfection reagent was added to the soluble microcarriers in the wells of the 6-well cell culture plate, and about 1.0 mL of transfection reagent was added to the spinner flask with intermittent agitation.

[0115] HEK cells were seeded in a portion of the foam. A portion of the foam was 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. The other portion of the foam was 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 used 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 portion in the wells of the 6-well cell culture plate, and approximately 1.0 mL of transfection reagent was added to the foam portion in the perfusion cartridge device.

[0116] After adding the transfection reagent, cells were collected from the soluble microcarrier and the foam portion, and reporter gene expression levels were detected by measuring the response of green fluorescent protein (GFP) as the reporter gene. The GFP positivity 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 in the cell after exposure to the transfection reagent. Figure 19 is a bar graph showing the GFP positivity rate of transfected HEK cells for each culture condition. As shown in the figures, bar 1610 represents the GFP positivity rate of transfected HEK cells cultured on the soluble foam portion in a 6-well cell culture plate; bar 1620 represents the GFP positivity rate of transfected HEK cells cultured on the soluble foam portion in a perfusion cartridge device; bar 1630 represents the GFP positivity rate of transfected HEK cells cultured on soluble microcarriers in a 6-well cell culture plate; and bar 1640 represents the GFP positivity rate of transfected HEK cells cultured on soluble microcarriers in a spinner flask. Cells cultured on soluble foam scaffolds coated with Corning Synthemax II-SC of this disclosure were observed to exhibit greater transfection efficiency than cells cultured on microcarriers. While not bound by any particular theory, it is thought 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.

[0117] Example 25 We demonstrated the production of adeno-associated virus (AAV) vectors on foam scaffolds formed according to the process of Example 11 and coated according to the process of Example 14. One million 293 AAV cells were seeded on first and second sets of foam scaffolds and transferred to a chamber of a radial flow perfusion cartridge device, where they were exposed to IMDM medium supplemented with 10% fetal bovine serum (FBS).

[0118] Approximately 24 hours later, cells derived from the first set of foam scaffolds were transfected with the AAV-2 helper-free packaging system (commercially available from Cell Biolabs, Inc., San Diego, California, USA) using the calcium phosphate transfection method. Similarly, approximately 24 hours later, cells derived from the second set of foam scaffolds were transfected with the AAV-2 helper-free packaging system using the PEI method. In the calcium phosphate method, cells were incubated for approximately 18 hours with a calcium phosphate / DNA complex prepared using a plasmid concentration of 6.4 μg / mL and a molar ratio of 1:1:1. In the PEI method, cells on the foam scaffolds were transfected with 2 μg plasmid / mL. Each PEI method used a PEI / DNA ratio of 2 / 1 and a plasmid molar ratio of 1:1:1.

[0119] A 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 sets of cells, the medium was removed after approximately 18 hours and replaced with fresh medium. The cells were then incubated further and harvested 72 hours after transfection. The foam scaffold was collected and lysed by adding a digestion solution containing approximately 50 U / mL of pectinase and approximately 5.0 mM EDTA. After lysis of the foam scaffold, the cells were collected using centrifugation and washed with dPBS. A portion of the cells were retained for flow cytometry analysis, and 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 of benzonase.

[0120] After incubation at 37°C for approximately 30 minutes, the virus extract was clarified by centrifugation at 14,000 rpm for approximately 5 minutes. Viral titers were measured using the AAV-2 ELISA assay (commercially available from PROGEN Biotechnik GmbH, located in Heidelberg, Germany).

[0121] Transfection efficiency was analyzed using flow cytometry, and good transfection efficiency was observed. Figure 20 is a bar graph showing the GFP positivity of transfected cells for each set of foam scaffolds. As shown, bar 1710 represents the GFP positivity of cells transfected using the calcium phosphate transfection method, and the transfection efficiency was measured to be approximately 86.4%; bar 1720 represents the GFP positivity of cells transfected using the PEI transfection method, and the transfection efficiency was measured to be approximately 73.8%.

[0122] Viral titers were also measured using an ELISA assay. Figure 21 is a bar graph showing the number of viral particles (vp) per set of foam scaffolds. As shown, bar 1810 represents viral particles from cells transfected using the calcium phosphate transfection method, and the result was 8.7 × 10⁶. 11 The number of virus particles was measured; bar 1820 represents the number of virus particles in cells transfected using the PEI transfection method, 5.7 × 10⁶. 11 A number of virus particles was measured. Figure 22 is a bar graph showing the number of virus particles per cell per set of foam scaffolds. As shown, bar 1910 represents the number of virus particles / cell in cells transfected using the calcium phosphate transfection method, and is 1.17 × 10⁶. 5 The number of virus particles / cells was measured; bar 1920 represents the number of virus particles / cells in cells transfected using the PEI transfection method, 6.8 × 10⁶. 4 Individual virus particles / cells were measured.

[0123] Finally, the functionality of the viral vector was evaluated by testing the ability of the extract to induce GFP expression in infected HEK293 cells. Viral vector infectivity was assessed by testing the ability of the extract to induce GFP expression in infected HEK293 cells. 5 Extracts prepared using individual virus particles / cells were evaluated by infecting HEK293 cells. Approximately 72 hours post-infection, the GFP positivity rate of infected HEK cells was measured by flow cytometry. Infection of cells derived from each set of foam scaffolds was observed. GFP expression was used to identify the efficiency of gene transfer, which ranged from approximately 10% to approximately 26% in this example. Thus, it was concluded that functional AAV vectors can be produced on the foam scaffolds disclosed herein. Figure 23 is a bar graph showing the percentage of cells showing GFP expression for each set of foam scaffolds after infection. As shown, bar 2010 represents the percentage of cells showing GFP expression for cells transfected using the calcium phosphate transfection method, with approximately 26% of cells showing GFP expression; bar 2020 represents the percentage of cells showing GFP expression for cells transfected using the PEI transfection method, with approximately 10% of cells showing GFP expression.

[0124] According to aspect (1) of the present disclosure, a soluble foam scaffold for cell culture is provided. The soluble foam scaffold comprises pectic acid; an ionotropic crosslinked polygalacturonic acid compound selected from at least one of partially esterified pectic acid, partially amidated pectic acid, and salts thereof; and at least one first water-soluble polymer having surface activity.

[0125] According to aspect (2) of the present disclosure, a soluble foam scaffold as described in aspect (1) is provided, further comprising an adhesive polymer coating.

[0126] According to aspect (3) of the present disclosure, a soluble foamed scaffold as described in aspect (2) is provided, wherein the adhesive polymer coating comprises a peptide.

[0127] According to aspect (4) of the present disclosure, a soluble foamed scaffold as described in aspect (2) is provided, 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.

[0128] According to aspect (5) of the present disclosure, a dissolvable foamed scaffold as described in aspect (2) is provided, wherein the adhesive polymer coating comprises "Synthemax" II-SC.

[0129] According to aspect (6) of the present disclosure, a soluble foamed scaffold according to any of aspects (1) to (5) is provided, wherein at least one first polymer has a hydrophilic-lipophilic balance (HLB) greater than about 8.

[0130] According to aspect (7) of the present disclosure, a soluble foamed scaffold according to any of aspects (1) to (6) is provided, wherein at least one first polymer has a hydrophilic-lipophilic balance (HLB) greater than about 10.

[0131] According to aspect (8) of the present disclosure, a soluble foamed scaffold according to any one of aspects (1) to (7) is provided, wherein at least one first polymer can have a hydrophilic-lipophilic balance (HLB) of about 10 to about 40.

[0132] According to aspect (9) of the present disclosure, a soluble foamed scaffold according to any one of aspects (1) to (8) is provided, wherein at least one first polymer comprises a cellulose derivative.

[0133] According to aspect (10) of the present disclosure, a soluble foamed scaffold as described in aspect (9) is provided, wherein the cellulose derivative is selected from the group consisting of hydroxyethylcellulose (HEC), hydroxypropylcellulose (HPC), methylcellulose (MC), hydroxyethylmethylcellulose (HEMC), and hydroxypropylmethylcellulose (HPMC).

[0134] According to aspect (11) of the present disclosure, a soluble foamed scaffold according to any one of aspects (1) to (8) is provided, wherein at least one first polymer comprises a protein.

[0135] According to aspect (12) of the present disclosure, a soluble foam scaffold as described in aspect (11) is provided, wherein the protein is selected from the group consisting of bovine serum albumin (BSA), gelatin, casein, and hydrophobin.

[0136] According to aspect (13) of the present disclosure, a soluble foamed scaffold according to any one of aspects (1) to (8) is provided, wherein at least one first polymer comprises a synthetic amphiphilic polymer.

[0137] According to aspect (14) of the present disclosure, a soluble foamed scaffold according to aspect (13) is provided, wherein the synthetic amphiphilic polymer comprises a poloxamer.

[0138] According to aspect (15) of the present disclosure, a soluble foamed scaffold according to any of aspects (1) to (14) is provided, comprising at least two first water-soluble polymers having surface activity.

[0139] According to aspect (16) of the present disclosure, a soluble foamed scaffold according to any one of aspects (1) to (15) is provided, further comprising at least one second polymer that does not have surface activity.

[0140] According to aspect (17) of the present disclosure, a soluble foamed scaffold according to aspect (16) is provided, wherein at least one second polymer comprises a synthetic polymer.

[0141] According to aspect (18) of the present disclosure, a soluble foamed scaffold as described in aspect (17) is provided, wherein the synthetic polymer is selected from the group consisting of polyethylene glycol, polyvinylpyrrolidone, polyvinyl alcohol, carboxyvinyl polymer, polyacrylic acid, polyacrylamide; homopolymers and copolymers of (2-hydroxypropyl)methacrylamide; polyvinyl methyl ether-maleic anhydride; and polyethylene oxide / polypropylene oxide block copolymer.

[0142] According to aspect (19) of the present disclosure, a soluble foamed scaffold according to aspect (16) is provided, wherein at least one second polymer comprises a semi-synthetic polymer.

[0143] According to aspect (20) of the present disclosure, a soluble foamed scaffold as described in aspect (19) is provided, wherein the semi-synthetic polymer is selected from the group consisting of dextran derivatives, carboxymethylcellulose, hydroxyethylcellulose and derivatives, methylcellulose and derivatives, ethylcellulosecellulose, ethylhydroxyethylcellulose, and hydroxypropylcellulose.

[0144] According to aspect (21) of the present disclosure, a soluble foamed scaffold according to aspect (16) is provided, wherein at least one second polymer comprises a natural polymer.

[0145] According to aspect (22) of the present disclosure, a soluble foamed scaffold as described in aspect (21) is provided, wherein the natural polymer is selected from the group consisting of starch, starch derivatives, curdlan, pullulan, gellan gum, xanthan gum, dextran, albumin, casein, casein salts, gelatin, agar, alginate, carrageenan, guar gum, guar gum derivatives, locust bean gum, hyaluronic acid, and chondroitin sulfate.

[0146] According to aspect (23) of the present disclosure, a soluble foamed scaffold according to any of aspects (1) to (22) is provided, further comprising a water-soluble plasticizer.

[0147] According to aspect (24) of the present disclosure, a soluble foamed scaffold as described in aspect (23) is provided, wherein the water-soluble plasticizer is selected from the group consisting of glycerol, polyhydric alcohol, sorbitol, ethylene glycol, propylene glycol, polyethylene glycol, and combinations thereof.

[0148] According to aspect (25) of the present disclosure, a soluble foamed scaffold according to any of aspects (23) to (24) is provided, comprising less than approximately 55% by mass of a water-soluble plasticizer.

[0149] According to aspect (26) of the present disclosure, a dissolvable foamed scaffold according to any of aspects (23) to (25) is provided, comprising about 15% to about 55% by mass of a water-soluble plasticizer.

[0150] According to aspect (27) of the present disclosure, a dissolvable foamed scaffold according to any of aspects (1) to (26) is provided, having a porosity of about 85% to about 96%.

[0151] According to aspect (28) of the present disclosure, a dissolvable foamed scaffold according to any of aspects (1) to (27) is provided, comprising an average pore diameter of about 50 μm to about 500 μm.

[0152] According to aspect (29) of the present disclosure, a dissolvable foamed scaffold according to any of aspects (1) to (28) is provided, comprising an average pore diameter of approximately 75 μm to approximately 450 μm.

[0153] According to aspect (30) of the present disclosure, a dissolvable foamed scaffold according to any of aspects (1) to (29) is provided, comprising an average pore diameter of about 100 μm to about 400 μm.

[0154] According to aspect (31) of this disclosure, approximately 0.40 g / cm³ 3 A dissolvable foamed scaffold according to any one of embodiments (1) to (30) is provided, having a wet density of less than .

[0155] According to aspect (32) of this disclosure, approximately 0.30 g / cm³ 3A dissolvable foamed scaffold according to any one of embodiments (1) to (31) is provided, having a wet density of less than .

[0156] According to aspect (33) of this disclosure, approximately 0.16 g / cm³ 3 ~Approx. 0.40g / cm 3 A dissolvable foamed scaffold is provided, comprising a wet density of the following, according to any one of embodiments (1) to (32).

[0157] According to aspect (34) of this disclosure, approximately 0.20 g / cm³ 3 A dissolvable foamed scaffold according to any of embodiments (1) to (33) is provided, containing a dry density of less than .

[0158] According to aspect (35) of this disclosure, approximately 0.10 g / cm³ 3 A dissolvable foamed scaffold according to any of embodiments (1) to (34) is provided, containing a dry density of less than .

[0159] According to aspect (36) of this disclosure, approximately 0.02 g / cm³ 3 ~Approx. 0.20g / cm 3 A dissolvable foamed scaffold is provided, including a dry density, according to any of embodiments (1) to (35).

[0160] According to aspect (37) of the present disclosure, a dissolvable foamed scaffold according to any of aspects (1) to (36) is provided, which includes an open structure.

[0161] According to aspect (38) of the present disclosure, a dissolvable foam scaffolding according to any of aspects (1) to (37) is provided, in which the dissolution of the dissolvable foam scaffolding is completed in less than approximately one hour.

[0162] According to aspect (39) of the present disclosure, a dissolvable foam scaffolding according to any of aspects (1) to (38) is provided, wherein the dissolution of the dissolvable foam scaffolding is completed in less than approximately 15 minutes.

[0163] A method for forming a soluble foamed scaffold is provided according to an aspect (40) of the present disclosure. The method includes forming a first aqueous mixture by adding a polygalacturonic acid compound selected from at least one of pectic acid, partially esterified pectic acid, partially amidated pectic acid, and salts thereof to an aqueous solution; forming a second aqueous mixture by adding at least one first water-soluble polymer having surface activity and a divalent metal salt to an aqueous solution; combining the first aqueous mixture and the second aqueous mixture to form a combined aqueous mixture; adding a gel inducer to the combined aqueous mixture; and introducing bubbles into the combined aqueous mixture to form a foamed scaffold.

[0164] According to an aspect (41) of the present disclosure, the method according to aspect (40) is provided, further comprising adding at least one second polymer that does not have surface activity to form a second aqueous mixture.

[0165] According to an aspect (42) of the present disclosure, the method according to aspect (41) is provided, wherein adding at least one first polymer and at least one second polymer includes adding about 35% to about 65% of at least one first polymer and about 35% to about 65% of at least one second polymer.

[0166] According to aspect (43) of the present disclosure, a method is provided according to any of aspects (40) to (42), wherein forming a second aqueous mixture comprises adding at least two first water-soluble polymers having surface activity.

[0167] According to aspect (44) of the present disclosure, a method is provided according to any one of aspects (40) to (43), 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 oxalates, tartrates, phosphates, carbonates, citrates, and combinations thereof.

[0168] According to aspect (45) of the present disclosure, a method is provided which is described in any of aspects (40) to (43), further comprising adding a water-soluble plasticizer to the second aqueous mixture in order to form a second aqueous mixture.

[0169] According to aspect (46) of the present disclosure, the method according to aspect (45) is provided, wherein the water-soluble plasticizer is selected from the group consisting of glycerol, polyhydric alcohol, sorbitol, ethylene glycol, propylene glycol, polyethylene glycol, and combinations thereof.

[0170] According to aspect (47) of the present disclosure, a method is provided according to any of aspects (45) to (46), wherein the water-soluble plasticizer constitutes less than about 55% by mass of the total solid additives of the second aqueous mixture.

[0171] According to aspect (48) of the present disclosure, a method is provided according to any of aspects (45) to (47), wherein the water-soluble plasticizer constitutes about 15% to about 55% by mass of the total solid additives of the second aqueous mixture.

[0172] According to aspect (49) of the present disclosure, a method is provided which is described in any of aspects (40) to (48), further comprising adding an emulsifier to the second aqueous mixture in order to form a second aqueous mixture.

[0173] According to aspect (50) of the present disclosure, a method is provided which is a method according to any one of aspects (40) to (49), further comprising adding at least one leaching solid to the second aqueous mixture in order to form a second aqueous mixture.

[0174] According to one aspect (51) of the present disclosure, the method according to aspect (50) is provided, wherein at least one leaching solid is selected from the group consisting of salts, biocompatible monosaccharides and disaccharides, and water-soluble proteins.

[0175] According to aspect (52) of the present disclosure, a method according to any one of aspects (40) to (51) is provided, wherein the gel inducer is selected from the group consisting of lactone lactic acid, lactone glycolate, glucono delta-lactone, and acid anhydride.

[0176] According to aspect (53) of the present disclosure, a method is provided which further comprises coating a soluble foam scaffold with an adhesive polymer coating, as described in any of aspects (40) to (52).

[0177] According to aspect (54) of the present disclosure, the method of aspect (53) is provided, wherein the adhesive polymer coating comprises a peptide.

[0178] According to aspect (55) of the present disclosure, the method according to aspect (53) is provided, 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.

[0179] According to aspect (56) of the present disclosure, the method according to aspect (53) is provided, wherein the adhesive polymer coating comprises "Synthemax" II-SC.

[0180] According to an aspect (57) of the present disclosure, a method for culturing cells on a soluble foam scaffold is provided. The method comprises seeding cells onto a soluble foam scaffold such that the cells enter the pores of the soluble foam scaffold, wherein the soluble scaffold comprises pectic acid; an ionotropically crosslinked polygalacturonic acid compound selected from at least one of partially esterified pectic acid, partially amidated pectic acid, and salts thereof; and at least one first water-soluble polymer having surface activity; and contacting the soluble foam scaffold with a cell culture medium.

[0181] One aspect (58) of the present disclosure provides a method of the present invention (57) wherein cells aggregate within the pores of a soluble foam scaffold to form spheroids.

[0182] According to aspect (59) of the present disclosure, a method according to aspect (57) or (58) is provided, wherein the soluble foam scaffold comprises an adhesive polymer coating, and seeding cells onto the soluble foam scaffold involves adhering the cells to the surface of the soluble foam scaffold.

[0183] According to aspect (60) of the present disclosure, a method is provided which includes contacting a soluble foam scaffold with a cell culture medium, and sinking a soluble foam scaffold into a cell culture medium, as described in any of aspects (57) to (59).

[0184] According to aspect (61) of the present disclosure, a method is provided which includes bringing a soluble foam scaffold into contact with a cell culture medium, and then continuously passing the cell culture medium over the soluble foam scaffold.

[0185] One aspect (62) of the present disclosure provides a method according to aspect (61), which includes continuously passing a cell culture medium over a soluble foam scaffold to remove at least a portion of the cell culture medium from contact with the soluble foam scaffold, and bringing the soluble foam scaffold into contact with fresh cell culture medium so that the volume of cell culture medium in contact with the soluble foam scaffold remains substantially constant.

[0186] A method for recovering cells from a soluble foam scaffold is provided according to an aspect (63) of the present disclosure. The method comprises digesting the soluble foam scaffold by exposing the soluble foam scaffold to an enzyme, wherein the soluble scaffold comprises a pectic acid; an ionotropically crosslinked polygalacturonic acid compound selected from at least one of partially esterified pectic acid, partially amidated pectic acid, and salts thereof; and at least one first water-soluble polymer having surface activity; and exposing the soluble foam scaffold to a chelating agent.

[0187] According to aspect (64) of the present disclosure, the method of aspect (63) is provided, wherein the enzyme comprises a non-proteolytic enzyme.

[0188] According to aspect (65) of the present disclosure, the method according to aspect (64) is provided, wherein the non-proteinase is selected from the group consisting of pectinases and pectinases.

[0189] According to aspect (66) of the present disclosure, a method is provided which involves digesting a soluble foam scaffold by exposing the soluble foam scaffold to an enzyme in a concentration of about 1 U to about 200 U.

[0190] According to aspect (67) of the present disclosure, a method is provided which includes exposing a soluble foam scaffold to about 1 mM to about 200 mM of a chelating agent, as described in any of aspects (63) to (66).

[0191] According to aspect (68) of the present disclosure, a foamed scaffold product formed from a composition is provided. The composition comprises pectic acid; an ionotropic crosslinked polygalacturonic acid compound selected from at least one of partially esterified pectic acid, partially amidated pectic acid, and salts thereof; at least one first water-soluble polymer having surface activity; and less than about 55% by mass of a water-soluble plasticizer.

[0192] According to aspect (69) of the present disclosure, a foamed scaffold product according to aspect (68) is provided, further comprising an adhesive polymer coating.

[0193] According to aspect (70) of the present disclosure, a foamed scaffold product according to aspect (69) is provided, wherein the adhesive polymer coating comprises a peptide.

[0194] According to aspect (71) of the present disclosure, a foamed scaffold product according to aspect (69) is provided, 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.

[0195] According to aspect (72) of the present disclosure, a foamed scaffold product according to aspect (69) is provided, wherein the adhesive polymer coating comprises "Synthemax" II-SC.

[0196] According to aspect (73) of the present disclosure, a foamed scaffold product according to any one of aspects (68) to (72) is provided, wherein at least one first polymer has a hydrophilic-lipophilic balance (HLB) greater than about 8.

[0197] According to aspect (74) of the present disclosure, a foamed scaffold product according to any one of aspects (68) to (73) is provided, wherein at least one first polymer has a hydrophilic-lipophilic balance (HLB) greater than about 10.

[0198] According to aspect (75) of the present disclosure, a foamed scaffold product according to any one of aspects (68) to (74) is provided, wherein at least one first polymer can have a hydrophilic-lipophilic balance (HLB) of about 10 to about 40.

[0199] According to aspect (76) of the present disclosure, a foamed scaffold product according to any one of aspects (68) to (75) is provided, wherein at least one first polymer comprises a cellulose derivative.

[0200] According to aspect (77) of the present disclosure, a foamed scaffold product according to aspect (76) is provided, wherein the cellulose derivative is selected from the group consisting of hydroxyethylcellulose (HEC), hydroxypropylcellulose (HPC), methylcellulose (MC), hydroxyethylmethylcellulose (HEMC), and hydroxypropylmethylcellulose (HPMC).

[0201] According to aspect (78) of the present disclosure, a foamed scaffold product according to any one of aspects (68) to (75) is provided, wherein at least one first polymer comprises a protein.

[0202] According to aspect (79) of the present disclosure, a foamed scaffold product according to aspect (78) is provided, wherein the protein is selected from the group consisting of bovine serum albumin (BSA), gelatin, casein, and hydrophobin.

[0203] According to aspect (80) of the present disclosure, a foamed scaffold product according to any one of aspects (68) to (75) is provided, wherein at least one first polymer comprises a synthetic amphiphilic polymer.

[0204] According to aspect (81) of the present disclosure, a foamed scaffold product according to aspect (80) is provided, wherein the synthetic amphiphilic polymer comprises a poloxamer.

[0205] According to aspect (82) of the present disclosure, a foamed scaffold product according to any one of aspects (68) to (81) is provided, comprising at least two first water-soluble polymers having surface activity.

[0206] According to aspect (83) of the present disclosure, a foamed scaffolding product according to any one of aspects (68) to (82) is provided, wherein the foamed scaffolding composition further comprises at least one second polymer that does not have surface activity.

[0207] According to an aspect (84) of the present disclosure, a foamed scaffold product according to aspect (83) is provided, wherein at least one second polymer comprises a synthetic polymer.

[0208] According to aspect (85) of the present disclosure, a foamed scaffold product as described in aspect (84) is provided, wherein the synthetic polymer is selected from the group consisting of polyethylene glycol, polyvinylpyrrolidone, polyvinyl alcohol, carboxyvinyl polymer, polyacrylic acid, polyacrylamide; homopolymers and copolymers of (2-hydroxypropyl)methacrylamide; polyvinyl methyl ether-maleic anhydride; and polyethylene oxide / polypropylene oxide block copolymer.

[0209] According to aspect (86) of the present disclosure, a foamed scaffold product according to aspect (83) is provided, wherein at least one second polymer comprises a semi-synthetic polymer.

[0210] According to aspect (87) of the present disclosure, a foamed scaffold product according to aspect (86) is provided, wherein the semi-synthetic polymer is selected from the group consisting of dextran derivatives, carboxymethylcellulose, hydroxyethylcellulose and derivatives, methylcellulose and derivatives, ethylcellulosecellulose, ethylhydroxyethylcellulose, and hydroxypropylcellulose.

[0211] According to an aspect (88) of the present disclosure, a foamed scaffold product according to aspect (83) is provided, wherein at least one second polymer comprises a natural polymer.

[0212] According to aspect (89) of the present disclosure, a foamed scaffold product as described in aspect (88) is provided, wherein the natural polymer is selected from the group consisting of starch, starch derivatives, curdlan, pullulan, gellan gum, xanthan gum, dextran, albumin, casein, casein salts, gelatin, agar, alginate, carrageenan, guar gum, guar gum derivatives, locust bean gum, hyaluronic acid, and chondroitin sulfate.

[0213] According to aspect (90) of the present disclosure, a foamed scaffold product according to any one of aspects (68) to (89) is provided, wherein the water-soluble plasticizer is selected from the group consisting of glycerol, polyhydric alcohol, sorbitol, ethylene glycol, propylene glycol, polyethylene glycol, and combinations thereof.

[0214] According to aspect (91) of the present disclosure, a foamed scaffold product according to any one of aspects (68) to (90) is provided, comprising about 15% to about 55% by mass of a water-soluble plasticizer.

[0215] According to aspect (92) of the present disclosure, a foamed scaffold product according to any of aspects (68) to (91) is provided, having a porosity of about 85% to about 96%.

[0216] According to aspect (93) of the present disclosure, a foamed scaffold product according to any of aspects (68) to (92) is provided, comprising an average pore diameter of about 50 μm to about 500 μm.

[0217] According to aspect (94) of the present disclosure, a foamed scaffold product according to any of aspects (68) to (93) is provided, comprising an average pore diameter of about 75 μm to about 450 μm.

[0218] According to aspect (95) of the present disclosure, a foamed scaffold product according to any of aspects (68) to (94) is provided, comprising an average pore diameter of about 100 μm to about 400 μm.

[0219] According to aspect (96) of this disclosure, approximately 0.40 g / cm³ 3 A foamed scaffold product according to any one of embodiments (68) to (95) is provided, which includes a wet density of less than .

[0220] According to aspect (97) of this disclosure, approximately 0.30 g / cm³ 3 A foamed scaffold product according to any one of embodiments (68) to (96) is provided, which includes a wet density of less than .

[0221] According to aspect (98) of this disclosure, approximately 0.16 g / cm³ 3~Approx. 0.40g / cm 3 A foamed scaffold product according to any one of embodiments (68) to (97) is provided, including a wet density.

[0222] According to aspect (99) of this disclosure, approximately 0.20 g / cm³ 3 A foamed scaffold product according to any one of embodiments (68) to (98) is provided, comprising a dry density of less than .

[0223] According to aspect (100) of this disclosure, approximately 0.10 g / cm³ 3 A foamed scaffold product according to any one of embodiments (68) to (99) is provided, which includes a dry density of less than .

[0224] According to aspect (101) of this disclosure, approximately 0.02 g / cm³ 3 ~Approx. 0.20g / cm 3 A foamed scaffold product according to any one of embodiments (68) to (100) is provided, including a dry density.

[0225] According to aspect (102) of the present disclosure, a foamed scaffold product according to any one of aspects (68) to (101) is provided, which includes an open structure.

[0226] While this disclosure includes a limited number of embodiments, those skilled in the art who are interested in this disclosure will recognize that other embodiments not departing from the scope of this disclosure may be devised.

[0227] Preferred embodiments of the present invention are described below in separate sections.

[0228] Embodiment 1 A soluble foam scaffold for cell culture, Pectic acid; an ionotropic cross-linked polygalacturonic acid compound selected from at least one of partially esterified pectic acid, partially amidated pectic acid, and salts thereof, A first water-soluble polymer having surface activity and A dissolvable foam scaffolding, including a material that can be dissolved.

[0229] Embodiment 2 The dissolvable foam scaffold according to Embodiment 1, further comprising a water-soluble plasticizer.

[0230] Embodiment 3 The dissolvable foam scaffold according to Embodiment 2, comprising less than about 55% by mass of a water-soluble plasticizer.

[0231] Embodiment 4 The dissolvable foam scaffold according to any one of Embodiments 1 to 3, further comprising an adhesive polymer coating.

[0232] Embodiment 5 The dissolvable foam scaffold according to Embodiment 4, wherein the adhesive polymer coating contains a peptide.

[0233] Embodiment 6 The dissolvable foam scaffold according to Embodiment 4, wherein the adhesive polymer coating contains a peptide selected from the group consisting of BSP, vitronectin, fibronectin, laminin, type I collagen, type IV collagen, denatured collagen, and mixtures thereof.

[0234] Embodiment 7 The dissolvable foam scaffold according to Embodiment 4, wherein the adhesive polymer coating contains "Synthemax" II-SC.

[0235] Embodiment 8 The dissolvable foam scaffold according to any one of Embodiments 1 to 7, wherein the at least one first polymer has a hydrophilic-lipophilic balance (HLB) exceeding about 8.

[0236] Embodiment 9 The dissolvable foam scaffold according to any one of Embodiments 1 to 8, wherein the at least one first polymer contains a cellulose derivative.

[0237] Embodiment 10 The dissolvable foam scaffold according to any one of Embodiments 1 to 8, wherein the at least one first polymer contains a protein.

[0238] Embodiment 11 A soluble foamed scaffold according to any one of embodiments 1 to 8, wherein the at least one first polymer comprises a synthetic amphiphilic polymer.

[0239] Embodiment 12 A soluble foamed scaffold according to any one of embodiments 1 to 11, comprising at least two first water-soluble polymers having surface activity.

[0240] Embodiment 13 A soluble foamed scaffold according to any one of embodiments 1 to 12, further comprising at least one second polymer that does not have surface activity.

[0241] Embodiment 14 The dissolvable foamed scaffold according to Embodiment 13, wherein the at least one second polymer comprises a synthetic polymer.

[0242] Embodiment 15 The dissolvable foamed scaffold according to Embodiment 13, wherein the at least one second polymer comprises a semi-synthetic polymer.

[0243] Embodiment 16 The dissolvable foamed scaffold according to Embodiment 13, wherein the at least one second polymer comprises a natural polymer.

[0244] Embodiment 17 A dissolvable foamed scaffold according to any of Embodiments 1 to 16, comprising an average pore size of approximately 50 μm to approximately 500 μm.

[0245] Embodiment 18 Approximately 0.40g / cm 3 A dissolvable foamed scaffold according to any of Embodiments 1 to 17, comprising a wet density of less than .

[0246] Embodiment 19 A dissolvable foamed scaffold according to any one of embodiments 1 to 18, including an open structure.

[0247] Embodiment 20 The dissolvable foamed scaffold according to any one of Embodiments 1 to 19, wherein digestion of the dissolvable foamed scaffold is completed in less than about 1 hour.

[0248] Embodiment 21 A method of forming a dissolvable foamed scaffold, the method comprising: forming a first aqueous mixture by adding a polygalacturonic acid compound selected from at least one of pectic acid; partially esterified pectic acid, partially amidated pectic acid, and salts thereof to an aqueous solution; forming a second aqueous mixture by adding at least one first water-soluble polymer having surface activity and a divalent metal salt to an aqueous solution; combining the first aqueous mixture with the second aqueous mixture to form a combined aqueous mixture; adding a gel inducer to the combined aqueous mixture; and introducing air bubbles into the combined aqueous mixture to form a foamed scaffold A method comprising.

[0249] Embodiment 22 The method according to Embodiment 21, wherein forming the second aqueous mixture further comprises adding a water-soluble plasticizer to the second aqueous mixture.

[0250] Embodiment 23 The method according to Embodiment 22, wherein the water-soluble plasticizer constitutes less than about 55% by mass of the total solid additives of the second aqueous mixture.

[0251] Embodiment 24 The method according to any one of Embodiments 21 to 23, wherein forming the second aqueous mixture further comprises adding at least one second polymer having no surface activity.

[0252] Embodiment 25 The method according to Embodiment 24, wherein adding at least one first polymer and at least one second polymer includes adding about 35% to about 65% of the at least one first polymer and about 35% to about 65% of the at least one second polymer.

[0253] Embodiment 26 The method according to any one of embodiments 21 to 25, wherein forming a second aqueous mixture involves adding at least two first water-soluble polymers having surface activity.

[0254] Embodiment 27 The aforementioned divalent metal salt, A cation selected from the group consisting of magnesium, calcium, zinc, strontium, barium, and combinations thereof, Anions selected from the group consisting of oxalates, tartrates, phosphates, carbonates, citrates, and combinations thereof. The method according to any one of embodiments 21 to 26, including the method described above.

[0255] Embodiment 28 The method according to any one of embodiments 21 to 27, further comprising adding an emulsifier to the second aqueous mixture to form a second aqueous mixture.

[0256] Embodiment 29 The method according to any one of embodiments 21 to 28, further comprising adding at least one leaching solid to the second aqueous mixture to form a second aqueous mixture.

[0257] Embodiment 30 The method according to Embodiment 29, wherein the at least one leaching solid is selected from the group consisting of salts, biocompatible monosaccharides and disaccharides, and water-soluble proteins.

[0258] Embodiment 31 The method according to any one of embodiments 21 to 30, wherein the gel inducer is selected from the group consisting of lactone lactic acid, lactone glycolate, glucono delta-lactone, and acid anhydride.

[0259] Embodiment 32 The method according to any one of embodiments 21 to 31, further comprising coating the aforementioned soluble foam scaffold with an adhesive polymer coating.

[0260] Embodiment 33 The method according to Embodiment 32, wherein the adhesive polymer coating contains a peptide.

[0261] Embodiment 34 The method according to Embodiment 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.

[0262] Embodiment 35 The method according to Embodiment 32, wherein the adhesive polymer coating includes "Synthemax" II-SC.

[0263] Embodiment 36 In a method for culturing cells using a soluble foam scaffold, the method is Seeding cells onto a soluble foam scaffold such that the cells enter the pores of the soluble foam scaffold, wherein the soluble scaffold is Pectic acid; an ionotropic cross-linked polygalacturonic acid compound selected from at least one of partially esterified pectic acid, partially amidated pectic acid, and salts thereof, A first water-soluble polymer having surface activity and Including sowing, and The soluble foam scaffold is brought into contact with the cell culture medium. Methods that include...

[0264] Embodiment 37 The method according to Embodiment 36, wherein cells aggregate within the pores of the soluble foam scaffold to form spheroids.

[0265] Embodiment 38 The method according to Embodiment 36 or 37, wherein the soluble foam scaffold comprises an adhesive polymer coating, and seeding cells onto the soluble foam scaffold involves adhering the cells to the surface of the soluble foam scaffold.

[0266] Embodiment 39 The method according to any one of embodiments 36 to 38, wherein contacting the soluble foam scaffold with a cell culture medium includes dipping the soluble foam scaffold into the cell culture medium.

[0267] Embodiment 40 The method according to any one of embodiments 36 to 39, wherein contacting the soluble foam scaffold with a cell culture medium includes continuously passing the cell culture medium over the soluble foam scaffold.

[0268] Embodiment 41 The method according to Embodiment 40, wherein the continuous passing of cell culture medium over the soluble foam scaffold removes at least a portion of the cell culture medium from contact with the soluble foam scaffold, and the soluble foam scaffold is brought into contact with fresh cell culture medium such that the volume of cell culture medium in contact with the soluble foam scaffold remains substantially constant.

[0269] Embodiment 42 In a method for recovering cells from a soluble foam scaffold, the method is The method involves digesting the soluble foam scaffold by exposing it to an enzyme; The aforementioned dissolvable scaffolding, Pectic acid; an ionotropic cross-linked polygalacturonic acid compound selected from at least one of partially esterified pectic acid, partially amidated pectic acid, and salts thereof, A first water-soluble polymer having surface activity and Including digestion, and Exposing the aforementioned soluble foamed scaffold to a chelating agent. Methods that include...

[0270] Embodiment 43 The method according to Embodiment 42, wherein the enzyme includes a non-protein-degrading enzyme.

[0271] Embodiment 44 The method according to Embodiment 43, wherein the non-protein-degrading enzyme is selected from the group consisting of pectin-degrading enzymes and pectinases.

[0272] Embodiment 45 The method according to any one of embodiments 42 to 44, wherein digesting the soluble foam scaffold comprises exposing the soluble foam scaffold to the enzyme in a concentration of about 1 U to about 200 U.

[0273] Embodiment 46 The method according to any one of embodiments 42 to 45, comprising exposing the dissolvable foamed scaffold to the chelating agent in a concentration of approximately 1 mM to approximately 200 mM.

[0274] Embodiment 47 A foamed scaffold product formed from the composition, Pectic acid; an ionotropic cross-linked polygalacturonic acid compound selected from at least one of partially esterified pectic acid, partially amidated pectic acid, and salts thereof, A first water-soluble polymer having surface activity, Water-soluble plasticizers in an amount of less than approximately 55% by mass and A foamed scaffolding product, including...

[0275] Embodiment 48 The foamed scaffold product according to Embodiment 47, comprising approximately 15% to 55% by mass of a water-soluble plasticizer.

[0276] Embodiment 49 The foamed scaffold product according to embodiment 47 or 48, further comprising an adhesive polymer coating.

[0277] Embodiment 50 The foamed scaffold product according to Embodiment 49, wherein the adhesive polymer coating contains a peptide.

[0278] Embodiment 51 The foamed scaffold product according to Embodiment 49, 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.

[0279] Embodiment 52 The foamed scaffold product according to Embodiment 49, wherein the adhesive polymer coating comprises "Synthemax" II-SC.

[0280] Embodiment 53 The foamed scaffold product according to any one of embodiments 47 to 52, wherein at least one of the first polymers has a hydrophilic-lipophilic balance (HLB) greater than about 8.

[0281] Embodiment 54 The foamed scaffold product according to any one of embodiments 47 to 53, wherein the at least one first polymer comprises a cellulose derivative.

[0282] Embodiment 55 The foamed scaffold product according to any one of embodiments 47 to 53, wherein the at least one first polymer comprises a protein.

[0283] Embodiment 56 The foamed scaffold product according to any one of embodiments 47 to 53, wherein the at least one first polymer comprises a synthetic amphiphilic polymer.

[0284] Embodiment 57 A foamed scaffold product according to any one of embodiments 47 to 56, comprising at least two first water-soluble polymers having surface activity.

[0285] Embodiment 58 The foamed scaffolding product according to any one of embodiments 47 to 57, wherein the foamed scaffolding composition further comprises at least one second polymer that does not have surface activity.

[0286] Embodiment 59 The foamed scaffold product according to Embodiment 58, wherein the at least one second polymer comprises a synthetic polymer.

[0287] Embodiment 60 The foamed scaffold product according to Embodiment 58, wherein the at least one second polymer comprises a semi-synthetic polymer.

[0288] Embodiment 61 The foamed scaffold product according to Embodiment 58, wherein the at least one second polymer comprises a natural polymer.

[0289] Embodiment 62 A foamed scaffold product according to any one of embodiments 47 to 61, comprising a porosity of approximately 85% to approximately 96%.

[0290] Embodiment 63 A foamed scaffold product according to any one of embodiments 47 to 62, comprising an average pore diameter of approximately 50 μm to approximately 500 μm.

[0291] Embodiment 64 Approximately 0.40g / cm 3 A foamed scaffold product according to any of embodiments 47 to 63, comprising a wet density of less than .

[0292] Embodiment 65 A foamed scaffold product according to any one of embodiments 47 to 64, including an open structure.

Claims

1. A foamed scaffold for cell culture, Pectic acid; an ionotropic cross-linked polygalacturonic acid compound selected from at least one of partially esterified pectic acid, partially amidated pectic acid, and salts thereof, A first water-soluble polymer having surface activity and A second polymer that does not have surface activity and Includes, The at least one first water-soluble polymer is a poloxamer, The at least one second polymer comprises dextran, The foamed scaffolding has an open structure and highly interconnected pores, The aforementioned foamed scaffolding has a porosity of 85% to 96%. Average pore size of 50 μm to 500 μm, 0.40 g / cm ３ Wet density less than 0.20 g / cm³ ３ Dry density less than Includes foam scaffolding.

2. The foamed scaffolding according to claim 1, further comprising an adhesive polymer coating.

3. The foamed scaffold according to claim 2, wherein the adhesive polymer coating contains a peptide.

4. The foamed scaffold according to claim 2, 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.

5. The foamed scaffold according to claim 2, wherein the adhesive polymer coating comprises a copolymer containing a peptide having an RGD sequence.

6. The foamed scaffolding according to any one of claims 1 to 5, further comprising a water-soluble plasticizer.

7. The foamed scaffolding according to claim 6, comprising less than approximately 55% by mass of a water-soluble plasticizer.

8. A method for forming a foamed scaffold, wherein the method is Pectic acid; forming a first aqueous mixture by adding a polygalacturonic acid compound selected from at least one of partially esterified pectic acid, partially amidated pectic acid, and salts thereof to an aqueous solution; A second aqueous mixture is formed by adding a first water-soluble polymer having surface activity, wherein the first water-soluble polymer is a poloxamer, a divalent metal salt, and dextran to an aqueous solution; The first aqueous mixture is combined with the second aqueous mixture to form a combined aqueous mixture; Adding a gel inducer to the aforementioned combined aqueous mixture; and A foam is obtained by introducing air bubbles into the aforementioned combined aqueous mixture; Leave the obtained foam at room temperature for one hour; The obtained foam is frozen by exposing it to a temperature of -80°C for about 16 hours, and then exposed to a temperature of -86°C and a pressure of 0.11 mbar (about 11 Pa) for 72 hours to form a foamed scaffold. Includes, The formed foamed scaffold has an open structure and highly interconnected pores, The foamed scaffolding that was formed was Porosity of 85% to 96%, Average pore size of 50 μm to 500 μm, 0.40 g / cm ３ Wet density less than 0.20 g / cm³ ３ Dry density less than Methods that include...

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