Kits and methods for producing digestible spheroid-stabilized hydrogels

Abstract

A kit for producing a spheroid-stabilized hydrogel in calcium-free or calcium-chelated cell culture medium includes: (a) a gelling agent comprising a polygalacturonic acid (PGA) compound or alginic acid compound containing at least one of (i) pectinic acid or a salt thereof, or (ii) a partially esterified pectinic acid or a salt thereof having an esterification degree of about 1 to about 40 mol %, (b) a crosslinker comprising a salt of a divalent ion, and (c) a proton donor comprising a lactone, ester, or other compound that hydrolyzes in aqueous solution to form an acid over a period of 10 minutes to 1 hour. The resulting spheroid-stabilized hydrogel and a method for producing the same are also provided.

Description

Related Applications

[0001] This application claims the benefit of priority under 35 U.S.C. § 120 of and relies upon U.S. Provisional Patent Application No. 62 / 909,963, filed October 3, 2019, and U.S. Provisional Patent Application No. 63 / 056,898, filed July 27, 2020, the entire contents of which are incorporated herein by reference. [Technical Field]

[0002] The present disclosure relates to digestible spheroid-stabilized hydrogels, methods for producing digestible spheroid-stabilized hydrogels, and kits for producing digestible spheroid-stabilized hydrogels. [Background technology]

[0003] Three-dimensional (hereinafter "3D") cell culture is an artificially created environment that allows cells to grow and / or interact (primarily with each other) in three dimensions. Compared to two-dimensional (hereinafter "2D") cell culture monolayers, 3D cell cultures provide improved cell-cell interactions that more closely mimic the natural microenvironment of tissue. For example, cells grown in 2D cell culture monolayers may adhere to the substrate on which they are cultured, while cells grown in 3D cell cultures may interact with each other rather than adhere to the substrate on which they are cultured.

[0004] Over the past decade, 3D cell culture has been used to grow a wide variety of cancerous and non-cancerous cell lines into spheroids or 3D cell colonies. Spheroids are used for 3D tissue modeling in the fields of drug discovery, toxicology, and regenerative medicine. Although many 3D cell culture substrates with spheroid-attracting geometries have been developed, transporting spheroids generated on such 3D cell culture substrates can be challenging. For example, spheroids tend to fuse together if direct spheroid-spheroid contact is established, even for moderate periods (e.g., approximately 1 hour). Therefore, spheroids transported in 3D cell culture substrates where individual spheroids are not physically separated can result in the formation of spheroid aggregates. Furthermore, spheroids transported in 3D cell culture substrates where individual spheroids are physically separated can also result in the formation of spheroid aggregates due to spheroid displacement during transport. Summary of the Invention [Problem to be solved by the invention]

[0005] Therefore, there is a continuing need to stabilize spheroids during transport. [Means for solving the problem]

[0006] In one embodiment, a digestible spheroid-stabilized hydrogel is disclosed, which comprises: (a) a gelling agent comprising a polygalacturonic acid (PGA) compound containing at least one of (i) pectinic acid or a salt thereof, or (ii) a partially esterified pectinic acid or a salt thereof having an esterification degree of about 1 to about 40 mol %, or an alginic acid compound; (b) a crosslinker comprising a salt of a divalent cation; and (c) a proton donor comprising a lactone, an ester, or another compound that hydrolyzes in aqueous solution to form an acid over a period of 10 minutes to 1 hour.

[0007] In another embodiment, a method for producing a digestible spheroid-stabilized hydrogel is disclosed. The method includes the steps of (a) providing a crosslinker and a proton donor together with an aqueous medium containing a gelling agent comprising a PGA compound or an alginic acid compound, the PGA compound comprising at least one of (i) pectinic acid or a salt thereof, or (ii) a partially esterified pectinic acid or a salt thereof having an esterification degree of about 1 to about 40 mol %, wherein the crosslinker comprises a salt of a divalent ion, and the proton donor comprises a lactone, ester, or other slowly decomposing acid, such as gluconolactone. The crosslinker and proton donor are provided together with the aqueous medium containing the PGA compound under conditions such that the PGA compound is crosslinked via the crosslinker and the proton donor to form a digestible spheroid-stabilized hydrogel.

[0008] In yet another embodiment, a kit for producing spheroid-stabilizing hydrogels in calcium-free or calcium-chelated cell culture medium is provided, comprising: (a) a gelling agent comprising alginic acid or a polygalacturonic acid (PGA) compound containing at least one of (i) pectinic acid or a salt thereof or (ii) a partially esterified pectinic acid or a salt thereof having a degree of esterification of about 1 to about 40 mole %, (b) a crosslinking agent comprising a salt of a divalent ion, and (c) a proton donor comprising a lactone, ester, or other compound that hydrolyzes in aqueous solution to form an acid over a period of 10 minutes to 1 hour.

[0009] It should be understood that both the foregoing general description and the following detailed description, which describe various embodiments, are intended to provide an overview or framework for understanding the nature and character of the claimed subject matter. The accompanying drawings are included to provide a further understanding of the various embodiments, and are incorporated into and constitute a part of this specification. The drawings illustrate various embodiments described herein and, together with the description, serve to explain the basic scheme and operation of the claimed subject matter. [Brief explanation of the drawings]

[0010] [Figure 1A] FIG. 1 is a schematic diagram showing the preparation of digestible spheroid-stabilized hydrogels, in which spheroids are cultured in a 3D cell culture substrate (i.e., a 3D cell culture flask) in cell culture medium. [Figure 1B] 1A is a schematic diagram showing the preparation of digestible spheroid-stabilized hydrogels, in which cell culture medium is removed from spheroids cultured in the 3D cell culture flask in Figure 1A. [Figure 1C] Schematic diagram showing the preparation of digestible spheroid-stabilized hydrogels, where digestible PGA spheroid-stabilized hydrogels are provided in an overlaid arrangement on spheroids cultured in the 3D cell culture flasks of Figure 1B. [Figure 2A] FIG. 1 is a schematic diagram showing the digestion of a digestible PGA spheroid-stabilized hydrogel, where the digestible PGA spheroid-stabilized hydrogel is provided in an overlaid arrangement on spheroids cultured in a 3D cell culture flask. [Figure 2B] FIG. 2B is a schematic diagram showing the digestion of a digestible PGA spheroid-stabilized hydrogel, in which a digestion solution is provided to the digestible PGA spheroid-stabilized hydrogel of FIG. 2A. [Figure 2C] FIG. 2B is a schematic diagram showing the digestion of digestible PGA spheroid-stabilized hydrogels, where the digestion solution in FIG. 2B is replaced with cell culture medium. [Figure 3A] Brightfield images of control HepG2 cells grown on a 2D cell culture monolayer in Leibovitz's L-15 medium without digestible PGA spheroid-stabilizing hydrogel. [Figure 3B] Fluorescence images of live control HepG2 cells grown on a 2D cell culture monolayer in Leibovitz's L-15 medium without digestible PGA spheroid-stabilizing hydrogels and simultaneously stained with red-fluorescent ethidium homodimer and green-fluorescent calcein AM. [Figure 3C]Fluorescence images of dead control HepG2 cells grown on a 2D cell culture monolayer in Leibovitz's L-15 medium without digestible PGA spheroid-stabilizing hydrogel and simultaneously stained with red fluorescent ethidium homodimer and green fluorescent calcein AM. [Figure 4A] Brightfield image of HepG2 cells grown on a 2D cell culture monolayer in Leibovitz's L-15 medium containing an overlaying digestible PGA spheroid-stabilizing hydrogel (1% wt / wt pectic acid). [Figure 4B] Fluorescence images of live HepG2 cells grown on a 2D cell culture monolayer in Leibovitz's L-15 medium containing an overlaid digestible PGA spheroid-stabilizing hydrogel (1% wt / wt pectic acid) and simultaneously stained with red-fluorescent ethidium homodimer and green-fluorescent calcein AM. [Figure 4C] Fluorescence images of dead control HepG2 cells grown on a 2D cell culture monolayer in Leibovitz's L-15 medium containing overlaid digestible PGA spheroid-stabilizing hydrogel (1% wt / wt pectic acid) and simultaneously stained with red fluorescent ethidium homodimer and green fluorescent calcein AM. [Figure 5A] Brightfield images of control HepG2 spheroids grown on a 3D cell culture substrate in Leibovitz's L-15 medium without digestible PGA spheroid-stabilizing hydrogel. [Figure 5B] Fluorescence images of live control HepG2 spheroids grown on a 3D cell culture substrate in Leibovitz's L-15 medium without digestible PGA spheroid-stabilizing hydrogel and simultaneously stained with red-fluorescent ethidium homodimer and green-fluorescent calcein AM. [Figure 5C] Fluorescence images of dead control HepG2 spheroids grown on a 3D cell culture substrate in Leibovitz's L-15 medium without digestible PGA spheroid-stabilizing hydrogel and simultaneously stained with red fluorescent ethidium homodimer and green fluorescent calcein AM. [Figure 6A]Brightfield images of HepG2 spheroids grown on a 3D cell culture substrate in Leibovitz's L-15 medium containing an overlaying digestible PGA spheroid-stabilizing hydrogel (1% wt / wt pectic acid). [Figure 6B] Fluorescence images of live HepG2 spheroids grown on a 3D cell culture substrate in Leibovitz's L-15 medium containing an overlaying digestible PGA spheroid-stabilizing hydrogel (1% wt / wt pectic acid) and simultaneously stained with red-fluorescent ethidium homodimer and green-fluorescent calcein AM. [Figure 6C] Fluorescence image of dead control HepG2 spheroids grown on a 3D cell culture substrate in Leibovitz's L-15 medium containing overlaid digestible PGA spheroid-stabilizing hydrogel (1% wt / wt pectic acid) and simultaneously stained with red fluorescent ethidium homodimer and green fluorescent calcein AM. [Figure 7A] 10 is a brightfield image of control HepG2 spheroids grown on a 3D cell culture substrate in Leibovitz's L-15 medium without digestible PGA spheroid-stabilized hydrogel after performing a drop test. [Figure 7B] Fluorescence images of live control HepG2 spheroids grown on 3D cell culture substrates in Leibovitz's L-15 medium without digestible PGA spheroid-stabilized hydrogels and simultaneously stained with red-fluorescent ethidium homodimer and green-fluorescent calcein AM after performing a drop test. [Figure 7C] Fluorescence images of dead control HepG2 spheroids grown on 3D cell culture substrates in Leibovitz's L-15 medium without digestible PGA spheroid-stabilized hydrogels and simultaneously stained with red fluorescent ethidium homodimer and green fluorescent calcein AM after performing a drop test. [Figure 8A] Brightfield images of HepG2 spheroids grown on a 3D cell culture substrate in Leibovitz's L-15 medium with overlaid digestible PGA spheroid-stabilizing hydrogel (1% wt / wt pectic acid) after performing a drop test. [Figure 8B] Fluorescence images of live HepG2 spheroids grown on a 3D cell culture substrate in Leibovitz's L-15 medium with overlaid digestible PGA spheroid-stabilizing hydrogel (1% wt / wt pectic acid) and simultaneously stained with red-fluorescent ethidium homodimer and green-fluorescent calcein AM after performing a drop test. [Figure 8C] Fluorescence images of dead control HepG2 spheroids grown on 3D cell culture substrates in Leibovitz's L-15 medium with overlaid digestible PGA spheroid-stabilizing hydrogel (1% wt / wt pectic acid) and simultaneously stained with red fluorescent ethidium homodimer and green fluorescent calcein AM after performing a drop test. [Figure 9A] FIG. 1 is a schematic diagram showing a 3D cell culture substrate comprising an array of spheroid-containing cell culture microwells in a corrugated configuration. [Figure 9B] FIG. 9B is a schematic diagram showing a cutaway of the 3D cell culture substrate of FIG. 9A. [Figure 10] FIG. 1 shows a culture flask with a bottom micropatterned with a 3D cell culture substrate containing an array of spheroid-containing cell culture microwells. [Figure 11] Figure 10 shows a close-up view of a 3D cell culture substrate containing an array of micropatterned spheroid-containing cell culture microwells on the bottom surface of a culture flask. DETAILED DESCRIPTION OF THE INVENTION

[0011] As used herein, the following terms shall have the definitions set forth below.

[0012] As used herein, the term "digestibility" refers to the ability of pectin polysaccharides to break down and / or degrade into their component galacturonic acid monomers, dimers, or other oligomers. For example, pectin polysaccharide-containing spheroid-stabilizing hydrogels are digestible via cleavage of their glycosidic linkages, e.g., cleavage of 1,4-α-D-galacturonic acid linkages. In embodiments, by decomposing and / or degrading the pectin polysaccharides of the pectin polysaccharide-containing spheroid-stabilizing hydrogel, spheroids overlaid with the pectin polysaccharide-containing spheroid-stabilizing hydrogel can be recovered with a viability of about 90% to about 100% compared to control spheroids, e.g., compared to the viability of spheroids not containing the overlaid pectin polysaccharide-containing spheroid-stabilizing hydrogel and / or the viability of spheroids containing the overlaid pectin polysaccharide-containing spheroid-stabilizing hydrogel before digestion.

[0013] As used herein, the term "spheroid" refers to an aggregate, cluster, and / or assembly of cells cultured to allow 3D growth. In contrast to cells cultured to allow 2D growth, such as in a monolayer, spheroids can retain 3D structure and / or more closely mimic the natural microenvironment of tissue.

[0014] As used herein, the terms "stabilize" and "stabilization" refer to the ability of a pectin polysaccharide-containing hydrogel to maintain spheroids at target locations upon application of force. For example, in the context of a 3D cell culture substrate, a pectin polysaccharide-containing hydrogel is stabilized to maintain spheroids in target microwells and / or their target capillary structures upon application of force. As another example, in the context of a 3D cell culture substrate, a pectin polysaccharide-containing hydrogel is stabilized to maintain about 90% to about 100% of spheroids in each target microwell and / or its capillary structure upon application of force, compared to control spheroids, e.g., compared to placing spheroids in each target microwell and / or capillary structure of a 3D cell culture substrate without an overlying pectin polysaccharide-containing hydrogel (where only about 60% of spheroids are retained in each microwell).

[0015] As used herein, the term "hydrogel" refers to a semi-solid bonded and / or cross-linked pectin polysaccharide matrix that is fully or partially swollen with water and / or cell culture medium. The bonds and / or cross-links in the pectin polysaccharide matrix can be physical and / or chemical in nature, for example, via ionic interactions, covalent bonds, van der Waals interactions, hydrogen bonding, chain entanglements, and / or self-association. The hydrogel may be capable of absorbing about 30% to 10,000% of its dry weight of water and / or water-miscible alcohol.

[0016] As used herein, the terms "improve" and "improved" refer to the improved viability of spheroids grown in a 3D cell culture substrate and covered with a pectin polysaccharide-containing hydrogel compared to control spheroids. For example, spheroids grown in a 3D cell culture substrate, covered with a pectin polysaccharide-containing hydrogel, and cultured at 4°C for 24 hours contained 84% fewer dead cells compared to control spheroids, such as the viability of spheroids grown in a 3D cell culture substrate without an overlay of a pectin polysaccharide-containing spheroid-stabilizing hydrogel.

[0017] As used herein, the term "cell culture medium" refers to a medium used in the context of culturing cells that supports cell growth, e.g., 3D cell growth, etc. Cell culture medium can include proteins, peptides, amino acids, purines, pyrimidines, nucleotides, phospholipid precursors, vitamins, energy sources (e.g., carbohydrates), inorganic ions (e.g., calcium, magnesium, iron, phosphate and / or sulfate), salts, buffering agents (e.g., phosphate and / or acetate), indicators of pH change (e.g., phenol red and / or bromo-cresol purple), and / or water. Components of the cell culture medium can be provided in a buffer solution that may include a pH and / or salt concentration that mimics the in vivo environment. In embodiments, the cell culture medium includes calcium-free or calcium-chelated cell culture medium. Examples of specific cell culture media include, but are not limited to, Basal Eagle Medium (hereinafter "BME"), Eagle's Minimum Essential Medium (i.e., Modified Eagle Medium, hereinafter "MEM"), Dulbecco's Modified Eagle Medium (hereinafter "DMEM"), Leibovitz's L-15 Medium (hereinafter "L-15"), Roswell Park Memorial Institute Medium (hereinafter "RPMI"), Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12 Medium (hereinafter "DMEM / F12"), Iscove's Modified Dulbecco's Medium (hereinafter "IMDM"), National Collection of Type Cultures Medium (hereinafter "NCTC"), and / or Osteogenic Induction Medium (hereinafter "OIM"). Calcium-free examples include DMEM from Thermo Fisher (catalog number 21068028).

[0018] As used herein, the term "gelation" refers to the formation of a gel through bonding and / or cross-linking of pectin polysaccharides. Gelation can result in the formation of a semi-solid bonded and / or cross-linked pectin polysaccharide matrix from a liquid containing pectin polysaccharides. Gelation can occur through physical bonding and / or cross-linking and / or chemical bonding and / or cross-linking.

[0019] As used herein, the term "superimposed arrangement" refers to a pectin polysaccharide-containing hydrogel that is formed in situ after application to a population of spheroids in a 3D cell culture substrate. For example, the pectin polysaccharide-containing hydrogel is in a superimposed arrangement where the pectin polysaccharide hydrogel is applied to a population of spheroids in the microwells and / or capillary structures of the 3D cell culture substrate under conditions suitable for the pectin polysaccharide matrix to gel and form the pectin polysaccharide-containing hydrogel.

[0020] Reference will now be made in detail to embodiments of digestible spheroid-stabilized hydrogels with reference to Figures 1C, 2A, and 9A-11. Thereafter, embodiments of methods and kits for producing digestible spheroid-stabilized hydrogels will be described in detail.

[0021] I. Digestible spheroid-stabilized hydrogel In one embodiment, a digestible spheroid-stabilized hydrogel is disclosed. The digestible spheroid-stabilized hydrogel may include: (a) a gelling agent including an alginate compound or a PGA compound including at least one of (i) pectinic acid or a salt thereof, or (ii) a partially esterified pectinic acid or a salt thereof having an esterification degree of about 1 to about 40 mol %, (b) a crosslinking agent including a salt of a divalent ion, and (c) a proton donor including a lactone, an ester, or other compound that hydrolyzes in aqueous solution to form an acid over a period of 10 minutes to 1 hour.

[0022] In an embodiment, the digestible spheroid-stabilized hydrogel comprises (a) a gelling agent comprising a PGA compound. The PGA compound can be as described in International Publication No. WO 2016 / 200888, the entire contents of which are incorporated herein by reference. In an embodiment, the PGA compound is a pectin polysaccharide composed of α-1,4-linked D-galacturonic acid monomers. The PGA compound can include (i) pectinic acid, which is a polymer of galacturonic acid; (ii) pectinic acid, which is a polymer of galacturonic acid in which the carboxyl groups are esterified (i.e., partially esterified pectinic acid); and / or (iii) a salt of pectinic acid and / or pectinic acid. In an embodiment, the salt of pectinic acid is pectinate.

[0023] In some embodiments, the gelling agent can include an alginate compound. The alginate compound can include alginic acid, which is a linear copolymer of homopolymer blocks of (1-4)-linked β-D-mannuronate (M) and its C-5 epimer α-L-guluronate (G) residues covalently linked in various sequences or blocks. In some embodiments, the alginate can include a salt of alginic acid (e.g., sodium alginate is the sodium salt of alginic acid, and potassium alginate is a compound that is the potassium salt of alginic acid).

[0024] In an embodiment, the PGA compound comprises pectic acid, hi an embodiment, the pectic acid has the formula:

[0025] [ka]

[0026] In Formula I, the value of n can vary based on the pectic acid used. In some embodiments, n can be from about 1 to about 100, or greater than about 100. In some particular embodiments, n is at least about 1 and can be less than or equal to about 50, less than or equal to about 40, less than or equal to about 30, or less than or equal to about 20. In particular embodiments, n is from about 8 to about 16.

[0027] In embodiments, the digestible spheroid-stabilized hydrogel comprises about 1% wt / wt to about 2% wt / wt, or about 1% wt / wt to about 3% wt / wt, or about 1% wt / wt to about 4% wt / wt, or about 1% wt / wt to about 5% wt / wt, or about 1% wt / wt to about 10% wt / wt, or about 1% wt / wt pectic acid. The digestible spheroid-stabilized hydrogel can contain higher % wt / wt pectic acid depending on the application, although it is contemplated that this increase in pectic acid may affect the digestibility of the hydrogel. Pectin can be formed by the hydrolysis of certain pectin esters. Pectin is a cell wall polysaccharide, and sources of pectin include, but are not limited to, citrus peel and apple peel. In embodiments, pectin is a predominantly linear polymer with a 1,4-linked α-D-galacturonic acid backbone randomly interrupted by 1,2-linked L-rhamnose. In embodiments, the average molecular weight of the pectin can range from about 50,000 daltons to about 200,000 daltons.

[0028] In embodiments, the PGA compound comprises pectinic acid (i.e., partially esterified pectinic acid). In embodiments, the pectinic acid has a degree of esterification of about 1 to about 40 mol %, or about 5 to about 30 mol %, or about 10 to about 20 mol %. In embodiments, the PGA compound comprises pectinic acid having a degree of esterification of about 1 to about 40 mol %. The pectinic acid or partially esterified pectinic acid can be esterified with methyl groups. Furthermore, the free carboxyl groups of the pectinic acid can be partially and / or completely neutralized with monovalent ions, such as sodium, potassium, and / or ammonium ions.

[0029] In embodiments, the digestible spheroid-stabilized hydrogel comprises about 0.5% wt / wt to about 2% wt / wt, or about 0.75% wt / wt to about 1.75% wt / wt, or about 1% wt / wt of pectinic acid. In embodiments, the digestible spheroid-stabilized hydrogel comprises about 0.5% wt / wt to about 2% wt / wt, or about 0.75% wt / wt to about 1.75% wt / wt, or about 1% wt / wt of pectinic acid esterified with a methyl group.

[0030] In embodiments, the PGA compound comprises a salt of pectinic acid and / or pectinic acid. Specific examples of salts of pectinic acid and / or pectinic acid include salts of monovalent cations, such as Na + and K. + The salts of the compounds of formula (I) can include, but should not be limited to, the salts of the compounds of formula (I).

[0031] In embodiments, the digestible spheroid-stabilizing hydrogel comprises about 0.5% wt / wt to about 2% wt / wt, or about 0.75% wt / wt to about 1.75% wt / wt, or about 1% wt / wt of pectinic acid and / or a salt of pectinic acid.

[0032] In some embodiments, the PGA compound comprises a mixture of pectinic acid, pectinic acid, and / or a salt of pectinic acid and / or pectinic acid. In alternative embodiments, the PGA compound comprises pure pectinic acid, pectinic acid, or a salt thereof. In some embodiments, the digestible spheroid-stabilized hydrogel comprises a blend of a PGA compound and a compatible polymer. Examples of specific compatible polymers that can be blended with the PGA compound include, but are not limited to, polysaccharides selected from dextran, substituted cellulose derivatives, alginic acid, starch, glycogen, arabinoxylan, and / or agarose; glycosaminoglycans selected from hyaluronic acid and / or chondroitin sulfate; proteins selected from elastin, fibroin, collagen, and / or derivatives thereof; and / or water-soluble synthetic polymers selected from polyalkylene glycols, poly(hydroxyalkyl(meth)acrylates), poly(meth)acrylamides, poly(N-vinyl-2-pyrrolidone), polyvinyl alcohol, and / or derivatives thereof. Polymers can be considered compatible in the digestible spheroid-stabilized hydrogel if their inclusion in a blend with a PGA compound does not impair the digestibility of the digestible spheroid-stabilized hydrogel.

[0033] In embodiments, the digestible spheroid-stabilized hydrogel includes (b) a crosslinker comprising a salt of a divalent ion. In embodiments, the crosslinker comprises a calcium salt. In embodiments, the calcium salt is selected from calcium carbonate (i.e., CaCO), calcium oxalate (i.e., CaCO), and / or calcium phosphate (i.e., Ca(PO)), although other calcium salts that are non-toxic to cells are also contemplated. In embodiments, the calcium salt is calcium carbonate. Other divalent salts that can dissociate under acidic conditions and have low solubility are also contemplated. In embodiments, the calcium salt has a water solubility of greater than about 0 mg / L to about 20 mg / L, or about 3 mg / L to about 20 mg / L, or about 6 mg / L to about 20 mg / L at 25°C. In embodiments, the crosslinker is an ionic crosslinker. Without being bound by theory, it is believed that the inclusion of an ionic crosslinker in the digestible spheroid-stabilized hydrogel crosslinks the PGA polymer chains to form a gel in solution. Additionally, the low solubility of the salt is believed to reduce or prevent premature or instantaneous gelation upon mixing.

[0034] In embodiments, the digestible spheroid-stabilized hydrogel includes (c) a proton donor, including a lactone, ester, or other compound that hydrolyzes in aqueous solution to form an acid over a period of 10 minutes to 1 hour. In embodiments, the proton donor acts as a dissociating agent to dissolve and / or slowly release a salt of a divalent ion in solution. For example, in embodiments where the crosslinker is a calcium salt, the proton donor dissolves calcium carbonate in solution and / or releases Ca. 2+ It can act as a dissociating agent, slowly releasing ions. Examples of suitable proton donors include lactones, esters, or other slowly dissolving acids, such as gluconolactone. In an embodiment, the proton donor includes gluconolactone. Gluconolactone has the formula:

[0035] [ka]

[0036] In embodiments, the PGA compound is crosslinked via a crosslinker and a proton donor to form a digestible spheroid-stabilizing hydrogel. In embodiments, the crosslinker, upon exposure to the proton donor, converts inorganic calcium ions, i.e., Ca 2+ More specifically, in embodiments, the crosslinking agent provides a source of inorganic calcium ions upon dissociation of a calcium salt, e.g., CaCO3, when exposed to gluconolactone. In embodiments, the digestible spheroid-stabilized hydrogel comprises a molar ratio of crosslinking agent to proton donor of about 0.7:2 to about 1:2, or about 1:2 to about 1:2.3. In embodiments, the digestible spheroid-stabilized hydrogel comprises a molar ratio of crosslinking agent to proton donor of about 1:2.

[0037] In embodiments, the digestible spheroid-stabilizing hydrogel further comprises components of a cell culture medium that support cell growth. In embodiments, the components of the cell culture medium support cell growth, such as 3D cell growth, and include a pH and / or salt concentration that mimics an in vivo environment. For example, the digestible spheroid-stabilizing hydrogel can include proteins, peptides, amino acids, purines, pyrimidines, nucleotides, phospholipid precursors, vitamins, energy sources (e.g., carbohydrates), inorganic ions (e.g., calcium, magnesium, iron, phosphate, and / or sulfate), salts, buffers (e.g., phosphate and / or acetate), indicators for pH changes (e.g., phenol red and / or bromo-cresol purple), and / or water.

[0038] In embodiments, the digestible spheroid-stabilized hydrogel comprises, by volume, about 50% water and 50% cell culture medium supporting cell growth to about 0% water and 100% cell culture medium, or about 50% DPBS and 50% cell culture medium to about 100% cell culture medium. In embodiments, the digestible spheroid-stabilized hydrogel comprises calcium-free cell culture medium or calcium-chelated cell culture medium.

[0039] 1C, 2A, 9A, and 9B, in embodiments, the digestible spheroid-stabilized hydrogel 300 is provided with a 3D cell culture substrate 100 containing spheroids 500, as described above. 3D cell culture substrates 100 suitable for forming spheroids are known to those skilled in the art. For example, the 3D cell culture substrate 100 can be as described in WO 2016 / 069892, the entire contents of which are incorporated herein by reference. Further 3D cell culture substrates 100 can be as described in U.S. Patent Application Publication Nos. 2004 / 0125266, 2012 / 0064627, 2014 / 0227784, 2017 / 0226455, WO 2008 / 106771, and WO 2014 / 165273. Each of these documents is incorporated herein by reference in its entirety.

[0040] In an embodiment, the 3D cell culture substrate 100 includes a frame 110 that defines at least one microwell 130. In an embodiment, the 3D cell culture substrate 100 includes a frame 110 that defines an array 150 of microwells. Each microwell 130 of the 3D cell culture substrate 100 can include at least one sidewall 132 (e.g., a sidewall), a well bottom 134 (e.g., a lowest point), and an open top 136 (e.g., an open top). In an embodiment, each microwell 130 is configured to contain an aqueous solution, such as cell culture medium. In an embodiment, the 3D cell culture substrate 100 includes a spheroid container. An example of a spheroid container is shown in WO 2019 / 014610, which is incorporated herein by reference.

[0041] 9A-9B, in embodiments, each microwell 130 has a spheroid-attracting geometry 170 and / or a microcapillary structure 170. In embodiments, each microwell 130 has a spheroid-attracting geometry 170, such as a rounded well bottom 134 (e.g., a hemisphere or concave surface found on the rounded well bottom 134). In embodiments, the spheroid-attracting geometry 170 is selected from pillars, discontinuous walls, a rounded well bottom, a concave well bottom, a pit, and / or a nib region. In embodiments, each microwell 130 is structured and / or arranged to include a spheroid-attracting geometry 170 that provides an environment conducive to the formation of spheroids 500 during culture.

[0042] 9A-9B , in embodiments, each microwell 130 has a microcapillary structure 170. Examples of suitable microcapillary structures 170 include, but are not limited to, mouth areas, ridges, crevices, pillars, discontinuous walls, corrugated walls, parabolic well shapes, and / or sinusoidal well shapes. In embodiments, the microcapillary structures 170 facilitate the evacuation of air when introducing a solution, such as a liquid solution, into the microwell 130. As shown in FIGS. 9A-9B , in embodiments, each microwell 130 includes a corrugated sidewall 132. Thus, in embodiments in which each microwell 130 includes a corrugated sidewall 132, the array 150 of microwells in the 3D cell culture substrate includes a row of individual microwells 130. In an embodiment, the corrugated sidewall 132 is a micro-capillary structure 170 that allows air to move from a first (narrower) area to a second, wider area upon introduction of a solution, e.g., a liquid solution, avoiding air pockets.

[0043] In embodiments, each microwell 130 comprises a surface, such as the frame 110, sidewalls 132 and / or well bottom 134, that is non-adhesive to cells so that the cells can interact with, and e.g., form spheroids on, the 3D cell culture substrate 100 rather than with the 3D cell culture substrate 100. Examples of non-adhesive materials that can form and / or apply such surfaces of the 3D cell culture substrate 100 include, but are not limited to, perfluorinated polymers, olefins, agarose and / or non-ionic hydrogels, e.g., polyacrylamide, polyethers, e.g., polyethylene oxide, and polyols, e.g., polyvinyl alcohol, etc.

[0044] In embodiments, each microwell 130 has a mouth region adjacent to an open top 136 that provides a more open area where the microwell 130 constricts and / or narrows to the well bottom 134. In embodiments, the mouth region of the open top 136 allows solutions introduced therein to flow to the well bottom 134, promoting cell aggregation and spheroid formation at the well bottom 134. Additionally, the mouth region of the open top 136 forms a transition between the annular interior surface between the mouth regions and the sidewall 132, which can provide a geometry that can prevent air pockets within the microwell 130. In embodiments, the mouth region provides a transition between the open top 136 and the sidewall 132 that is not at a 90° angle, which can reduce the formation of air bubbles within the microwell 130. In some embodiments, the height of the sidewall 132 (e.g., from the open top 136 to the well bottom 134) is 100% or more of the diameter of the microwell 130 at the mouth region of the open top 136. In some embodiments, the height of the sidewall 132 (e.g., from the open top 136 to the well bottom 134) is about 100% to about 400%, or about 110% to about 375%, or about 120% to about 350%, or about 130% to about 325%, or about 140% to about 300%, or about 150% to about 275%, or about 160% to about 250%, or about 170% to about 225%, or about 180% to about 200%, or about 190% of the diameter of the microwell 130 at the mouth region of the open top 136.

[0045] 10-11, in an embodiment, the 3D cell culture substrate 100 comprises and / or is included as part of a cell culture vessel 700. For example, the cell culture vessel 700 can comprise the 3D cell culture substrate 100 as a surface thereof. The cell culture vessel 700 can be selected from a multi-layer plate, a dish, a flask, a tube, a multi-layer flask, a soft-sided flask, and / or a bag.

[0046] 10 , in an embodiment, the cell culture vessel 700 is a flask. The flask can include a top surface 710, at least one sidewall 730 extending from the 3D cell culture substrate 100 to the top surface 710, and a cell culture chamber 750 defined by the top surface 710, the at least one sidewall 730, and / or the 3D cell culture substrate 100. In an embodiment, the flask includes a port 770. In a further embodiment, the flask includes a port 770 having a screw top 790. The port 770 can be defined by the top surface 710, the at least one sidewall 730, and / or the 3D cell culture substrate 100. In an embodiment, the port 770 functions to remove cells and / or cell culture medium from the cell culture chamber 750 through the use of appropriate tubing and / or connections.

[0047] In embodiments, the 3D cell culture substrate 100 can be formed from materials including, but not limited to, glass, polycarbonate, polymethylpentene, K-resin, polymethylmethacrylate, polystyrene, polystyrene butadiene copolymer, polyester, polyethylene, polypropylene, polycyclic olefin, polyvinyl chloride, polysulfone, fluoropolymer, polyamide, fully hydrogenated styrene polymer, polycarbonate polydimethylsiloxane (i.e., PDMS) copolymer, and / or cyclic olefin copolymer.

[0048] In embodiments, the digestible spheroid-stabilized hydrogel 300 comprises a 3D cell culture substrate 100 containing spheroids 500. In embodiments, the spheroids 500 are disposed within a microwell array 150 of the 3D cell culture substrate 100. In embodiments, the digestible spheroid-stabilized hydrogel 300 comprises a 3D cell culture substrate 100 containing spheroids 500 such that the spheroids 500 are covered by the digestible spheroid-stabilized hydrogel 300. In embodiments, the digestible spheroid-stabilized hydrogel 300 improves the survival rate of the spheroids 500 in the 3D cell culture substrate 100 and / or stabilizes the spheroids 500 at a targeted location in the 3D cell culture substrate 100. For example, when the digestible spheroid-stabilized hydrogel 300 comprises a 3D cell culture substrate 100 containing spheroids 500, the digestible spheroid-stabilized hydrogel 300 can stabilize individual spheroids 500 in target microwells 130 within the 3D cell culture substrate 100 such that the spheroids 500 remain in the same microwells 130 in which they were formed and / or placed prior to application of the digestible spheroid-stabilized hydrogel 300.

[0049] In embodiments, spheroids 500 can be formed from natural cell types and / or genetically modified cell types. In embodiments, spheroids 500 are formed from somatic cells, stem cells, progenitor cells, such as embryonic stem cells, and / or induced pluripotent stem cells, in any desired differentiation state, such as pluripotency, multipotency, committed fate, and / or immortalization. In embodiments, spheroids 500 are formed from disease cells and / or disease model cells, such as cancerous cells and / or transformed cells induced to a hyperproliferative state.

[0050] In embodiments, spheroids 500 are formed from cells derived from any desired tissue and / or organ type, including, but not limited to, cells from the adrenal gland, bladder, blood vessels, bone, bone marrow, brain, cartilage, cervix, endometrium, esophagus, gastrointestinal tract, immune system (e.g., T lymphocytes, B lymphocytes, leukocytes, macrophages, and / or dendritic cells), liver, lung, lymphatic vessels, muscle (e.g., cardiac muscle), nerve, ovary, pancreas (e.g., islet cells), pituitary, prostate, kidney, saliva, skin, tendon, testis, and / or thyroid cells. In embodiments, spheroids 500 are formed from hepatocytes. In embodiments, spheroids 500 are formed from mammalian cells, such as human, mouse, rat, rabbit, dog, cat, cow, pig, chicken, goat, and / or horse cells. In embodiments, spheroids 500 are formed from one or more cell types cultured to allow 3D cell growth. In embodiments, spheroids 500 are formed from a single cell type and / or a mixture of cell types.

[0051] Having described in detail an embodiment of the digestible spheroid-stabilized hydrogel 300, reference will now be made in detail to an embodiment of a method for producing the digestible spheroid-stabilized hydrogel 300, with reference to Figures 1A-2C. Thereafter, an embodiment of a kit for producing the digestible spheroid-stabilized hydrogel 300 will be described in detail.

[0052] II. Methods for Producing Digestive Spheroid-Stabilized Hydrogels In one embodiment, a method for producing a digestible spheroid-stabilized hydrogel 300 is disclosed. The method can include the step of (a) providing a crosslinker and a proton donor together with an aqueous medium containing a gelling agent comprising a PGA compound or an alginic acid compound, the PGA compound comprising at least one of (i) pectinic acid or a salt thereof, or (ii) a partially esterified pectinic acid or a salt thereof having an esterification degree of about 1 to about 40 mol %, where the crosslinker comprises a salt of a divalent ion, and the proton donor comprises a lactone, ester, or other compound that hydrolyzes in aqueous solution to form an acid over a period of 10 minutes to 1 hour. The crosslinker and proton donor are provided together with the aqueous medium containing the gelling agent comprising a PGA compound under conditions such that the PGA compound is crosslinked via the crosslinker and the proton donor to form a digestible spheroid-stabilized hydrogel 300.

[0053] In an embodiment, the method includes (a) providing a crosslinker and a proton donor together with an aqueous medium containing a gelling agent, including a PGA compound. The crosslinker, proton donor, and gelling agent are as described above with respect to the digestible spheroid-stabilized hydrogel 300. More specifically, the compositions and amounts of the crosslinker, proton donor, and gelling agent utilized are as described above with respect to the digestible spheroid-stabilized hydrogel 300. For example, the crosslinker can be a calcium salt, the proton donor can be gluconolactone, and the gelling agent, including a PGA compound, can be pectic acid. As a further example, a molar ratio of crosslinker to proton donor of about 2:1 and about 1% weight / weight of pectic acid as the PGA compound can be provided together with the aqueous medium.

[0054] In embodiments, the method includes (a) providing a crosslinker and a proton donor together with an aqueous medium containing a gelling agent, including a PGA compound. In embodiments, the aqueous medium includes a cell culture medium. In embodiments, the cell culture medium includes a calcium-free cell culture medium or a calcium-chelated cell culture medium. In embodiments, the cell culture medium is selected from BME, MEM, DMEM, L-15, RPMI, DMEM / F12, IMDM, NCTC, and / or OIM. In embodiments, the cell culture medium is L-15. In embodiments, the cell culture medium is selected so that the method can be performed without the need for the addition of carbon dioxide, i.e., CO2, to maintain a suitable cell culture pH. For example, without being bound by theory, it is believed that using L-15 cell culture medium as the aqueous medium allows the method to be performed without the addition of carbon dioxide. The cell culture medium, together with the crosslinker, proton donor, and PGA compound, can be provided in a volume of about 300 μL to about 10 mL, or about 300 μL to about 3 mL, or about 1 mL to about 10 mL, depending on the size of the container. In an embodiment, the PGA compound is provided as a separate solution from the cell culture medium. For example, the PGA compound can be provided as a separate solution from the calcium-free medium and / or the calcium-chelated medium. In an embodiment, the PGA compound can be provided as a separate solution from the calcium-free medium and / or the calcium-chelated medium and mixed therewith.

[0055] In embodiments, a crosslinker and a proton donor are provided in step (a) together with an aqueous medium containing a gelling agent comprising a PGA compound under conditions such that the PGA compound is crosslinked via the crosslinker and the proton donor to form a digestible spheroid-stabilized hydrogel 300. In embodiments, the conditions under which the PGA compound is crosslinked via the crosslinker and the proton donor include, but are not limited to, the components of the method, i.e., the amount and / or concentration of reactants, the temperature of the method, the pH of the method, the mixing method employed, the volume, purity of reactants, and / or the reaction time of the method. In embodiments, the amounts and / or concentrations of the components of the method are as described above. In embodiments, the method is carried out at ambient temperature and / or room temperature. In some embodiments, the method is carried out at a temperature ranging from about 0°C to about 35°C or from about 5°C to 32°C. In embodiments, the method can be carried out to maintain an appropriate cell culture pH. For example, the method can be carried out to maintain a pH of about 6.5 to about 8 or about 7 to about 7.5. In embodiments, the method can be carried out for a reaction time sufficient for gelation to occur. For example, this method can be carried out with a reaction time of about 30 seconds to about 5 minutes or more, depending on the type of container, the number of stacked containers, the concentration of PGA, or the number of encapsulated spheroids. Without being bound by theory, it is believed that the reaction time for gelation to occur may depend on the amount and / or concentration of the crosslinker and / or proton donor. For example, a lower amount and / or lower concentration of the crosslinker and / or proton donor may result in a longer reaction time for gelation to occur. Conversely, as another example, a higher amount and / or higher concentration of the crosslinker and / or proton donor may result in a shorter reaction time for gelation to occur. It is further noted that gelation can be diffusion-limited by crosslinking at the surface of the digestible spheroid-stabilized hydrogel 300.

[0056] Gelation can occur through physical and / or chemical bonds and / or crosslinks. In embodiments, gelation of the digestible spheroid-stabilized hydrogel 300 occurs through ionic bonds and / or crosslinks of the PGA compound. Ionic bonds and / or crosslinks of the PGA compound can be achieved through the use of an ionic crosslinker, such as a calcium salt. As described above with respect to the digestible spheroid-stabilized hydrogel 300, the crosslinker can provide a source of calcium ions upon exposure to a proton donor. In embodiments, the proton donor, e.g., gluconolactone, hydrolyzes to gluconic acid in a pH- and temperature-rate-dependent manner upon exposure to the crosslinker, as described above, to provide a source of calcium ions. In embodiments, the crosslinker and proton donor are provided in a molar ratio of about 2:1.

[0057] In an embodiment, the step of (a) providing a crosslinker and a proton donor together with an aqueous medium containing a gelling agent comprising a PGA compound includes (1) mixing the crosslinker with an aqueous medium containing a gelling agent comprising a PGA compound to form an aqueous solution and / or (2) providing gluconolactone in an aqueous solution to initiate crosslinking of the PGA compound. Regarding (1), in an embodiment, the step of mixing the crosslinker with an aqueous medium containing a gelling agent comprising a PGA compound, such as a cell culture medium, to form an aqueous solution includes vortexing to provide a substantially homogeneous aqueous solution. Also, regarding (1), in an embodiment, the gelling agent comprising a PGA compound is provided together with and / or added to the aqueous medium, such as a cell culture medium, before providing the crosslinker thereto. Furthermore, regarding (1), in an embodiment, the crosslinker is mixed before providing the aqueous medium and the gelling agent comprising a PGA compound.

[0058] With respect to (2), in embodiments, crosslinking of the PGA compound is initiated by providing gluconolactone to the aqueous solution formed in (1). In embodiments, providing gluconolactone to the aqueous solution formed in (1) comprises mixing gluconolactone with the aqueous solution.

[0059] In an embodiment, the method for producing the digestible spheroid-stabilized hydrogel 300 further includes (b) providing, prior to gelling of the digestible spheroid-stabilized hydrogel 300, an aqueous medium comprising a crosslinker, a proton donor, and a gelling agent comprising the PGA compound of (a), and a 3D cell culture substrate 100 comprising spheroids 500. The 3D cell culture substrate 100 and spheroids 500 can be as described above with respect to the digestible spheroid-stabilized hydrogel 300. In an embodiment, the spheroids 500 are disposed within the microwell array 150 of the 3D cell culture substrate 100. With respect to (b), in embodiments, providing the 3D cell culture substrate 100 with an aqueous medium comprising a crosslinker, a proton donor, and a gelling agent comprising the PGA compound of (a) comprises applying the aqueous medium comprising the crosslinker, a proton donor, and a gelling agent comprising the PGA compound of (a) to at least one microwell 130 and / or the array of microwells 150 comprising spheroids 500. In embodiments, the digestible spheroid-stabilized hydrogel 300 is formed in situ upon application to at least one microwell 130 and / or the array of microwells 150 comprising spheroids 500. In this manner, gelation of the aqueous solution of (a) does not occur prior to application to the 3D cell culture substrate 100. Also, in this manner, the digestible spheroid-stabilized hydrogel 300 can be formed in a stacked arrangement on and / or in at least one microwell 130 and / or the array of microwells 150 of the 3D cell culture substrate 100.

[0060] 1C and 2A, a digestible spheroid-stabilized hydrogel 300 prepared according to the aforementioned method is shown. Specifically, the digestible spheroid-stabilized hydrogels 300 prepared according to the described method are shown in a stacked arrangement.

[0061] 1A-1B, spheroids 500 can be cultured in a 3D cell culture substrate 100 before preparing and / or layering a digestible spheroid-stabilized hydrogel 300 as described above. Specifically, as shown in FIG. 1A, spheroids 500 can be cultured in the 3D cell culture substrate 100 as described above, in a cell culture medium 900 also as described above. Then, with reference to FIG. 1B, the cell culture medium 900 can be removed from the 3D cell culture substrate 100 before preparing and / or layering a digestible spheroid-stabilized hydrogel 300 as described above. The method for preparing and / or layering a digestible spheroid-stabilized hydrogel 300 can then be carried out as described above and as shown in FIGS. 1C and 2A.

[0062] 2B-2C, after preparing and / or layering digestible spheroid-stabilized hydrogel 300 as described above, digestible spheroid-stabilized hydrogel 300 can be digested. For example, digestible spheroid-stabilized hydrogel 300 can be digested by providing and / or adding digestive agent 1100, such as digestion solution 1100. In embodiments, digestive agent 1100 and / or digestion solution 1100 include an agent for degrading digestible spheroid-stabilized hydrogel 300. For example, digestive agent 1100 and / or digestion solution 1100 can include at least one pectinase, at least one alginate lyase (or alginase), and / or at least one chelating agent. As a further example, digestive agent 1100 and / or digestion solution 1100 can include at least one pectinase and / or at least one chelating agent. As a further example, the digestive agent 1100 and / or digestive solution 1100 can include at least one arginase and / or at least one chelating agent.

[0063] In embodiments, the pectinase and / or arginase can be selected from hydrolases, lyases, and / or esterases. The pectinase and / or arginase can function to digest and / or degrade the digestible spheroid-stabilized hydrogel 300 through its catalytic activity. For example, the pectinase and / or arginase can function to dissociate the digestible spheroid-stabilized hydrogel 300, releasing spheroids therefrom. In embodiments, the pectinase is produced from Aspergillus niger. According to embodiments, arginase can be isolated from a variety of sources, including seaweed, marine mollusks (Littorina spp., Haliotis spp., Turbo cornutus), and a wide variety of marine and terrestrial bacteria. In the examples of the embodiments of the present disclosure, alginate lyase (product number: A1603, CAS number: 9024-15-1) commercially available from Sigma-Aldrich was used. In embodiments, pectinase and / or arginase is provided with and / or added to the digestible spheroid-stabilized hydrogel 300 at a concentration of about 10 U / ml to about 1000 U / ml, about 20 U / ml to about 500 U / ml, about 50 U / ml to about 200 U / ml, or about 100 U / ml.

[0064] In embodiments, the chelating agent may be selected from ethylenediaminetetraacetic acid (i.e., EDTA), cyclohexanediaminetetraacetic acid (i.e., CDTA), ethylene glycol tetraacetic acid (i.e., EGTA), citric acid, and / or tartaric acid. In embodiments, the chelating agent is EDTA. In some embodiments, the chelating agent is BAPTA. In embodiments, pectinase is mixed with the chelating agent before providing the digestion solution 1100 with the digestible spheroid-stabilized hydrogel 300. In embodiments, the pectinase mixed with the chelating agent, i.e., the digestion solution, is provided with and / or added to the digestible spheroid-stabilized hydrogel 300 in a volume approximately twice that of the digestible spheroid-stabilized hydrogel 300. For example, 4 mL of digestion solution may be provided with and / or added to a microwell 130 containing 2 mL of digestible spheroid-stabilized hydrogel 300.

[0065] In embodiments, the digestion solution 1100 is provided with the digestible spheroid-stabilized hydrogel 300 for a digestion reaction time. For example, the digestion solution 1100 can be provided with the digestible spheroid-stabilized hydrogel 300 for a reaction time sufficient to allow the digestible spheroid-stabilized hydrogel 300 to decompose and / or degrade into its constituent galacturonic acid monomers. Without being bound by theory, it is believed that the reaction time for digestion to occur can be determined by the amount and / or concentration of the gelling agent, including the PGA compound. For example, a lower amount and / or lower concentration of the PGA compound may result in a shorter reaction time for digestion to occur. Conversely, as another example, a higher amount and / or higher concentration of the PGA compound may result in a shorter reaction time for digestion to occur. The digestible spheroid-stabilized hydrogel 300 can be digested in the 3D cell culture substrate 100 containing the spheroids 500 to recover the spheroids 500. In embodiments, the digestible spheroid-stabilized hydrogel 300 is degraded and / or degraded, allowing for the recovery of viable spheroids 500 that are covered by the digestible spheroid-stabilized hydrogel 300.

[0066] 2B-2C, after digestion and / or recovery of the spheroids 500, the digestion solution 1100 can be replaced with cell culture medium 900. Specifically, the digestion solution 1100 can be removed and the cell culture medium 900 can be provided with and / or added to the spheroids 500 disposed within the 3D cell culture substrate 100.

[0067] Having described in detail an embodiment of a method for producing the digestible spheroid-stabilized hydrogel 300, reference will now be made in detail to an embodiment of a kit for producing the digestible spheroid-stabilized hydrogel 300.

[0068] III. Kit for producing spheroid-stabilized hydrogels In embodiments, a kit for preparing and / or manufacturing a spheroid-stabilized hydrogel 300 in calcium-free or calcium-chelated cell culture medium is disclosed. The kit can include: (a) a gelling agent including alginic acid or a PGA compound including at least one of (i) pectinic acid or a salt thereof, or (ii) a partially esterified pectinic acid or a salt thereof having an esterification degree of about 1 to about 40 mole %; (b) a crosslinker including a salt of a divalent ion; and (c) a proton donor including a lactone, ester, or other compound that hydrolyzes in aqueous solution to form an acid over a period of 10 minutes to 1 hour. In embodiments, the kit also includes (d) a 3D cell culture substrate 100.

[0069] The 3D cell culture substrate 100, gelling agent, crosslinking agent, and proton donor are as described above with respect to the digestible spheroid-stabilized hydrogel 300 and / or the method for producing the digestible spheroid-stabilized hydrogel 300. For example, the gelling agent comprising a PGA compound can be pectic acid, the crosslinking agent can be a calcium salt, and the proton donor can be gluconolactone. As another example, the 3D cell culture substrate 100 can be a spheroid container.

[0070] In embodiments, the 3D cell culture substrate 100 comprises an array 150 of microwells comprising at least one capillary structure 170 and / or spheroid-attracting geometry 170. In embodiments, the at least one capillary structure 170 is selected from a mouth region, a ridge, a cleft, a pillar, a discontinuous wall, a wavy wall, a parabolic well shape, and / or a sinusoidal well shape. In embodiments, the spheroid-attracting geometry 170 is selected from a pillar, a discontinuous wall, a rounded well bottom, a concave well bottom, a pit, and / or a nib region. In embodiments, the 3D cell culture substrate 100 comprises and / or is included as part of a cell culture vessel 700. The cell culture vessel 700 can be selected from a multi-layer plate, a dish, a flask, a tube, a multi-layer flask, a soft-sided flask, and / or a bag.

[0071] In embodiments, the kit for preparing and / or manufacturing the digestible spheroid-stabilized hydrogel 300 further includes a cell culture medium. In embodiments, the cell culture medium is a calcium-free cell culture medium or a calcium-chelated cell culture medium. The cell culture medium is as described above with respect to the method for preparing and / or manufacturing the digestible spheroid-stabilized hydrogel and / or the digestible spheroid-stabilized hydrogel 300. In embodiments, the PGA compound is a separate solution from the cell culture medium. For example, the PGA compound can be provided as a separate solution from the calcium-free medium and / or the calcium-chelated medium. In embodiments, the PGA compound can be provided as a separate solution from the calcium-free medium and / or the calcium-chelated medium and mixed therewith.

[0072] In embodiments, the kit for preparing and / or manufacturing digestible spheroid-stabilized hydrogel 300 further includes a digestive agent for degrading digestible spheroid-stabilized hydrogel 300. In embodiments, the digestive agent includes pectinase and / or a chelating agent. The pectinase and chelating agent are as described above with respect to digestible spheroid-stabilized hydrogel 300 and / or the method for manufacturing digestible spheroid-stabilized hydrogel 300. In embodiments, the chelating agent is selected from EDTA, CDTA, ETGA, citric acid, and / or tartaric acid. In embodiments, the chelating agent is EDTA.

[0073] In embodiments, the kit further includes instructions for providing guidance regarding the use of the 3D cell culture substrate 100, the gelling agent comprising a PGA compound, the crosslinking agent, the proton donor, the cell culture medium, and / or the digestion agent. For example, the kit can include instructions regarding a method for producing the digestible spheroid-stabilized hydrogel 300 described above and / or a method for digesting the digestible spheroid-stabilized hydrogel 300. In embodiments, the kit includes instructions for (a) providing a crosslinking agent and a proton donor together with an aqueous medium comprising a gelling agent comprising a PGA compound and associated reaction conditions and / or digesting the digestible spheroid-stabilized hydrogel 300 with the digestion agent 1100 and associated reaction conditions, as described above with respect to the method for producing the digestible spheroid-stabilized hydrogel 300. It is understood that the instructions convey the use of the 3D cell culture substrate 100, the gelling agent comprising a PGA compound, the crosslinking agent, the proton donor, the cell culture medium, and / or the digestion agent described above.

[0074] It should now be understood that various embodiments of digestible spheroid-stabilized hydrogels, methods for making digestible spheroid-stabilized hydrogels, and kits for preparing and / or making digestible spheroid-stabilized hydrogels are described herein, and that such embodiments can be utilized in combination with various other embodiments.

[0075] In a first aspect, the present disclosure provides a digestible spheroid-stabilized hydrogel comprising: (a) a gelling agent comprising a polygalacturonic acid (PGA) compound including at least one of alginic acid, or (i) pectinic acid or a salt thereof, or (ii) a partially esterified pectinic acid or a salt thereof having a degree of esterification of about 1 to about 40 mol %; (b) a crosslinking agent comprising a salt of a divalent ion; and (c) a proton donor comprising a lactone, ester, or other compound that hydrolyzes in aqueous solution to form an acid over a period of 10 minutes to 1 hour, wherein the PGA compound is crosslinked via the crosslinking agent and the proton donor to form the digestible spheroid-stabilized hydrogel.

[0076] In a second aspect, the present disclosure provides a digestible spheroid-stabilized hydrogel according to the first aspect, wherein the PGA compound comprises pectic acid.

[0077] In a third aspect, the present disclosure provides a digestible spheroid-stabilized hydrogel according to the first or second aspect, wherein the salt of a divalent ion comprises a calcium salt.

[0078] In a fourth aspect, the present disclosure provides the digestible spheroid-stabilized hydrogel according to any one of the first to third aspects, wherein the salt of the divalent ion comprises a calcium salt selected from calcium carbonate, calcium sulfate, calcium phosphate, or a combination thereof.

[0079] In a fifth aspect, the present disclosure provides the digestible spheroid-stabilized hydrogel according to any one of the first to fourth aspects, wherein the salt of a divalent ion comprises calcium carbonate.

[0080] In a sixth aspect, the present disclosure provides a digestible spheroid-stabilized hydrogel according to any one of the first to fifth aspects, wherein the digestible spheroid-stabilized hydrogel comprises about 1% weight / weight of a PGA compound and a molar ratio of crosslinker to proton donor of about 2:1.

[0081] In a seventh aspect, the present disclosure provides a digestible spheroid-stabilized hydrogel according to any one of the first to sixth aspects, wherein the digestible spheroid-stabilized hydrogel comprises calcium carbonate as a salt of a divalent ion, about 1% weight / weight of pectic acid, gluconolactone as a proton donor, and a molar ratio of calcium carbonate to gluconolactone of about 2:1.

[0082] In an eighth aspect, the present disclosure provides a digestible spheroid-stabilized hydrogel according to any one of the first to seventh aspects, wherein the digestible spheroid-stabilized hydrogel further comprises a component of a cell culture medium that supports cell growth.

[0083] In a ninth aspect, the present disclosure provides the digestible spheroid-stabilized hydrogel according to any one of the first to eighth aspects, wherein the digestible spheroid-stabilized hydrogel further comprises a calcium-free or calcium-chelated cell culture medium.

[0084] In a tenth aspect, the present disclosure provides a digestible spheroid-stabilized hydrogel according to any one of the first to ninth aspects, wherein the digestible spheroid-stabilized hydrogel is provided together with a three-dimensional (3D) cell culture substrate comprising spheroids.

[0085] In an eleventh aspect, the present disclosure provides a digestible spheroid-stabilized hydrogel according to any one of the first to tenth aspects, wherein the digestible spheroid-stabilized hydrogel is provided together with a three-dimensional (3D) cell culture substrate comprising spheroids, and the digestible spheroid-stabilized hydrogel improves the survival rate of the spheroids in the 3D cell culture substrate and / or stabilizes the spheroids at a target location in the 3D cell culture substrate.

[0086] In a twelfth aspect, the present disclosure provides a digestible spheroid-stabilized hydrogel according to any one of the first to eleventh aspects, wherein the digestible spheroid-stabilized hydrogel is provided together with a spheroid container.

[0087] In a thirteenth aspect, the present disclosure provides the digestible spheroid-stabilized hydrogel according to any one of the first to twelfth aspects, wherein the salt of the divalent ion comprises a calcium salt having a water solubility of greater than 0 mg / L to about 20 mg / L at 25°C.

[0088] In a fourteenth aspect, the present disclosure provides the digestible spheroid-stabilized hydrogel according to any one of the first to thirteenth aspects, wherein the proton donor comprises gluconolactone.

[0089] In a fifteenth aspect, the present disclosure provides a method for producing a digestible spheroid-stabilized hydrogel, comprising: (a) providing a crosslinker and a proton donor together with an aqueous medium containing a gelling agent comprising a polygalacturonic acid (PGA) compound comprising at least one of (i) pectinic acid or a salt thereof, or (ii) partially esterified pectinic acid or a salt thereof having a degree of esterification of about 1 to about 40 mole % or alginic acid, under conditions whereby the PGA compound is crosslinked via the crosslinker and the proton donor to form a digestible spheroid-stabilized hydrogel, wherein the crosslinker comprises a salt of a divalent ion, and the proton donor comprises a lactone, ester, or other compound that hydrolyzes in aqueous solution to form an acid over a period of 10 minutes to 1 hour.

[0090] In a sixteenth aspect, the present disclosure provides a method for producing a digestible spheroid-stabilized hydrogel according to the fifteenth aspect, wherein the salt of the divalent ion comprises a calcium salt.

[0091] In a seventeenth aspect, the present disclosure provides a method for producing a digestible spheroid-stabilized hydrogel according to the fifteenth or sixteenth aspect, wherein the PGA compound comprises pectic acid and the salt of a divalent ion comprises calcium carbonate.

[0092] In an eighteenth aspect, the present disclosure provides a method for producing a digestible spheroid-stabilized hydrogel according to any one of the fifteenth to seventeenth aspects, wherein the aqueous medium comprises a cell culture medium.

[0093] In a nineteenth aspect, the present disclosure provides a method for producing a digestible spheroid-stabilized hydrogel according to any one of the fifteenth to eighteenth aspects, wherein the aqueous medium comprises calcium-free or calcium-chelated cell culture medium.

[0094] In a twentieth aspect, the present disclosure provides a method for producing a digestible spheroid-stabilized hydrogel according to any one of the fifteenth to nineteenth aspects, wherein the step (a) of providing a crosslinker and a proton donor together with an aqueous medium comprising a gelling agent comprising a PGA compound comprises: (1) mixing the crosslinker with an aqueous medium comprising a gelling agent comprising a PGA compound to form an aqueous solution; and (2) initiating crosslinking of the PGA compound by providing a proton donor to the aqueous solution.

[0095] In a twenty-first aspect, the present disclosure provides a method for producing a digestible spheroid-stabilized hydrogel according to any one of the fifteenth to twentieth aspects, wherein the step (a) of providing a crosslinker and a proton donor together with an aqueous medium comprising a gelling agent comprising a PGA compound comprises: (1) mixing the crosslinker with the aqueous medium comprising the gelling agent comprising a PGA compound to form an aqueous solution; and (2) mixing the proton donor with the aqueous solution to initiate crosslinking of the PGA compound.

[0096] In a twenty-second aspect, the present disclosure provides a method for producing a digestible spheroid-stabilized hydrogel according to any one of the fifteenth to twenty-first aspects, wherein the digestible spheroid-stabilized hydrogel comprises a PGA compound that is about 1% weight / weight pectic acid and a molar ratio of crosslinker to proton donor of about 2:1.

[0097] In a twenty-third aspect, the present disclosure provides a method for producing a digestible spheroid-stabilized hydrogel according to any one of the fifteenth to twenty-second aspects, further comprising the step of providing an aqueous medium comprising a crosslinker, a proton donor, and a gelling agent comprising the polygalacturonic acid (PGA) compound of (a) and a three-dimensional (3D) cell culture substrate comprising spheroids, prior to gelation of the digestible spheroid-stabilized hydrogel, such that the digestible spheroid-stabilized hydrogel is formed in situ.

[0098] In a twenty-fourth aspect, the present disclosure provides a method for producing a digestible spheroid-stabilized hydrogel according to the twenty-third aspect, wherein the 3D cell culture substrate comprises an array of microwells comprising at least one capillary structure and a spheroid-attracting geometry, spheroids are disposed in the array of microwells, and step (b) comprises applying to at least one of the microwells an aqueous medium comprising a crosslinker, a proton donor, and a gelling agent comprising the polygalacturonic acid (PGA) compound of (a).

[0099] In a twenty-fifth aspect, the present disclosure provides a method for producing a digestible spheroid-stabilized hydrogel according to the twenty-third or twenty-fourth aspect, wherein the digestible spheroid-stabilized hydrogel is formed in a layered configuration.

[0100] In a twenty-sixth aspect, the present disclosure provides a method for producing a digestible spheroid-stabilized hydrogel according to any one of the fifteenth to twenty-fifth aspects, further comprising providing a pectinase or an arginase.

[0101] In a twenty-seventh aspect, the present disclosure provides a method for producing a digestible spheroid-stabilized hydrogel according to the twenty-sixth aspect, wherein pectinase or arginase is added to dissociate the hydrogel and release spheroids therefrom.

[0102] In a twenty-eighth aspect, the present disclosure provides a method for producing a digestible spheroid-stabilized hydrogel according to any one of the fifteenth to twenty-seventh aspects, wherein the proton donor comprises gluconolactone.

[0103] In a twenty-ninth aspect, the present disclosure provides a kit for preparing and / or producing a digestible spheroid-stabilized hydrogel, comprising: (a) a gelling agent comprising an alginic acid, or a polygalacturonic acid (PGA) compound including at least one of (i) pectinic acid or a salt thereof, or (ii) a partially esterified pectinic acid or a salt thereof having a degree of esterification of about 1 to about 40 mole %, (b) a crosslinking agent comprising a salt of a divalent ion, and (c) a proton donor comprising a lactone, ester, or other compound that hydrolyzes in aqueous solution to form an acid over a period of 10 minutes to 1 hour.

[0104] In a thirtieth aspect, the present disclosure provides a kit for preparing and / or producing the digestible spheroid-stabilized hydrogel according to the twenty-ninth aspect, further comprising (d) a 3D cell culture substrate.

[0105] In a thirty-first aspect, the present disclosure provides a kit for preparing and / or producing a digestible spheroid-stabilized hydrogel according to the thirtieth aspect, wherein the 3D cell culture substrate has an array of microwells comprising at least one capillary structure and a spheroid-attracting geometry.

[0106] In a thirty-second aspect, the present disclosure provides a kit for preparing and / or producing a digestible spheroid-stabilized hydrogel according to the thirty or thirty-first aspect, wherein the 3D cell culture substrate comprises at least a portion of a cell culture vessel.

[0107] In a thirty-third aspect, the present disclosure provides a kit for preparing and / or producing a digestible spheroid-stabilized hydrogel according to the thirty-second aspect, wherein the cell culture vessel is selected from a multi-layer plate, a dish, a flask, a tube, a multi-layer flask, a soft-sided flask or a bag.

[0108] In a thirty-fourth aspect, the present disclosure provides a kit for preparing and / or producing a digestible spheroid-stabilized hydrogel according to any one of the thirty-first to thirty-third aspects, wherein at least one capillary structure of the 3D cell culture substrate is selected from a ridge, a cleft, a pillar, a discontinuous wall, a wavy wall, a mouth, a parabolic well shape, a sinusoidal well shape, or a combination thereof.

[0109] In a thirty-fifth aspect, the present disclosure provides a kit for preparing and / or manufacturing a digestible spheroid-stabilized hydrogel according to any one of the thirty-first to thirty-fourth aspects, wherein the spheroid-attracting geometry comprises pillars, discontinuous walls, wavy walls, rounded well bottoms, concave well bottoms, pits, nib regions, or combinations thereof.

[0110] In a thirty-sixth aspect, the present disclosure provides a kit for preparing and / or producing a digestible spheroid-stabilized hydrogel according to any one of the thirty to thirty-fifth aspects, wherein the 3D cell culture substrate is a spheroid container.

[0111] In a thirty-seventh aspect, the present disclosure provides a kit for preparing and / or producing the digestible spheroid-stabilized hydrogel according to any one of the twenty-ninth to thirty-sixth aspects, wherein the gelling agent comprising a PGA compound comprises pectic acid.

[0112] In a thirty-eighth aspect, the present disclosure provides a kit for preparing and / or producing a digestible spheroid-stabilized hydrogel according to any one of the twenty-ninth to thirty-seventh aspects, wherein the salt of the divalent ion comprises a calcium salt.

[0113] In a thirty-ninth aspect, the present disclosure provides a kit for preparing and / or producing the digestible spheroid-stabilized hydrogel according to any one of the twenty-ninth to thirty-eighth aspects, wherein the salt of the divalent ion comprises a calcium salt selected from calcium carbonate, calcium sulfate, calcium phosphate, or a combination thereof.

[0114] In a fortieth aspect, the present disclosure provides a kit for preparing and / or producing a digestible spheroid-stabilized hydrogel according to any one of the twenty-ninth to thirty-ninth aspects, wherein the salt of the divalent ion is calcium carbonate.

[0115] In a forty-first aspect, the present disclosure provides a kit for preparing and / or producing a digestible spheroid-stabilized hydrogel according to any one of the twenty-ninth to fortieth aspects, wherein the salt of the divalent ion is a calcium salt having a water solubility of about 0 mg / L to about 20 mg / L at 25°C.

[0116] In a forty-second aspect, the present disclosure provides a kit for preparing and / or producing the digestible spheroid-stabilized hydrogel of any one of the twenty-ninth to forty-first aspects, further comprising a digestive agent for degrading the digestible spheroid-stabilized hydrogel, the digestive agent comprising pectinase or arginase and a chelating agent selected from ethylenediaminetetraacetic acid (EDTA), cyclohexanediaminetetraacetic acid (CDTA), ethylene glycol tetraacetic acid (ETGA), citric acid, tartaric acid, or a combination thereof.

[0117] In a forty-third aspect, the present disclosure provides a kit for preparing and / or producing the digestible spheroid-stabilized hydrogel according to the forty-second aspect, wherein the chelating agent comprises ethylenediaminetetraacetic acid (EDTA).

[0118] In a forty-fourth aspect, the present disclosure provides a kit for preparing and / or producing the digestible spheroid-stabilized hydrogel according to any one of the twenty-ninth to forty-third aspects, further comprising a cell culture medium.

[0119] In a forty-fifth aspect, the present disclosure provides a kit for preparing and / or producing a digestible spheroid-stabilized hydrogel according to the forty-fourth aspect, wherein the cell culture medium comprises calcium-free or calcium-chelated cell culture medium.

[0120] In a forty-sixth aspect, the present disclosure provides a kit for preparing and / or producing a digestible spheroid-stabilized hydrogel according to the forty-fourth or forty-fifth aspect, wherein the PGA compound is mixed with a cell culture medium.

[0121] In a forty-seventh aspect, the present disclosure provides a kit for preparing and / or producing a digestible spheroid-stabilized hydrogel according to the forty-fourth or forty-fifth aspect, wherein the PGA compound is in a solution separate from the cell culture medium.

[0122] In a forty-eighth aspect, the present disclosure provides a kit for preparing and / or producing a digestible spheroid-stabilized hydrogel according to any one of the twenty-ninth to forty-seventh aspects, wherein the proton donor comprises gluconolactone. [Example]

[0123] The embodiments described herein will be further clarified by the following examples.

[0124] Example 1 Effect of layered digestible PGA spheroid-stabilized hydrogels on cell viability Experimental Protocol: We investigated the effect of layered digestible PGA spheroid-stabilized hydrogels on the viability of HepG2 cells. More specifically, we investigated the effect of layered digestible PGA spheroid-stabilized hydrogels on the viability of HepG2 cells grown on 2D cell culture monolayers and spheroids grown on 3D cell culture substrates.

[0125] 2D cell culture monolayer Corning T-75 flasks (approximately 3 x 10 cells per flask) 6HepG2 cells (ATCC; HB-8065) ​​were prepared for seeding onto 2D cell culture substrates by growing them in cell culture medium (1000 cells / well). The cell culture medium was minimal essential medium with GlutaMAX and Eagle's salts (ThermoFisher) containing 1x penicillin-streptomycin (ThermoFisher) and 10% fetal bovine serum (ThermoFisher). HepG2 cells were grown in T-75 flasks to approximately 90% confluence. The cell culture medium was then removed from the T-75 flask, and 2.5 mL of 0.25% trypsin-ethylenediaminetetraacetic acid (i.e., EDTA) solution (ThermoFisher) was added to the T-75 flask. The T-75 flask was incubated at 37°C for 5 min using a standard tissue culture incubator at 37°C, 5% CO2, and approximately 95% relative humidity. 12.5 mL of minimum essential medium (i.e., MEM; catalog number 11095080, Thermo Fisher Scientific, Waltham, MA) containing 10% fetal bovine serum (i.e., FBS; catalog number 35-010-CV, Corning, Incorporated, Corning, NY) was added to a T-75 flask to form a mixture. The mixture was then pipetted up and down several times to break up any clusters of HepG2 cells. 20 μL of the mixture was then placed in a cellometer (Cellometer Auto T4, Nexcelom, Lawrence, MA), and HepG2 cells were counted per mL of the mixture. HepG2 cells were then cultured at approximately 4 × 10 cells per mL in 35 mL of MEM + 10% FBS. 5 The cells were diluted to a final concentration of 1000 x g.

[0126] To prepare HepG2 cells for seeding into 2D cell culture substrates, HepG2 cells were seeded onto 2D cell culture substrates (Corning Inc., Catalog No. 430641U cell culture flasks) by aliquoting 5 mL of HepG2 cells diluted in MEM + 10% FBS per well. The HepG2 cells were left at room temperature for 30 minutes and then incubated. The HepG2 cells in the 2D cell culture substrates were then incubated overnight (approximately 16 hours) at 37°C in an incubator (Fisher Scientific, NAPCO Series 8000 DH CO2 incubator) with 5% CO2 and 95% relative humidity.

[0127] Digestive PGA spheroid-stabilized hydrogels (hereinafter "PGA hydrogels") were prepared and layered onto L-15 medium (ATCC® 30-2008™, catalog number 21083027, Thermo Fisher Scientific). In preparation for the layered PGA hydrogels, any remaining liquid was removed from the wells of the 2D cell culture substrate, and 1 mL of polygalacturonic acid (i.e., PGA or pectic acid; Sigma Aldrich #P3850) in L-15 medium was added to each well of the 2D cell culture substrate and incubated at room temperature for 5 minutes. PGA was added to Leibovitz's medium at a PGA concentration of 1% w / w. The PGA in the L-15 medium was then removed from the wells.

[0128] PGA hydrogels were prepared by adding PGA in L-15 medium to a 15 mL conical tube (Corning, Inc.). PGA was added to a concentration of 1% w / w in L-15 medium. Calcium carbonate (i.e., CaCO3; Sigma Aldrich Product No. 795445G) was then added to a concentration of 1 mg / mL. The CaCO3 was thoroughly mixed to avoid settling at the bottom of the 15 mL conical tube, and then the PGA in L-15 medium was added. The 15 mL conical tube was then vortexed briefly (approximately 2 seconds) to ensure that the CaCO3 was evenly distributed throughout the PGA in L-15 medium. Glucono delta-lactone (i.e., GDL; Sigma Aldrich) was then added to the CaCO3 evenly distributed throughout the PGA in L-15 medium. GDL was added to a final concentration of approximately 3.56 mg / mL. The 15 mL conical tube was then inverted several times to ensure proper mixing of the PGA, CaCO3, and GDL in L-15 medium. After inversion, 1–2 mL of the mixture of PGA, CaCO3, and GDL in L-15 medium was added to each well of the 2D cell culture substrate. Gelation of the mixture of PGA, CaCO3, and GDL in L-15 medium was allowed to occur at room temperature for approximately 30 minutes to form layered PGA hydrogels. To provide control wells, instead of adding the mixture of PGA, CaCO3, and GDL in L-15 medium, 1–2 mL (approximately 220 μL / cm) was added. 2 ) L-15 medium was added to control wells.

[0129] The 2D cell culture substrate was then stored overnight in a refrigerator at a temperature of about 2°C to about 8°C.

[0130] Live / dead cell staining was then performed according to the following protocol. 2D cell culture substrates were removed from storage at 5°C and warmed to room temperature for approximately 15 minutes. An equal volume (1–2 mL, depending on the volume of PGA / CaCO3 and GDL mixture added to the well) of calcium- and magnesium-free Dulbecco's phosphate-buffered saline (i.e., DPBS; ThermoFisher) was then added to each well of the 2D cell culture substrate and incubated for 5 minutes. The DPBS was then removed from each well, taking care not to remove any of the overlying PGA hydrogel.

[0131] A live / dead cell staining solution was prepared by adding 15 μL of red-fluorescent ethidium homodimer and 7.5 mL of green-fluorescent calcein AM to 15 mL of DPBS and mixing thoroughly using the LIVE / DEAD™ Viability / Cytotoxicity Kit for Mammalian Cells (Cat. No. L3224, Thermo Fisher Scientific). Red-fluorescent ethidium homodimer indicates loss of plasma membrane integrity; an intact plasma membrane is a distinguishing feature of live cells. In contrast, green-fluorescent calcein AM indicates intracellular esterase activity; ubiquitous intracellular esterase activity is a distinguishing feature of live cells. In this way, live cells can be distinguished from dead cells by simultaneous staining with red-fluorescent ethidium homodimer and green-fluorescent calcein AM.

[0132] An equal volume of the prepared live / dead cell staining solution was then added to the overlaid PGA hydrogel in each well (i.e., 1 mL of the prepared live / dead cell staining solution was added to 1 mL of PGA hydrogel). The added live / dead cell staining solution was then incubated at 5°C for 2 hours. After incubation, the cells were examined under a Nikon ECLIPSE Ti bright-field microscope and a Nikon ECLIPSE Ti fluorescent microscope to visualize live cells (i.e., cells exhibiting green fluorescence) and dead cells (i.e., cells exhibiting red fluorescence).

[0133] 3D cell culture T-75 flasks (approximately 3 x 10 cells per flask) 6 HepG2 cells were prepared for seeding onto 3D cell culture substrates by growing them in cell culture medium at 1000 x 1000 cells per well. HepG2 cells were grown in a T-75 flask until approximately 90% confluent. The cell culture medium was then removed from the T-75 flask, and 2.5 mL of 0.25% trypsin-EDTA solution was added to the T-75 flask. The T-75 flask was incubated at 37°C in a CO2 incubator with 5% CO2 and 95% relative humidity for 5 minutes. 12.5 mL of MEM (catalog number 11095080, Thermo Fisher Scientific) containing 10% FBS (catalog number 35-010-CV, Corning, Incorporated) was added to the T-75 flask to form a mixture. The mixture was then pipetted up and down several times to disrupt any clusters of HepG2 cells. 20 μL of the mixture was then placed in a cellometer (Nexcelom, Lawrence, MA) and HepG2 cells were counted per mL of the mixture. HepG2 cells were then cultured at approximately 3.2 × 10 cells per mL in 35 mL of MEM + 10% FBS. 5 The cells were diluted to a final concentration of 1000 x g.

[0134] To prepare HepG2 cells for seeding into the 3D cell culture substrate, HepG2 cells were seeded onto the 3D cell culture substrate by aliquoting 5 mL of HepG2 cells diluted in MEM + 10% FBS per well. Each well of the 3D cell culture substrate contained approximately 1,300 microcavities. Approximately 1,200 HepG2 cells were seeded into each microcavity. The HepG2 cells were left at room temperature for 30 minutes and then incubated. The HepG2 cells in the 3D cell culture substrate were then incubated at 37°C in an incubator with 5% CO2 and 95% RH for 3 days to form spheroids (approximately 1,500 cells / spheroid).

[0135] PGA hydrogels were prepared and layered on L-15 medium (catalog number 21083027, Thermo Fisher Scientific). In preparation for the layered PGA hydrogel, any remaining liquid was removed from the wells of the 3D cell culture substrate, taking care not to aspirate any of the spheroids. Then, 1 mL of PGA in L-15 medium was added to each well of the 3D cell culture substrate and incubated at room temperature for 5 minutes. The PGA in L-15 medium was then removed from the wells, again taking care not to aspirate any of the spheroids.

[0136] PGA hydrogels were prepared by adding PGA in L-15 medium to a 15 mL conical tube. PGA was added to a concentration of 1% w / w in the L-15 medium. CaCO3 was then added to a concentration of 1 mg / mL. The CaCO3 was mixed thoroughly to avoid settling at the bottom of the 15 mL conical tube, and then the PGA in L-15 medium was added. The 15 mL conical tube was then vortexed briefly (approximately 2 seconds) to ensure that the CaCO3 was evenly distributed throughout the PGA in the L-15 medium. GDL was added to the CaCO3 that was evenly distributed throughout the PGA in the L-15 medium. GDL was added to a final concentration of approximately 3.56 mg / mL. The 15 mL conical tube was then inverted several times to ensure proper mixing of the PGA, CaCO3, and GDL in the L-15 medium. After inversion, 2–3 mL of a mixture of PGA, CaCO3, and GDL in L-15 medium was added to each well of the 3D cell culture substrate. The mixture of PGA, CaCO3, and GDL in L-15 medium was allowed to gel for approximately 30 minutes at room temperature to form a PGA hydrogel. To provide a control well, instead of adding the mixture of PGA, CaCO3, and GDL in L-15 medium, 1 mL of L-15 medium was added to the control well, incubated for 5 minutes, removed, and then 2–3 mL of L-15 medium was added to the control well.

[0137] The 3D cell culture substrate was then stored overnight in a refrigerator at a temperature of about 2°C to about 8°C.

[0138] Live / dead cell staining was then performed according to the following protocol: 3D cell culture substrates were removed from storage at 5°C and allowed to warm to room temperature for approximately 15 minutes. An equal volume (1-2 mL, depending on the volume of PGA / CaCO3 and GDL mixture added to the well) of DPBS was then added to each well of the 3D cell culture substrate and incubated for 5 minutes. The DPBS was then removed from each well, taking care not to remove any of the PGA hydrogel.

[0139] A live / dead cell staining solution was prepared by adding 15 μL of red-fluorescent ethidium homodimer and 7.5 mL of green-fluorescent calcein AM to 15 mL of DPBS and mixing thoroughly using the LIVE / DEAD™ Viability / Cytotoxicity Kit for Mammalian Cells (Cat. No. L3224, Thermo Fisher Scientific). Red-fluorescent ethidium homodimer indicates loss of plasma membrane integrity; an intact plasma membrane is a distinguishing feature of live cells. In contrast, green-fluorescent calcein AM indicates intracellular esterase activity; ubiquitous intracellular esterase activity is a distinguishing feature of live cells. In this way, live cells can be distinguished from dead cells by simultaneous staining with red-fluorescent ethidium homodimer and green-fluorescent calcein AM.

[0140] An equal volume of the prepared live / dead cell staining solution was then added to the PGA hydrogel in each well (i.e., 1 mL of the prepared live / dead cell staining solution was added to 1 mL of PGA hydrogel). The added live / dead cell staining solution was then incubated at 5°C for 2 hours. The cells were then examined under a bright-field microscope and a fluorescent microscope to visualize live cells (green fluorescence) and dead cells (red fluorescence).

[0141] Results. Referring to Figures 3A-3C and 4A-4C, HepG2 cells grown on 2D cell culture monolayers containing overlaid PGA hydrogels exhibited higher viability (see, e.g., Figures 4B-4C) than control HepG2 cells grown on 2D cell culture substrates without PGA hydrogels (see, e.g., Figures 3B-3C). Referring to Figures 5A-5C and 6A-6C, HepG2 spheroids grown on 3D cell culture substrates containing overlaid PGA hydrogels exhibited higher viability (see, e.g., Figures 5B-5C) than control HepG2 spheroids grown on 3D cell culture substrates without PGA hydrogels (see, e.g., Figures 6B-6C). Considering this improved viability, without being bound by theory, it is believed that the overlaid PGA hydrogel provided cell preservation properties to HepG2 cells during incubation.

[0142] Example 2 Drop testing of spheroids grown on 3D cell culture substrates containing overlaid PGA hydrogels Experimental Protocol. To mimic spheroid transport, a drop test was performed on HepG2 spheroids grown on a 3D cell culture substrate containing an overlaid PGA hydrogel. HepG2 spheroids grown on a 3D cell culture substrate containing an overlaid PGA hydrogel were prepared as described in the experimental protocol of Example 1. Live / dead cell staining was also performed as described in the experimental protocol of Example 1. Prior to the drop test, the overall health of the cells on the control plate was observed by live / dead staining of the cells in the 3D microcavity plate. The test plate was incubated with a live / dead solution overlaid on the PGA hydrogel at 5°C for 2 hours to allow the solution to penetrate the hydrogel layer and the cells, after which they were visualized. Additionally, control HepG2 spheroids grown on a 3D cell culture substrate without a PGA hydrogel were prepared as described in the experimental protocol of Example 1. Live / dead cell staining was also performed as described in the experimental protocol of Example 1 and above. The 3D cell culture substrate containing HepG2 spheroids and overlaid PGA hydrogel was wrapped in a layer of bubble wrap (approximately 2 inches thick) and packaged in a cardboard shipping box. The 3D cell culture substrate containing control HepG2 spheroids without PGA hydrogel was also wrapped in a layer of bubble wrap (approximately 2 inches thick) and packaged in a cardboard shipping box.

[0143] A drop test was then performed by dropping the cardboard shipping box from a height of approximately 1.5 m 5 to 10 times. The 3D cell culture substrates packaged within the cardboard shipping box were retrieved after the drop test was performed, and the 3D cell culture substrates were visually inspected with the naked eye and under bright-field and fluorescent microscopes to determine the displacement of the overlaid PGA hydrogel and / or spheroids.

[0144] Results. Referring to Figures 7A-7C and 8A-8C, the microcavities of the 3D cell culture substrate containing the overlaid PGA hydrogel exhibited higher retention of HepG2 spheroids after the drop test (see, e.g., Figures 8A-8C) compared to the microcavities of the control 3D cell culture substrate without PGA hydrogel (see, e.g., Figures 7A-7C). Also shown in Figures 7A-7C, the wells of the 3D cell culture substrate containing the overlaid PGA hydrogel remained intact after the drop test, with the HepG2 spheroids remaining therein. Given this retention, and without being bound by theory, it is believed that HepG2 spheroids provided in the 3D cell culture substrate containing the overlaid PGA hydrogel would not displace and / or form uncontrolled aggregates during transport.

[0145] Example 3 Recovery of HepG2 cells grown on 2D cell culture monolayers and spheroids grown on 3D cell culture substrates containing overlaid PGA hydrogels Experimental Protocol. Spheroids were harvested from 3D cell culture substrates containing overlaid PGA hydrogels. HepG2 cells grown on 2D cell culture monolayers containing overlaid PGA hydrogels were prepared as described in the experimental protocol of Example 1. HepG2 spheroids grown on 3D cell culture substrates containing overlaid PGA hydrogels were also prepared as described in the experimental protocol of Example 1. Next, a PGA digestion solution was prepared. For digestion of HepG2 cells grown on 2D cell culture monolayers containing overlaid PGA hydrogels, a PGA digestion solution was prepared by adding pectinase (Sigma-Aldrich) to a final concentration of 200 U / mL and EDTA to a final concentration of 20 mM to DPBS. For the digestion of HepG2 spheroids grown on 3D cell culture substrates containing overlaid PGA hydrogels, a PGA digestion solution was prepared by adding pectinase (Sigma Aldrich) to a final concentration of 100 U / mL and EDTA to a final concentration of 10 mM to DPBS.

[0146] Any remaining liquid was then removed from the wells of the 2D and 3D cell culture substrates. A volume of 2D cell culture monolayer PGA digestion solution equal to the volume of the PGA hydrogel was then added to each well of the 2D cell culture substrate. For example, for a well containing 2 mL of layered PGA hydrogel, a corresponding 2 mL of 2D cell culture monolayer PGA digestion solution was added to the well. In this way, a final concentration of 100 U / mL pectinase and 10 mM EDTA was achieved in each well of the 2D cell culture substrate. A volume of 3D cell culture substrate PGA digestion solution equal to twice the volume of the PGA hydrogel was added to each well of the 3D cell culture substrate. For example, for a well containing 2 mL of layered PGA hydrogel, 4 mL of 3D cell culture substrate PGA digestion solution was added to the well. The PGA digestion solution was then incubated in the wells at room temperature for 30 minutes. According to embodiments of the present disclosure, it is contemplated that the exact time required for digestion will depend, if any, on the specific concentrations of the reagents. In some embodiments, this time can range from about 15 minutes to about 1 hour. During this period, the PGA digestion solution acts to digest and solubilize the PGA matrix, breaking down polygalacturonic acid to monogalacturonic acid.

[0147] Any remaining liquid was then removed from the wells, and 2 mL of DPBS was added to each well. Instead of adding DPBS (e.g., to perform spheroid harvesting), cell culture medium can be added to continue spheroid cell culture.

[0148] Those skilled in the art will appreciate that changes could be made in the embodiments described above without departing from the broad inventive concept thereof. It is understood, therefore, that the disclosure is not limited to the particular embodiments disclosed, but that it is intended to cover modifications within the spirit and scope of the disclosure as defined by the appended claims.

[0149] All cited documents are incorporated herein by reference. The citation of any document should not be construed as an admission that it is prior art with respect to the present disclosure.

[0150] It should be further understood that where the terms "comprising" and / or "including" are used in describing various embodiments, those skilled in the art will understand that in some specific instances, embodiments may alternatively be described using the terms "consisting essentially of" or "consisting of."

[0151] It will be understood that every maximum numerical limitation given throughout this specification includes every lower numerical limitation, as if such lower numerical limitations were expressly written herein. Every minimum numerical limitation given throughout this specification will include every higher numerical limitation, as if such higher numerical limitations were expressly written herein. Every numerical range given throughout this specification will include every narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were all expressly written herein.

[0152] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the claimed subject matter belongs. The terminology used in the description herein is for the purpose of describing particular embodiments only and is not intended to be limiting. As used in this specification and the appended claims, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly dictates otherwise.

[0153] The term "about," as used in describing embodiments of the present disclosure, with respect to the dimensions and / or ranges of components and / or amounts, concentrations, volumes, process temperatures, process times, yields, flow rates, pressures, viscosities, and / or values ​​and ranges thereof of ingredients in a composition, such as a digestible spheroid-stabilizing hydrogel, refers to variations in the numerical quantities that may arise, for example, through typical measuring and / or handling techniques for preparing materials, compositions, ingredients of compositions, etc., inadvertent errors in procedures, and / or differences in the manufacture, source, and / or purity of starting materials and / or ingredients used to carry out the methods. The term "about" also encompasses amounts that differ due to degradation of compositions and / or mixtures having particular initial concentrations, as well as amounts that differ due to mixing and / or processing of compositions and / or mixtures having particular initial concentrations.

[0154] Preferred embodiments of the present invention will be described below in detail.

[0155] Embodiment 1 1. A kit for producing a spheroid-stabilized hydrogel in calcium-free or calcium-chelated cell culture medium, comprising: (a)(i) pectic acid or a salt thereof, or (ii) Partially esterified pectic acid or its salt having an esterification degree of about 1 to about 40 mol % A gelling agent containing at least one of a polygalacturonic acid (PGA) compound or an alginic acid compound; (b) a cross-linking agent comprising a salt of a divalent ion; (c) a proton donor, including a lactone, ester, or other compound that hydrolyzes in aqueous solution to form an acid over a period of 10 minutes to 1 hour; Includes a kit.

[0156] Embodiment 2 2. The kit of embodiment 1, wherein said PGA compound is admixed with said calcium-free or calcium-chelated cell culture medium.

[0157] Embodiment 3 2. The kit of embodiment 1, wherein said PGA compound is in a separate solution from said calcium-free or calcium-chelated cell culture medium.

[0158] Embodiment 4 2. The kit of embodiment 1, wherein the proton donor is gluconolactone.

[0159] Embodiment 5 2. The kit of embodiment 1, wherein the cross-linking agent is a calcium salt.

[0160] Embodiment 6 6. The kit of embodiment 5, wherein the calcium salt is selected from calcium carbonate, calcium sulfate, calcium phosphate, or a combination thereof.

[0161] Embodiment 7 6. The kit of embodiment 5, wherein the calcium salt comprises calcium carbonate.

[0162] Embodiment 8 6. The kit of embodiment 5, wherein the calcium salt has a water solubility of greater than 0 mg / L to about 20 mg / L at 25°C.

[0163] Embodiment 9 2. The kit of embodiment 1, wherein the kit further comprises a three-dimensional (3D) cell culture substrate.

[0164] Embodiment 10 10. The kit of embodiment 9, wherein said 3D cell culture substrate has an array of microwells comprising at least one capillary structure and a spheroid-attracting geometry.

[0165] Embodiment 11 10. The kit of embodiment 9, wherein said 3D cell culture substrate comprises at least a portion of a cell culture vessel.

[0166] Embodiment 12 2. The kit of embodiment 1, wherein the kit further comprises a spheroid container.

[0167] Embodiment 13 2. The kit of embodiment 1, further comprising a digestion agent for degrading the spheroid-stabilizing hydrogel, wherein the digestion agent comprises pectinase and a chelating agent selected from ethylenediaminetetraacetic acid (EDTA), cyclohexanediaminetetraacetic acid (CDTA), ethylene glycol tetraacetic acid (ETGA), citric acid, tartaric acid, or a combination thereof.

[0168] Embodiment 14 1. A method for producing a digestible spheroid-stabilized hydrogel, said method comprising: under conditions in which a polygalacturonic acid (PGA) compound is crosslinked via a crosslinker and a proton donor to form the digestible spheroid-stabilizing hydrogel; (a) a cross-linking agent including a calcium salt and a proton donor including gluconolactone, (i) pectic acid or a salt thereof, or (ii) Partially esterified pectic acid or its salt having an esterification degree of about 1 to about 40 mol % together with an aqueous medium containing a gelling agent comprising a PGA compound or an alginic acid compound, A method comprising:

[0169] Embodiment 15 15. The method of embodiment 14, wherein the method further comprises adding pectinase or alginate lyase to dissociate the hydrogel and release spheroids from the hydrogel.

[0170] Embodiment 16 15. The method of embodiment 14, wherein said aqueous medium comprises cell culture medium.

[0171] Embodiment 17 providing the crosslinker and the proton donor together with the aqueous medium containing the gelling agent comprising the PGA compound, (1) mixing the crosslinker with the aqueous medium containing the PGA compound to form an aqueous solution; (2) initiating cross-linking of the PGA compound by providing the gluconolactone with the aqueous solution; 15. The method of embodiment 14, comprising:

[0172] Embodiment 18 The digestible spheroid-stabilized hydrogel is about 1% weight / weight of said PGA compound, said Pectic acid; a molar ratio of the crosslinker to the proton donor of about 2:1; 15. The method of embodiment 14, comprising:

[0173] Embodiment 19 The method comprises: (b) providing the aqueous medium containing the crosslinker, the proton donor, and the gelling agent comprising the polygalacturonic acid (PGA) compound of (a) and a three-dimensional (3D) cell culture substrate containing spheroids, prior to gelation of the digestible spheroid-stabilized hydrogel, such that the digestible spheroid-stabilized hydrogel is formed in situ. 15. The method of embodiment 14, further comprising:

[0174] Embodiment 20 the 3D cell culture substrate has an array of microwells comprising at least one capillary structure and a spheroid-attracting geometry; the spheroids are disposed in the array of microwells; the providing step (b) comprises applying the aqueous medium comprising the crosslinker, the proton donor, and the gelling agent comprising the polygalacturonic acid (PGA) compound of (a) to at least one of the microwells; The method of embodiment 19.

Claims

1. 1. A kit for producing a spheroid-stabilized hydrogel in calcium-free or calcium-chelated cell culture medium, comprising: (a)(i) pectic acid or a salt thereof, or (ii) Partially esterified pectic acid or salt thereof having a degree of esterification of about 1 to about 40 mole percent. a gelling agent containing at least one of a polygalacturonic acid (PGA) compound or an alginic acid compound; (b) a cross-linking agent comprising a salt of a divalent ion; (c) a proton donor, including a lactone, ester, or other compound that hydrolyzes in aqueous solution to form an acid over a period of 10 minutes to 1 hour; and The kit includes a three-dimensional (3D) cell culture substrate for forming a layered PGA hydrogel; wherein 3D cell cultures within the microcavities of the 3D cell culture substrate comprising the overlaid PGA hydrogel exhibit a higher retention of the 3D cell culture after a drop test than 3D cell cultures within the microcavities of the 3D cell culture substrate without the overlaid PGA hydrogel.

2. the PGA compound is mixed with the calcium-free or calcium-chelated cell culture medium; or the PGA compound is in a separate solution from the calcium-free or calcium-chelated cell culture medium; The kit of claim 1.

3. the cross-linking agent is a calcium salt, the calcium salt is selected from calcium carbonate, calcium sulfate, calcium phosphate, or a combination thereof; and / or the calcium salt has a water solubility of greater than 0 mg / L to about 20 mg / L at 25°C; The kit of claim 1.

4. the 3D cell culture substrate has an array of microwells comprising at least one capillary structure and a spheroid-attracting geometry; and / or the 3D cell culture substrate comprises at least a portion of a cell culture vessel; The kit of claim 1.

5. The kit comprises: Spheroid container; a digestive agent for degrading the spheroid-stabilized hydrogel, the digestive agent comprising pectinase and a chelating agent selected from ethylenediaminetetraacetic acid (EDTA), cyclohexanediaminetetraacetic acid (CDTA), ethylene glycol tetraacetic acid (ETGA), citric acid, tartaric acid, or a combination thereof; or A combination of these 2. The kit of claim 1, comprising:

6. 1. A method for producing a digestible spheroid-stabilized hydrogel, said method comprising: under conditions in which a polygalacturonic acid (PGA) compound is crosslinked via a crosslinker and a proton donor to form the digestible spheroid-stabilizing hydrogel; (a) a cross-linking agent comprising a calcium salt and a proton donor comprising gluconolactone; (i) pectic acid or a salt thereof, or (ii) Partially esterified pectic acid or salt thereof having a degree of esterification of about 1 to about 40 mole percent. together with an aqueous medium containing a gelling agent comprising a PGA compound or an alginic acid compound, (b) providing the aqueous medium containing the crosslinker, the proton donor, and the gelling agent comprising the polygalacturonic acid (PGA) compound of (a) and a three-dimensional (3D) cell culture substrate containing spheroids prior to gelation of the digestible spheroid-stabilized hydrogel such that the digestible spheroid-stabilized hydrogel is formed in situ; applying the aqueous medium containing the crosslinker, the proton donor, and the gelling agent containing the polygalacturonic acid (PGA) compound of (a) to at least one of a plurality of microwells of the 3D cell culture substrate to form the digestible spheroid-stabilizing hydrogel in situ. Including, wherein a three-dimensional (3D) cell culture in a microcavity of a microwell of the 3D cell culture substrate comprising a PGA hydrogel overlaid with the digestible spheroid-stabilized hydrogel exhibits a higher retention of the 3D cell culture after a drop test than a 3D cell culture in a microcavity of a microwell of a 3D cell culture substrate not comprising a PGA hydrogel overlaid with the digestible spheroid-stabilized hydrogel.

7. Step (a) of providing the crosslinker and the proton donor together with the aqueous medium containing the gelling agent comprising the PGA compound, (1) mixing the crosslinker with the aqueous medium containing the PGA compound to form an aqueous solution; (2) initiating cross-linking of the PGA compound by providing the aqueous solution to the gluconolactone; 7. The method of claim 6, comprising:

8. The digestible spheroid-stabilized hydrogel is about 1% weight / weight of the PGA compound, which is pectic acid; a molar ratio of the crosslinker to the proton donor of about 2:1; 7. The method of claim 6, comprising:

9. the 3D cell culture substrate has an array of microwells comprising at least one capillary structure and a spheroid-attracting geometry; The spheroids are disposed in the array of microwells. The method of claim 6.

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