Cell preservation liquid and cell preservation method

Abstract

The present invention relates to: a preservation method for adherent cells which comprises refrigerated preservation of adherent cells in a non-frozen state in a cell preservation liquid that contains a high polymer; a cultivation method for adherent cells which comprises two-dimensional culturing of adherent cells after preservation via said preservation method; and a cell preservation liquid which includes a high polymer that is suitable for use in these methods.

Description

Cell preservation solution and cell preservation method

[0001] The present invention relates to a cell preservation solution and a cell preservation method that enable long-term refrigerated preservation of adherent cells.

[0002] Plate culture of primary human hepatocytes (PHH) is widely used in the development of new drugs, including pharmacokinetic and toxicity analysis studies. It is known that when PHH are plate-cultured, the expression level of drug-metabolizing enzymes decreases as the cell density decreases (Non-Patent Document 1). To maintain the properties of PHH in vitro for a long period of time, it is important to maintain the cultured cells at as high a density as possible.

[0003] Commercially available frozen PHHs are classified into two types: plateable grade (adherent cells) that can be cultured on a culture substrate, and suspension grade (floating cells) that cannot be cultured on a substrate. Plateable grade PHHs can generally be cultured on a plate for four weeks or more, allowing them to be used in vitro for extended periods. This makes them more useful than suspension grade PHHs, which can only be used for approximately eight hours after thawing. However, a method for selectively producing plateable grade frozen PHHs has not yet been established, and it is said that only about 30% of commercially available frozen PHHs are plateable grade.

[0004] On the other hand, when PHH are suspended in a preservation solution and placed under refrigerated conditions (4°C, on ice), they can be stored for a short period of time while maintaining their high ability to adhere to a culture substrate, but PHH stored in a refrigerator for a long period of time lose their ability to adhere to a culture substrate. When cells such as PHH are transported long distances, for example, from Japan to Europe by air, a door-to-door storage time of at least 48 to 72 hours, preferably about 96 hours, is required. Therefore, there is a need for the development of a cell preservation method that enables adherent cells such as PHH to maintain their high ability to adhere to a culture substrate even after long-term unfrozen storage.

[0005] Non-patent document 2 reports that the viability and ability to adhere to collagen I plates of PHHs stored in a variety of organ preservation solutions under refrigeration were maintained for up to approximately 24 hours, but significantly decreased with longer storage times.

[0006] Non-Patent Document 3 reports that PHHs were refrigerated in a cell preservation solution with a special composition containing an iron chelating agent, followed by heating, to mitigate cell death due to low-temperature stress. However, the cell preservation solution used in Non-Patent Document 3 has a complex composition and is difficult to reconstitute.

[0007] Patent Document 1 discloses a method for culturing vascular smooth muscle cells by culturing the cells in suspension in a medium composition containing deacylated gellan gum or a salt thereof. However, it does not disclose the ability of cells stored in the medium composition under refrigeration to adhere to a culture substrate, nor does it mention any effects on cells other than vascular smooth muscle cells.

[0008] Patent Document 2 discloses that good viability can be maintained for a long period of time by storing cells or tissues in a non-frozen state in a liquid composition containing deacylated gellan gum or a salt thereof and an acidic polysaccharide such as alginic acid. However, it does not disclose the ability of cells stored in the liquid composition under refrigeration to attach to a culture substrate.

[0009] International Publication WO2016 / 121896 International Publication WO2019 / 049985

[0010] Yamasaki et al., PLOS ONE, 15(9): (2020) e0237809Duret et al., Cell Transplantation, Vol. 24, pp. 2541-2555 (2015)Pless et al., Cell Transplantation, Vol. 21, pp. 23-37 (2012)

[0011] An objective of the present invention is to provide a cell preservation solution and a cell preservation method that enable long-term refrigerated preservation of adherent cells while maintaining their ability to attach to a culture substrate.A further objective of the present invention is to provide a cell culture method that enables two-dimensional culture of adherent cells after long-term refrigerated preservation while maintaining cellular properties.

[0012] As a result of extensive research to solve the above-mentioned problems, the inventors discovered that when adhesive cells were stored in a cell preservation solution containing a high molecular weight polymer such as deacylated gellan gum or a salt thereof for a long period of time under refrigeration, the cells did not settle to the bottom of the container but remained suspended, and their ability to adhere to the culture substrate in subsequent two-dimensional culture was significantly improved, which led to the completion of the present invention.

[0013] That is, the present invention includes the following: [1] A method for preserving adherent cells, which comprises refrigerating and preserving adherent cells in a cell preservation solution containing a high molecular weight polymer in an unfrozen state.

[0014] [2] The method according to [1] above, wherein the high molecular weight polymer comprises deacylated gellan gum or a salt thereof.

[0015] [3] The method according to [1] or [2] above, wherein the adherent cells are refrigerated and stored while retaining their ability to adhere to a culture substrate in two-dimensional culture.

[0016] [4] The method according to any one of [1] to [3] above, wherein the adherent cells are refrigerated for up to 100 hours.

[0017] [5] The method according to any one of [1] to [4] above, wherein the adherent cells are refrigerated for 70 hours or more.

[0018] [6] The method according to any one of [1] to [5] above, wherein the adherent cells are hepatocytes.

[0019] [7] The method according to any one of [1] to [6] above, wherein the adherent cells are in a suspended state in a cell preservation solution.

[0020] [8] The method according to any one of [1] to [7] above, wherein the cell preservation solution further contains an iron chelating agent.

[0021] [9] The method according to [8] above, wherein the iron chelating agent comprises deferoxamine.

[0022]

[10] A method for culturing adherent cells, comprising refrigerating and preserving the adherent cells by the method according to any one of [1] to [9] above, and then two-dimensionally culturing the adherent cells.

[0023]

[11] The method according to

[10] above, wherein the adherent cells are seeded on a collagen-coated culture substrate and two-dimensionally cultured.

[0024]

[12] The method according to

[10] or

[11] above, wherein refrigerated adherent cells are subjected to a heating treatment and then subjected to two-dimensional culture.

[0025]

[13] A cell preservation solution containing a high molecular weight polymer for preserving adherent cells in a non-frozen state under refrigeration.

[0026]

[14] The cell preservation solution described in

[13] above, wherein the high molecular weight polymer comprises deacylated gellan gum or a salt thereof.

[0027]

[15] The cell preservation solution according to

[13] or

[14] above, for refrigerated preservation of adherent cells while maintaining their ability to adhere to a culture substrate in two-dimensional culture.

[0028]

[16] The cell preservation solution according to any one of

[13] to

[15] above, for refrigerating and preserving adherent cells for up to 100 hours.

[0029]

[17] The cell preservation solution according to any one of

[13] to

[16] above, wherein the adherent cells are hepatocytes.

[0030]

[18] The cell preservation solution according to any one of

[13] to

[17] above, further comprising an iron chelating agent.

[0031] This specification includes the disclosure of Japanese Patent Application No. 2023-186162, from which the present application claims priority.

[0032] According to the present invention, it is possible to store adherent cells for a long period of time under refrigeration while maintaining their ability to adhere to a culture substrate. According to the present invention, it is also possible to two-dimensionally culture adherent cells after long-term refrigeration storage while maintaining their cellular properties.

[0033] Figure 1 shows photographs of cells in UW solution and UW+FP solution 24 hours after refrigeration. Figure 2 shows the viable cell recovery rate (A) and viability (B) of cells refrigerated for 24, 48, 72, or 96 hours in UW solution and UW+FP solution. Figure 3 shows the plating index (plating index) of cells refrigerated for 24, 48, 72, or 96 hours in UW solution and UW+FP solution 24 hours after plating. Figure 4 shows phase-contrast images (B) of cells refrigerated for 24, 48, 72, or 96 hours in UW solution and UW+FP solution 24 hours after plating. For comparison, a phase-contrast image (A) of freshly isolated HepaSH cells from humanized liver is also shown. The white bar in the lower right corner of each photograph represents a 200 μm scale. Figure 5 shows the ATP content (A) of cells refrigerated for 24 hours in UW solution and UW+FP solution, the ROS activity (B) measured by the mean fluorescence intensity derived from DCFH-DA, and the LDH activity (C) in the medium 4 hours after plating. Figure 6 shows phase-contrast images of cells plated on days 1, 3, 5, and 7 after refrigerated storage in UW+FP solution for 72 hours. For comparison, phase-contrast images of freshly isolated HepaSH cells immediately after isolation from humanized liver are also shown. The white bar in the lower right corner of each photograph represents a 200 μm scale. Figure 7 shows the drug-metabolizing enzyme activity of cells plated on days 1 (A), 4 (B), and 8 (C) after refrigerated storage in UW+FP solution for 72 hours. In the figure, 1A2, 2C9, 2C19, 2D6, and 3A4 / 5 represent phenacetin O-deethylation activity by CYP1A2, diclofenac 4'-hydroxylation activity by CYP2C9, omeprazole 5'-hydroxylation activity by CYP2C19, metoprolol O-demethylation activity by CYP2D6, and midazolam 1'-hydroxylation activity by CYP3A4 / 5, respectively. Figure 8 is a photograph showing the results of immunostaining of the liver 5 weeks after transplantation of HepaSH cells refrigerated in UW+FP solution for 72 hours and re-transplanted into the liver of a TK-NOG-hIL6 mouse after liver injury induction.A: H&E staining; B: staining with anti-human mitochondrial antibody. Figure 9 shows the results of flow cytometry analysis of cells isolated from the livers of TK-NOG-hIL6 mice 6 weeks after transplantation of HepaSH cells refrigerated for 72 hours in UW+FP solution. The cells were then seeded onto a plate and maintained in a culture medium. The white bar in the lower right corner of the photograph in Figure 9B represents a 200 μm scale. Figure 10 shows the adhesion efficiency of cells refrigerated for 24 or 96 hours in UW solution, UW+FP solution, UW+Def solution, or UW+FP&Def solution 24 hours after seeding onto a plate. Figure 11 shows phase-contrast images of cells refrigerated for 24 or 96 hours in UW solution, UW + FP solution, UW + Def solution, or UW + FP & Def solution, 24 hours after seeding onto plates. The white bar in the lower right corner of each photograph represents a 200 μm scale. Figure 12 shows the results of a permeation test of fluorescent substances added to the medium after 7 days of maintenance culture on transwell plates for cells refrigerated for 96 hours in UW solution, UW + FP solution, or UW + FP & Def solution. Figure 13 shows phase-contrast images of cells refrigerated for 96 hours in UW solution (B), UW + FP solution (C), or UW + FP & Def solution (D) on the 7th day of plate culture. For comparison, a phase-contrast image (A) of freshly isolated HepaSH cells from humanized liver is also shown. Figure 14 shows phase-contrast images of cells refrigerated for 72 hours in UW or UW+FP solution, followed by pre-warming or non-pre-warming (control) treatment, seeded onto a collagen I-coated 24-well plate 24 hours later. Figure 15 shows the viable cell recovery (A), viability (B), ROS activity (C), and retention efficiency (D) of cells refrigerated for 72 hours in UW or UW+FP solution, followed by pre-warming or non-pre-warming treatment. Open bars: no pre-warming; shaded bars: pre-warming. Figure 16 shows retention efficiency of cells refrigerated for 96 hours in UW+FP solution (control) or UW+FP solution with Def (UW+FP&Def solution), followed by pre-warming or non-pre-warming (control).Figure 17 shows phase-contrast photographs of cells that were refrigerated for 96 hours in UW+FP solution (control) or UW+FP solution with Def added (UW+FP&Def solution), and then pre-warmed or not pre-warmed, 24 hours after seeding onto plates.

[0034] The present invention will be described in detail below.

[0035] The present invention provides a cell preservation solution containing a polymeric polymer. The present invention also provides a method for preserving adherent cells, particularly adherent cells, in an unfrozen state in the presence of a polymeric polymer, more specifically in a cell preservation solution containing a polymeric polymer. The present invention also provides a method for culturing adherent cells, comprising two-dimensionally culturing adherent cells preserved according to such a preservation method.

[0036] In particular, the present invention relates to a method for preserving adherent cells, which comprises preserving adherent cells in a cell preservation solution containing a high molecular weight polymer under refrigeration in an unfrozen state.

[0037] In the present invention, the term "cell preservation solution" refers to a liquid composition suitable for stably preserving cells while maintaining their viability. The cell preservation solution of the present invention is different from a liquid medium suitable for cell growth. In a preferred embodiment, the "cell preservation solution" of the present invention may be a cell preservation solution for non-cryopreservation or refrigerated preservation. The "cell preservation solution" of the present invention is not a cell preservation solution for cryopreservation. The "cell preservation solution" of the present invention comprises an aqueous solvent and a polymer.

[0038] The "cell preservation solution" of the present invention may be prepared based on a commercially available or existing cell preservation solution or organ preservation solution, or may be newly prepared. In one embodiment, the "cell preservation solution" of the present invention is a non-cryopreservative solution such as Belzer UW （Ｒ） It may also be prepared using cold preservative solution (UW solution) as a base. （Ｒ）The cold preservation solution (UW solution) has the following composition: hydroxyethyl starch (pentafraction) 50 g / L, lactobionic acid (as lactone) 35.83 g / L, potassium dihydrogen phosphate 3.4 g / L, magnesium sulfate heptahydrate 1.23 g / L, raffinose pentahydrate 17.83 g / L, adenosine 1.34 g / L, allopurinol 0.136 g / L, total glutathione 0.922 g / L, potassium hydroxide 5.61 g / L, sodium hydroxide / hydrochloric acid (added to adjust the pH to 7.4 at 20° C.), and water. In one embodiment, Belzer UW solution （Ｒ） A solution having the composition of a cold preservation solution (UW solution) or a solution containing the same and also containing a high molecular weight polymer can be used as the cell preservation solution in the present invention.

[0039] In the present invention, a high-molecular-weight polymer refers to a polymer having a weight-average molecular weight of 10,000 or more. The molecular weight of a high-molecular-weight polymer can be determined, for example, by gel permeation chromatography (GPC) in terms of pullulan. Examples of high-molecular-weight polymers used in the present invention include, but are not limited to, polysaccharides. In a preferred embodiment, the polysaccharide used as the high-molecular-weight polymer of the present invention may be an acidic polysaccharide having an anionic functional group. Specific examples of high-molecular-weight polymers include, but are not limited to, those composed of one or more selected from the group consisting of deacylated gellan gum, gellan gum, hyaluronic acid, rhamsan gum, diutan gum, xanthan gum, carrageenan, xanthan gum, hexuronic acid, fucoidan, pectin, pectic acid, pectinic acid, alginic acid, heparan sulfate, heparin, heparitin sulfate, keratosulfate, chondroitin sulfate, dermatan sulfate, and rhamnan sulfate, as well as salts thereof. With respect to the polymer, the "salt" may be, for example, a salt of an alkali metal such as lithium, sodium, or potassium; a salt of an alkaline earth metal such as calcium, barium, or magnesium; a salt of aluminum, zinc, copper, or iron; an ammonium salt; a salt with an organic amine; or a salt with an amino acid. In a more preferred embodiment, the polymer of the present invention comprises deacylated gellan gum or a salt thereof. In one embodiment, the cell preservation solution of the present invention contains only deacylated gellan gum or a salt thereof as the polymer. In another embodiment, the cell preservation solution of the present invention contains, in addition to deacylated gellan gum or a salt thereof, another polymer such as another polysaccharide, for example, alginic acid, pectin, or pectic acid, as the polymer. In another embodiment, the cell preservation solution of the present invention contains, in addition to deacylated gellan gum or a salt thereof, as the polymer, an acidic polysaccharide (e.g., alginic acid, pectin, or pectic acid) or a salt thereof that maintains a random coil state in a divalent metal cation medium and can be crosslinked via divalent metal ions.When the cell preservation solution of the present invention contains, in addition to deacylated gellan gum or a salt thereof, an acidic polysaccharide (e.g., alginic acid, pectin, or pectic acid) or a salt thereof that maintains a random coil state in a divalent metal cation medium and can be crosslinked via divalent metal ions as a polymer, it may further contain a divalent metal cation such as calcium ion. The deacylated gellan gum or a salt thereof may be phosphorylated. The weight-average molecular weight of the deacylated gellan gum or a salt thereof is preferably 10,000 to 50,000,000, more preferably 1,000,000 to 10,000,000. Commercially available deacylated gellan gum or a salt thereof may be used. In one embodiment, the Polymer FP series manufactured by Nissan Chemical Industries, Ltd., such as FP001, may be used. In one embodiment, FCeM. （Ｒ） FP001 solution included as a component of the Preparation Kit (Nissan Chemical, product code 385-07981) can be used as the high molecular weight polymer reagent. In another embodiment, "KELCOGEL (registered trademark of CP Kelco) CG-LA" manufactured by Sansho Co., Ltd. may be used as the deacylated gellan gum or a salt thereof.

[0040] In one embodiment, the cell preservation solution containing the polymer of the present invention may contain the polymer, for example, deacylated gellan gum or a salt thereof, at a concentration of 0.001 to 1% (W / V), preferably 0.005 to 0.3% (W / V), more preferably 0.01 to 0.3% (W / V), for example, 0.01 to 0.03% (W / V) or 0.015 to 0.03% (W / V). The concentration of deacylated gellan gum or a salt thereof can be expressed as a value converted to free deacylated gellan gum. In this specification, % (W / V) means weight / volume %.

[0041] In the present invention, adherent cells refer to cells that can be maintained in a state of adhering (attaching) to a culture substrate. On the other hand, suspension cells refer to cells that cannot adhere (attach) to a culture substrate. Suspension cells gradually lose their function when cultured in a liquid medium, making it difficult to maintain and culture them for a long period of time. Maintenance culture refers to culturing cells while maintaining their morphology and function. Adherent cells can be successfully used in two-dimensional and three-dimensional culture.

[0042] The adherent cells used in the present invention may be derived from any organ or tissue, for example, hepatocytes (i.e., adherent hepatocytes), but are not limited thereto. The adherent cells may be any animal cells, for example, mammalian cells such as primate cells, canine cells, or feline cells. The adherent cells may be human cells or non-human animal cells, for example, non-human mammalian cells (non-human primate cells). In a preferred embodiment, the adherent cells may be human hepatocytes (i.e., human adherent hepatocytes).

[0043] The adhesive cells used in the present invention are preferably, but not limited to, primary cells. The adhesive cells used in the present invention may or may not be immortalized cells. The adhesive cells used in the present invention may also be cells induced to differentiate from stem cells (e.g., iPS cells, mesenchymal stem cells, etc.). In the present invention, "primary cells" refers to cells collected or isolated from biological tissue that can only proliferate (divide) a finite number of times. In the present invention, "primary cells" not only refer to cells collected or isolated from natural biological tissue, but also include cells isolated from tissues or organs reconstructed by transplanting primary cells into the bodies of non-human animals with a deficient or reduced immune response to humans, such as immunodeficient or immunotolerant animals (e.g., into the liver after liver damage induction). Examples of non-human animals with a deficient or reduced immune response to humans that are used for reconstructing tissues or organs from primary cells include, but are not limited to, non-human vertebrates with a deficient or reduced immune response to humans, as described in the specification of International Publication WO2020 / 122178, for example, TK-NOG-hIL6 mice (hyper-immunodeficient mice in which a human thymidine kinase gene and a human IL-6 gene have been introduced in an expressible manner into NOG mice derived from NOD / SCID mice and in which the IL2 receptor γ-chain gene has been knocked out).

[0044] The concentration of adherent cells in the cell preservation solution containing the above-mentioned polymer during preservation may be any cell concentration, but in one embodiment, it is preferably 1 x 10 ６ ~1x10 ８ cells / mL, more preferably 5 x 10 ６ ~7x10 ７ cells / mL, e.g., 1 x 10 ７ It may be a concentration of cells / mL.

[0045] In the method of the present invention, adherent cells are preserved in a cell preservation solution containing the above-mentioned polymer, and preferably refrigerated in an unfrozen state. In the present invention, "refrigerated storage" refers to storage at a temperature above 0°C to 10°C, typically 2 to 5°C, for example, 4°C.

[0046] According to the present invention, adherent cells can be preserved in a non-frozen state (e.g., refrigerated) for an extended period of time in a cell preservation solution containing a polymer. The time for which adherent cells are preserved in a non-frozen state in a cell preservation solution containing a polymer according to the present invention may be, but is not limited to, up to 100 hours, for example, up to 96 hours, or up to 72 hours. According to the present invention, the time for which adherent cells are preserved in a non-frozen state in a cell preservation solution containing a polymer may be 1 hour or more, typically 24 hours or more, preferably 40 hours or more, for example, 48 hours or more, 70 hours or more, or 72 hours or more, or may be 1 to 100 hours, 24 to 100 hours, 40 to 100 hours, 48 ​​to 100 hours, 70 to 100 hours, 72 to 100 hours, 1 to 96 hours, 24 to 96 hours, 40 to 96 hours, 48 ​​to 96 hours, 70 to 96 hours, 72 to 96 hours, 72 to 100 hours, or 24 to 72 hours.

[0047] In the cell preservation solution of the present invention, the adherent cells are preferably in a suspended state, and in this case, the adherent cells can be refrigerated and preserved in a suspended state. In the present invention, it is believed that maintaining the suspended state of the cells in the preservation solution without settling reduces ischemic stress, thereby enhancing the preservation effect.

[0048] In the method of the present invention, adherent cells are stored in a non-frozen state (e.g., refrigerated) in a cell preservation solution containing the above-mentioned high molecular weight polymer, allowing the cells to be stably preserved without impairing their ability to adhere to the culture substrate.

[0049] In the present invention, the "ability to adhere" of adherent cells to a culture substrate refers to the ability of the adherent cells to attach to the culture substrate, reach a confluent state, and maintain that confluent state when the adherent cells are seeded on the culture substrate and cultured in two dimensions. In the present invention, the "confluent state" refers to a state in which the seeded adherent cells occupy 80% or more of the surface area of ​​the culture surface of the culture substrate (80% or more confluency). Meanwhile, in the present invention, "adhesion" refers to the attachment of cells to the culture substrate in a culturable state.

[0050] In a preferred embodiment, adherent cells stored in a refrigerated, unfrozen state in a cell preservation solution containing the above-mentioned polymer retain their ability to adhere to a culture substrate, and therefore can be suitably used for solid-phase culture on a culture substrate, particularly two-dimensional culture (also known as monolayer culture), after refrigerated storage in the cell preservation solution. Therefore, the adherent cell preservation method of the present invention can be a method for refrigerating and preserving adherent cells in a refrigerated, unfrozen state in a cell preservation solution containing the above-mentioned polymer while retaining their ability to adhere to a culture substrate in two-dimensional culture. Alternatively, the adherent cell preservation method of the present invention can be a method for preserving adherent cells to be subjected to two-dimensional culture (e.g., plate culture) after refrigerated storage in a refrigerated, unfrozen state in a cell preservation solution containing the above-mentioned polymer.

[0051] When primary hepatocytes (PHH) are suspended in a conventional cell preservation solution and placed under refrigerated conditions (4°C, on ice), they can be stored for a short period of time while maintaining their high ability to adhere to the culture substrate. However, prolonged refrigerated storage reduces the ability of PHH to adhere to the culture substrate, resulting in a state where gaps exist between the cells (non-confluent state). PHH cultured in a state where gaps exist between the cells (non-confluent state) undergo changes in cell morphology and also lose hepatocyte characteristics (e.g., drug-metabolizing enzyme activity). By refrigerating and preserving PHH in an unfrozen state in a cell preservation solution containing the above-described polymeric polymer according to the method of the present invention, adherent cells, including hepatocytes, can retain their ability to adhere to the culture substrate, thereby maintaining other cellular characteristics of the adherent cells. In a preferred embodiment, adherent cells stored in a cell preservation solution containing the above-described polymeric polymer maintain their cell morphology. In a preferred embodiment, adherent cells stored in a cell preservation solution containing the above-described polymer maintain their cell-specific enzyme activity. For example, when adherent cells, such as hepatocytes, are refrigerated and preserved as described above, their cell morphology is maintained, and drug-metabolizing enzyme activity is maintained. Examples of drug-metabolizing enzyme activity include, but are not limited to, cytochrome P450 enzyme activities such as phenacetin O-deethylation activity by CYP1A2, diclofenac 4'-hydroxylation activity by CYP2C9, omeprazole 5'-hydroxylation activity by CYP2C19, metoprolol O-demethylation activity by CYP2D6, and midazolam 1'-hydroxylation activity by CYP3A4 / 5. Furthermore, adherent cells preserved under refrigeration in a cell preservation solution containing the above-described polymer maintain a healthier state, as indicated by, for example, an increase in intracellular ATP levels, and exhibit suppressed increases in ROS activity, which indicates ischemic stress. Adherent cells refrigerated in a cell preservation solution containing the above-mentioned polymer exhibit enhanced plating efficiency (plating index) on the culture substrate on which they are seeded after refrigerated storage. Refrigerated storage of adherent cells in a cell preservation solution containing the above-mentioned polymer is thought to significantly reduce ischemic stress on the adherent cells, which also leads to the above-mentioned effects.

[0052] In the present invention, an iron chelating agent may be further added to the cell preservation solution containing the above-mentioned polymer. In the present invention, a cell preservation solution further containing an iron chelating agent in addition to the above-mentioned polymer can be used. The iron chelating agent may include, but is not limited to, deferoxamine and / or ferrostatin. The iron chelating agent may be added to the cell preservation solution containing the above-mentioned polymer at a concentration of preferably 10 μM to 5 mM, for example, 100 μM to 1 mM, or 300 μM to 700 μM, but is not limited to these ranges. By further adding an iron chelating agent to the cell preservation solution containing the above-mentioned polymer, the plating efficiency (plating index) of adherent cells is further enhanced, and the plating ability of adherent cells is improved.

[0053] The adherent cells refrigerated according to the above-described adherent cell preservation method retain their high ability to adhere to a culture substrate, and are therefore suitable for two-dimensional culture. The present invention also provides a method for culturing adherent cells, comprising refrigerating the adherent cells in a cell preservation solution containing the above-described polymer according to the above-described adherent cell preservation method, and then two-dimensionally culturing the adherent cells.

[0054] Such a method for culturing adherent cells may include refrigerating and preserving the adherent cells using a method for preserving adherent cells, seeding the adherent cells on a culture substrate, and performing two-dimensional culture.

[0055] In the present invention, a culture substrate refers to a solid-phase material or substance to which cells can adhere and function as a scaffold during culture. Examples of culture substrates include, but are not limited to, culture plates such as multiwell plates, dishes, flasks, bottles, glass slides, cover slips, films, membranes, porous carriers, hollow fibers, and fibers. Culture substrates may be made of any cell scaffold material, including, but not limited to, glass; plastics such as polystyrene, polyethylene terephthalate, polysulfone, polyethersulfone, and polycarbonate; metals such as silver and gold; metal oxides such as indium-tin oxide; and ceramics. The surface (culture surface) of the culture substrate may also be coated with a scaffold material such as collagen, elastin, fibronectin, vitronectin, laminin, or gelatin.

[0056] In one embodiment, adherent cells preserved in the cell preservation solution containing the polymer can be seeded on a collagen-coated culture substrate and cultured in two dimensions. Examples of collagen used for coating include, but are not limited to, collagen I and collagen nanofiber.

[0057] Two-dimensional culture is a monolayer culture on a culture substrate (solid phase). When the surface of the culture substrate on which the adherent cells are seeded is flat, the two-dimensional culture is called a plate culture. Two-dimensional culture of adherent cells stored in a cell preservation solution on a culture substrate may be a plate culture.

[0058] Two-dimensional culture of adherent cells may be performed under typical two-dimensional culture conditions. Two-dimensional culture is preferably performed under heated conditions. In one embodiment, two-dimensional culture can be performed at a temperature of preferably 30 to 40°C, more preferably 35 to 40°C, even more preferably 35 to 38°C, and typically 37°C. In one embodiment, two-dimensional culture can be performed for, for example, 1 hour to 6 months, preferably 10 hours to 3 months, or 20 hours to 2 weeks.

[0059] In the method of the present invention, adherent cells refrigerated and stored in a cell preservation solution containing the above-mentioned polymer may be subjected to a heating treatment and then subjected to two-dimensional culture. The heating treatment may be any heating treatment that can reduce cold stress on cells. The heating treatment can be carried out by suspending the adherent cells refrigerated and stored in a cell preservation solution containing the above-mentioned polymer in a liquid medium such as a seeding medium, and then incubating the cells at preferably 30 to 40°C, more preferably 35 to 40°C, even more preferably 35 to 38°C, and typically 37°C, for 20 to 60 minutes, for example, 30 to 60 minutes. By subjecting the refrigerated-stored adherent cells to a heating treatment and then subjecting them to two-dimensional culture, cold stress during refrigerated storage of the adherent cells can be reduced and the plating efficiency (plating index) can be improved.

[0060] The present invention also provides a cell preservation solution containing the above-described polymer, which is suitable for use in the cell preservation method and cell culture method of the present invention. The cell preservation solution containing the above-described polymer of the present invention is suitable for preserving cells, particularly adherent cells, in an unfrozen state for a long period of time (e.g., refrigerated storage). The cell preservation solution containing the above-described polymer of the present invention may be used to preserve adherent cells in an unfrozen state for up to 100 hours, for example, up to 96 hours, or up to 72 hours, under refrigeration. The cell preservation solution of the present invention containing the above-mentioned high molecular weight polymer may be used to refrigerate and preserve adherent cells in an unfrozen state for 1 hour or more, typically 24 hours or more, preferably 40 hours or more, for example, 48 hours or more, 70 hours or more, or 72 hours or more, or alternatively, 1 to 100 hours, 24 to 100 hours, 40 to 100 hours, 48 ​​to 100 hours, 70 to 100 hours, 72 to 100 hours, 1 to 96 hours, 24 to 96 hours, 40 to 96 hours, 48 ​​to 96 hours, 70 to 96 hours, 72 to 96 hours, 72 to 100 hours, or 24 to 72 hours.

[0061] The cell preservation solution containing the polymer of the present invention may also be used for refrigerating and preserving adherent cells while maintaining their ability to adhere to a culture substrate in two-dimensional culture. The cell preservation solution containing the polymer of the present invention may be used for preserving adherent cells to be subjected to two-dimensional culture after long-term refrigerated storage in an unfrozen state.

[0062] The cell preservation solution containing the polymer of the present invention can maintain the ability of adherent cells to adhere to a culture substrate during refrigerated storage and can be used to improve the preservation of adherent cells during refrigerated storage. The cell preservation solution containing the polymer of the present invention is particularly suitable for refrigerated storage of hepatocytes, including human hepatocytes.

[0063] The present invention will be described in more detail below using examples, although the technical scope of the present invention is not limited to these examples.

[0064] In the following examples, all p values ​​in statistical analyses were calculated by paired t-tests of two groups, and p<0.05 was considered to indicate a significant difference.

[0065] [Example 1] Preparation of a cell preservation solution containing a high-molecular-weight polymer and preservation of cells in the preservation solution FCeM containing a high-molecular-weight polymer FP001 solution as a component （Ｒ） A preparation kit (Nissan Chemical, product code 385-07981) was used to prepare the cell preservation solution. The high polymer FP001 solution contains deacylated gellan gum. （Ｒ） Cold preservation solution (Preservation Solutions, Inc.; purchased from Astellas Pharma; product code 01544782; hereinafter also referred to as UW solution) was used as the base of the cell preservation solution.

[0066] FCeM （Ｒ） According to the instructions in the Preparation Kit's user's manual, FP001 solution was added to UW solution and mixed to prepare a cell preservation solution UW+FP solution (final concentration of high molecular weight polymer: 0.02% (W / V)). The prepared UW+FP solution was used for cell preservation as described below without dilution.

[0067] To confirm the cell preservation effect in UW+FP solution, human hepatocyte HepaSH cells (Uehara et al., Biochemical and Biophysical Research Communications, 663 (2023) 132-141; Central Institute for Experimental Animals, Japan) were used. HepaSH cells are isolated from humanized livers reconstructed by transplanting human primary hepatocytes (PHH) into the livers of hyperimmunodeficient TK-NOG-hIL6 mice (Uehara et al., Biochemical and Biophysical Research Communications, 663 (2023) 132-141; International Publication WO 2020 / 122178) after liver damage induction. Six samples of HepaSH cells isolated from six different mice were used.

[0068] HepaSH cells were added to UW solution or UW+FP solution at a concentration of 1 x 10 ７ After preparing a cell suspension at a concentration of 1000 cells / mL, the suspension was stored in a 15 mL centrifuge tube (Greiner Bio-One) at 4°C for 96 hours (refrigerated storage). Figure 1 shows the state of the cells in the storage solution 24 hours after the start of storage. Cells suspended in the UW solution precipitated, but cells suspended in the UW+FP solution did not precipitate and remained floating even after 24 hours of storage.

[0069] During the 96-hour storage period, cells in UW solution or UW+FP solution were sampled every 24 hours. Trypan blue solution (Thermo Fisher Scientific, product code 15250061) was added to the obtained cell samples, and the viable and total cell counts were counted using a hemocytometer (FMG, product code 521-10). The ratio of the viable cell count at each sampling time point to the viable cell count at the start of storage (viable cell recovery rate; % Recovery) and the ratio of the viable cell count to the total cell count (including live and dead cells) at each sampling time point (viability rate; % Viability) were calculated. The results are shown in Figure 2. There was no significant difference in the viable cell recovery rate or viability of cells stored in UW+FP solution compared to UW solution, indicating that the addition of the high-molecular-weight polymer FP does not affect these.

[0070] In addition, cells in the UW solution or the UW+FP solution were sampled every 24 hours during the 96-hour storage period, and the ability of the cells to settle on the scaffold after storage was tested. ６ The cells were suspended in a seeding medium (William's E medium + 10% fetal bovine serum, 100 U / mL penicillin, 100 mg / mL streptomycin, 5 mg / mL insulin) at a concentration of 100 cells / mL, and the suspension was seeded at 0.5 mL / well on a collagen I-coated 24-well plate (Corning; product code 356408). ２ Incubator (37°C, 5% CO ２ ) overnight. 24 hours after seeding, the medium was replaced with a seeding medium containing the nuclear staining fluorescent dye Hoechst 33342 (Dojindo Research Institute, product code H342). After 30 minutes of incubation for staining, phase-contrast images were taken using EVOS cell imaging systems (Thermo Fisher Scientific) to measure the average number of nuclei per area (within a 200 μm grid). For comparison, freshly isolated HepaSH cells from humanized livers were suspended in seeding medium and seeded onto collagen I-coated 24-well plates in the same manner as above. After 24 hours of culture, they were stained and the average number of nuclei was measured. The ratio of the average number of nuclei 24 hours after seeding of cells preserved in the cell preservation solution to the average number of nuclei (100%) 24 hours after seeding of HepaSH cells immediately after isolation was calculated and used as the plating index. The results are shown in Figures 3 and 4.

[0071] Throughout the storage period, cells stored in UW+FP solution exhibited higher fixation efficiency than cells stored in UW solution, with the fixation efficiency remaining at approximately 80% even after 72 hours of storage (Figure 3). Phase contrast images (Figure 4) also revealed that cells stored in UW solution for 48 hours or longer had gaps between the cells when plated and did not maintain a confluent state, whereas cells stored in UW+FP solution maintained a confluent state with no gaps between the cells even after 72 hours of storage.

[0072] It was shown that by storing cells in a preservation solution containing a high molecular weight polymer, the cell's ability to adhere can be maintained even after long-term storage.

[0073] Example 2 Reduction of ischemic stress in cells preserved in a high molecular weight polymer-containing preservation solution HepaSH cells (30 specimens) were cultured in UW solution or UW+FP solution at 1 x 10 ７ The cells were suspended at a concentration of 1000 cells / mL and stored for 24 hours at 4° C. After storage at 4° C. for 24 hours, the number of viable cells was counted.

[0074] Next, the amount of ATP per 1,000 cells after storage at 4°C for 24 hours was measured using CellTiter-Glo. （Ｒ） Viability was determined using a Promega 2.0 Cell Viability Assay and measured by measuring luminescence with an EnSpire multimode plate reader (PerkinElmer). The results are shown in Figure 5A. Cells preserved in UW+FP solution showed statistically significantly higher ATP levels than those preserved in UW solution. During cell death, intracellular ATP levels decrease. These results suggest that preservation in the presence of high-molecular-weight polymers maintains cells in a healthier state, even after prolonged storage.

[0075] In addition, cells stored at 4°C for 24 hours were measured for the amount of reactive oxygen species (ROS) (ROS activity), an indicator of ischemic stress in the cells after storage, using a total ROS detection kit (Dojindo Research Institute, product code R252). After 24 hours of storage at 4°C, the cells were stained with an anti-HLA antibody (BD, product code 555555) and the Highly Sensitive DCFH-DA dye included in the kit according to the kit's instructions, and then suspended in loading buffer (1x) supplemented with propidium iodide (PI). The stained cells were analyzed using a FACS Aria II cell sorter (BD). The mean fluorescence intensity (FITC mean) derived from DCFH-DA was calculated for PI-negative (indicating viable cells) and HLA-positive (indicating human hepatocytes) cell populations and compared between UW solution and UW+FP solution. The results are shown in Figure 5B. The FITC mean was statistically significantly higher for cells preserved in UW solution compared to cells preserved in UW+FP solution. In this assay, the greater the amount of intracellular ROS accumulation, the higher the FITC mean. That is, cells preserved in UW solution showed increased ROS accumulation, indicating enhanced ischemic stress. These results indicate that preservation in the presence of a high-molecular-weight polymer reduces ischemic stress in cells.

[0076] Furthermore, after 24 hours of storage at 4°C, cells were suspended in the above-mentioned seeding medium, seeded onto collagen I-coated 24-well plates, and cultured for 4 hours. The culture supernatant was then collected. Lactate dehydrogenase (LDH) activity in the collected culture supernatant was measured as an indicator of cell death using the CytoTox-ONE Homogeneous Membrane Integrity Assay (Promega, product code G7890) according to the manufacturer's instructions. Fluorescence measurements were performed using an Infinite F200F Pro plate reader (TECAN). The fluorescence intensity measured for the culture supernatant was normalized to that measured in unused seeding medium (control medium). The results are shown in Figure 5C. Cells stored in UW solution had statistically significantly higher LDH activity than cells stored in UW+FP solution. When cell death increases, LDH leaks from cells into the medium, increasing the LDH activity of the culture supernatant. In other words, storage in UW solution increased cell death after seeding. It is believed that storage in UW solution increased cell death after seeding due to increased ischemic stress. These results indicate that storage in the presence of a high-molecular-weight polymer can reduce cell death after seeding on a culture substrate due to increased ischemic stress.

[0077] [Example 3] Maintenance of cell characteristics after preservation in cell preservation solution HepaSH cells (four specimens) were cultured in UW+FP solution at 1 x 10 ７ The cells were suspended at a concentration of 1000 cells / mL and stored at 4°C for 72 hours. The cells thus stored in a refrigerator for 72 hours were seeded onto a collagen I-coated 24-well plate and cultured for 8 days (maintenance culture). As a control, HepaSH cells immediately after isolation from humanized liver (freshly isolated HepaSH cells) were seeded onto a collagen I-coated 24-well plate and similarly cultured for 8 days (maintenance culture). Four hours after seeding, the medium was replaced with Cellartis （Ｒ） Power ＴＭThe medium was then changed to Primary HEP medium (Takara Bio, product code Y20020), and the medium was replaced with the same medium on day 1 (the day after seeding), day 3, day 5, and day 7 of culture. Figure 6 shows phase contrast images of the cells on days 1, 3, 5, and 7 of culture. Throughout the culture period up to day 7, there was no difference in cell morphology depending on whether or not they had been preserved in the cell preservation solution, and the confluent state was maintained in plate culture.

[0078] During the culture period up to day 7, the activities of major drug-metabolizing enzymes in the cells were measured on days 1, 4, and 8 of culture. HepaSH cells harvested on days 1, 4, and 8 of culture were incubated for 1 hour at 37°C in Williams' Medium E without Phenol Red (Thermo Fisher Scientific, product code A12176) supplemented with phenacetin (a substrate for CYP1A2) (100 μM), diclofenac (a substrate for CYP2C9) (40 μM), omeprazole (a substrate for CYP2C19) (10 μM), metoprolol (a substrate for CYP2D6) (5 μM), and midazolam (a substrate for CYP3A4 / 5) (5 μM), which are typical substrates of the drug-metabolizing enzyme group human cytochrome P450. Metabolites contained in the medium were then measured using a liquid chromatography tandem mass spectrometer. The metabolites of these substrates were measured as indicators of the activity of each drug-metabolizing enzyme, including acetaminophen due to the O-deethylation of phenacetin by CYP1A2, 4'-hydroxydiclofenac due to the 4'-hydroxylation of diclofenac by CYP2C9, 5'-hydroxyomeprazole due to the 5'-hydroxylation of omeprazole by CYP2C19, O-demethylated metoprolol due to the O-demethylation of metoprolol by CYP2D6, and 1'-hydroxymidazolam due to the 1'-hydroxylation of midazolam by CYP3A4 / 5. ＴＭThe cells were dissolved in MT Cell Lysis Reagent (Sigma-Aldrich, product code C3228), and protein concentration was quantified using a TaKaRa BCA Protein Assay Kit (Takara Bio, product code T9300A). The drug-metabolizing enzyme activity was expressed as the amount of metabolite produced per unit time and per unit protein weight. Figure 7 shows the drug-metabolizing enzyme activity expressed as the amount of metabolite per unit protein weight. A comparison with the drug-metabolizing enzyme activity in non-refrigerated cells showed that HepaSH cells refrigerated for 72 hours in UW+FP solution maintained drug-metabolizing enzyme activity throughout the culture period, up to day 8 of culture.

[0079] These results demonstrate that cells preserved in the presence of high molecular weight polymers can maintain their cellular properties even after long-term refrigerated storage. In particular, maintaining the activity of drug-metabolizing enzymes in hepatocytes is extremely important for the preservation and transportation of hepatocytes.

[0080] Furthermore, 1x10 ７HepaSH cells were suspended at a concentration of 1000 cells / mL and stored at 4°C for 72 hours. They were then transplanted via the spleen into the livers of TK-NOG-hIL6 mice after liver injury induction. Five weeks after transplantation, the livers were harvested and formalin-fixed. Paraffin sections were prepared and stained with hematoxylin and eosin (H&E) and immunostained with an anti-human mitochondrial antibody (Merck, product code MAB1273). Images were captured using a NanoZoomer slide scanner (Hamamatsu Photonics). The results are shown in Figure 8. It was demonstrated that the majority of the recipient mouse livers had been replaced with human mitochondria-positive human hepatocytes (Figures 8A and B). Furthermore, HepaSH cells were reisolated 6 weeks after transplantation from another TK-NOG-hIL6 mouse into which HepaSH cells stored in a refrigerator for 72 hours had been transplanted after liver injury induction. The isolated cells were stained with anti-HLA and anti-H2kD antibodies (BD, product code 553566) and analyzed by flow cytometry. More than 96% of the isolated cells were HLA-positive human hepatocytes (Figure 9A). Re-isolated HepaSH cells were seeded on collagen I-coated plates, enabling them to be cultured in the same manner as the cells before transplantation (Figure 9B). These results demonstrate that hepatocytes preserved in the presence of high-molecular-weight polymers retain their liver reconstitution capacity even after prolonged refrigerated storage.

[0081] [Example 4] Effects of Addition of Iron Chelators and Pre-warming 1) Addition of Iron Chelators We hypothesized that an iron chelator, which removes free iron ions from cells, would be effective in preventing ferroptosis, an iron-dependent cell death. Therefore, we investigated whether the addition of an iron chelator to a cell preservation solution, in addition to a high-molecular-weight polymer, would further improve the retention ability of cells preserved in the cell preservation solution.

[0082] HepaSH cells (9 samples) were cultured at 1 × 10 in UW solution or UW + FP solution with or without the addition of an iron chelating agent (final concentration 500 μM, deferoxamine; hereinafter also referred to as Def). ７The cells were suspended at a concentration of 1000 cells / mL and stored (refrigerated) at 4°C for 24 hours or 96 hours. Hereinafter, the UW solution with Def added will be referred to as UW+Def solution, and the UW+FP solution with Def added will be referred to as UW+FP&Def solution.

[0083] The cells after refrigeration were seeded onto collagen I-coated 24-well plates, and 24 hours after seeding, they were stained and the average number of nuclei was measured. From the measured average number of nuclei, the plating efficiency (plating index) was calculated in the same manner as in Example 1.

[0084] The results are shown in Figures 10 and 11. After 24 hours of refrigeration, cells stored in UW+FP, UW+Def, and UW+FP&Def showed statistically significant increases in fixation efficiency compared to UW solution (no addition), but no additive effect was observed with the combination of high-molecular-weight polymer and Def (Figure 10). On the other hand, after 96 hours of refrigeration, cells stored in UW+FP, UW+Def, and UW+FP&Def showed statistically significant increases in fixation efficiency compared to UW solution (no addition). Furthermore, the combination of high-molecular-weight polymer and Def in UW+FP&Def showed statistically significant increases in fixation efficiency compared to the addition of high-molecular-weight polymer or Def alone (Figure 10). Furthermore, especially after 96 hours of refrigeration, cells stored in UW or UW + Def solution did not maintain a confluent state in subsequent plate culture, whereas cells stored in UW + FP or UW + FP & Def solution maintained a confluent state in plate culture, as shown by phase contrast images (Figure 11). The combined use of a polymer that can prevent both ischemic stress and ferroptosis and an iron chelator maintained the cell adhesion ability, enabling confluent culture, even after 96 hours of storage in the cell preservation solution.

[0085] HepaSH cells were cultured at 1 × 10 in UW solution, UW + FP solution, or UW + FP & Def solution. ７The cells were suspended at a concentration of 1000 cells / mL and stored at 4°C for 96 hours. After suspending in seeding medium, the cells were seeded onto rat collagen I (Corning, product code 354236)-coated Transwell plates (Corning, product code 354495) for 7 days of maintenance culture (plate culture). On day 7 of culture, medium containing PE-streptavidin (BD, product code 554061) was added to the upper layer of the Transwell. The lower layer of the medium was collected 15, 30, and 60 minutes after the addition of PE-streptavidin, and fluorescence was measured using an Infinite F200F Pro plate reader (TECAN). In this assay, if the plate culture was not maintained at a confluent state, PE-streptavidin migrated from the upper layer to the lower layer through the gaps between the cells, resulting in the detection of fluorescence from the fluorescent dye PE (phycoerythrin) in the medium.

[0086] For comparison, fluorescence measurements were also performed in a well (Fr) seeded with freshly isolated HepaSH cells from humanized livers and in a well without cells (no cells). The measured fluorescence intensity was normalized to the value measured in unused seeding medium (control medium). The fluorescence intensity measurement results are shown in Figure 12. Figure 13 shows a phase-contrast image of cells on day 7 of plate culture.

[0087] In cells stored in UW solution for 96 hours, intercellular gaps were observed in phase-contrast images (Figure 13B), and fluorescence was detected 15 minutes after the addition of PE-streptavidin (Figure 12). In contrast, in cells stored in UW+FP solution or UW+FP&Def solution for 96 hours, intercellular gaps were not observed in phase-contrast images (Figures 13C and 13D). Similar to freshly isolated HepaSH cells (Fr), almost no fluorescence was detected even 30 minutes after the addition of PE-streptavidin (Figure 12). These results demonstrate that HepaSH cells stored for 96 hours in the presence of high-molecular-weight polymers or high-molecular-weight polymers and iron chelators maintained confluency even after 7 days of plate culture.

[0088] 2) Alleviating cold stress by pre-warming We hypothesized that cell death after seeding could be suppressed by suspending cryopreserved cells in seeding medium, incubating at 37°C for 30-60 minutes, and then seeding them onto collagen I plates. Therefore, we investigated the effect of adding a high molecular weight polymer to the cell preservation solution and also by pre-warming the cells before seeding.

[0089] HepaSH cells (10 specimens) were cultured at 1 × 10 in UW solution or UW + FP solution in the same manner as in Example 1. ７ The cells were suspended at a concentration of 1000 cells / mL and stored at 4°C for 72 hours, after which half of the suspension was collected and suspended in 10 times the volume of seeding medium. The cells were then pre-warmed by incubating them in a thermostatic water bath at 37°C for 30 minutes.

[0090] The pre-warmed cells were stained with trypan blue and counted using a hemocytometer to calculate the viable cell recovery rate and viability, according to the method described in Example 1. Furthermore, phase-contrast images were taken 24 hours after seeding, and the plating efficiency (plating index) was calculated according to the method described in Example 1. ROS activity was measured according to the method described in Example 2. As a control, a similar preservation test and measurement were performed, except that the pre-warming treatment was not performed. The results are shown in Figures 14 and 15.

[0091] Pre-warming tended to decrease the viable cell recovery rate for cells stored in UW solution, but it did not change much for cells stored in UW + FP solution (Fig. 15A). Because pre-warming promotes the death of damaged cells that have accumulated ischemic stress, the viable cell recovery rate decreased for cells stored in UW solution due to accumulated ischemic stress, whereas the viable cell recovery rate did not decrease for cells stored in UW + FP solution due to less accumulated ischemic stress and fewer damaged cells (Fig. 15A).

[0092] On the other hand, prewarming showed a statistically significant improvement in viability for cells preserved in both UW and UW+FP solutions (Figure 15B). Furthermore, prewarming also showed a statistically significant reduction in ROS activity and an increase in engraftment efficiency for cells preserved in both UW and UW+FP solutions (Figures 15C and 15D). Prewarming also reduces cold stress on cells, which is thought to be responsible for the improved viability, reduced ROS activity, and increased engraftment efficiency.

[0093] These results indicate that pre-warming treatment alleviates the cold stress of cells after refrigerated storage, even in the presence of high molecular weight polymers.

[0094] 3) Addition of iron chelating agent and pre-warming to alleviate cold stress HepaSH cells (6 specimens) were cultured at 1x10 in UW + FP solution or UW + FP & Def solution. ７ The cells were suspended at a concentration of 1000 cells / mL and stored at 4°C for 96 hours. After that, the cells were suspended in 10x the volume of seeding medium and pre-warmed by incubating at 37°C for 30 minutes in a thermostatic water bath. The pre-warmed cells were seeded onto collagen I-coated 24-well plates and cultured overnight. Phase-contrast images were taken 24 hours after seeding, and the plating efficiency (plating index) was calculated according to the method described in Example 1. The results are shown in Figures 16 and 17. Both the addition of an iron chelator and pre-warming were shown to statistically significantly enhance plating efficiency. When the addition of an iron chelator and pre-warming were combined, the plating efficiency was statistically significantly enhanced compared to the control (UW+FP solution, without iron chelator or pre-warming), but no additive or synergistic effects were observed due to the combination of the addition of an iron chelator and pre-warming. Phase contrast images showed that all cells stored under refrigeration were plated in a confluent state (FIG. 17).

[0095] Examples 1 to 4 show that ischemic stress in cells that occurs during preservation in a cell preservation solution becomes apparent after the cells are seeded on a culture substrate after preservation and culture is initiated. Examples 1 to 4 show that when cells that survived despite ischemic stress during preservation in a cell preservation solution were seeded on a culture substrate after preservation and cultured, cell death was accelerated and the cell retention efficiency in culture on the culture substrate was reduced. Furthermore, it was shown that the present invention can significantly improve such cell death and the reduction in retention efficiency.

[0096] The present invention enables adherent cells to maintain their ability to attach to a culture substrate during long-term refrigerated storage in an unfrozen state, thereby enabling long-distance transportation, such as air transportation, of adherent cells to be subjected to two-dimensional culture after refrigerated storage.

[0097] All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety.

Claims

1. A method for preserving adherent cells, comprising refrigerating and preserving adherent cells in a cell preservation solution containing a high molecular weight polymer in a non-frozen state.

2. The method of claim 1, wherein the high molecular weight polymer comprises deacylated gellan gum or a salt thereof.

3. The method of claim 1, wherein the adherent cells are stored refrigerated while retaining their ability to attach to a culture substrate in two-dimensional culture.

4. The method of claim 1, wherein the adherent cells are refrigerated for up to 100 hours.

5. The method of claim 1, wherein the adherent cells are refrigerated for 70 hours or more.

6. The method of claim 1, wherein the adherent cells are hepatocytes.

7. The method of claim 1, wherein the adherent cells are in suspension in a cell preservation solution.

8. The method of claim 1, wherein the cell preservation solution further comprises an iron chelator.

9. The method of claim 8, wherein the iron chelator comprises deferoxamine.

10. A method for culturing adherent cells, comprising refrigerating and preserving the adherent cells by the method according to any one of claims 1 to 9, and then two-dimensionally culturing the adherent cells.

11. The method according to claim 10, wherein the adherent cells are seeded on a collagen-coated culture substrate and cultured in two dimensions.

12. The method according to claim 10, wherein the refrigerated adherent cells are subjected to a heating treatment and then to two-dimensional culture.

13. A cell preservation solution containing a high molecular weight polymer for refrigerated preservation of adherent cells in a non-frozen state.

14. The cell preservation solution according to claim 13, wherein the high molecular weight polymer comprises deacylated gellan gum or a salt thereof.

15. The cell preservation solution according to claim 13, for refrigerated preservation of adherent cells while retaining their ability to attach to a culture substrate in two-dimensional culture.

16. The cell preservation solution according to claim 13, for refrigerated preservation of adherent cells for up to 100 hours.

17. The cell preservation solution according to claim 13, wherein the adherent cells are hepatocytes.

18. The cell preservation solution of claim 13, further comprising an iron chelating agent.

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