WO2024107344A1 - Structures en mousse solubles pour la culture cellulaire et leurs procédés de fabrication - Google Patents

Structures en mousse solubles pour la culture cellulaire et leurs procédés de fabrication Download PDF

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WO2024107344A1
WO2024107344A1 PCT/US2023/036731 US2023036731W WO2024107344A1 WO 2024107344 A1 WO2024107344 A1 WO 2024107344A1 US 2023036731 W US2023036731 W US 2023036731W WO 2024107344 A1 WO2024107344 A1 WO 2024107344A1
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scaffold
dissolvable
foam scaffold
dissolvable foam
foam
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PCT/US2023/036731
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English (en)
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Vinalia TJONG
Yue Zhou
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Corning Incorporated
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    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/0062—General methods for three-dimensional culture
    • C—CHEMISTRY; METALLURGY
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/0068—General culture methods using substrates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2510/00—Genetically modified cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2510/00—Genetically modified cells
    • C12N2510/02—Cells for production
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2513/00—3D culture
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/70—Polysaccharides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2535/00—Supports or coatings for cell culture characterised by topography
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2537/00—Supports and/or coatings for cell culture characterised by physical or chemical treatment
    • C12N2537/10—Cross-linking

Definitions

  • the present disclosure generally relates to dissolvable cell culture materials.
  • the present disclosure relates to dissolvable foam scaffolds for cell culture and methods of making such dissolvable foam scaffolds.
  • 3D cell culture in contrast to 2D culture, more accurately represents the environment experienced by cells in-vivo, and it has been demonstrated that cell responses in 3D cultures are more similar to in-vivo behavior than the cell responses in 2D cultures.
  • the additional dimensionality of 3D cultures is believed to lead to the differences in cellular responses because not only does it influence the spatial organization of the cell surface receptors engaged in interactions with surrounding cells, but it also induces physical constraints to cells. These spatial and physical aspects in 3D cultures are believed to affect the signal transduction from the outside to the inside of cells, and ultimately influence gene expression and cellular behavior.
  • 3D culture technologies which simulate the natural 3D environment of cells.
  • Some bioreactors include a carrier in the form of a stationary packing material forming a fixed or packed bed for promoting cell adhesion and growth.
  • the arrangement of the packing material of the fixed bed affects local fluid, heat, and mass transport, and usually is very dense to maximize cell cultivation in a given space.
  • Yet another 3D cell culture technology is a porous 3D matrix or scaffold which promotes the growth and proliferation of the cultured cells within pores and other interior spaces of the matrix.
  • a protease treatment may be used to harvest the cells.
  • commonly used harvesting procedures such as protease treatment, subject the cells to harsh conditions which may damage cell structure and function.
  • protease treatment alone often causes only a limited amount of cell detachment.
  • the problem results from the densely packed nature of the fixed bed material which makes it more difficult to circulate the protease agent throughout the bed and increase the yield of cells harvested.
  • it can be difficult to circulate the protease agent through interior spaces of the 3D matrix, which in turn makes it difficult to dislodge cells during the harvest process. This difficulty is compounded by the presence of extracellular macromolecules secreted by the cultured cells that serve to attach the cells to the surface of the fixed bed material or to the surface of the matrix.
  • a dissolvable foam scaffold for cell culture includes an ionotropically crosslinked polygalacturonic acid compound selected from at least one of: pectic acid; partially esterified pectic acid, partially amidated pectic acid and salts thereof; and a water-soluble polymer having surface activity and having a hydrophilic-lipophilic balance (HLB) of greater than about 20.
  • HLB hydrophilic-lipophilic balance
  • the HLB of the water-soluble polymer is greater than or equal to about 22.
  • the dissolvable foam scaffold is made from a simplified formulation that contains 0 wt.% of a water soluble plasticizer.
  • the dissolvable foam scaffold does not contain glycerol, sorbitol, ethylene glycol, propylene glycol, polyethylene glycol. According to aspects of embodiments, the dissolvable foam scaffold only includes a single water-soluble polymer having surface activity, and may not include any polymer having no surface activity.
  • a method for forming a dissolvable foam scaffold 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 a water-soluble polymer having surface activity and a divalent metal salt to an aqueous solution; combining the first aqueous mixture with second first aqueous mixture to form a combined aqueous mixture; adding a gel inducing agent to the combined aqueous mixture to form a foaming solution; and introducing gas bubbles into the foaming solution to form a foam scaffold.
  • 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 a water-soluble polymer having
  • the water-soluble polymer has a hydrophilic -lipophilic balance (HLB) of greater than about 20.
  • HLB hydrophilic -lipophilic balance
  • forming the second aqueous mixture does not include adding a water soluble plasticizer to the second aqueous mixture, and may not include adding a second polymer having no surface activity.
  • the method does not comprise adding an emulsifying agent to any of the first aqueous mixture, the second aqueous mixture, the combined aqueous mixture, and the foaming solution.
  • the method includes adding a first amount of the gel inducing agent to the aqueous mixture before foaming and adding a second amount of the gel inducing agent to the aqueous mixture during or after foaming.
  • a ratio of the second amount of gel inducing agent to the first amount of gel inducing agent is greater than about 2, or about 7 or greater.
  • a method for culturing cells on a dissolvable foam scaffold includes seeding cells on a dissolvable foam scaffold such that cells enter pores of the dissolvable foam scaffold, the dissolvable scaffold comprising: an ionotropically crosslinked polygalacturonic acid compound selected from at least one of: pectic acid; partially esterified pectic acid, partially amidated pectic acid and salts thereof; and a water-soluble polymer having surface activity and having a hydrophilic-lipophilic balance (HLB) of greater than about 20; and contacting the dissolvable foam scaffold with cell culture medium.
  • the dissolvable foam scaffold has 0 wt.% of a water soluble plasticizer, and does not include polymer having no surface activity.
  • a foamed scaffold product is provided herein.
  • the foamed scaffold product is formed from a composition including an ionotropically crosslinked polygalacturonic acid compound selected from at least one of: pectic acid; partially esterified pectic acid, partially amidated pectic acid and salts thereof; at least one first water-soluble polymer having surface activity and having a hydrophilic-lipophilic balance (HLB) of greater than about 20; and 0 wt. % water soluble plasticizer.
  • HLB hydrophilic-lipophilic balance
  • Figure 1 is a perspective view of a dissolvable foam scaffold in accordance with the present disclosure
  • Figure 2 shows an SEM picture of a foam scaffold prepared in Example 1;
  • Figure 3 shows an SEM picture of a foam scaffold prepared in Example 2.
  • Figure 4 shows an SEM picture of a foam scaffold prepared in Example 3.
  • Figure 5 shows an SEM picture of a foam scaffold prepared in Example 4.
  • Figure 6 shows an SEM picture of a foam scaffold prepared in Example 5.
  • Figure 7 shows an SEM picture of a foam scaffold prepared in Example 6
  • Figure 8 shows an SEM picture of a foam scaffold prepared in Example 7
  • Figure 9 shows an SEM picture of a foam scaffold prepared in Example 8
  • Figure 10 shows an SEM picture of a foam scaffold prepared in Example 9
  • Figure 11 shows an SEM picture of a foam scaffold prepared in Example 10.
  • Figure 12 shows an SEM picture of a foam scaffold prepared in Example 11
  • Figure 13 shows four foams prepared in Example 12 where varied amounts of a plasticizer were added to form the four foam scaffolds
  • Figure 14 shows an SEM picture of a foam scaffold prepared in Example 13
  • Figure 15 shows spheroids formed in the pores of a foam scaffold prepared in
  • Figure 16 shows cells adhered to the foam scaffold prepared in Example 14;
  • Figure 17 shows cells adhered to the foam scaffold prepared in Example 15;
  • Figure 18 shows cells after six days of expansion adhered to the foam scaffold prepared in Example 15;
  • Figure 19 is a bar graph showing the GFP-positive percentage of transfected HEK cells for each of the culture conditions of Example 24;
  • Figure 20 is a bar graph showing GFP-positive percentage of transfected cells per set of foam scaffold of Example 25;
  • Figure 21 is a bar graph showing the number of viral particles (vp) per set of foam scaffold of Example 25;
  • Figure 22 is a bar graph showing the number of viral particles per cell obtained per set of foam scaffold of Example 25.
  • Figure 23 is a bar graph showing the fraction of cells exhibiting GFP expression for each of the sets of foam scaffolds of Example 25 after infection.
  • Figure 24 shows an SEM picture of a foam scaffold prepared in Example 26. DETAILED DESCRIPTION
  • Embodiments of the present disclosure relate to dissolvable foam scaffolds for cell culture and methods of making such dissolvable foam scaffolds. Embodiments of the present disclosure further relate to methods of cell culture of adherent cells, cell aggregates, or spheroids, in dissolvable foam scaffolds. Furthermore, embodiments of the present disclosure relate to bioreactors systems including dissolvable foam scaffolds. As will become clearer in the discussions below, foam scaffolds as disclosed herein are described as being dissolvable and insoluble. As used herein, the term “insoluble” is used to refer to a material or combination of materials that is not soluble, and that remains crosslinked, under conventional cell culture conditions which include, for example, cell culture media.
  • dissolvable is used to refer to a material or combination of materials that is digested when exposed to an appropriate concentration of an enzyme that digests or breakdowns the material or combination of materials.
  • Dissolvable foam scaffolds as described herein are porous scaffolds having an open pore architecture and highly interconnected pores. The pores of the scaffolds provide a protected environment for the culturing of cells where the cell-to-cell interactions and formation of ECM in a 3D fashion are aided.
  • the dissolvable foam scaffolds may be completely digested which allows for harvesting cells without damaging the cells using protease treatment and/or mechanical harvesting techniques.
  • FIG. 1 is a perspective view of a dissolvable foam scaffold 10 in accordance with the present disclosure.
  • dissolvable foam scaffold 10 is a porous foam that includes an open pore architecture.
  • Dissolvable foam scaffold 10 has a porosity of from about 85% to about 96% and an average pore size diameter of between about 50 pm and about 500 pm.
  • Dissolvable foam scaffold 10 provides a protected environment within the pores of the foam scaffold for the culturing of cells. Additionally, dissolvable foam scaffold 10 is also dissolvable when exposed to an appropriate enzyme that digests or breakdowns the material which facilitates harvesting of the cells cultured in the scaffold without damaging the cells.
  • Dissolvable foam scaffolds as described herein include at least one ionotropically crosslinked polysaccharide.
  • polysaccharides possess attributes beneficial to cell culture applications.
  • Polysaccharides are hydrophillic, non-cytotoxic and stable in culture medium. Examples include pectic acid, also known as polygalacturonic acid (PGA), or salts thereof, partly esterified pectic acid or salts thereof, or partly amidated pectic acid or salts thereof.
  • Pectic acid can be formed via hydrolysis of certain pectin esters.
  • Pectins are cell wall polysaccharides and in nature have a structural role in plants. Major sources of pectin include citrus peel (e.g., peels from lemons and limes) and apple peel.
  • Pectins are predominantly linear polymers based on a 1,4-linked alpha-D-galacturonate backbone, interrupted randomly by 1,2-linked L-rhamnose. The average molecular weight ranges from about 50,000 to about 200,000 Daltons.
  • the polygalacturonic acid chain of pectin may be partly esterified, e.g., methyl groups and the free acid groups may be partly or fully neutralized with monovalent ions such as sodium, potassium, or ammonium ions.
  • Polygalacturonic acids partly esterified with methanol are called pectinic acids, and salts thereof are called pectinates.
  • the degree of methylation (DM) for high methoxyl (HM) pectins can be, for example, from 60 to 75 mol% and those for low methoxyl (LM) pectins can be from 1 to 40 mol%.
  • the degree of esterification of partly esterified polygalacturonic acids as 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 therebetween. Without wishing to be bound by any particular theory, it is believed that a minimum amount of free carboxylic acid groups (not esterified) facilitates a degree of ionotropic crosslinking which allow for the formation of a dissolvable scaffold which is insoluble.
  • the polygalacturonic acid chain of pectin may be partly amidated.
  • Polygalacturonic acids partly amidated pectin may be produced, for example, by treatment with ammonia.
  • Amidated pectin contains carboxyl groups (-COOH), methyl ester groups (-COOCHs), and amidated groups (-CONH2). The degree of amidation may vary and may be, for example, from about 10% to about 40% amidated.
  • dissolvable foam scaffolds as described herein may include a mixture of pectic acid and partly esterified pectic acid. Blends with compatible polymers may also be used.
  • pectic acid and/or partly esterified pectic acid may be mixed with other polysaccharides such as dextran, substituted cellulose derivatives, alginic acid, starches, glycogen, arabinoxylans, agarose, etc.
  • Glycosaminoglycans like hyaluronic acid and chondroitin sulfate, or various proteins such as elastin, fibrin, silk fibroin, collagen and their derivatives can be also used.
  • Water soluble synthetic polymers can be also blended with pectic acid and/or partly esterified pectic acid.
  • Exemplary water soluble synthetic polymers include, but are not limited to, polyalkylene glycol, poly(hydroxyalkyl(meth)acrylates), poly(meth)acrylamide and derivatives, poly(N- vinyl-2-pyrrolidone), and polyvinyl alcohol.
  • dissolvable foam scaffolds as described herein may further include at least one first polymer.
  • the at least one first polymer is water soluble, non-ionotropically crosslinkable and has surface activity.
  • surface activity refers to the activity of an agent to lower or eliminate the surface tension (or interfacial tension) between two liquids or between a liquid and a solid or between gas and liquid.
  • the at least one first polymer may have a hydrophilic-lipophilic balance (HLB) of greater than about 8 or even greater than about 10.
  • HLB hydrophilic-lipophilic balance
  • the at least one first polymer may have an HLB of between about 8 and about 40 or between about 10 and about 40.
  • the at least one first polymer may have an HLB of between about 8 and about 15, or even between about 10 and about 12.
  • HLB provides a reference for the lipophilic or hydrophilic degree of a polymer. A larger HLB value indicates stronger hydrophilicity, while a smaller HLB value indicates a stronger lipophilicity.
  • the HLB value varies in the range of from 1 to 40 and the hydrophilic -lipophilic transition is often considered to be between about 8 and about 10. When the HLB value is less than the hydrophilic-lipophilic transition, the material is lipophilic, and when the HLB value is greater than the hydrophilic- lipophilic transition the material is hydrophilic.
  • Exemplary first polymers in accordance with embodiments of the present disclosure may be any of cellulose derivatives, proteins, synthetic amphiphilic polymers, and combinations thereof.
  • Exemplary cellulose derivatives include, but are not limited to, hydroxyethylcellulose (HEC), hydroxypropylcellulose (HPC), methylcellulose (MC), hydroxy ethylmethylcellulose (HEMC), and hydroxypropyl-methylcellulose (HPMC).
  • Exemplary proteins include, but are not limited to, bovine serum albumin (BSA), gelatine, casein and hydrophobins.
  • Exemplary synthetic amphiphilic polymers include, but are not limited to, a poloxamer available under the trade name Synperonics® (commercially available from Croda International, Snaith, United Kingdom), a poloxamer available under the trade name Pluronics® (commercially available from BASF Corp., Parsippany, NJ) and a poloxamer available under the trade name Kolliphor® (commercially available from BASF Corp., Parsippany, NJ).
  • Synperonics® commercially available from Croda International, Snaith, United Kingdom
  • Pluronics® commercially available from BASF Corp., Parsippany, NJ
  • Kolliphor® commercially available from BASF Corp., Parsippany, NJ.
  • Dissolvable foam scaffolds as described herein may further include at least one second polymer.
  • the at least one second polymer is water soluble and has no surface activity.
  • Exemplary second polymers may be any of synthetic polymers, semisynthetic polymers, natural polymers and combinations thereof.
  • Exemplary synthetic polymers include, but are not limited to, polyethylene glycol, polyvinyl pyrrolidone, polyvinyl alcohol, carboxyvinyl polymer, polyacrylic acid, polyacrylamide, homopolymer and copolymer of N- (2-Hydroxypropyl) methacrylamide, polyvinyl methyl ether-maleic anhydride, and polyethylene oxide/polypropylene oxide block copolymers.
  • Exemplary semisynthetic polymers include, but are not limited to, dextran derivatives, carboxymethyl cellulose, hydroxyethyl cellulose and derivatives, methylcellulose and derivatives, ethylcellulose cellulose, ethyl hydroxyethyl cellulose, and hydroxypropyl cellulose.
  • Exemplary natural polymers include, but are not limited to, starch and starch derivatives, polymers obtained by microbial fermentation such as curdlan, pullulan and gellan gum, xanthan gum, dextran, proteins such as albumin, casein and caseinates, gelatin, seaweed extracts such as agar, alginates and carrageenan, seed extracts such as guar gum and derivatives and locust bean gum, hyaluronic acid, and chondroitin sulfate.
  • polymers obtained by microbial fermentation such as curdlan, pullulan and gellan gum, xanthan gum, dextran, proteins such as albumin, casein and caseinates, gelatin, seaweed extracts such as agar, alginates and carrageenan, seed extracts such as guar gum and derivatives and locust bean gum, hyaluronic acid, and chondroitin sulfate.
  • Dissolvable foam scaffolds as described herein may be crosslinked to increase their mechanical strength and to prevent the dissolution of the scaffolds when placed in contact with cell culture medium.
  • Crosslinking may be performed by ionotropic gelation as described below wherein ionotropic gelation is based on the ability of poly electrolytes to crosslink in the presence of multivalent counter ions to form crosslinked scaffolds.
  • ionotropic gelation of the polysaccharide of the dissolvable foam scaffolds is the result of strong interactions between divalent cations and the polysaccharide.
  • scaffolds as described herein are porous foam scaffolds.
  • Foam scaffolds as described herein may have a porosity of from about 85% to about 96%.
  • foam scaffolds as described herein may have a porosity of from about 91% to about 95%, or about 94% to about 96%.
  • the term “porosity” refers to the measure of open pore volume in the dissolvable scaffold and is referred to in terms of % porosity, wherein % porosity is the percent of voids in the total volume of the dissolvable foam scaffold.
  • Foam scaffolds as described herein may have an average pore size diameter of between about 50 pm and about 500 pm.
  • average pore size diameter may be between about 75 pm and about 450 pm, or between about 100 pm and about 400 pm, or even between 150 pm and about 350 pm and all values therebetween.
  • Scaffolds as described herein may have a wet density of less than about 0.40 g/cc.
  • scaffolds as described herein may have a wet density of less than about 0.35 g/cc, or less than about 0.30 g/cc, or less than about 0.25 g/cc.
  • Scaffolds as described herein may have a wet density of between about 0.16 g/cc and about 0.40 g/cc, or between about 0.16 g/cc and about 0.35 g/cc, or between about 0.16 g/cc and about 0.30 g/cc, or even between about 0.16 g/cc and about 0.25 g/cc, and all values therebetween.
  • Scaffolds as described herein may have a dry density of less than about 0.20 g/cc.
  • scaffolds as described herein may have a dry density of less than about 0.15 g/cc, or less than about 0.10 g/cc, or less than about 0.05 g/cc.
  • Scaffolds as described herein may have a dry density of between about 0.02 g/cc and about 0.20 g/cc, or between about 0.02 g/cc and about 0.15 g/cc, or between about 0.02 g/cc and about 0.10 g/cc, or even between about 0.02 g/cc and about 0.05 g/cc, and all values therebetween.
  • Open pores allow for cellular access on both sides of the scaffold and allow for liquid flow and transport of nutrients through the dissolvable scaffold.
  • Partially open pores allow for cellular access on one side of the scaffold, but mass transport of nutrients and waste products is limited to diffusion.
  • Closed pores have no openings and are not accessible by cells or by mass transport of nutrients and waste products.
  • Dissolvable foam scaffolds as described herein have an open pore architecture and highly interconnected pores. Generally, the open pore architecture and highly interconnected pores enable migration of cells into the pores of the dissolvable foam scaffolds and also facilitate enhanced mass transport of nutrients, oxygen and waste products.
  • the open pore architecture also influences cell adhesion and cell migration by providing a high surface area for cell-to-cell interactions and space for ECM regeneration.
  • Dissolvable foam scaffolds as described herein are digested when exposed to an appropriate enzyme that digests or breakdowns the material.
  • Non-proteolytic enzymes suitable for digesting the foam scaffolds, harvesting cells, or both include pectinolytic enzymes or pectinases, which are a heterogeneous group of related enzymes that hydrolyze the pectic substances.
  • Pectinases polygalacturonase are enzymes that break down complex pectin molecules to shorter molecules of galacturonic acid.
  • PectinexTM ULTRA SP-L commercially available from Novozyme North American, Inc., Franklinton, NC
  • PectinexTM ULTRA SP- L contains mainly polygalacturonase, (EC 3.2.1.15) pectintranseliminase (EC 4.2.2.2) and pectinesterase (EC: 3.1.1.11).
  • the EC designation is the Enzyme Commission classification scheme for enzymes based on the chemical reactions the enzymes catalyze.
  • digestion of the dissolvable foam scaffolds also includes exposing the scaffold to a divalent cation chelating agent.
  • exemplary chelating agents include, but are not limited to, ethylenediaminetetraacetic acid (EDTA), cyclohexanediaminetetraacetic (CDTA), ethylene glycol tetraacetic acid (EGTA), citric acid and tartaric acid.
  • the time to complete digestion of dissolvable foam scaffolds as described herein may be less than about 1 hour.
  • the time to complete digestion of foam scaffolds may be less than about 45 minutes, or less than about 30 minutes, or less than about 15 minutes, or between about 1 minute and about 25 minutes, or between about 3 minutes and about 20 minutes, or even between about 5 minutes and about 15 minutes.
  • scaffolds as described herein may further include an adhesion polymer coating.
  • the adhesion polymer may include peptides.
  • Exemplary peptides may include, but are not limited to BSP, vitronectin, fibronectin, laminin, Type I and IV collagen, denatured collagen (gelatin), and like peptides, and mixtures thereof. Additionally, the peptides may be those having an RGD sequence.
  • the coating may be, for example, Synthemax® II-SC (commercially available from Coming, Incorporated, Coming, NY).
  • the adhesion polymer may include an extracellular matrix.
  • the coating may be, for example, Matrigel® (commercially available from Coming, Incorporated, Coming, NY).
  • Methods as described herein may include forming a first aqueous mixture which includes dissolving a polysaccharide in an aqueous solution.
  • Polysaccharides may be those as described above, such as pectic acid or salts thereof, partly esterified pectic acid or salts thereof, or partly amidated pectic acid or salts thereof, and blends of such polysaccharides.
  • Methods for forming dissolvable foam scaffolds as described herein may further include forming a second aqueous mixture including a water insoluble divalent metal salt in an aqueous solution.
  • Metals of the divalent metal salts may include, but are not limited to, magnesium, calcium, zinc, strontium, barium, and like cations, and combinations thereof.
  • Anions of the divalent metal salts may include, but are not limited to, oxalates, tartrates, phosphates, carbonates, citrates, and like organic and inorganic anions, and combinations thereof.
  • forming a second aqueous mixture may further include adding the at least one first polymer as described above to the second aqueous mixture.
  • methods as described herein may further include adding the at least one second polymer as described above to the second aqueous mixture.
  • the at least one first polymer and the at least one second polymer may be added to the second aqueous mixture separately or may be added to the second aqueous mixture together.
  • the mixture may include about 50% of the at least one first polymer and about 50% of the at least one second polymer.
  • the mixture may include between about 35% and about 65% (and all values therebetween) of the at least one first polymer and between about 35% and about 65% (and all values therebetween) of the at least one second polymer.
  • forming a second aqueous mixture may further include adding a water soluble plasticizer to the second aqueous mixture.
  • Plasticizers as described herein are non-toxic and do not affect the solubility of the polysaccharides of the dissolvable foam scaffolds.
  • a plasticizer provides flexibility and softness to the resulting foam such that the resulting foam is soft and pliable.
  • Plasticizers as 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 wt.
  • adding a water soluble plasticizer to the second aqueous mixture may include adding less than about 50 wt. %, or less than about 40 wt. %, or less than about 30 wt. %, or less than about 25 wt. %, or between about 15 wt. % and about 55 wt. % ,or between about 15 wt. % and about 50 wt. %, or between about 15 wt. % and about 40 wt. %, or between about 15 wt. % and about 30 wt. %, or between about 15 wt. % and about 25 wt.
  • total solid additives added to form the second aqueous mixture refers to all of the components of the aqueous mixture except for water.
  • forming a second aqueous mixture may further include adding an emulsifying agent to the second aqueous mixture.
  • Emulsifying agents as described herein may include, but are not limited to, Tween® 20, Tween® 80 (each commercially available from commercially available from Croda International, Snaith, United Kingdom).
  • forming a second aqueous mixture may further include adding at least one leachable solid to the second aqueous mixture.
  • Leachable solids as described herein include materials that reinforce or create pores during the formation of the foam scaffold.
  • Leachable solids may be, but are not limited to, nontoxic leachable materials such as salts, biocompatible mono and disaccharides and water-soluble proteins.
  • Exemplary salts include, but are not limited to, sodium chloride, potassium chloride, calcium chloride, sodium tartrate, sodium citrate, and the like.
  • Exemplary biocompatible mono 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.
  • each of the materials described above in relation to the second aqueous mixture may all be optionally added to the second aqueous mixture and may be added to the second aqueous mixture in any order with the possibility that two or more of the materials may be added to the second aqueous mixture simultaneously.
  • forming a second aqueous mixture includes adding a leachable solid to an aqueous solution including a divalent metal salt and mixing the aqueous mixture to facilitate the dissolution of the leachable solid dissolves in the aqueous mixture.
  • the at least one first polymer, the at least one second polymer and/or the water soluble plasticizer are subsequently added to the second aqueous mixture.
  • Methods for forming a dissolvable foam scaffold as described herein may further include, subsequent to forming the first and second aqueous mixtures, combining the second aqueous mixture with the first aqueous mixture to form a combined aqueous mixture.
  • a foam may be formed from the combined aqueous mixture by introducing gas bubbles into the aqueous mixture through mixing, beating, agitating, aerating, whipping, injecting or other mechanical actions.
  • the gas may be for example, but not limited to, air, nitrogen, helium, hydrogen, argon, carbon dioxide or other inert gas.
  • the method for forming a dissolvable foam scaffold may further include adding a gel inducing agent to the combined aqueous mixture.
  • the gel inducing agent may be an acid that provides a buffering action and/or materials that slowly generate acid.
  • Exemplary acids include, but are not limited to, lactic acid lactone, glycolic acid lactone, glucono delta lactone and acid anhydrides.
  • Methods for forming a dissolvable foam scaffold as described herein may further include, coating the dissolvable foam scaffold with an adhesion polymer coating.
  • Coating the dissolvable foam scaffold may include exposing the scaffold to an aqueous solution having an adhesion polymer in the aqueous solution.
  • the adhesion polymer may include peptides.
  • Exemplary peptides may include, but are not limited to BSP, vitronectin, fibronectin, laminin, Type I and IV collagen, denatured collagen (gelatin), and like peptides, and mixtures thereof. Additionally, the peptides may be those having an RGD sequence.
  • the coating may be, for example, Synthemax® II-SC (commercially available from Coming, Incorporated, Coming, NY).
  • any type of cell may be cultured on the dissolvable foam scaffolds including, but not limited to, immortalized cells, primary culture cells, cancer cells, stem cells (e.g., embryonic or induced pluripotent), etc.
  • the cells may be mammalian cells, avian cells, piscine cells, etc.
  • the cells may be of any tissue type including, but not limited to, kidney, fibroblast, breast, skin, brain, ovary, lung, bone, nerve, muscle, cardiac, colorectal, pancreas, immune (e.g., B cell), blood, etc.
  • the cells may be in any cultured form in the bag including disperse (e.g., freshly seeded), confluent, 2- dimensional, 3 -dimensional, spheroid, etc.
  • Culturing cells on a dissolvable foam scaffold may include seeding cells on the dissolvable foam scaffold. Seeding cells on a dissolvable foam scaffold may include contacting the scaffold with a solution containing the cells. During seeding cells on the dissolvable foam scaffold, the cells enter the pores of the dissolvable foam scaffold. Where the dissolvable foam scaffold includes an adhesion polymer coating, cells may enter the pores of the dissolvable foam scaffold and attach to the scaffold material.
  • Culturing cells on dissolvable foam scaffolds may further include contacting the scaffolds with cell culture medium.
  • contacting the scaffolds with cell culture medium includes placing cells to be cultured on the scaffolds in an environment with medium in which the cells are to be cultured.
  • Contacting the scaffolds with cell culture medium may include pipetting cell culture medium onto the scaffolds, or submerging the scaffolds in cell culture medium, or passing cell culture media over the scaffolds in a continuous manner.
  • continuous refers to culturing cells with a consistent flow of cell culture medium into and out of the cell culture environment.
  • Such passing cell culture media over the scaffolds in a continuous manner may include submerging the scaffolds in cell culture medium for a predetermined period of time, then removing at least some of the cell culture medium after the predetermined period of time and adding fresh cell culture medium such that the volume of cell culture medium in contact with the dissolvable foam scaffold remains substantially constant.
  • Cell culture medium may be removed and replaced according to any predetermined schedule. For example, at least some of the cell culture medium may be removed and replaced every hour, or every 12 hours, or every 24 hours, or every 2 days, or every 3 days, or every 4 days, or every 5 days.
  • Cell culture medium may be for example, but is not limited to, sugars, salts, amino acids, serum (e.g., fetal bovine serum), antibiotics, growth factors, differentiation factors, colorant, or other desired factors.
  • exemplary cell culture medium includes Dulbecco’s Modified Eagle Medium (DMEM), Ham’s F12 Nutrient Mixture, Minimum Essential Media (MEM), RPMI Medium, Iscove's Modified Dulbecco’s Media (IMDM) MesencultTM-XF medium, and the like.
  • Methods for harvesting cells from dissolvable foam scaffolds as described herein may include digesting the dissolvable foam scaffold by exposing the dissolvable foam scaffold to an enzyme.
  • non- proteolytic enzymes suitable for digesting the foam scaffolds, harvesting cells, or both include pectinolytic enzymes or pectinases, which are a heterogeneous group of related enzymes that hydrolyze the pectic substances.
  • PectinexTM ULTRA SP-L commercially available from Novozyme North American, Inc., Franklinton, NC
  • PectinexTM ULTRA SP-L contains mainly polygalacturonase, (EC 3.2.1.15) pectintranseliminase (EC 4.2.2.2) and pectinesterase (EC: 3.1.1.11).
  • the EC designation is the Enzyme Commission classification scheme for enzymes based on the chemical reactions the enzymes catalyze.
  • Exposing the dissolvable foam scaffold to an enzyme may include exposing the scaffold to enzyme concentrations of between about 1 and about 200 U.
  • the method may include exposing the scaffold to enzyme concentrations of between about 2 U and about 150 U, or between about 5 U and about 100 U, or even between about 10 U and about 75 U, and all values therebetween.
  • Methods for harvesting cells as described herein may further include exposing the material to a chelating agent.
  • exemplary chelating agents include, but are not limited to, ethylenediaminetetraacetic acid (EDTA), cyclohexanediaminetetraacetic (CDTA), ethylene glycol tetraacetic acid (EGTA), citric acid and tartaric acid.
  • Exposing the dissolvable foam scaffold to a chelating agent may include exposing the scaffold to chelating agent concentrations of between about 1 mM and about 200 mM.
  • the method may include exposing the scaffold to chelating agent concentrations of between about 10 mM and about 150 mM, or between about 20 mM and about 100 mM, or even between about 25 mM and about 50 mM, and all values therebetween.
  • a first aqueous mixture containing 2.0 wt. % polygalacturonic acid (PGA) was prepared by dissolving about 162 grams of polygalacturonic acid sodium salt in demineralized water in an oil bath set at a temperature of 104°C. The aqueous mixture was cooled to room temperature.
  • a second aqueous mixture was prepared by adding about 1.06 grams of CaCOs to about 24.52 grams of ultrapure water in the bowl of a KitchenAid mixer equipped with a wire loop whip. About 0.125 grams of TWEEN® 20 (commercially available from Sigma-Aldrich, St. Louis, MO) was also added to the bowl of the KitchenAid mixer.
  • sucrose was then added to the mixer bowl and mixed to facilitate dissolution of the sucrose in the second aqueous mixture.
  • About 7.5 grams glycerol, about 1.94 grams Methocel HPMC Culminal 724 and the first aqueous mixture were added to form a combined aqueous mixture in the mixing bowl and mixed at a stir speed (speed 1 of the KitchenAid mixer) for about 5 minutes.
  • the combined aqueous mixture was then whipped at a fast whipping speed (speed 10 of the KitchenAid mixer) for about 20 minutes to introduce air into the combined aqueous mixture.
  • a solution of about 3.77 grams of gluconolactone (GDL) in about 30 mb of water was added to the mixing bowl and whipping was continued for about 1 minute.
  • GDL gluconolactone
  • FIG. 1 An opaque white foam having a wet foam density of about 0.25 g/cc was obtained following the process discussed above.
  • the foam was left uncovered in the mixing bowl at room temperature for about 1 hour to allow time for crosslinking to take place within the foam.
  • the foam was then exposed to temperatures of about -80°C for about 16 hours to freeze the foam and then exposed to a temperature of -86°C and a pressure of 0. 11 mbar for about 72 hours.
  • the resulting foam was observed to have a dry foam density of about 0.04 to about 0.045 g/cc and was observed to be porous with highly interconnected pores.
  • Figure 2 shows an SEM picture of the foam prepared in this Example 1.
  • the composition of the resulting foam was determined to include about 1.3 wt. % PGA, about 0.78 wt. % HPM and a total solid content of about 2.08 wt. %.
  • Example 2 The process as described in Example 1 was repeated except the second aqueous mixture was prepared by adding about 0.53 grams. The resulting foam was observed to be porous with highly interconnected pores.
  • Figure 3 shows an SEM picture of the foam prepared in this Example 2.
  • a first aqueous mixture containing 2.0 wt. % polygalacturonic acid (PGA) was prepared by dissolving about 162 grams of polygalacturonic acid sodium salt in demineralized water in an oil bath set at a temperature of 104°C. The aqueous mixture was cooled to room temperature.
  • a second aqueous mixture was prepared by adding about 7.5 grams of glycerol to ultrapure water and heating under a microwave at 800W for about 30 seconds. About 0.97 grams of bovine gelatin was cold-swelled in about 5.8 mb of ultrapure water and then added to the second aqueous mixture and stirred until dissolution was observed.
  • the second aqueous mixture was then sonicated for about 1 minute and then transferred to the bowl of a KitchenAid mixer equipped with a wire loop whip.
  • About 17.5 grams sucrose and about 0.97 grams Methocel HPMC Culminal 724 were then added to the bowl of the KitchenAid mixer and the aqueous mixture was stirred for about 5 minutes.
  • the first aqueous mixture containing 2.0 wt. % PGA were added to form a combined aqueous mixture in the mixing bowl and mixed at a stir speed (speed 1 of the KitchenAid mixer) for about 3 minutes.
  • the combined aqueous mixture was then whipped at a fast whipping speed (speed 10 of the KitchenAid mixer) for about 20 minutes to introduce air into the combined aqueous mixture. While continuing to whip the combined aqueous mixture, a solution of about 3.77 grams of gluconolactone (GDL) in about 30 mL of water was added to the mixing bowl and whipping was continued for about 1 minute.
  • GDL gluconolactone
  • Example 3 The process as described in Example 3 was repeated except that about 0.97 grams of porcine gelatin, instead of bovine gelatin, was cold-swelled in about 5.8 mb of ultrapure water and then added to the second aqueous mixture and stirred until dissolution was observed.
  • the resulting foam was observed to have a wet foam density of about 0.21 g/cc and a dry foam density of about 0.06 g/cc and was observed to be porous with highly interconnected pores.
  • Figure 5 shows an SEM picture of the foam prepared in this Example 4.
  • Example 5 The process as described in Example 1 was repeated except the first aqueous mixture was prepared by dissolving about 172 grams of 3.0 wt. % polygalacturonic acid sodium salt in demineralized water in an oil bath set at a temperature of 104°C. Additionally, Methocel HPMC Culminal 724 was omitted from the second aqueous mixture. The resulting foam was observed to have a wet foam density of about 0.40 g/cc and a dry foam density of about 0.11 g/cc. The foam was observed to be less porous than the foam formed in Example 1 and the pores were less interconnected than the pores of the foam formed in Example 1.
  • FIG. 6 shows an SEM picture of the foam prepared in this Example 5. Although a foam having a wet density of greater than about 0.40 g/cc and a dry density of greater than about 0.11 g/cc was demonstrated as being able to support culturing of cells, foams having a wet density of less than about 0.40 g/cc and a dry density of less than about 0.11 g/cc exhibited improved cell culturing conditions as compared to foams having wet and dry densities such as those possessed by the foam as formed in this Example 5.
  • Example 2 The process as described in Example 1 was repeated except the first aqueous mixture was prepared by dissolving about 162 grams of 1.59 wt. % polygalacturonic acid sodium salt in demineralized water in an oil bath set at a temperature of 104°C. Additionally, the second aqueous mixture was prepared by adding 0.53 grams of CaCO 3 to about 24.52 grams of ultrapure water and about 2.58 grams Methocel HPMC Culminal 724 was added to the second aqueous mixture. The resulting foam was observed to have a wet foam density of about 0.36 g/cc and a dry foam density of about 0.09 g/cc. Figure 7 shows an SEM picture of the foam prepared in this Example 6.
  • Example 7 The process as described in Example 1 was repeated except that sucrose was not added to the second aqueous mixture. The resulting foam was observed to have a wet foam density of about 0.23 g/cc and a dry foam density of about 0.03 g/cc.
  • Figure 8 shows an SEM picture of the foam prepared in this Example 7.
  • Example 2 The process as described in Example 1 was repeated except the first aqueous mixture was prepared by dissolving about 162 grams of 2.59 wt. % polygalacturonic acid sodium salt in demineralized water in an oil bath set at a temperature of 104°C. Additionally, the second aqueous mixture was prepared by adding 0.97 grams Methocel HPMC Culminal 724 to the second aqueous mixture. The ratio of PGA to Methocel HPMC Culminal 724 in the combined aqueous mixture was controlled to be 81/19. The resulting foam was observed to have a wet foam density of about 0.18 g/cc and a dry foam density of about 0.057 g/cc. Figure 9 shows an SEM picture of the foam prepared in this Example 8.
  • Example 1 The process as described in Example 1 was repeated except the second aqueous mixture was prepared by adding 1.94 grams Pluronic® P123, instead of Methocel HPMC Culminal 724, to the second aqueous mixture.
  • the ratio of PGA to Pluronic P123 in the combined aqueous mixture was controlled to be 62.5/37.5.
  • the resulting foam was observed to have a wet foam density of about 0.18 g/cc and a dry foam density of about 0.03 g/cc and was observed to have a greater porosity than the foam as formed in Example 1.
  • Figure 11 shows an SEM picture of the foam prepared in this Example 10.
  • Example 12 shows an SEM picture of the foam prepared in this Example 11.
  • Example 11 The process as described in Example 11 was repeated to form multiple foams in which varied amounts of glycerol were added to the second aqueous mixture.
  • a first foam of this example 6.5 grams of glycerol was added the second aqueous mixture.
  • Glycerol constituted 19 wt. % of the total solid additives added to form the combined aqueous mixture.
  • the resulting foam was observed to have a wet foam density of about 0.19 g/cc and a dry foam density of about 0.055 g/cc.
  • 7.5 grams of glycerol was added the second aqueous mixture.
  • Glycerol constituted 22 wt.
  • Glycerol constituted 52 wt. % of the total solid additives added to form the combined aqueous mixture.
  • the resulting foam was observed to have a wet foam density of about 0.26 g/cc and a dry foam density of about 0.35 g/cc.
  • the four foams of Example 12 illustrate the effect of the amount of plasticizer on density and porosity when added during the formation of the foams. Slower drying, greater density and less porosity were observed with increasing plasticizer content. Also, as shown in Figure 13, the generally cylindrical shape of the foam was lost as the amount of glycerol increased above about 20-25 wt. % of the total solid additives added to form the second aqueous mixture. It was observed that porosity and interconnectivity of the pores of the foam was greatest when the amount of plasticizer added to the second aqueous mixture was less than about 52 wt. % of the total solid additives added to form the second aqueous mixture.
  • Example 13 The process as described in Example 1 was repeated except the second aqueous mixture was prepared by adding a 50:50 weight ratio blend of Pluronic Pl 27 and Dextran. The resulting foam was observed to have a porosity and interconnectivity of pores similar to the foam prepared in Example 11.
  • Figure 14 shows an SEM picture of the foam prepared in this Example 13.
  • Synthemax® II-SC aqueous ethanol solution About 4.0 mb of the 250 pg/ml Synthemax® II-SC aqueous ethanol solution was added to each of the wells and the plates were then left undisturbed at room temperature for about 1.5 hours. Excess solution was removed from the wells and the foams were washed once with about 5.0 mb of a 70% aqueous ethanol.
  • the Synthemax® II-SC coating was then crosslinked by adding about 4.0 mb of a 0.05% v/v glutaraldehyde in 70% aqueous ethanol (prepared by mixing 40pl of 25% glutaraldehyde solution in 6.0 mb water and 14 mb ethanol). The plates were left undisturbed at room temperature for about 1.5 hours to allow time for crosslinking to occur. The foam was then rinsed three times with ultrapure water.
  • Foams formed in accordance with each of the processes in Examples 1-12 were coated with gelatin.
  • a 0. 1 wt. % gelatin solution was prepared by swelling about 500 mg of gelatin powder (from porcine skin) in ultrapure water followed by dissolution and homogenization in 20 mb of ultrapure water.
  • Each of the foams which were about 2-3 mm thick and had diameters of about 22 mm, were placed in separate wells of Polystyrene 6-Well Cell Culture Plates. About 4.0 mL of the gelatin solution was added to each of the wells and the plates were then left undisturbed at room temperature for about 1.5 hours.
  • Vero cells (ATCC® CCL-81, commercially available from ATCC, Manassas, VA) were cultured on cell culture plates in IMDM medium supplemented with 10% fetal bovine serum (PBS). The foam was cut into portions that were about 2-3 mm thick and had diameters of about 22 mm. The foam portions were sanitized in 70% aqueous ethanol for about 5.0 minutes, then placed in separate wells of a 6-Well Ultra-Low Attachment Cell Culture Plate. The foam portions were washed twice in ultrapure water and once in IMDM medium. Excess medium was removed from the wells before seeding.
  • IMDM medium fetal bovine serum
  • Vero cells were harvested from the cell culture plates using trypsin, resuspended in IMDM medium and 150 pL containing about 100,000 cells were seeded in each of the foam portions which were positioned in the wells of the 6-Well Cell Culture Plate.
  • the 6- Cell Culture Plate was placed in a cell culture incubator and, after about 2.0 hours, about 3.0 mL of IMDM medium was added to each well. After about 18 hours in the cell culture incubator, the foam portions were visualized using phase contrast microscopy. An image obtained from the phase contrast microscopy is depicted in Figure 15 which shows that the cells did not adhere to the uncoated foam portions, but instead formed spheroids in the pores of the foam portions.
  • the dissolvable foam scaffolds of the present disclosure may be utilized to culture spheroids or non-adherent cells.
  • hMSC Passage 2 (commercially available from RoosterBio Inc., Frederick, MD) were cultured on cell culture plates in MesencultTM-XF medium (a serum-free medium commercially available from STEMCELL Technologies, Vancouver, BC, Canada). The foam was cut into portions that were about 2-3 mm thick and had diameters of about 22 mm. The foam portions were sanitized in 70% aqueous ethanol for about 5.0 minutes, then placed in separate wells of a 6-Well Ultra-Low Attachment Cell Culture Plate. The foam portions were washed twice in ultrapure water and once in the MesencultTM-XF medium. Excess medium was removed from the wells before seeding.
  • hMSC were harvested from the cell culture plates using trypsin, re-suspended in MesencultTM-XF medium and 150 pL containing about 100,000 cells were seeded in each of the foam portions which were positioned in the wells of the 6-Well Cell Culture Plate.
  • the 6-Well Cell Culture Plate was placed in a cell culture incubator and, after about 2.0 hours, about 3.0 mb of MesencultTM-XF medium was added to each well. After about 18 hours in the cell culture incubator, cells were stained with 1 pg/mL Calcein-AM and visualized using fluorescence microscopy.
  • FIG. 16 An image obtained from the fluorescence microscopy is depicted in Figure 16 which shows that the cells were able to adhere to the foam portions, and spread within the pores of the foam portion.
  • the dissolvable foam scaffolds coated with Coming® Synthemax® II-SC of the present disclosure may be utilized to culture adherent cells in addition to spheroids or non-adherent cells and that the scaffolds support cell culture in serum-free medium.
  • hMSC Passage 2 as used in Example 17 were cultured on cell culture plates in IMDM medium supplemented with 10% fetal bovine serum (FBS). The foam was cut into portions that were about 2-3 mm thick and had diameters of about 22 mm. The foam portions were sanitized in 70% aqueous ethanol for about 5.0 minutes, then placed in separate wells of a 6-Well Ultra-Low Attachment Cell Culture Plate. The foam portions were washed twice in ultrapure water and once in the MesencultTM-XF medium.
  • FBS fetal bovine serum
  • hMSC were harvested from the cell culture plates using trypsin, re-suspended in IMDM medium and 150 pL containing about 100,000 cells were seeded in each of the foam portions which were positioned in the wells of the 6-Well Cell Culture Plate.
  • the 6- Well Cell Culture Plate was placed in a cell culture incubator and, after about 2.0 hours, about 3.0 mb of IMDM medium was added to each well. After about 18 hours in the cell culture incubator, cells were stained with Ipg/mL Calcein-AM and visualized using fluorescence microscopy.
  • FIG 17 An image obtained from the fluorescence microscopy is depicted in Figure 17 which shows that the cells were able to adhere to the foam portions, and spread within the pores of the foam portion. Expansion of the hMSC cells was allowed to proceed on the foam for six days and again visualized using fluorescence microscopy. An image obtained from the fluorescence microscopy after six days is depicted in Figure 18 which shows cells adhered to the foam portions, and further spread within the pores of the foam portion.
  • the dissolvable foam scaffolds coated with gelatin of the present disclosure may be utilized to culture adherent cells in addition to spheroids or non-adherent cells and that the scaffolds support cell culture in serum -containing medium.
  • Vero cells as used in Example 16 were cultured on Tissue Culture Treated (TCT) plates in IMDM medium supplemented with 10% fetal bovine serum (FBS).
  • TCT Tissue Culture Treated
  • FBS fetal bovine serum
  • the foam was cut into portions that were about 2-3 mm thick and had diameters of about 22 mm.
  • the foam portions were sanitized in 70% aqueous ethanol for about 5.0 minutes, then placed in separate wells of a Polystyrene 6-Well Cell Culture Plate. The foam portions were washed twice in ultrapure water and once in the IMDM medium. Excess medium was removed from the wells before seeding.
  • Vero cells were harvested from the TCT plates using trypsin and re-suspended in IMDM medium. Some foam portions were seeded with 150 pL containing about 25,000 cells and other foam portions were seeded with 150 pL containing about 50,000 cells, wherein the foam portions were positioned in the well of the 6-Well Cell Culture Plate.
  • the 6-Well Cell Culture Plate was placed in a cell culture incubator for about 6 days. After about 6 days, the medium was removed and the foams were dissolved by the addition to each of the wells of about 2.0 mb of a digestion solution containing about 50 U/mL pectinase and about 5.0 mM EDTA. The foam was observed to dissolve in under 5.0 minutes. Following dissolution of the foams, cells were counted using the trypan blue exclusion protocol as detailed in “Protocol for Performing a Trypan Blue Viability Test: Technical Reference Guide.” Lonza Cologne GmbH, September 2012, retrieved from:
  • hMSC Passage 2 as used in Example 17 were cultured on cell culture plates in MesencultTM-XF medium.
  • the foam was cut into portions that were about 2- 3 mm thick and had diameters of about 22 mm.
  • the foam portions were sanitized in 70% aqueous ethanol for about 5.0 minutes, then placed in separate wells of a Polystyrene 6-Well Cell Culture Plate.
  • the foam portions were washed twice in ultrapure water and once in the MesencultTM-XF medium. Excess medium was removed from the wells before seeding.
  • hMSC were harvested from the cell culture plates using trypsin, re-suspended in MesencultTM-XF medium and 150 pL containing about 100,000 cells were seeded in each of the foam portions which were positioned in the wells of the 6-Well Cell Culture Plate.
  • the 6-Well Cell Culture Plate was placed in a cell culture incubator for about 3 days.
  • osteocyte differentiation medium osteocyte differentiation medium, chondrocyte differentiation medium, and adipocyte differentiation medium (commercially available under the trade name StemProTM Osteogenesis Differentiation Kit, StemProTM Chondrogenesis Differentiation Kit and StemProTM Adipogenesis Differentiation Kit respectfully from Thermo Fisher Scientific, Waltham, MA) was added to the wells, wherein each type of differentiation medium was added to foam portions in different wells than the other types of differentiation medium.
  • foam portions exposed to osteocyte differentiation medium were stained with Alizarin Red
  • foam portions exposed to chondrocyte differentiation medium were stained with Alcian Blue
  • foam portions exposed to adipocyte differentiation medium were stained with Oil Red O.
  • hSMC cultured on dissolvable foam scaffolds coated with gelatin of the present disclosure maintain chondrogenic, osteogenic and adipogenic differentiation.
  • cells cultured on dissolvable foam scaffolds of the present disclosure exhibit biological responses similar to biological responses of cells in-vivo.
  • hMSC Passage 2 as used in Example 17 were cultured on cell culture plates in MesencultTM-XF medium.
  • the foam was cut into portions that were about 2-3 mm thick and had diameters of about 22 mm.
  • the foam portions were sanitized in 70% aqueous ethanol for about 5.0 minutes, then placed in separate wells of a 6- Well Ultra-Low Attachment Cell Culture Plate.
  • the foam portions were washed twice in ultrapure water and once in the MesencultTM-XF medium. Excess medium was removed from the wells before seeding.
  • hMSC were harvested from the cell culture plates using trypsin, re-suspended in MesencultTM-XF medium and 150 pL containing about 100,000 cells were seeded in each of the foam portions which were positioned in the wells of the 6-Well Cell Culture Plate.
  • the 6-Well Cell Culture Plate was placed in a cell culture incubator for about 7 days and medium was removed and replaced with fresh medium on day 4. After about 7 days, the medium was removed and the foams were dissolved by the addition to each of the wells of about 2.0 mb of a digestion solution containing about 50 U/mL pectinase and about 5.0 mM EDTA. The foam was observed to dissolve in under 5.0 minutes.
  • hMSC Human Mesenchymal Stem Cells
  • a 6-Well Cell Culture Plate containing scaffolds as formed in accordance with Example 2 was removed from the incubator and the scaffolds were transferred to separate bioreactors.
  • the other 6-Well Cell Culture Plates remained in the incubator. All of the scaffolds were kept under their respective conditions for about 6 days and were then dissolved by the addition of a digestion solution containing about 50 U/mL pectinase and about 5.0 mM EDTA. Following dissolution of the foams, cells were counted using the trypan blue exclusion protocol previously described. It was observed that the expansion of cells on the dissolvable foam scaffolds coated with Coming® Synthemax® II- SC of the present disclosure was similar under static conditions as under dynamic conditions.
  • Multi-passage culturing of Human Mesenchymal Stem Cells was investigated. As described herein, the passage number of a cell culture is a record of the number of times the culture has been subcultured, i.e. harvested and reseeded. Thus, multipassage culturing in relation to the present disclosure describes a process wherein cells are harvested from one dissolvable foam scaffold and reseeded on a different dissolvable foam scaffold. hMSC were harvested from the cell culture plates using trypsin, re-suspended in MesencultTM-XF medium and 150 pL containing about 100,000 cells were seeded in a first set of foam scaffolds as formed in Example 2 and as coated in Example 14.
  • the scaffolds were placed in MesencultTM-XF medium and placed in a cell culture incubator for about 6 days. After about 6 days, the scaffolds were dissolved by the addition of a digestion solution containing about 50 U/mL pectinase and about 5.0 mM EDTA. Following dissolution of the foams, cells were counted using the trypan blue exclusion protocol previously described. A solution containing about 100,000 cells that were harvested from the first set of scaffolds was then used to seed a second set of foam scaffolds as formed in Example 2 and as coated in Example 14. The scaffolds were placed in MesencultTM-XF medium and placed in a cell culture incubator for about 7 days.
  • the scaffolds were dissolved by the addition of a digestion solution containing about 50 U/mL pectinase and about 5.0 mM EDTA. Following dissolution of the foams, cells were counted using the trypan blue exclusion protocol. A solution containing about 100,000 cells harvested from the second set of scaffolds was then used to seed a third set of foam scaffolds as formed in Example 2 and as coated in Example 14. The scaffolds were placed in MesencultTM-XF medium and placed in a cell culture incubator for about 7 days. After about 7 days, the scaffolds were dissolved by the addition of a digestion solution containing about 50 U/mL pectinase and about 5.0 mM EDTA.
  • HEK Human embryonic kidney
  • Example 11 Human embryonic kidney
  • Example 14 Transfection of Human embryonic kidney (HEK) cells on a foam scaffold formed in accordance with the process of Example 11 and coated in accordance with the process of Example 14 was compared to the transfection of HEK cells on Coming® Synthemax® II-SC Dissolvable Microcarriers (commercially available from Coming Incorporated, Coming, NY). HEK cells were seeded on the Dissolvable Microcarriers. A first set of Dissolvable Microcarriers were placed in 6-Well Cell Culture Plates and exposed to IMDM medium supplemented with 10% fetal bovine serum (FBS). The 6-Well Cell Culture Plates were placed in a cell incubator and expanded in static conditions for about 3 days.
  • FBS fetal bovine serum
  • a second set of Dissolvable Microcarriers were placed in a disposable spinner flask and suspended in IMDM medium supplemented with 10% fetal bovine semm (FBS) for about 3 days with intermittent stirring (stir for 15 minutes every 2.0 hours). After about 3 days, about 150 pL of a transfection reagent was added to the Dissolvable Microcarriers in the wells of the 6-Well Cell Culture Plates and about 1.0 mb of a transfection reagent was added to the spinner flask with continued intermittent stirring.
  • FBS fetal bovine semm
  • FIG. 1 Portions of foam were seeded with HEK cells. Some foam portions were placed in 6-Well Cell Culture Plates, exposed to IMDM medium supplemented with 10% fetal bovine serum (FBS) and then the 6-Well Culture Plates were placed in a cell incubator for about 3 days. Other foam portions were placed in a chamber of a radial flow perfusion cartridge device for about 3 days. The perfusion cartridge device allowed for the continuous removal of spent IMDM medium supplemented with 10% fetal bovine serum (FBS) and addition of fresh IMDM medium supplemented with 10% fetal bovine serum (FBS) from the chamber. After about 3 days, about 150 pL of a transfection reagent was added to the foam portions in the wells of the 6-Well Cell Culture Plates and about 1.0 m of a transfection reagent was added to the foam portions in the perfusion cartridge device.
  • FBS fetal bovine serum
  • transfection efficiency refers to the percentage of cells that have a given nucleic acid or biologically active molecule present within the cell after exposure to a transfection reagent.
  • Figure 19 is a bar graph showing the GFP-positive percentage of the transfected HEK cells for each of the culture conditions.
  • bar 1610 represents the GFP-positive percentage of the transfected HEK cells cultured on the dissolvable foams portions in the 6- Well Cell Culture Plates
  • bar 1620 represents the GFP -positive percentage of the transfected HEK cells cultured on the dissolvable foams portions in the perfusion cartridge device
  • bar 1630 represents the GFP-positive percentage of the transfected HEK cells cultured on Dissolvable Microcarriers in 6-Well Cell Culture Plates
  • bar 1640 represents the GFP- positive percentage of the transfected HEK cells cultured on Dissolvable Microcarriers in the spinner flask.
  • AAV Adeno-Associated Virus
  • cells from the first set of foam scaffolds were transfected with an AAV-2 Helper Free Packaging System (commercially available from Cell Biolabs, Inc., San Diego, CA) using a Calcium Phosphate transfection method.
  • cells from the second set of foam scaffolds were transfected with an AAV-2 Helper Free Packaging System using a PEI method.
  • the Calcium Phosphate method cells were incubated for about 18 hours with calcium phosphate/ DNA complexes prepared with a plasmid concentration of 6.4 pg/mL and a 1: 1: 1 molar ratio of plasmids.
  • the radial flow perfusion cartridge devices were operated at a perfusion rate of 20 mL/min for about 2 hours and then at 5 mL/min for about 16 hours. For all sets of cells, medium was removed and replaced with fresh medium after about 18 hours. Cells were then incubated further and harvested 72 hours after transfection.
  • the foam scaffolds were collected and dissolved by the addition of a digestion solution containing about 50 U/mL pectinase and about 5.0 mM EDTA. Following dissolution of the foam scaffolds, cells were collected using centrifugation and washed in dPBS.
  • a portion of the cells were kept for flow cytometry analysis, and the remaining cells were lyzed using a 20mM tris pH8, 150mM NaCl, 0.1% sodium deoxycholate buffer supplemented with 50U/mL benzonase.
  • viral extracts were clarified by centrifugation for about 5 min at 14,000 rpm.
  • Viral titers were measured using the AAV-2 ELISA assay (commercially available from PROGEN Biotechnik GmbH, Heidelberg, Germany).
  • FIG. 20 is a bar graph showing the GFP-positive percentage of the transfected cells for each of the sets of foam scaffolds.
  • bar 1710 represents the GFP-positive percentage of the cells transfected using the Calcium Phosphate transfection method in which transfection method in which transfection efficiency was measured to be about 86.4%
  • bar 1720 represents the GFP-positive percentage of the cells transfected using the PEI transfection method in which transfection efficiency was measured to be about 73.8%.
  • Figure 21 is a bar graph showing the number of viral particles (vp) per set of foam scaffold.
  • bar 1810 represents the viral particles of the cells transfected using the Calcium Phosphate transfection method in which 8.7 x 10 11 viral particles were measured; and bar 1820 represents viral particles of the cells transfected using the PEI transfection method in which 5.7 x 10 11 viral particles were measured.
  • Figure 22 is a bar graph showing the number of viral particles per cell per set of foam scaffold.
  • bar 1910 represents the viral particles/cell of the cells transfected using the Calcium Phosphate transfection method in which 1.17 x 10 5 viral particles/cell were measured; and bar 1920 represents viral particles/cell of the cells transfected using the PEI transfection method in which 6.8 x 10 4 viral particles/cell were measured.
  • the functionality of the viral vectors was assessed by testing the extracts capacity to induce GFP expression in the infected HEK 293 cells.
  • Viral vector infectivity was assessed by infecting HEK 293 cells with extracts prepared using 10 5 viral particles/cell to infect. About 72 hours after infection, GFP-positive percentage of the infected HEK cells was measured by flow cytometry. Infection was observed for cells from each of the sets of foam scaffolds. GFP expression was used to identify the efficiency of gene transfer, which for the present example ranged from about 10% to about 26%. As such, it was concluded that functional AAV vectors can be produced on foam scaffolds as disclosed herein.
  • Figure 23 is a bar graph showing the fraction of cells exhibiting GFP expression for each of the sets of foam scaffolds after infection.
  • bar 2010 represents the fraction of cells exhibiting GFP expression for the cells transfected using the Calcium Phosphate transfection method in which about 26% of the cells exhibited GFP expression
  • bar 2020 represents the fraction of cells exhibiting GFP expression for the cells transfected using the PEI transfection method in which about 10% of the cells showed GFP expression.
  • Some of the dissolvable foams disclosed herein use various components (e.g., sucrose, glycerol, dextran, and others) that are added to facilitate the foaming process and account for a significant amount (e.g., by weight) of the solution used in the foaming process.
  • those components can take up to 90% of the final weight of the foam.
  • Most of those components is not covalently associated with the foam structure and will dissociate from the foam during coating or may need to be removed before cell culture. In cell culture, if not removed, those materials can potentially change the cell media composition, block cell binding epitopes, and reduce cell attachment. After released into cell culture media, those materials can significantly change the media osmotic pressure.
  • surface active molecules or foaming agents such as surfactants
  • Pluronic® P123 may be used in the foaming process, as disclosed in some of the above examples.
  • the above examples also still use other components such as sucrose, dextran, glycerol, and Tween® 20 for foaming.
  • sucrose, dextran, glycerol, and Tween® 20 for foaming.
  • pore size is important for the flow dynamics and total surface area. It has been observed that by using smaller size calcium carbonate particles as the source for gelation, much smaller size of pores was observed. It is believed that the smaller size calcium carbonate particles can lead to better distribution and easier release of calcium during mixing before acid being added, which increases the viscosity of the PGA solution. This leads to the foam cells being difficult to merge together to form large pores. However, faster release of calcium during foaming can also cause the PGA to become over-crosslinked before mixing completely and can damage the foam structure. It is desirable to be able to control pore size by control the viscosity of PGA solution during foaming but to also prevent uncontrolled over-crosslinking.
  • Example 26 the use of a plasticizer (e.g., glycerol), a foaming enhancement agent or sugar (e.g., sucrose), and a foaming enhancement agent in the form of a non-surface active polymer (e.g., dextran) are eliminated by using a high-HLB surface active polymer (such as, e.g., Poloxamer 407 or Pluronic® Fl 27) in place of two surfactants used in previous examples (e.g., a surface active polymer such as P123 and an emulsifying agent such as Tween® 20).
  • a high-HLB surface active polymer such as, e.g., Poloxamer 407 or Pluronic® Fl 27
  • a surface active polymer such as P123 and an emulsifying agent such as Tween® 20.
  • the new formulation removes non-critical materials, which may in some cases be undesirable in the finished dissolvable foam product. This elimination of materials also greatly simplifies the process and reduces the cost.
  • a large amount of leachable materials are eliminated from foam structure — materials which could otherwise potentially change cell media osmotic pressure and block cell binding epitopes during application. This will enable the elimination of a separate coating step in the future and enable direct usage of the foams in cell culture.
  • the simplified formulation provides tunable viscosity without adding additional components. This makes future optimization of the foaming process and control of foam structure easier and cheaper.
  • the simplified formulation can benefit a continuous foaming process by using a shear thinning property of the material.
  • the foaming formulation according to an example embodiment from some the above examples included a relatively long list of materials, as shown below in Table 1.
  • the simplified formulation of this Example 26 eliminates the plasticizer (e.g., glycerol), the emulsifying agent or second polymer having surface activity (e.g., Tween® 20), and other foaming enhancers or polymers having no surface activity (e.g., sucrose and dextran).
  • the simplified formulation in Table 1 also uses a poloxamer (e.g., Fl 27) of higher HLB than that of the P 123 used in the comparative example (Pluronic® Pl 23 has a HLB of 8 compared to the HLB of 22 for Pluronic® F127).
  • Most of the additional ingredients in the Comparative Example were used to provide physical properties to enable the foaming process, as highlighted in the Table 1.
  • These additional ingredients (Tween® 20, glycerol, sucrose, and dextran) account for about 80% of the total weight (excluding water) of the formulation in the Comparative Example.
  • Table 2 below compares the weight percentages of the components in the Comparative Example and Simplified Formulation of Table 1. Those materials may no value for final product and may need to be removed during a subsequent coating process. If the foam article is used in cell culture, those materials need be removed to prevent significant change of osmotic pressure or obstruction of cell binding epitopes.
  • Pluronic® F127 can replace a mixed surfactant of Pluronic® P123 and Tween® 20. It also enables good foaming without adding any sucrose, glycerol, or dextran. Measurements showed that the desired amount of porosity in the foam can be comparable to that of the earlier examples disclosed herein (including the Comparative Example).
  • Figure 24 shows an SEM image of a foam formed from the simplified formulation of Example 26, which has comparable pore structure as the foam made from the other Examples herein with more complex formulations.
  • Pl 23 is a paste material which is difficult to aliquot. It is also difficult to be dissolved in water and the dissolving process can take overnight.
  • F127 is powder which can be easily weighed out during formulation and the dissolution can be completed in one hour. Those properties can benefit manufacturing process.
  • the PGA molecules can be partially crosslinked and the viscosity of the solution increased. This enables creation of a shear-thinning and dissolvable material for 3D bioprinting.
  • amount of calcium ion was added to bind to about 10% of carboxyl groups in the PGA molecules, the solution increased viscosity by lOOOx with shear rate of 1/s, which was about 10 times of the glycerol (1.4 x 10 3 mPa*s).
  • the viscosity drops to 1/100 and became 1/10 of the glycerol.
  • the reduction of viscosity can be recovered quickly after the shear force removed.
  • By adjusting the amount of calcium it is possible to further tune the range of viscosity and shear-thinning response.
  • a small amount of calcium can be added to increase the viscosity of the PGA solution before the start foaming. This can help increase the viscosity and provide shear thinning attributes. Under shear of mixing blades, the solution reduces viscosity which helps incorporate air bubble into the form. When mixing is stopped, the solution viscosity quickly increases which helps stabilize the foam and prevent liquid drainage. This can further eliminate the need of foaming stabilizer materials such as glycerol, sucrose, and Dextran. By controlling the solution viscosity, the speed of bubble merging can be increased or decreased, and it is possible to change the pore size in the foam. The shear thinning property can also benefit a continuous foaming process.
  • part of the GDL can be introduced before foaming (e.g., the 0.5 g in Table 1), which lead to partially release of calcium from calcium carbonate and partially crosslink of PGA.
  • rest GDL can be added (e.g., the 3.5 g in Table 1) or an acid molecule such as acetic acid can be introduced in the form of a vapor to complete the gelation process.
  • Aspect 1 pertains to a dissolvable foam scaffold for cell culture comprising: an ionotropically crosslinked polygalacturonic acid compound selected from at least one of: pectic acid; partially esterified pectic acid, partially amidated pectic acid and salts thereof; and a water-soluble polymer having surface activity and having a hydrophilic-lipophilic balance (HLB) of greater than about 20.
  • an ionotropically crosslinked polygalacturonic acid compound selected from at least one of: pectic acid; partially esterified pectic acid, partially amidated pectic acid and salts thereof; and a water-soluble polymer having surface activity and having a hydrophilic-lipophilic balance (HLB) of greater than about 20.
  • HLB hydrophilic-lipophilic balance
  • Aspect 2 pertains to the dissolvable foam scaffold of Aspect 1, wherein the HLB of the water-soluble polymer is greater than or equal to about 22.
  • Aspect 3 pertains to the dissolvable foam scaffold of Aspect 1 or 2, wherein the dissolvable foam scaffold comprising 0 wt.% of a water soluble plasticizer.
  • Aspect 4 pertains to the dissolvable foam scaffold of any of Aspects 1-3, wherein the dissolvable foam scaffold does not comprise glycerol, sorbitol, ethylene glycol, propylene glycol, polyethylene glycol.
  • Aspect 5 pertains to the dissolvable foam scaffold of Aspects 1-4, further comprising an adhesion polymer coating.
  • Aspect 6 pertains to the dissolvable foam scaffold of Aspect 5, wherein the adhesion polymer coating comprises peptides.
  • Aspect 7 pertains to the dissolvable foam scaffold of Aspect 5, wherein the adhesion polymer coating comprises peptides selected from the group consisting of BSP, vitronectin, fibronectin, laminin, Type I collagen, Type IV collagen, denatured collagen and mixtures thereof.
  • Aspect 8 pertains to the dissolvable foam scaffold of Aspect 5, wherein the adhesion polymer coating comprises Synthemax® II-SC.
  • Aspect 9 pertains to the dissolvable foam scaffold of any of Aspects 1-8, wherein the at least one first polymer comprises a protein.
  • Aspect 10 pertains to the dissolvable foam scaffold of any of Aspects 1-8, wherein the at least one first polymer comprises a synthetic amphiphilic polymer.
  • Aspect 11 pertains to the dissolvable foam scaffold of any of Aspects 1-10, wherein the dissolvable foam scaffold only comprises a single water-soluble polymer having surface activity.
  • Aspect 12 pertains to the dissolvable foam scaffold of any of Aspects 1-11, wherein the dissolvable foam scaffold does not comprise a polymer having no surface activity.
  • Aspect 13 pertains to the dissolvable foam scaffold of any of Aspects 1-12, wherein the dissolvable foam scaffold does not comprise dextran.
  • Aspect 14 pertains to the dissolvable foam scaffold of any of Aspects 1-13, wherein the dissolvable foam scaffold consists of: the ionotropically crosslinked polygalacturonic acid compound, and the water-soluble polymer having surface activity.
  • Aspect 15 pertains to the dissolvable foam scaffold of any of Aspects 1-13, wherein the dissolvable foam scaffold consists of: the ionotropically crosslinked polygalacturonic acid compound, the water-soluble polymer having surface activity, and the adhesion polymer coating.
  • Aspect 16 pertains to the dissolvable foam scaffold of any of Aspects 1-5, comprising an average pore size diameter of between about 50 pm and about 500 pm.
  • Aspect 17 pertains to the dissolvable foam scaffold of any of Aspects 1-16, comprising an open pore architecture.
  • Aspect 18 pertains to the dissolvable foam scaffold of any of Aspects 1-17, wherein digestion of the dissolvable foam scaffold is complete in less than about 1 hour.
  • Aspect 19 pertains to a method for forming a dissolvable foam 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 a water-soluble polymer having surface activity and a divalent metal salt to an aqueous solution; combining the first aqueous mixture with second first aqueous mixture to form a combined aqueous mixture; adding a gel inducing agent to the combined aqueous mixture to form a foaming solution; and introducing gas bubbles into the foaming solution to form a foam scaffold, wherein the water-soluble polymer has a hydrophilic-lipophilic balance (HLB) of greater than about 20.
  • HLB hydrophilic-lipophilic balance
  • Aspect 21 pertains to the method of Aspect 19 or 20, wherein forming the second aqueous mixture does not comprise adding a water soluble plasticizer to the second aqueous mixture.
  • Aspect 22 pertains to the method of any of Aspects 19-21, wherein forming the second aqueous mixture does not comprise adding a second polymer having no surface activity.
  • Aspect 23 pertains to the method of any of Aspects 19-22, wherein forming the second aqueous mixture comprises only adding a single water-soluble polymer having surface activity.
  • Aspect 24 pertains to the method of any of Aspects 19-23, wherein the divalent metal salt comprises: a cation selected from the group consisting of magnesium, calcium, zinc, strontium, barium, and combinations thereof; and an anion selected from the group consisting of oxalates, tartrates, phosphates, carbonates, citrates, and combinations thereof.
  • 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.
  • Aspect 25 pertains to the method of any of Aspects 19-24, wherein the method does not comprise adding an emulsifying agent to any of the first aqueous mixture, the second aqueous mixture, and the combined aqueous mixture, and the foaming solution.
  • Aspect 26 pertains to the method of any of Aspects 19-25, wherein forming a second aqueous mixture further comprises adding at least one leachable solid to the second aqueous mixture.
  • Aspect 27 pertains to the method of Aspect 26, wherein the at least one leachable solid is selected from the group consisting of salts, and water-soluble proteins.
  • Aspect 28 pertains to the method of any of Aspects 19-27, wherein the gel inducing agent is selected from the group consisting of lactic acid lactone, glycolic acid lactone, glucono delta lactone, and acid anhydrides.
  • Aspect 29 pertains to the method of any of Aspects 19-28, wherein adding the gel inducing agent to the combined aqueous mixture comprises adding a first amount of the gel inducing agent to the aqueous mixture before foaming and adding a second amount of the gel inducing agent to the aqueous mixture during or after foaming.
  • Aspect 30 pertains to the method of Aspect 29, wherein a ratio of the second amount of gel inducing agent to the first amount of gel inducing agent is greater than about 2.
  • Aspect 31 pertains to the method of Aspect 30, wherein the ratio is about 7 or greater.
  • Aspect 32 pertains to the method of Aspects 19-31, further comprising coating the dissolvable foam scaffold with an adhesion polymer coating.
  • Aspect 33 pertains to the method of Aspect 32, wherein the adhesion polymer coating comprises peptides.
  • Aspect 34 pertains to the method of Aspect 32, wherein the adhesion polymer coating comprises peptides selected from the group consisting of BSP, vitronectin, fibronectin, laminin, Type I collagen, Type IV collagen, denatured collagen and mixtures thereof.
  • Aspect 35 pertains to the method of Aspect 32, wherein the adhesion polymer coating comprises Synthemax® II-SC.
  • Aspect 36 pertains to the method of any of Aspects 19-35, wherein foaming solution comprises more than 10 wt.% of the polygalacturonic acid compound.
  • Aspect 37 pertains to the method of Aspect 36, wherein the foaming solution comprises more than 30 wt.% of the polygalacturonic acid compound.
  • Aspect 38 pertains to the method of any of Aspects 19-37, wherein the foaming solution comprises more than 3 wt.% of the water-soluble polymer having surface activity.
  • Aspect 39 pertains to the method of Aspect 38, wherein the foaming solution comprises more than about 10 wt.% of the water-soluble polymer having surface activity.
  • Aspect 40 pertains to the method of any of Aspects 19-39, wherein the foaming solution comprises more than 2 wt.% of the divalent metal salt.
  • Aspect 41 pertains to the method of Aspect 40, wherein the foaming solution comprises more than about 5 wt.% of the divalent metal salt.
  • Aspect 42 pertains to the method of any of Aspects 19-41, wherein the foaming solution comprises more than 10 wt.% of the gel inducing agent.
  • Aspect 43 pertains to the method of Aspect 42, wherein the foaming solution comprises more than 40 wt.% of the gel inducing agent.
  • Aspect 44 pertains to a method for culturing cells on a dissolvable foam scaffold, the method comprising: seeding cells on a dissolvable foam scaffold such that cells enter pores of the dissolvable foam scaffold, the dissolvable scaffold comprising: an ionotropically crosslinked polygalacturonic acid compound selected from at least one of: pectic acid; partially esterified pectic acid, partially amidated pectic acid and salts thereof; and a water- soluble polymer having surface activity and having a hydrophilic -lipophilic balance (HLB) of greater than about 20; and contacting the dissolvable foam scaffold with cell culture medium.
  • an ionotropically crosslinked polygalacturonic acid compound selected from at least one of: pectic acid; partially esterified pectic acid, partially amidated pectic acid and salts thereof; and a water- soluble polymer having surface activity and having a hydrophilic -lipophilic balance (HLB) of greater than about 20
  • HLB hydrophilic -lip
  • Aspect 45 pertains to the method of Aspect 44, wherein the HLB of the water-soluble polymer is greater than or equal to about 22.
  • Aspect 46 pertains to the method of Aspect 44 or 45, wherein the dissolvable foam scaffold further comprising 0 wt.% of a water soluble plasticizer.
  • Aspect 47 pertains to the method of any of Aspects 44-46, wherein the dissolvable foam scaffold does not comprise glycerol, sorbitol, ethylene glycol, propylene glycol, or polyethylene glycol.
  • Aspect 48 pertains to the method of any of Aspects 44-47, wherein the dissolvable foam scaffold does not comprise a polymer having no surface activity.
  • Aspect 49 pertains to the method of any of Aspects 44-48, wherein the dissolvable foam scaffold consists of: the ionotropically crosslinked polygalacturonic acid compound, and the water-soluble polymer having surface activity.
  • Aspect 50 pertains to the method of any of Aspects 44-48, wherein cells aggregate in the pores of the dissolvable foam scaffold to form spheroids.
  • Aspect 51 pertains to the method of any of Aspects 44-48 and 50, wherein the dissolvable foam scaffold comprises an adhesion polymer coating and wherein seeding cells on a dissolvable foam scaffold comprises adhering cells to the surface of the dissolvable foam scaffold.
  • Aspect 52 pertains to the method of Aspect 51, wherein the dissolvable foam scaffold consists of: the ionotropically crosslinked polygalacturonic acid compound, the water-soluble polymer having surface activity, and the adhesion polymer coating.
  • Aspect 53 pertains to the method of any of Aspects 44-48 and 50-52, wherein contacting the dissolvable foam scaffold with cell culture medium comprises submerging the dissolvable foam scaffold in cell culture medium.
  • Aspect 54 pertains to the method of any of Aspects 44-53, wherein contacting the dissolvable foam scaffold with cell culture medium comprises continuously passing cell culture medium over the dissolvable foam scaffold.
  • Aspect 55 pertains to the method of Aspect 54, wherein continuously passing cell culture medium over the dissolvable foam scaffold comprises removing at least some of the cell culture medium from contact with the dissolvable foam scaffold and contacting the dissolvable foam scaffold with fresh cell culture medium such that the volume of cell culture medium in contact with the dissolvable foam scaffold remains substantially constant.
  • Aspect 56 pertains to the method of any of Aspects 44-55, further comprising digesting the dissolvable foam scaffold by exposing the dissolvable foam scaffold to an enzyme; and exposing the dissolvable foam scaffold to a chelating agent.
  • Aspect 57 pertains to the method of Aspect 56, wherein the enzyme comprises a non- proteolytic enzyme.
  • Aspect 58 pertains to the method of Aspect 57, wherein the non-proteolytic enzyme is selected from the group consisting of pectinolytic enzymes and pectinases.
  • Aspect 59 pertains to the method of any of Aspects 56-58, wherein digesting the dissolvable foam scaffold comprises exposing the dissolvable foam scaffold to between about 1 U and about 200 U of the enzyme.
  • Aspect 60 pertains to the method of any of Aspects 56-59 comprising exposing the dissolvable foam scaffold to between about 1 mM and about 200 mM of the chelating agent.
  • Aspect 61 pertains to a foamed scaffold product formed from a composition
  • a composition comprising: an ionotropically crosslinked polygalacturonic acid compound selected from at least one of: pectic acid; partially esterified pectic acid, partially amidated pectic acid and salts thereof; at least one first water-soluble polymer having surface activity and having a hydrophilic-lipophilic balance (HLB) of greater than about 20; and 0 wt. % water soluble plasticizer.
  • an ionotropically crosslinked polygalacturonic acid compound selected from at least one of: pectic acid; partially esterified pectic acid, partially amidated pectic acid and salts thereof; at least one first water-soluble polymer having surface activity and having a hydrophilic-lipophilic balance (HLB) of greater than about 20; and 0 wt. % water soluble plasticizer.
  • HLB hydrophilic-lipophilic balance
  • Aspect 62 pertains to the foamed scaffold product of Aspect 61, further comprising an adhesion polymer coating.
  • Aspect 63 pertains to the foamed scaffold product of Aspect 62, wherein the adhesion polymer coating comprises peptides.
  • Aspect 64 pertains to the foamed scaffold product of Aspect 62, wherein the adhesion polymer coating comprises peptides selected from the group consisting of BSP, vitronectin, fibronectin, laminin, Type I collagen, Type IV collagen, denatured collagen and mixtures thereof.
  • Aspect 65 pertains to the foamed scaffold product of Aspect 62, wherein the adhesion polymer coating comprises Synthemax® II-SC.
  • Aspect 66 pertains to the foamed scaffold product of any of Aspects 61-65, wherein the at least one first polymer comprises a cellulose derivative.
  • Aspect 67 pertains to the foamed scaffold product of any of Aspects 61-65, wherein the at least one first polymer comprises a protein.
  • Aspect 68 pertains to the foamed scaffold product of any of Aspects 61-65, wherein the at least one first polymer comprises a synthetic amphiphilic polymer.
  • Aspect 69 pertains to the foamed scaffold product of any of Aspects 61-68, wherein the dissolvable foam scaffold only comprises a single water-soluble polymer having surface activity.
  • Aspect 70 pertains to the foamed scaffold product of any of Aspects 61-69, wherein the dissolvable foam scaffold does not comprise a polymer having no surface activity.
  • Aspect 71 pertains to the foamed scaffold product of any of Aspects 61-70 comprising a porosity of between about 85% and about 96%.
  • Aspect 72 pertains to the foamed scaffold product of any of Aspects 61-71 comprising an average pore size diameter of between about 50 pm and about 500 pm.
  • Aspect 73 pertains to the foamed scaffold product of any of Aspects 61-72 comprising an open pore architecture.
  • Aspect 74 pertains to the foamed scaffold product of any of Aspects 61-73, wherein the composition does not comprise an emulsifying agent.
  • Aspect 75 pertains to the foamed scaffold product of any of Aspects 61-74, wherein the composition does not comprise glycerol, sorbitol, ethylene glycol, propylene glycol, polyethylene glycol, dextran, or sucrose.
  • Aspect 76 pertains to the foamed scaffold product of any of Aspects 61-75, wherein the composition comprises more than 10 wt.% of the polygalacturonic acid compound.
  • Aspect 77 pertains to the foamed scaffold product of Aspect 76, wherein the composition comprises more than 30 wt.% of the polygalacturonic acid compound.
  • Aspect 78 pertains to the foamed scaffold product of any of Aspects 61-77, wherein the composition comprises more than 3 wt.% of the water-soluble polymer having surface activity.
  • Aspect 79 pertains to the foamed scaffold product of Aspect 78, wherein the composition comprises more than about 10 wt.% of the water-soluble polymer having surface activity.
  • Aspect 80 pertains to the foamed scaffold product of any of Aspects 61-79, wherein the composition comprises more than 2 wt.% of the divalent metal salt.
  • Aspect 81 pertains to the foamed scaffold product of Aspect 80, wherein the composition comprises more than about 5 wt.% of the divalent metal salt.
  • Aspect 82 pertains to the foamed scaffold product of any of Aspects 61-81, wherein the composition comprises more than 10 wt.% of the gel inducing agent.
  • Aspect 83 pertains to the foamed scaffold product of Aspect 82, wherein the composition comprises more than 40 wt.% of the gel inducing agent.

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Abstract

La présente invention concerne une structure en mousse soluble pour la culture cellulaire. La structure en mousse soluble comprend un composé d'acide polygalacturonique réticulé de manière ionotropique choisi parmi au moins un des éléments suivants : acide pectique ; acide pectique partiellement estérifié, acide pectique partiellement amidé et leurs sels ; et un polymère hydrosoluble ayant une activité de surface et présentant un équilibre hydrophile-lipophile (HLB) supérieur à environ 20.
PCT/US2023/036731 2022-11-14 2023-11-03 Structures en mousse solubles pour la culture cellulaire et leurs procédés de fabrication WO2024107344A1 (fr)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019104069A1 (fr) * 2017-11-21 2019-05-31 Corning Incorporated Échafaudages solubles de mousse pour la culture cellulaire et procédés pour leur fabrication
WO2022076519A1 (fr) * 2020-10-08 2022-04-14 Corning Incorporated Procédés et systèmes de récolte et de réensemencement de culture cellulaire utilisant des substrats solubles

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019104069A1 (fr) * 2017-11-21 2019-05-31 Corning Incorporated Échafaudages solubles de mousse pour la culture cellulaire et procédés pour leur fabrication
WO2022076519A1 (fr) * 2020-10-08 2022-04-14 Corning Incorporated Procédés et systèmes de récolte et de réensemencement de culture cellulaire utilisant des substrats solubles

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
"Protocol for Performing a Trypan Blue Viability Test: Technical Reference Guide.", September 2012, LONZA COLOGNE GMBH

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