Cell preservation liquid and cell preservation method

Abstract

To provide a cell preservation liquid and a cell preservation method which enable long-term refrigerated preservation of adherent cells while maintaining adherence capability to a culture substrate.SOLUTION: 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-molecular-weight; a cultivation method for adherent cells which comprises two-dimensional culturing of adherent cells after preservation via the preservation method; and a cell preservation liquid which includes a high-molecular-weight that is suitable for use in these methods.SELECTED DRAWING: None

Description

[Technical field]

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

[0002] Plate culture of primary human hepatocytes (PHH) is widely used in the development process of new drugs, such as pharmacokinetic tests and toxicity analysis tests. It is known that when PHH are plate cultured, the expression level of drug-metabolizing enzymes decreases when the cell density is low (Non-Patent Document 1). In order to maintain the characteristics 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] There are two types of commercially available frozen PHH: 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 PHH can generally be cultured on a substrate for more than four weeks and can be used in vitro for a long period of time, making them more useful than suspension grade PHH that can only be used for about eight hours after thawing. However, a method for selectively producing plateable grade frozen PHH has not yet been established, and it is said that only about 30% of commercially available frozen PHH are plateable grade.

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

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

[0006] Non-Patent Document 3 reports that PHHs were refrigerated in a cell preservation solution of a special composition containing an iron chelating agent, and then heated to alleviate 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 a suspension culture medium composition containing deacylated gellan gum or a salt thereof. However, it does not disclose the ability of the cells stored in a refrigerated medium composition to adhere to a culture substrate, nor does it mention the effects on cells other than vascular smooth muscle cells.

[0008] Patent Document 2 discloses that cells or tissues can be maintained in a good viability for a long period of time by storing them 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, there is no disclosure about the ability of cells stored in the liquid composition under refrigeration to attach to a culture substrate. [Prior art documents] [Patent documents]

[0009] [Patent Document 1] International Publication WO2016 / 121896 [Patent Document 2] International Publication WO2019 / 049985 [Non-patent literature]

[0010] [Non-Patent Document 1] Yamasaki et al., PLOS ONE, 15(9): (2020) e0237809 [Non-Patent Document 2] Duret et al., Cell Transplantation, Vol. 24, pp. 2541-2555 (2015) [Non-Patent Document 3] Pless et al., Cell Transplantation, Vol. 21, pp. 23-37 (2012) Summary of the Invention [Problem to be solved by the invention]

[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 retaining their ability to adhere 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 retaining cell characteristics. [Means for solving the problem]

[0012] As a result of extensive research to solve the above-mentioned problems, the inventors discovered that when adherent cells were refrigerated for long periods of time in a cell preservation solution containing a high molecular weight polymer such as deacylated gellan gum or a salt thereof, 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 increased, thereby completing the present invention.

[0013] That is, the present invention includes the following. [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 according to [1] above, wherein the polymer comprises deacylated gellan gum or a salt thereof. [3] The method according to [1] or [2] above, wherein the adherent cells are refrigerated while retaining their ability to adhere to a culture substrate in two-dimensional culture. [4] The method according to any one of the above [1] to [3], wherein the adherent cells are refrigerated for up to 100 hours. [5] The method according to any one of [1] to [4] above, wherein the adherent cells are refrigerated for 70 hours or more. [6] The method according to any one of the above [1] to [5], wherein the adherent cells are hepatocytes. [7] The method according to any one of the above [1] to [6], wherein the adherent cells are in a suspended state in a cell preservation solution. [8] The method according to any one of [1] to [7] above, wherein the cell preservation solution further contains an iron chelating agent. [9] The method according to [8] above, wherein the iron chelating agent comprises deferoxamine.

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

[11] The method according to

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

[12] The method according to

[10] or

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

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

[14] The cell preservation solution described in

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

[15] The cell preservation solution according to

[13] or

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

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

[13] to

[15] above, for refrigerated preservation of adherent cells for up to 100 hours.

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

[13] to

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

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

[13] to

[17] above, further comprising an iron chelating agent. Effect of the Invention

[0014] According to the present invention, it is possible to store adherent cells for a long time in a refrigerator 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 storage in a refrigerator while maintaining their cellular characteristics. [Brief description of the drawings]

[0015] [Figure 1] FIG. 1 is a set of photographs showing the state of cells in UW solution and UW+FP solution 24 hours after the start of refrigerated storage. [Diagram 2] FIG. 2 shows the viability (A) and viability (B) of cells refrigerated and stored in UW solution or UW+FP solution for 24 hours, 48 ​​hours, 72 hours, or 96 hours. [Diagram 3] FIG. 3 shows the plating index 24 hours after plating of cells that had been refrigerated in UW solution or UW+FP solution for 24 hours, 48 ​​hours, 72 hours, or 96 hours. [Figure 4] Figure 4 shows a phase contrast image (B) of cells refrigerated for 24, 48, 72, or 96 hours in UW or UW+FP solution 24 hours after seeding on a plate. For comparison, a phase contrast image (A) of freshly isolated HepaSH cells from a humanized liver is also shown. The white bar at the bottom right of each photograph is a scale bar of 200 μm. [Diagram 5] Figure 5 shows the ATP content in cells refrigerated for 24 hours in UW solution and UW+FP solution (A), ROS activity measured using the mean fluorescence intensity derived from DCFH-DA (B), and LDH activity in the medium 4 hours after seeding on a plate (C). [Figure 6] Figure 6 shows phase contrast images of cells seeded on a plate after refrigerated storage in UW+FP solution for 72 hours on the 1st, 3rd, 5th, and 7th days of maintenance culture. For comparison, phase contrast images of HepaSH cells freshly isolated from humanized liver are also shown. The white bar at the bottom right of each photograph is a scale bar of 200 μm. [Figure 7]Figure 7 shows the drug metabolizing enzyme activity in cells refrigerated for 72 hours in UW+FP solution, seeded on a plate, and maintained in culture for 1 (A), 4 (B), and 8 (C) days. 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] Figure 8 shows the results of immunostaining of the liver 5 weeks after transplantation of HepaSH cells refrigerated for 72 hours in UW+FP solution into the liver of a TK-NOG-hIL6 mouse after induction of liver damage. A: H&E staining, B: staining with anti-human mitochondrial antibody. [Figure 9] Figure 9 shows the results of flow cytometry analysis of cells isolated from the liver 6 weeks after transplantation of HepaSH cells refrigerated for 72 hours in UW+FP solution into the liver of TK-NOG-hIL6 mice after induction of liver injury (A), and a photograph showing the state of maintenance culture of the reisolated HepaSH cells after seeding them on a plate (B). The white bar at the bottom right of the photograph in Figure 9B is a scale bar of 200 μm. [Figure 10] FIG. 10 shows the fixation efficiency 24 hours after seeding onto plates of cells that had been refrigerated for 24 or 96 hours in UW solution, UW+FP solution, UW+Def solution, or UW+FP&Def solution. [Figure 11] Figure 11 shows phase contrast images of cells refrigerated for 24 or 96 hours in UW, UW+FP, UW+Def, or UW+FP&Def solutions, taken 24 hours after seeding onto a plate. The white bar at the bottom right of each image is a 200 μm scale bar. [Figure 12] FIG. 12 shows the results of a permeation test of fluorescent substances added to the medium after cells that had been refrigerated for 96 hours in UW solution, UW+FP solution, or UW+FP&Def solution were maintained in a transwell for 7 days. [Figure 13]13 is a photograph showing a phase contrast image of cells on the 7th day of plate culture after refrigerated storage for 96 hours in UW solution (B), UW+FP solution (C), or UW+FP&Def solution (D). For comparison, a phase contrast image (A) of HepaSH cells immediately after isolation from humanized liver (freshly isolated) is also shown. [Figure 14] FIG. 14 is a photograph showing phase contrast images of cells that were refrigerated for 72 hours in UW solution or UW+FP solution, and then seeded onto a collagen I-coated 24-well plate with or without pre-warming treatment. [Figure 15] Figure 15 shows the viable cell recovery rate (A), viability (B), ROS activity (C), and fixation efficiency (D) of cells that were pre-warmed or not pre-warmed after refrigerated storage in UW solution or UW+FP solution for 72 hours. Open bars: no pre-warming, shaded bars: pre-warming. [Figure 16] FIG. 16 shows the fixation efficiency of cells that were pre-warmed or not pre-warmed after refrigerated storage for 96 hours in UW+FP solution (control) or UW+FP solution containing Def (UW+FP&Def solution). [Figure 17] Figure 17 is a photograph showing phase contrast images of cells that were refrigerated for 96 hours in UW+FP solution (control) or UW+FP solution with added Def (UW+FP&Def solution), and then pre-warmed or not pre-warmed, 24 hours after seeding onto a plate. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] The present invention will be described in detail below. The present invention provides a cell preservation solution containing a polymer. The present invention also provides a method for preserving adherent cells, which comprises preserving cells, particularly adherent cells, in a non-frozen state in the presence of a polymer, more specifically, in a cell preservation solution containing a polymer. Furthermore, the present invention also provides a method for culturing adherent cells, which comprises two-dimensionally culturing the adherent cells preserved according to such a preservation method.

[0017] In particular, the present invention relates to 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 a non-frozen state.

[0018] 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-frozen storage or refrigerated storage. The "cell preservation solution" of the present invention is not a cell preservation solution for frozen storage.

[0019] 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-cryopreservation solution such as Belzer UW (R) It may also be prepared using cold preservation solution (UW solution) as a base. (R) The composition of the cold preservation solution (UW solution) is as follows: 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 pH to 7.4 at 20° C.), and water. In one embodiment, the Belzer UW solution is (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.

[0020] In the present invention, the term "high molecular weight polymer" refers to a polymer having a weight average molecular weight of 10,000 or more. The molecular weight of the high molecular weight polymer can be determined, for example, by gel permeation chromatography (GPC) in terms of pullulan. Examples of the high molecular weight polymer 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. Specifically, examples of the high molecular weight polymer 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, and salts thereof. The "salt" with respect to the polymer 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, but is not limited thereto. 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 comprises only deacylated gellan gum or a salt thereof as the polymer. In another embodiment, the cell preservation solution of the present invention comprises, in addition to deacylated gellan gum or a salt thereof, another polymer, for example, another polysaccharide such as alginic acid, pectin, or pectinic acid, as the polymer. In another embodiment, the cell preservation solution of the present invention comprises, in addition to deacylated gellan gum or a salt thereof, an acidic polysaccharide (e.g., alginic acid, pectin, or pectinic acid) or a salt thereof that maintains a random coil state in a divalent metal cation medium and can be crosslinked via a divalent metal ion, as the polymer.When the cell preservation solution of the present invention contains, as a high molecular weight 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 a divalent metal ion in addition to deacylated gellan gum or a salt thereof, 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 may be preferably 10,000 to 50,000,000, more preferably 1,000,000 to 10,000,000. As the deacylated gellan gum or a salt thereof, a commercially available product may be used. In one embodiment, the polymer FP series manufactured by Nissan Chemical Industries, Ltd., for example, FP001, may be used. In one embodiment, FCeM. (R) FP001 solution included as a component of Preparation Kit (Nissan Chemical, product code 385-07981) can be used as a 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 its salt.

[0021] 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 in terms of the free deacylated gellan gum. In the present specification, % (W / V) means weight / volume %.

[0022] In the present invention, adhesive 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 during culture in a liquid medium, so it is difficult to maintain and culture them for a long period of time. Note that maintenance culture refers to culturing cells while maintaining their morphology and function. Adherent cells can be successfully used in two-dimensional culture and three-dimensional culture.

[0023] 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, dog cells, and feline cells. The adherent cells may be human cells or non-human cells, for example, non-human animal cells such as non-human mammalian cells (non-human primate cells). In a preferred embodiment, the adherent cells may be human hepatocytes (i.e., human adherent hepatocytes).

[0024] The adhesive cells used in the present invention are preferably primary cells, but are not limited thereto. The adhesive cells used in the present invention may be immortalized cells or may not be immortalized cells. The adhesive cells used in the present invention may be cells induced to differentiate from stem cells (e.g., iPS cells, mesenchymal stem cells, etc.). In the present invention, the term "primary cells" refers to cells collected or isolated from biological tissues and capable of proliferation (division) only a limited number of times. In the present invention, the term "primary cells" refers not only to cells collected or isolated from natural biological tissues, but also to cells isolated from tissues or organs reconstructed by transplanting primary cells into the body (e.g., into the liver after induction of liver damage) of non-human animals with a defective or reduced immune response to humans, such as immunodeficient animals or immunotolerant animals. Non-human animals with deficient or reduced immune responses to humans to be used for reconstructing tissues or organs from primary cells include, but are not limited to, non-human vertebrates with deficient or reduced immune responses to humans described in the specification of International Publication WO2020 / 122178, such as TK-NOG-hIL6 mice (hyperimmunodeficient mice in which a non-human animal human thymidine kinase gene and a human IL-6 gene have been introduced in an expressible manner into a NOG mouse derived from a NOD / SCID mouse in which the IL2 receptor γ-chain gene has been knocked out).

[0025] The concentration of the adherent cells in the cell preservation solution containing the above-mentioned polymer during preservation may be any cell concentration. In one embodiment, the concentration is preferably 1x10 6 ~1x10 8 cells / mL, more preferably 5x10 6 ~7x10 7 Cells / mL, e.g., 1x10 7 It may be at a concentration of cells / mL.

[0026] In the method of the present invention, the adherent cells are preserved in a cell preservation solution containing the above-mentioned polymer, and preferably refrigerated in a non-frozen state. In the present invention, "refrigerated storage" means storage at a temperature of more than 0°C to 10°C, typically 2 to 5°C, for example, 4°C.

[0027] In the present invention, the adherent cells can be preserved in a non-frozen state (e.g., refrigerated) for a long period of time in a cell preservation solution containing a polymer. The time for preserving the adherent cells in a non-frozen state in a cell preservation solution containing a polymer according to the present invention is not limited to the following, and may be up to 100 hours, for example, up to 96 hours, or up to 72 hours. The time for preserving the adherent cells in a non-frozen state in a cell preservation solution containing a polymer according to the present invention 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 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.

[0028] 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 the cells do not settle in the preservation solution and are maintained in a suspended state, thereby reducing ischemic stress and improving the preservation effect.

[0029] 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, thereby enabling the cells to be stably preserved without compromising their ability to adhere to the culture substrate.

[0030] In the present invention, the "ability to adhere" of adherent cells to a culture substrate means the ability of the adherent cells to adhere to a culture substrate, reach a confluent state, and maintain the confluent state when the adherent cells are seeded on the culture substrate and cultured two-dimensionally. In the present invention, the "confluent state" means a state in which the seeded adherent cells occupy 80% or more of the surface area of ​​the culture surface of the culture substrate (confluency of 80% or more). Meanwhile, in the present invention, "adhesion" means that the cells adhere to the culture substrate in a culturable state.

[0031] In a preferred embodiment, the adherent cells refrigerated in the cell preservation solution containing the above-mentioned polymer maintain their ability to adhere to a culture substrate, and therefore can be suitably used for solid-phase culture on a culture substrate, particularly for two-dimensional culture (also called monolayer culture), after refrigerated storage in the cell preservation solution. Therefore, the method for preserving the adherent cells of the present invention can be a method for preserving the adherent cells in a cell preservation solution containing the above-mentioned polymer in a non-frozen state while maintaining their ability to adhere to a culture substrate in two-dimensional culture. Alternatively, the method for preserving the adherent cells of the present invention can be a method for preserving the adherent cells to be subjected to two-dimensional culture (e.g., plate culture) after refrigerated storage in the cell preservation solution containing the above-mentioned polymer in a non-frozen state.

[0032] When primary hepatocytes (PHH) are suspended in a conventional cell preservation solution and placed under refrigerated conditions (4°C, on ice), they can be preserved for a short time while maintaining a high ability to adhere to the culture substrate. However, if the cells are preserved in a refrigerated state for a long time, the ability of the PHH to adhere to the culture substrate decreases, and the cells are in a state where there are gaps between the cells (non-confluent state). PHH cultured in a state where there are gaps between the cells (non-confluent state) changes cell morphology and loses characteristics as hepatocytes (drug metabolizing enzyme activity, etc.). By preserving the cells in a refrigerated state in a cell preservation solution containing the above-mentioned polymeric polymer in a non-frozen state according to the method of the present invention, the adhesive cells including hepatocytes can retain their ability to adhere to the culture substrate, and as a result, other cell characteristics of the adhesive cells can also be maintained. In a preferred embodiment, the cell morphology is maintained in the adhesive cells preserved in a refrigerated state in the cell preservation solution containing the above-mentioned polymeric polymer. In a preferred embodiment, the enzyme activity specific to the cells is maintained in the adhesive cells preserved in a refrigerated state in the cell preservation solution containing the above-mentioned polymeric polymer. For example, when the adhesive cells, which are hepatocytes, are refrigerated as described above, the cell morphology is maintained and the drug metabolizing enzyme activity is maintained. Examples of the 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. In addition, the adhesive cells refrigerated and stored in the cell preservation solution containing the above-mentioned polymer maintain a healthier state, as shown by, for example, an increase in the amount of ATP in the cells, and show an inhibition of the increase in ROS activity indicating ischemic stress. The adhesive cells refrigerated and stored in the cell preservation solution containing the above-mentioned polymer show an increase in the plating efficiency (plating index) on the culture substrate seeded after refrigerated storage. It is believed that by refrigerating and preserving adherent cells in a cell preservation solution containing the above-mentioned high molecular weight polymer, the ischemic stress on the adherent cells can be significantly reduced, which also leads to the above-mentioned effects.

[0033] 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, a further increase in the plating efficiency (plating index) of the adhesive cells is observed, and the plating ability of the adhesive cells is improved.

[0034] The adherent cells refrigerated according to the above-mentioned method for preserving adherent cells retain a high ability to adhere to a culture substrate, and therefore can be suitably used 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-mentioned polymer according to the above-mentioned method for preserving adherent cells, and then two-dimensionally culturing the adherent cells.

[0035] 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 onto a culture substrate, and performing two-dimensional culture.

[0036] In the present invention, the culture substrate means a solid-phase material or substance to which cells can adhere during culture and function as a scaffold. The culture substrate may be, but is not limited to, a culture plate such as a multi-well plate, a dish (culture dish), a flask, a bottle, a slide glass, a cover glass, a film, a membrane, a porous carrier, a hollow fiber, a fiber, etc. The culture substrate may be, but is not limited to, a material made of any cell scaffold material such as glass, polystyrene, polyethylene terephthalate, polysulfone, polyethersulfone, polycarbonate, etc., metals such as silver and gold, metal oxides such as indium-tin oxide, ceramics, etc. The culture substrate may also have a surface (culture surface) coated with a scaffold material such as collagen, elastin, fibronectin, vitronectin, laminin, gelatin, etc.

[0037] In one embodiment, the adherent cells preserved in the cell preservation solution containing the above-mentioned polymer can be seeded on a culture substrate coated with collagen and cultured in two dimensions. Examples of collagen used for coating include, but are not limited to, collagen I, collagen nanofiber, etc.

[0038] 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. The two-dimensional culture on the culture substrate of the adherent cells refrigerated in the cell preservation solution may be a plate culture.

[0039] The two-dimensional culture of the adhesive cells may be performed based on the usual culture conditions for two-dimensional culture. The two-dimensional culture is preferably performed under heated conditions. In one embodiment, the two-dimensional culture can be performed at preferably 30 to 40°C, more preferably 35 to 40°C, even more preferably 35 to 38°C, typically 37°C. In one embodiment, the 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.

[0040] In the method of the present invention, the adhesive cells refrigerated in the cell preservation solution containing the polymer may be subjected to a heating treatment and then to two-dimensional culture. The heating treatment may be any heating treatment that can reduce low-temperature stress of the cells. The heating treatment can be carried out by suspending the adhesive cells refrigerated in the cell preservation solution containing the 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, typically 37°C, for 20 to 60 minutes, for example 30 to 60 minutes. By subjecting the refrigerated-stored adhesive cells to a heating treatment and then to two-dimensional culture, the low-temperature stress during refrigerated storage of the adhesive cells can be reduced and the plating efficiency (plating index) can be improved.

[0041] The present invention also provides a cell preservation solution containing a polymer as described above, which is preferably used in the cell preservation method and cell culture method of the present invention. The cell preservation solution containing the polymer of the present invention is suitable for preserving cells, particularly adhesive cells, in a non-frozen state for a long period of time (e.g., refrigerated storage). The cell preservation solution containing the polymer of the present invention may be for preserving adhesive cells in a non-frozen state for up to 100 hours, for example, up to 96 hours, or up to 72 hours, refrigerated storage. The cell preservation solution containing the above-mentioned high molecular weight polymer of the present invention may be for refrigerated preservation of adherent cells in a non-frozen 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 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.

[0042] The cell preservation solution containing the polymer of the present invention may also be for refrigerating and preserving adhesive 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 for preserving adhesive cells to be subjected to two-dimensional culture after long-term refrigerated storage in a non-frozen state.

[0043] The cell preservation solution containing the polymer of the present invention can be used to maintain the ability of adhesive cells to adhere to a culture substrate during refrigerated storage and to improve the preservation of adhesive 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. EXAMPLES

[0044] The present invention will be described in more detail below with reference to examples, although the technical scope of the present invention is not limited to these examples.

[0045] In the following Examples, all p values ​​in statistical analyses were calculated by paired t-tests of two groups, with p<0.05 indicating a significant difference.

[0046] [Example 1] Preparation of a polymer-containing cell preservation solution and preservation of cells in the solution FCeM containing polymer FP001 liquid as a component (R) A preparation kit (Nissan Chemical, product code 385-07981) was used to prepare the cell preservation solution. The polymer FP001 solution contains deacylated gellan gum. Belzer UW (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.

[0047] FCeM (R)Following the instructions in the Preparation Kit's user 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.

[0048] 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 cells isolated from humanized livers reconstructed by transplanting human primary hepatocytes (PHH) into the liver of hyperimmunodeficient TK-NOG-hIL6 mice (Uehara et al., Biochemical and Biophysical Research Communications, 663 (2023) 132-141., International Publication WO2020 / 122178) after induction of liver damage. Here, six samples of HepaSH cells isolated from six different mice were used.

[0049] HepaSH cells were added to UW solution or UW+FP solution at 1x10 7 After making a cell suspension at a concentration of 1000 cells / mL, a 15 mL centrifuge tube (Greiner Bio-One) was used as a storage container and stored 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. The cells suspended in the UW solution settled, but the cells suspended in the UW+FP solution did not settle and remained floating even after 24 hours of storage.

[0050] 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 number of live cells and total cells were counted using a hemocytometer (FMG, product code 521-10). The ratio of the number of live cells at each sampling time point to the number of live cells at the start of storage (viable cell recovery rate; % Recovery) and the ratio of the number of live cells to the total number of cells (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 and viability of cells stored in UW+FP solution compared to UW solution, indicating that the addition of high molecular weight polymer FP does not affect these.

[0051] In addition, the 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 attach to the scaffold after storage was tested. The cell samples were 1x10 6The cells were suspended in 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, seeded at 0.5 mL / well on a collagen I-coated 24-well plate (Corning; product code 356408) and cultured overnight in a CO2 incubator (37°C, 5% CO2). 24 hours after seeding, the medium was replaced with seeding medium containing the nuclear staining fluorescent dye Hoechst 33342 (Dojindo Science Institute, product code H342), and the cells were incubated for 30 minutes for staining. Phase contrast images were then taken using EVOS cell imaging systems (Thermo Fisher Scientific) to measure the average number of nuclei per fixed area (within a 200 μm grid). For comparison, freshly isolated HepaSH cells from humanized liver were suspended in a seeding medium and seeded on a collagen I-coated 24-well plate in the same manner as above, and the average number of nuclei was measured after 24 hours of culture. The ratio of the average number of nuclei 24 hours after seeding of cells preserved in a cell preservation solution to the average number of nuclei 24 hours after seeding of freshly isolated HepaSH cells (100%) was calculated and used as the plating efficiency (plating index). The results are shown in Figures 3 and 4.

[0052] Throughout the storage period, cells stored in UW+FP solution showed higher fixation efficiency than cells stored in UW solution, and the fixation efficiency was about 80% even after 72 hours of storage (Figure 3). Phase contrast images (Figure 4) also showed that cells stored in UW solution had gaps between the cells when plate cultured for 48 hours or more, and did not maintain a confluent state, whereas cells stored in UW+FP solution maintained a confluent state with no gaps between the cells and could be plate cultured even after 72 hours of storage.

[0053] It was shown that by storing cells in a preservation solution containing a high molecular weight polymer, the cells' adhesion ability can be maintained even after long-term storage.

[0054] [Example 2] Reduction of ischemic stress in cells preserved in a preservation solution containing a high molecular weight polymer HepaSH cells (30 specimens) were cultured in UW solution or UW+FP solution at 1x10 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.

[0055] 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 the 2.0 Cell Viability Assay (Promega) and 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 compared to UW solution. During the process of cell death, the amount of ATP in the cells decreases. This indicates that preservation in the presence of high molecular weight polymers keeps cells in a healthier state even after long-term storage.

[0056] In addition, the amount of reactive oxygen species (ROS) (ROS activity), which is an indicator of ischemic stress in cells after storage, was measured using a total ROS detection kit (Dojindo Research Institute, product code R252) for cells stored at 4°C for 24 hours. The cells stored at 4°C for 24 hours 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 instruction manual, and then suspended in loading buffer (Loading buffer; 1x) containing 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 in the PI-negative (indicating live cells) and HLA-positive (indicating human hepatocytes) cell population was calculated and compared between the UW solution and the UW+FP solution. The results are shown in Figure 5B. In cells preserved in UW solution, FITCmean was statistically significantly higher than in cells preserved in UW+FP solution. In this assay, the greater the amount of ROS accumulated in the cells, the higher the FITCmean. In other words, in cells preserved in UW solution, the amount of ROS accumulated increased, indicating enhanced ischemic stress. This result indicates that preservation in the presence of high molecular weight polymers reduces ischemic stress in cells.

[0057] Furthermore, the cells stored at 4°C for 24 hours were suspended in the above-mentioned seeding medium, seeded on a collagen I-coated 24-well plate, and cultured for 4 hours, after which the culture supernatant was collected. Lactate dehydrogenase (LDH) activity in the collected culture supernatant was measured as an indicator of cell death using CytoTox-ONE Homogeneous Membrane Integrity Assay (Promega, product code G7890) according to the manufacturer's instructions. For fluorescence measurement, a plate reader Infinite F200F Pro (TECAN) was used. The fluorescence intensity measured for the culture supernatant was normalized to the measured value in unused seeding medium (control medium). The results are shown in Figure 5C. The LDH activity was statistically significantly higher in cells stored in UW solution compared to cells stored in UW+FP solution. When cell death is enhanced, LDH leaks from within the cells into the medium, resulting in higher LDH activity in the culture supernatant. In other words, when stored in UW solution, cell death was enhanced after seeding. It is believed that preservation in UW solution enhanced cell death after seeding due to enhanced ischemic stress. This result indicates that preservation in the presence of a high molecular weight polymer can reduce cell death after seeding on a culture substrate due to enhanced ischemic stress.

[0058] [Example 3] Maintenance of cell characteristics after storage in cell preservation solution HepaSH cells (four specimens) were cultured in UW+FP solution at 1x10 7 The cells were suspended at a concentration of 10 ... (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 culture day 1 (the day after seeding), days 3, 5, and 7. 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.

[0059] In addition, the activity of major drug metabolizing enzymes in cells on the 1st, 4th, and 8th days of culture was measured during the culture period up to the 7th day. HepaSH cells harvested on the 1st, 4th, and 8th days of culture were incubated at 37°C for 1 hour with Williams' Medium E, no Phenol Red (Thermo Fisher Scientific, product code A12176) supplemented with phenacetin (substrate for CYP1A2) (100 μM), diclofenac (substrate for CYP2C9) (40 μM), omeprazole (substrate for CYP2C19) (10 μM), metoprolol (substrate for CYP2D6) (5 μM), and midazolam (substrate for CYP3A4 / 5) (5 μM), which are typical substrates of the drug metabolizing enzyme group human cytochrome P450. The 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 metabolites produced per unit time and unit protein weight. Figure 7 shows the drug-metabolizing enzyme activity expressed as the amount of metabolites per unit protein weight. By comparing with the drug-metabolizing enzyme activity in cells not refrigerated, it was shown that HepaSH cells refrigerated for 72 hours in UW+FP solution maintained drug-metabolizing enzyme activity throughout the culture period up to the 8th day of culture.

[0060] These results indicate that cells preserved in the presence of polymers can retain their cellular properties even after long-term refrigerated storage. In particular, it is very important for hepatocytes to maintain their drug-metabolizing enzyme activity for the preservation and transportation of hepatocytes.

[0061] In addition, add 1x10 7HepaSH cells suspended at a concentration of 1000 cells / mL and stored at 4°C for 72 hours were transplanted via the spleen into the liver of TK-NOG-hIL6 mice after induction of liver damage. Five weeks after transplantation, the liver was harvested and formalin-fixed, then paraffin sections were prepared, stained with hematoxylin and eosin (H&E) and immunostained with an anti-human mitochondrial antibody (Merck, product code MAB1273), and images were acquired using a slide scanner NanoZoomer (Hamamatsu Photonics). The results are shown in Figure 8. It was shown that most of the recipient mouse liver was replaced with human hepatocytes that were positive for human mitochondria (Figures 8A and B). In addition, HepaSH cells were reisolated 6 weeks after transplantation from another individual TK-NOG-hIL6 mouse into which HepaSH cells stored in a refrigerator for 72 hours were transplanted into the liver after induction of liver damage. 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). By seeding the reisolated HepaSH cells on collagen I-coated plates, it was possible to perform fixation culture in the same way as with the cells before transplantation (Figure 9B). This result showed that hepatocytes preserved in the presence of a high molecular weight polymer maintained the ability to reconstitute the liver even after long-term refrigerated storage.

[0062] [Example 4] Effects of adding iron chelating agent and pre-warming 1) Addition of iron chelating agents We hypothesized that an iron chelator that removes free iron ions from cells would be effective in preventing ferroptosis, an iron-dependent cell death. Therefore, we investigated whether the attachment ability of cells preserved in a cell preservation solution could be further improved by adding an iron chelator in addition to a polymer to the cell preservation solution.

[0063] HepaSH cells (9 samples) were cultured at 1x10 in UW solution or UW+FP solution with or without the addition of an iron chelating agent (final concentration 500 μM, deferoxamine; hereafter also referred to as Def). 7The cells were suspended at a concentration of 1000 cells / mL and stored (refrigerated) for 24 hours or 96 hours at 4°C. In the following, the UW solution with Def added is referred to as UW+Def solution, and the UW+FP solution with Def added is referred to as UW+FP&Def solution.

[0064] The cells after refrigerated storage were seeded on a collagen I-coated 24-well plate, 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.

[0065] The results are shown in Figures 10 and 11. In cells stored in a refrigerator for 24 hours, a statistically significant increase in fixation efficiency was observed when stored in UW+FP solution, UW+Def solution, and UW+FP&Def solution compared to UW solution (without addition), but no additive effect was observed by the combined use of high molecular weight polymer and Def (Figure 10). On the other hand, in cells stored in a refrigerator for 96 hours, a statistically significant increase in fixation efficiency was observed when stored in UW+FP solution, UW+Def solution, and UW+FP&Def solution compared to UW solution (without addition), and in addition, the combined use of high molecular weight polymer and Def in UW+FP&Def solution showed a statistically significant increase in fixation efficiency compared to the addition of high molecular weight polymer or Def alone (Figure 10). Furthermore, in particular, when cells were stored in UW solution or UW+Def solution after 96 hours of refrigeration, they did not maintain a confluent state in the subsequent plate culture, whereas when cells were stored in UW+FP solution or UW+FP&Def solution, the phase contrast images showed that the cells maintained a confluent state in plate culture (Figure 11). The combined use of a polymer that can avoid both ischemic stress and ferroptosis and an iron chelator showed that the adhesion ability of cells that enables confluent culture was maintained even after 96 hours of storage in the cell preservation solution.

[0066] HepaSH cells were also cultured at 1x10 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, then suspended in seeding medium and seeded on a transwell (Corning, product code 354495) coated with rat collagen I (Corning, product code 354236) for 7 days of maintenance culture (plate culture). On the 7th day of culture, medium containing PE streptavidin (BD, product code 554061) was added to the upper layer of the transwell, and the lower layer medium was collected 15, 30, and 60 minutes after the addition of PE streptavidin, and the fluorescence was measured using a plate reader Infinite F200F Pro (TECAN). In this assay, if the confluent state is not maintained in the plate culture, PE streptavidin will migrate from the upper layer to the lower layer through the gaps between the cells, and the fluorescence derived from the fluorescent dye PE (phycoerythrin) will be detected in the medium.

[0067] For comparison, fluorescence measurements were performed in the same manner on wells (Fr) seeded with freshly isolated HepaSH cells immediately after isolation from humanized liver, and on wells (no cells) that had not been seeded with cells. The measured fluorescence intensity was normalized to the measured value in unused seeding medium (control medium). The results of the fluorescence intensity measurements are shown in Figure 12. Figure 13 shows a phase contrast image of the cells on the 7th day of plate culture.

[0068] In the cells stored in UW solution for 96 hours, gaps between cells were observed in the phase contrast image (Fig. 13B), and fluorescence was detected 15 minutes after the addition of PE-streptavidin (exposure to cells) (Fig. 12). In contrast, in the cells stored in UW+FP solution and UW+FP&Def solution for 96 hours, gaps between cells were not observed in the phase contrast image (Fig. 13C and D), and almost no fluorescence was detected even 30 minutes after the addition of PE-streptavidin, as in the case of HepaSH cells (Fr) immediately after isolation (Fig. 12). These results indicate that HepaSH cells stored in a refrigerator for 96 hours in the presence of a high molecular weight polymer or a high molecular weight polymer and an iron chelator maintained a confluent state even after 7 days of plate culture.

[0069] 2) Pre-warming to reduce cold stress We thought that cell death after seeding could be suppressed by suspending the cells after cryopreservation in a seeding medium, incubating at 37°C for 30-60 minutes, and then seeding them on collagen I plates. Therefore, we investigated the effect of adding a high molecular weight polymer to the cell preservation solution, as well as heating the cells before seeding (pre-warming).

[0070] HepaSH cells (10 samples) were cultured in UW solution or UW+FP solution at 1x10 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 harvested and suspended in 10 times the volume of seeding medium. The cells were then pre-warmed by incubating at 37°C for 30 minutes in a thermostatic water bath.

[0071] For the cells that had been pre-warmed, the number of live cells and the total number of cells were counted using trypan blue staining and a hemocytometer according to the method described in Example 1, and the recovery rate and survival rate of live cells were calculated. Furthermore, according to the method described in Example 1, phase contrast images were taken 24 hours after seeding, the plating efficiency (plating index) was calculated, and ROS activity was measured according to the method described in Example 2. As a control, a similar storage test and measurement were performed except that the pre-warming treatment was not performed. The results are shown in Figures 14 and 15.

[0072] Pre-warming tended to decrease the viable cell recovery rate in cells preserved in UW solution, but there was almost no change in the viable cell recovery rate in cells preserved in UW+FP solution (Fig. 15A). Since pre-warming promotes the death of damaged cells that have accumulated ischemic stress, it is thought that the viable cell recovery rate decreased in cells preserved in UW solution due to the accumulation of ischemic stress, but the viable cell recovery rate did not decrease in cells preserved in UW+FP solution because there was less accumulation of ischemic stress and there were fewer damaged cells (Fig. 15A).

[0073] On the other hand, the viability of cells preserved in both UW and UW+FP solutions was statistically significantly improved by pre-warming (Fig. 15B). Furthermore, the pre-warming treatment reduced ROS activity and enhanced fixation efficiency statistically significantly for cells preserved in both UW and UW+FP solutions (Fig. 15C and D). Pre-warming also has the effect of reducing low-temperature stress on cells, which is thought to have led to the improvement in viability, reduction in ROS activity, and enhancement of fixation efficiency.

[0074] 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.

[0075] 3) Addition of iron chelating agents and pre-warming to mitigate cold stress HepaSH cells (6 samples) were cultured at 1x10 in UW+FP solution or UW+FP&Def solution. 7 The cells were suspended at a concentration of 10 ... Phase contrast images showed that all cells stored under refrigeration were plated in a confluent state (FIG. 17).

[0076] In Examples 1 to 4, it is shown that ischemic stress in cells occurring during storage in a cell preservation solution becomes apparent after the cells after storage are seeded on a culture substrate and culture is started. Examples 1 to 4 show that when cells that survived despite being subjected to ischemic stress during storage in a cell preservation solution are seeded on a culture substrate after storage and cultured, cell death is accelerated and the cell adhesion efficiency in culture on the culture substrate is reduced, and further, it is shown that such cell death and reduction in adhesion efficiency can be significantly improved by the present invention. [Industrial Applicability]

[0077] 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 transport, of adherent cells to be subjected to two-dimensional culture after refrigerated storage.

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

10. A method for culturing adherent cells, comprising refrigerating and storing 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 preserving adherent cells in a non-frozen state under refrigeration.

14. The cell preservation solution according to claim 13 , wherein the 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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