Method for transporting spheroid

Abstract

To provide a method capable of transporting a spheroid containing mesenchymal stem cells with reduced decrease in cell survival rate.SOLUTION: A spheroid transport method according to the present invention comprises a step of transporting a spheroid containing mesenchymal stem cells at 40°C or lower, the spheroid being suspended in a solution within a syringe.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method for transporting spheroids. [Background technology]

[0002] In order to administer cells to the affected area of ​​a patient, the cells must be transported from the facility where they were prepared to the hospital, and it is important to maintain the quality of the cells during transportation. As a method for preserving or transporting stem cells while maintaining cell viability, for example, a method using a gelling agent is known (Patent Document 1). [Prior art documents] [Patent documents]

[0003] [Patent Document 1] Japanese Patent Application Publication No. 2023-133621 Summary of the Invention [Problem to be solved by the invention]

[0004] However, no studies have been conducted on methods for transporting spheroids containing mesenchymal stem cells. Therefore, an object of the present invention is to transport spheroids containing mesenchymal stem cells while suppressing a decrease in cell viability. [Means for solving the problem]

[0005] The present invention has the following aspects. [1] A step of transporting spheroids containing mesenchymal stem cells at 40°C or less, A method for transporting spheroids, wherein the spheroids are suspended in a solution in a syringe. [2] The method according to [1], wherein the solution is a solution that can be administered to a living body. [3] The method according to [2], wherein the solution contains a sodium salt, a potassium salt, and a calcium salt. [4] The method according to [3], wherein the solution is Ringer's solution, lactated Ringer's solution, acetated Ringer's solution, or bicarbonate Ringer's solution. [5] The method according to [4], wherein the solution is lactated Ringer's solution. [6] The method according to any one of [1] to [5], wherein the mesenchymal stem cells are derived from adipose tissue. [7] The method according to any one of [1] to [6], further comprising the step of culturing mesenchymal stem cells adherently on a substrate containing a polyimide resin to obtain the spheroids, prior to the step of transporting the spheroids.

[0006] The present invention also has the following aspects. [8] A step of storing the spheroids containing mesenchymal stem cells at 40°C or less; A method for storing spheroids, wherein the spheroids are suspended in a solution in a syringe. [9] The method according to [8], wherein the solution is a solution that can be administered to a living body.

[10] The method according to [9], wherein the solution contains a sodium salt, a potassium salt, and a calcium salt.

[11] The method according to

[10] , wherein the solution is Ringer's solution, lactated Ringer's solution, acetated Ringer's solution, or bicarbonate Ringer's solution.

[12] The method according to

[11] , wherein the solution is lactated Ringer's solution.

[13] The method according to any one of [8] to

[12] , wherein the mesenchymal stem cells are derived from adipose tissue.

[14] The method according to any one of [8] to

[13] , comprising a step of obtaining the spheroids by adhesion culturing mesenchymal stem cells on a substrate containing a polyimide resin, prior to the step of storing the spheroids.

[15] The method according to any one of [8] to

[14] , wherein the storage of the spheroids involves transportation. [Effects of the Invention]

[0007] According to the present invention, spheroids containing mesenchymal stem cells can be transported while suppressing a decrease in cell viability. [Brief explanation of the drawings]

[0008] [Figure 1] The observed images and cell viability of spheroids stored at 4°C or 37°C for the specified time periods are shown. [Figure 2] The cell viability of spheroids stored at 4°C or 37°C for the specified time periods is shown. [Figure 3] The image shows spheroids transported at 5±3°C for 72 hours. DETAILED DESCRIPTION OF THE INVENTION

[0009] A method for transporting spheroids according to one aspect of the present invention includes transporting spheroids containing mesenchymal stem cells suspended in a solution in a syringe at 40°C or below. This method enables the transport of spheroids containing mesenchymal stem cells while suppressing a decrease in cell viability, shape changes during transport, and cell differentiation.

[0010] The mesenchymal stem cells are not particularly limited, and examples thereof include adipose-derived stem cells, bone marrow-derived stem cells, umbilical cord-derived stem cells, and dental pulp-derived stem cells. The mesenchymal stem cells may be mesenchymal stem cells derived from humans or non-human animals. The mesenchymal stem cells may be, for example, adipose-derived stem cells, more specifically, human adipose-derived stem cells (AdSCs).

[0011] As used herein, the term "spheroid" refers to a cell aggregate (cell mass), and includes three-dimensional cell aggregates. Spheroids are preferably approximately spherical. The size of the spheroid is not particularly limited, and the spheroid may have a diameter of, for example, 10 to 1500 μm, 10 to 1000 μm, or 10 to 800 μm. Here, the diameter of the spheroid is measured by a standard method using, for example, image analysis software or a particle size distribution analyzer. The method for producing spheroids is not particularly limited, and for example, spheroids can be obtained by adhesion culture of mesenchymal stem cells on a substrate containing a polyimide resin, as described below.

[0012] Although spheroids containing mesenchymal stem cells may contain cells other than mesenchymal stem cells, they preferably contain mesenchymal stem cells at 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, 98% or more, or 99% or more of the total number of cells. More preferably, spheroids containing mesenchymal stem cells consist solely of mesenchymal stem cells. Examples of cells other than mesenchymal stem cells include precursor cells differentiated from mesenchymal stem cells and differentiated cells differentiated from the precursor cells.

[0013] As used herein, "transporting" a spheroid refers to moving the spheroid from a desired starting point to a desired destination over a predetermined period of time. The time taken for transportation may be, for example, 1 hour or more, 3 hours or more, 6 hours or more, 13 hours or more, 24 hours or more, 48 hours or more, or 72 hours or more. From the viewpoint of maintaining a high cell viability, the time taken for transportation may be 72 hours or less, 48 ​​hours or less, 24 hours or less, 13 hours or less, 6 hours or less, or 3 hours or less.

[0014] The solution in which the spheroids are suspended is not particularly limited as long as it is a solution in which cells can survive. A solution in which cells can survive means a solution having conditions such as osmotic pressure and pH that allow cells to survive. The osmotic pressure may be, for example, 150 to 750 mOsm / L, 250 to 380 mOsm / L (i.e., an isotonic solution), or 250 to 270 mOsm. The pH may be, for example, 3.5 to 8.5, 5 to 7.5, or 6 to 7.5.

[0015] From the perspective of being ready-to-use, i.e., allowing spheroids to be administered to patients as is after transportation, the solution in which spheroids are suspended is preferably a solution that can be administered to a living body. A solution that can be administered to a living body means a solution that is physiologically acceptable in a living body and does not typically exhibit unacceptable toxic reactions upon administration. The living body may be a mammal such as a human, and more specifically, a patient requiring administration of spheroids. Examples of solutions that can be administered to a living body include solutions used as infusions, such as extracellular fluid replacement solutions, hypotonic electrolyte solutions, peripheral intravenous nutrition solutions, hyperalimentation solutions, and plasma volume expanders. These solutions contain sodium salts, potassium salts, and calcium salts. Examples of extracellular fluid replacement solutions include Ringer's solution, lactated Ringer's solution, acetated Ringer's solution, and bicarbonate Ringer's solution. The solution in which spheroids are suspended is preferably Ringer's solution, lactated Ringer's solution, acetated Ringer's solution, or bicarbonate Ringer's solution, and more preferably lactated Ringer's solution.

[0016] The temperature during transportation is 40°C or lower, preferably 37°C or lower, more preferably 15°C or lower, and even more preferably 10°C or lower. The temperature during transportation may be 8°C or lower or 4°C or lower. The lower limit of the temperature is not particularly limited as long as it is a temperature at which the cells can be kept suspended, i.e., a temperature at which the solution containing the suspended cells does not freeze. Depending on the composition of the solution, the temperature may be, for example, -3°C or higher, 0°C or higher, above 0°C, 1°C or higher, 2°C or higher, 4°C or higher, or 8°C or higher. More specifically, the temperature may be, for example, 1 to 10°C, 1 to 4°C, 2 to 8°C, or 4 to 10°C. When transportation is performed at a temperature of 37°C or higher, transportation is preferably performed while maintaining constant conditions such as temperature and carbon dioxide concentration using a transportation system such as Cellporter (manufactured by Corefront Co., Ltd.).

[0017] The syringe may be a syringe used for administering a drug solution to a patient and may be made of glass or plastic. By storing a spheroid suspension in the syringe and transporting it, the spheroids can be administered to a patient directly by simply attaching an injection needle to the syringe at the destination.

[0018] The method for transporting spheroids of the present invention may further comprise the step of culturing mesenchymal stem cells in an adherent manner on a substrate containing a polyimide resin to obtain the above-mentioned spheroids.

[0019] Polyimide resin is a cell adhesive material that exhibits moderate cell adhesion, and thus, cells can be cultured in an adherent state by using a substrate containing polyimide resin. "Adherent culture" is a concept that contrasts with "suspension culture," and is a culture method in which cells or spheroids are cultured in an adherent state on the culture surface of a substrate. Here, "adhesion" refers to a state in which cells or spheroids are bound to the culture surface of the substrate via cell-substrate adhesion molecules contained in the extracellular matrix (ECM), and the cells or spheroids do not float in the medium even when the medium is gently shaken.

[0020] An example of the polyimide resin is a polyimide resin containing a structural unit represented by the following formula (I): In the polyimide resin, the chemical structure represented by formula (I) may be different for each structural unit of the resin, or may be the same. [ka] In formula (I), X 0 represents a tetravalent organic group which is an acid dianhydride residue, and Y 0 represents a divalent organic group that is a residue of a diamine compound. 0 and Y 0 At least one of these preferably contains one or more fluorine atoms and preferably contains one or more ether bonds and / or thioether bonds.

[0021] A preferred example of the structural unit represented by formula (I) is a structural unit represented by formula (II). [ka] In formula (II), X represents an oxygen atom, a sulfur atom, or a divalent organic group; Y represents a divalent organic group; Z 1 , Z 2 , Z 3 , Z 4 , Z 5 , and Z 6 each independently represents a hydrogen atom, a fluorine atom, a chlorine atom, a bromine atom, or an iodine atom, p is 0 or 1. In formula (II), when p=0, X may not be present (in other words, the left and right benzene rings may be directly bonded), but when p=1, the left and right benzene rings are bonded via X. 1 , Z 2 , Z 3 , Z 4 , Z 5 , and Z 6 At least one of them preferably contains one or more fluorine atoms.

[0022] Polyimide resins are typically obtained by imidizing polyamic acids obtained by polymerizing one or more acid dianhydrides and one or more diamines. Polyimide resins may contain polyamic acids as part of their chemical structure. Polyimide resins can be produced by known methods. For example, a two-stage synthesis method can be used. The two-stage synthesis method for polyimide resins involves synthesizing a polyamic acid as a precursor and converting the polyamic acid into polyimide acid. The polyamic acid precursor may be a polyamic acid derivative. Examples of polyamic acid derivatives include polyamic acid salts, polyamic acid alkyl esters, polyamic acid amides, polyamic acid derivatives from bismethylidene pyromellitide, polyamic acid silyl esters, and polyamic acid isoimides. Examples of polyimides include polyimides composed of acid anhydrides such as pyromellitic dianhydride, biphenyltetracarboxylic dianhydride, and benzophenonetetracarboxylic dianhydride, and diamines such as oxydiamine, paraphenylenediamine, metaphenylenediamine, and benzophenonediamine.

[0023] From the viewpoint of good spheroid formation, fluorinated polyimide resins are preferred as polyimide resins. Fluorinated polyimide resins are, in other words, fluorine-containing polyimide resins. Examples of fluorinated polyimide resins include 4,4'-hexafluoroisopropylidenediphthalic anhydride (6FDA) / 1,4-bis(aminophenoxy)benzene (TPEQ) copolymer, 6FDA / 1,3-bis(4-aminophenoxy)benzene (TPER) copolymer, 6FDA / 4,4'-oxydiphthalic anhydride (ODPA) / TPEQ copolymer, 4,4'-(4,4'-isopropylidenediphenoxy)diphthalic acid (BPADA) / 2,2-bis[4-(4-aminophenoxy)phenyl]hexafluoroisopropylidenediphthalic anhydride ... The copolymer may be a 6FDA / 2,2-bis(4-(4-aminophenoxy)phenyl)propane (HFBAPP) copolymer, a 6FDA / 2,2'-bis(trifluoromethyl)benzidine (TFMB) copolymer, a 6FDA / 4,4'-diaminodiphenyl ether (ODA) copolymer, a 6FDA / 4,4'-bis(4-aminophenoxy)biphenyl (BAPB) copolymer, or a 6FDA / 2,2-bis(4-(4-aminophenoxy)phenyl)sulfone (BAPS) copolymer.

[0024] The weight average molecular weight of the polyimide resin is, for example, 5,000 to 2,000,000, preferably 8,000 to 1,000,000, and more preferably 20,000 to 500,000. In this specification, the weight average molecular weight is measured by the following method.

[0025] (Measurement of weight average molecular weight) Device: HCL-8220GPC (Tosoh Corporation) Column: TSKgel Super AWM-H Eluent (LiBr·H2O, N-methylpyrrolidone with phosphoric acid): 0.01 mol / L Measurement method: A 0.5% by mass solution is prepared using an eluent, and the molecular weight is calculated based on a calibration curve prepared using polystyrene.

[0026] The thickness of the substrate is not particularly limited and may be, for example, 10 to 5000 μm or 100 to 2000 μm. The area of ​​the substrate (total area of ​​the upper surface of the substrate and the openings) is not particularly limited and may be, for example, 0.01 to 10000 cm. 2 or 0.03 to 5000 cm 2 It may be.

[0027] In one embodiment, the substrate may include a plurality of recesses each having an opening. The bottom surface of each recess may be a cell-adhesive surface made of polyimide resin. The cell-adhesive surface is a surface to which cells can adhere at certain adhesion points when they settle on the surface in a culture medium.

[0028] The number of recesses is not particularly limited, and 2 The number of recesses per unit area may be 1 or more, 10 or more, 20 or more, 30 or more, or 50 or more, and may be 1,000 or less, 500 or less, 300 or less, 200 or less, or 100 or less. The total number of recesses may be, for example, 1 or more, 10 or more, 100 or more, 1,000 or more, 10,000 or more, or 50,000 or more.

[0029] The shape of the opening of the recess is not particularly limited and may be, for example, a circle, a polygon, or an ellipse. The diameter of the opening may be, for example, 2000 μm or less, 10 to 2000 μm, 10 to 1000 μm, 10 to 700 μm, 10 to 600 μm, or 10 to 500 μm. In this specification, the diameter of a certain portion refers to the diameter of a circle circumscribing the portion, i.e., the maximum length of the portion.

[0030] The shape of the bottom of the recess is not particularly limited and may be, for example, a circle, a polygon, or an ellipse. The shape of the bottom may be the same as or different from the shape of the opening. The diameter of the bottom may be the same as or different from the diameter of the opening, and may be smaller or larger than the diameter of the opening. The diameter of the bottom may be, for example, 10 to 2000 μm, 10 to 1000 μm, 10 to 700 μm, 10 to 600 μm, 10 to 500 μm, 10 to 400 μm, or 10 to 300 μm. The bottom of the recess may be a flat surface or a smooth surface.

[0031] The distance between adjacent openings, i.e., the length of the gap, is not particularly limited and may be, for example, 800 μm or less, 700 μm or less, 600 μm or less, 500 μm or less, 300 μm or less, 200 μm or less, or 100 μm or less.

[0032] The depth of the recesses is not particularly limited, and may be, for example, 100 nm to 500 nm, 10 μm to 1000 μm, or 10 μm to 300 μm.

[0033] Of the entire surface of the substrate, the surfaces other than the bottom surfaces of the recesses, i.e., the inner surfaces of the recesses and the top surface of the substrate (which can also be said to be the periphery of the recesses), may be cell-nonadhesive. A cell-nonadhesive surface is a surface to which cells do not adhere at all, or to which cells temporarily adhere weakly but then naturally detach. More specifically, of the entire surface of the substrate, the surfaces other than the bottom surfaces of the recesses may be composed of a cell-nonadhesive material.

[0034] The non-cell-adhesive substance is not particularly limited as long as it does not bind to molecules such as proteins and sugar chains present on the surface of cells. Examples of non-cell-adhesive substances include polyethylene glycol and its derivatives, 2-methacryloyloxyethyl phosphorylcholine (MPC) and its derivatives, compounds containing polyhydroxyethyl methacrylate (poly-HEMA) and its derivatives, or polymers of these compounds, compounds containing segmented polyurethane (SPC) and its derivatives, proteins such as albumin, and sugar chains to which cells do not adhere (agarose, cellulose, etc.). These substances can be used singly or in combination. Among these, MPC and its derivatives or polymers thereof are preferred, with MPC polymers being more preferred, from the standpoints of adhesion to cell-adhesive sites, simplification of the substrate production process, and improvement of the uniformity of spheroids obtained by culture.

[0035] In another embodiment, the substrate may be a cell culture sheet having a micropattern formed of a cell-adhesive surface and a cell-nonadhesive surface made of polyimide resin. For example, the cell culture sheet may have a micropattern formed of a polyimide resin layer and a mask of a cell-nonadhesive material provided on the surface of the layer. The micropattern may be, for example, recesses. In this case, the depth of the recesses depends on the thickness of the mask of the cell-nonadhesive material. Therefore, the lower limit of the depth of the recesses is the minimum thickness at which cells can recognize the nonadhesive nature of the mask. The depth of the recesses may be, for example, less than 10 μm, and may be 100 nm to 500 nm. The number of recesses, the shape and diameter of the openings and bottoms of the recesses, and the distance between the recesses may be the same as in the above embodiment.

[0036] The formation of the fine pattern may be carried out by, for example, microcontact printing, spin coating, casting, roll coating, die coating, gravure coating, spray coating, bar coating, flexographic printing, dip coating, inkjet printing, or patterning. The patterning method is a method of forming a fine pattern by injecting a desired substance into gaps in an uneven structure formed on the surface of a base material by utilizing capillary force generated in the gaps.

[0037] The medium used for culturing may be any medium containing components necessary for the proliferation of mesenchymal stem cells, and may be, for example, a basal medium such as Eagle's minimum essential medium (EMEM), Dulbecco's modified Eagle's medium (DMEM), α-minimum essential medium (α-MEM), Glasgow's minimum essential medium (GMEM), Iscove's modified Dulbecco's medium (IMDM), Roswell Park Memorial Institute (RPMI) 1640 medium, Ham's F-12 medium, MCDB medium, or Williams' medium E, to which components such as serum, growth factors, differentiation-inducing factors, antibiotics, hormones, amino acids, sugars, or salts may be added as necessary.

[0038] The culture temperature is not particularly limited, but is usually about 25 to 40° C. The relative humidity during culture is not particularly limited, and may be, for example, 40 to 95% RH.

[0039] The culture time is not particularly limited and can be determined appropriately depending on the cell proliferation rate and the desired size of the spheroids. The culture time may be, for example, 4 hours to 30 days (4 hours to 720 hours), 1 day to 14 days (24 hours to 336 hours), or 1 day to 7 days (24 hours to 168 hours).

[0040] It is preferable to degas the substrate before culturing cells. The method of degassing is not particularly limited, and common methods such as spraying, pipetting, shaking, heating and cooling, centrifugation, vacuum degassing, and ultrasonic treatment can be used.

[0041] The above describes a method for transporting spheroids, but the method can also be used for storing spheroids. Specifically, one aspect of the present invention relates to a method for storing spheroids, which includes a step of storing spheroids containing mesenchymal stem cells at 40°C or below, where the spheroids are suspended in a solution in a syringe. This method allows spheroids containing mesenchymal stem cells to be stored while suppressing a decrease in cell viability. Furthermore, shape changes and cell differentiation during storage can also be suppressed.

[0042] As used herein, "storing" a spheroid refers to holding the spheroid in a container (syringe) for a predetermined period of time to maintain its state (shape, function, properties, etc.). Storage may involve transportation (specifically, transportation of a syringe containing the spheroid) or not. For example, transporting spheroids from a facility where the spheroids were prepared to a hospital where the patient is staying for administration to the patient's diseased site is considered storage involving transportation, as long as the purpose is to maintain the spheroid state. The storage time may be, for example, 1 hour or more, 3 hours or more, 6 hours or more, 13 hours or more, 24 hours or more, 48 hours or more, or 72 hours or more. From the viewpoint of maintaining a high cell viability, the storage time may be 72 hours or less, 48 ​​hours or less, 24 hours or less, 13 hours or less, 6 hours or less, or 3 hours or less.

[0043] Details of the spheroids, the solution for suspending the spheroids, and the syringe are as described in the description of the method for transporting spheroids.

[0044] The storage temperature is 40°C or lower, preferably 37°C or lower, more preferably 15°C or lower, and even more preferably 10°C or lower. The storage temperature may be 8°C or lower or 4°C or lower. The lower limit of the temperature is not particularly limited as long as it is a temperature at which the cells can be kept suspended, i.e., a temperature at which the cell suspension solution does not freeze. Depending on the composition of the solution, the temperature may be, for example, -3°C or higher, 0°C or higher, above 0°C, 1°C or higher, 2°C or higher, 4°C or higher, or 8°C or higher. More specifically, the temperature may be, for example, 1 to 10°C, 1 to 4°C, 2 to 8°C, or 4 to 10°C. When storing at a temperature of 37°C or higher, it is preferable to maintain constant conditions such as temperature and carbon dioxide concentration using a transportation system such as Cellporter (manufactured by Corefront Co., Ltd.).

[0045] The method for storing spheroids of the present invention may further include a step of culturing mesenchymal stem cells on a substrate containing a polyimide resin to obtain the above-mentioned spheroids. Details of this step are as described in the description of the method for transporting spheroids. [Example]

[0046] <Spheroid production> Frozen human adipose-derived stem cells (catalog number: PT-5006, purchased from Lonza) were thawed in a 37°C water bath and added to 9 mL of basal medium (trade name: KBM ADSC-1, manufactured by Kohjin Bio Co., Ltd.) containing 5% FBS and 1% antibiotics. After centrifugation at 210 × g for 5 minutes, the supernatant was removed and the cells were dispersed in the basal medium. This cell suspension was transferred to two or three culture flasks (culture area 225 cm). 2 ) to 30 mL (1.0 × 10 6Each flask was added with 10 mL of KBM ADSC-1 medium containing 5% FBS and 1% antibiotics, and the medium was transferred to a tube. After centrifugation at 210 × g for 5 minutes, the cells were suspended in 4 mL of basal medium containing 1% antibiotics (KBM ADSC-2, Kohjin Bio Co., Ltd.). The number of cells was counted, and the concentration was adjusted to 1.0 × 10 6 The concentration was adjusted to 100 cells / mL. Cells were seeded at 500 cells / well onto a culture substrate that had been degassed using the method described below. The substrate was left to stand in a safety cabinet for 15 minutes, then placed in a 5% (v / v) CO2 incubator at 37°C and cultured for 3 days to produce spheroids.

[0047] The culture substrate used to produce spheroids was a substrate with a hole (cavity) having a circular opening of approximately 300 μm in diameter and a bottom surface. This substrate contained 6FDA / TPEQ copolymer (weight average molecular weight: 180,000) on the bottom surface, and the rest of the surface was coated with MPC polymer. The details of the substrate are as follows: Number of holes: 42,000 (base material 1cm 2 (449 pieces per box) Hole bottom: smooth Distance between openings: 200μm Hole depth: 300μm Base material area (total area of ​​the top surface of the base material and openings): 93.6 cm 2

[0048] The substrate was degassed before use as follows: First, approximately 40 mL of PBS was added to the substrate and pipetted repeatedly, and the substrate was left to stand in a 5% (v / v) CO2 incubator at 37°C for approximately 15 minutes. Next, after pipetting again, the PBS was aspirated, and 40 mL of KBM ADSC-2 medium was added to the substrate. The substrate was then left to stand overnight in a 5% (v / v) CO2 incubator at 37°C. The medium was removed from the substrate immediately before use.

[0049] <Test Example 1> <Storage of spheroids> After spheroid formation, the medium was removed from the culture substrate, and 20 mL of lactated Ringer's solution (trade name: Lactec (registered trademark) Injection, manufactured by Otsuka Pharmaceutical Factory, Inc.) was added. The spheroids were detached by repeated pipetting. The resulting spheroid suspension was collected. 20 mL of Lactec Injection was added again to the substrate, and the spheroids were detached by pipetting. The spheroid suspension was collected. The substrate was centrifuged at 210 × g for 3 minutes, and the spheroids were suspended in 40 mL of Lactec Injection. The substrate was centrifuged again at 210 × g for 3 minutes, and the spheroids were suspended in 5–6 mL of Lactec Injection. The resulting spheroid suspension was dispensed into 1.5 mL Eppendorf tubes. The Eppendorf tubes were stored in a 4°C refrigerator or a 37°C 5% (v / v) CO2 incubator for 24 hours.

[0050] <Spheroid evaluation> During storage, the spheroids were observed for morphology and cell viability was measured. Cell viability was measured as follows: First, the Eppendorf tube containing the spheroids was centrifuged at 500 × g for 1 minute, and the supernatant was removed. The spheroids were then suspended in 500 μL of cell detachment solution (product name: Accumax™, manufactured by Promocell) and incubated in a 37°C incubator for 10 minutes, with pipetting every 2 minutes. Then, 1 mL of KBM ADSC-1 medium was added, and the mixture was centrifuged at 210 × g for 5 minutes. The supernatant was removed, and the cells were suspended in 500 μL of Lactec Injection. The cell suspension was passed through a cell strainer (manufactured by Falcon), and the resulting filtrate was used to calculate cell viability using the trypan blue exclusion method according to the following formula: Cell viability (%) = [1.00 - (number of blue cells / total number of cells)] x 100

[0051] The results are shown in Figures 1 and 2. The spheroid shape and cell viability were highly maintained after storage at 37°C for 3 hours and at 4°C for 24 hours.

[0052] <Test Example 2> After spheroid production, the spheroids were recovered from the culture substrate and suspended in 5-6 mL of Lactec solution using the same method as in Test Example 1. The resulting spheroid suspension was sealed in a syringe and transported at 5±3°C for 0-72 hours. The spheroids were recovered 0, 24, 48, or 72 hours after the start of transportation, and cell viability was measured using a method similar to Test Example 1. Stem cell markers were also measured using flow cytometry. The results are shown in Table 1. The spheroids obtained after 0 and 72 hours were observed for morphology and subjected to RT-qPCR expression analysis of stemness genes (Oct-4 and Nanog). The results are shown in Figure 3 and Table 1. The results of the gene expression analysis were calculated by the ΔΔCt method to calculate the relative expression level (2 -ΔΔCt β-actin was used as an endogenous control gene, and StepOne™ (Applied Biosystems) was used as the thermal cycler.

[0053] [Table 1]

[0054] The cell viability after syringe sealing was 90% or higher at all times. The abundance ratio of negative markers was approximately 1.5% at all times, while the abundance ratio of positive markers was 99% or higher. Furthermore, the spheroid shape and stemness gene expression were maintained even after 72 hours of transportation. In other words, spheroids could be transported from 0 to 72 hours without changing the cell viability, the abundance ratio of stem cell marker-expressing cells, or the expression level of stemness genes, while maintaining their shape.

Claims

1. Transporting the spheroids containing mesenchymal stem cells at 40°C or less, A method for transporting spheroids, wherein the spheroids are suspended in a solution in a syringe.

2. The method of claim 1 , wherein the solution is a solution that can be administered to a living body.

3. The method of claim 2 , wherein the solution is a solution containing sodium, potassium, and calcium salts.

4. 4. The method of claim 3, wherein the solution is Ringer's solution, lactated Ringer's solution, acetated Ringer's solution, or bicarbonated Ringer's solution.

5. 5. The method of claim 4, wherein the solution is lactated Ringer's solution.

6. The method of claim 1 or 2, wherein the mesenchymal stem cells are derived from adipose tissue.

7. The method according to claim 1 or 2, further comprising the step of culturing mesenchymal stem cells in an adherent manner on a substrate containing a polyimide resin to obtain the spheroids, prior to the step of transporting the spheroids.

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