Method for producing cells into which exogenous mitochondria have been introduced.

Abstract

Provided is a production method of cells into which exogenous mitochondria have been introduced, the production method comprising culturing recipient cells using cell culture equipment in which mitochondria isolated from donor cells are coated on the culture surface, and incorporating the mitochondria into the recipient cells. Also provided are cells produced by the production method, the cells having exogenous mitochondria introduced thereinto. Also provided are a cell culture substrate usable in the aforesaid production method and a method for manufacturing the same, and a cell culture kit.

Description

Technical Field

[0001] The present invention relates to a method for producing cells into which exogenous mitochondria have been introduced.

Background Art

[0002] Mitochondria are one of the organelles present in eukaryotic cells and play important roles in adenosine triphosphate (ATP) production and apoptosis. In recent years, it has been reported that introducing exogenous mitochondria into transplant cells improves cell function and enhances the effect of cell transplantation therapy (see, for example, Non-Patent Document 1), and various methods for introducing exogenous mitochondria into cells have been studied.

[0003] As methods for introducing exogenous mitochondria into cells, (1) a method of incubating exogenous mitochondria together with recipient cells (see, for example, Patent Document 1); (2) a method of modifying the surface of exogenous mitochondria with a cell membrane-permeable peptide and then incubating with recipient cells (see, for example, Non-Patent Document 2); (3) a method of introducing exogenous mitochondria into recipient cells by microinjection (see, for example, Patent Document 2); etc. are known.

[0004] However, the method (1) above has the problem that although the operation is simple, the introduction efficiency is low because the surface of mitochondria is negatively charged. Although the method (2) above has a higher introduction efficiency than the method (1), in addition to the laborious modification operation of mitochondria, there is a concern that cells may be damaged by the cell membrane-permeable peptide, which is a cationic substance. Further, although the method (3) above can surely introduce mitochondria into recipient cells, in addition to the need for advanced techniques for the operation, there is a limit to the number of target recipient cells. Furthermore, damage to cells is also a concern in the method (3).

Prior Art Documents

Patent Documents

[0005] [Patent Document 1] Special Publication No. 2021-532095 [Patent Document 2] Special Publication No. 2019-500395 [Non-patent literature]

[0006] [Non-Patent Document 1] Guo, Y. et al., Stem Cell Res. Ther., 11, 245, 2020 [Non-Patent Document 2] Maeda, H. et al., J. Cell. Mol. Med., 24, 5007-5020, 2020 [Overview of the project] [Problems that the invention aims to solve]

[0007] Therefore, the object of the present invention is to provide a novel method for producing cells into which exogenous mitochondria have been introduced. [Means for solving the problem]

[0008] The following embodiments are specific means for solving the above problems. <1> A method for producing cells into which exogenous mitochondria have been introduced, A production method comprising culturing recipient cells using cell culture equipment coated with mitochondria isolated from donor cells, and allowing the recipient cells to take up the mitochondria. <2> The cell culture equipment is a cell culture vessel or a cell culture carrier. <1> The production method described above. <3> <1> or <2> Cells into which exogenous mitochondria have been introduced, produced by the production method described above. <4> The amount of protein from the introduced exogenous mitochondria was 2.0 × 10⁶ 5 The amount is 0.6 μg or more per cell. <3> The cells described. <5> Cell culture equipment with isolated mitochondria coated on the culture surface. <6> A cell culture vessel or cell culture carrier, <5> Cell culture substrate as described above. <7> A method for producing a cell culture vessel coated with isolated mitochondria, A manufacturing method comprising adding a liquid containing isolated mitochondria to the cell culture vessel and centrifuging the cell culture vessel to adhere the mitochondria to the culture surface of the cell culture vessel. <8> A cell culture kit comprising a cell culture substrate and a liquid containing isolated mitochondria. [Effects of the Invention]

[0009] According to the present invention, a novel method for producing cells into which exogenous mitochondria have been introduced can be provided. [Brief explanation of the drawing]

[0010] [Figure 1] This figure shows mitochondria that have adhered to the inner bottom surface of a dish after being added to the dish and incubated for a predetermined time, labeled with green fluorescent protein (GFP). [Figure 2A] This figure shows the fluorescence intensity of the supernatant after centrifugation of a suspension of GFP-labeled mitochondria suspended in phosphate-buffered saline (PBS) using various centrifugal forces for 10 minutes. [Figure 2B] This figure shows the fluorescence intensity of a pellet suspension obtained by centrifuging a suspension of GFP-labeled mitochondria in PBS for 10 minutes using various centrifugal forces, and then resuspending the resulting mitochondrial precipitate (pellet) in PBS. [Figure 3] This figure shows the mitochondria adhering to the inner bottom surface of the wells when centrifugation of the plate was performed after adding mitochondria stained with carboxyfluorescein succinimimidyl ester (CSFE) to the plate, and when centrifugation was not performed. [Figure 4]This figure shows the geometric mean fluorescence intensity (gMFI) of cells cultured using two plates: one in which GFP-labeled mitochondria were added to a plate, followed by plate centrifugation and incubation for a predetermined time to coat the inner bottom surface of the wells, and another in which mitochondria were coated by incubation only without plate centrifugation. The cells were then analyzed by flow cytometry. [Figure 5] These are confocal laser microscope images showing the mitochondria taken up by cells when C3H10T1 / 2 cells were seeded on a plate coated with GFP-labeled mitochondria, and when C3H10T1 / 2 cells were seeded on a plate without mitochondrial coating, and then mitochondria were added to the adhered C3H10T1 / 2 cells. [Figure 6] This figure shows the geometric mean fluorescence intensity of C3H10T1 / 2 cells after flow cytometry analysis, comparing two cases: when C3H10T1 / 2 cells were seeded on a plate coated with GFP-labeled mitochondria, and when C3H10T1 / 2 cells were seeded on a plate without mitochondrial coating, and then mitochondria were added to the adhered C3H10T1 / 2 cells. [Figure 7] This figure shows the relative cell count after culture when C3H10T1 / 2 cells were seeded on a mitochondrial-coated plate, and when C3H10T1 / 2 cells were seeded on an uncoated plate and then mitochondria were added to the adhered C3H10T1 / 2 cells. [Figure 8] This figure shows the amount of ATP produced by C3H10T1 / 2 cells when seeded on a mitochondrial-coated plate. [Figure 9] This figure shows the relative cell count after culturing C3H10T1 / 2 cells on plates coated with various numbers of mitochondria. [Figure 10]This figure shows the relative cell count after culturing oligomycin-treated C3H10T1 / 2 cells on plates coated with various numbers of mitochondria. [Figure 11A] This figure shows in vivo imaging images of mice after subcutaneous administration of mitochondrial-transformed NanoLuc luciferase-expressing C3H10T1 / 2 cells or untreated NanoLuc luciferase-expressing C3H10T1 / 2 cells into the back. [Figure 11B] This figure shows the plasma luciferase activity when mitochondrial transgenic NanoLuc luciferase-expressing C3H10T1 / 2 cells or untreated NanoLuc luciferase-expressing C3H10T1 / 2 cells were subcutaneously administered to the back of mice. [Figure 12A] This figure shows serum AST levels when mitochondrial transgenic NanoLuc luciferase-expressing C3H10T1 / 2 cells or untreated NanoLuc luciferase-expressing C3H10T1 / 2 cells were intravenously administered to carbon tetrachloride (CCl4) induced liver injury model mice. [Figure 12B] This figure shows serum ALT levels when mitochondrial transgenic NanoLuc luciferase-expressing C3H10T1 / 2 cells or untreated NanoLuc luciferase-expressing C3H10T1 / 2 cells were intravenously administered to CCl4-induced liver injury model mice. [Figure 13] This figure shows the relative cell counts after culture when various cells (C3H10T1 / 2 cells, Hepa1-6 cells, HEK293 cells, HaCaT cells) were seeded on plates coated with mitochondria, and when the same cells were seeded on plates without mitochondria coating. [Figure 14A] This figure shows the amount of mitochondrial protein incorporated per 2.0 × 10⁵ cells when HEK293 cells were seeded on a plate coated with mitochondria, and when mitochondria were added after HEK293 cells were seeded on a plate. [Figure 14B]This figure shows the amount of mitochondrial protein taken up per 2.0 × 10⁵ cells when HaCaT cells were seeded on a plate coated with mitochondria, and when mitochondria were added after HaCaT cells were seeded on the plate. [Figure 15] This figure shows the relative cell count after culturing C3H10T1 / 2 cells in the presence of polyethyleneimine (PEI) at various concentrations. [Modes for carrying out the invention]

[0011] The method for producing cells into which exogenous mitochondria have been introduced according to this embodiment (hereinafter also simply referred to as the "production method") includes culturing recipient cells using cell culture equipment coated with mitochondria isolated from donor cells on the culture surface, and allowing the recipient cells to take up mitochondria (exogenous mitochondria).

[0012] "Donor cells" refer to cells that provide mitochondria, and "recipient cells" refer to cells into which mitochondria are introduced. Donor cells and recipient cells may be cells containing normal mitochondria, or they may be cells containing dysfunctional mitochondria with mutated mitochondrial DNA. When cells containing dysfunctional mitochondria are used as recipient cells, at least a portion of the endogenous mitochondria may be removed beforehand using conventionally known methods.

[0013] The species from which donor and recipient cells originate are not particularly limited, as long as they are species that contain mitochondria within their cells; they may be animals or plants. Examples of species include mammals such as mice, rats, dogs, sheep, monkeys, and humans. Donor and recipient cells may originate from different individuals of the same species, or from different species.

[0014] The cell types of donor and recipient cells are not particularly limited. If the donor and recipient cells are animal cells, examples of cell types include muscle cells, hepatocytes, fibroblasts, epithelial cells, nerve cells, adipocytes, and mesenchymal stem cells. The donor and recipient cells may be of the same species or different species.

[0015] Any method can be used to isolate mitochondria from donor cells, and commercially available kits can be used if necessary. Known methods for isolating mitochondria from donor cells include lysing the donor cells and then isolating the mitochondrial fraction by centrifugation, and creating pores in the cell membrane of the donor cells and then isolating the mitochondrial fraction by centrifugation. Among these, from the viewpoint of isolating mitochondria with minimal damage, the method of creating pores in the cell membrane of the donor cells and then isolating the mitochondrial fraction is preferred, and in particular, the method of creating pores in the cell membrane of the donor cells using SLO and then isolating the mitochondrial fraction is preferred (see, for example, Shibata, T. et al., Biochem. Biophys. Res. Commun., 463, 563-568, 2015).

[0016] Conventional cell culture equipment capable of culturing recipient cells can be used as cell culture equipment for coating isolated mitochondria. Examples of cell culture equipment include cell culture vessels such as dishes, plates, and flasks; and cell culture carriers such as microcarriers. Examples of materials for cell culture equipment include glass; synthetic polymers such as polyethylene, polypropylene, and polystyrene; natural polymers such as cellulose and collagen; and metals. The cell culture equipment may also be a cell culture vessel for producing cell sheets in which a temperature-responsive polymer is immobilized on the culture surface. Examples of temperature-responsive polymers include poly(N-isopropylacrylamide).

[0017] The isolated mitochondria are coated onto the culture surface of the cell culture equipment. The "culture surface" can be any surface that can come into contact with recipient cells during culture; for example, the inner bottom surface of a cell culture vessel, or the outer surface of a cell culture carrier.

[0018] Any method can be used to coat the culture surface of cell culture equipment with isolated mitochondria, as long as the mitochondria adhere to the culture surface. Typically, the mitochondria can adhere to the culture surface simply by allowing the solution containing the isolated mitochondria to stand in contact with the culture surface. The standing time is preferably 12 hours or more, and more preferably 24 hours or more. The solution in which the isolated mitochondria are suspended is not particularly limited and may be a cell culture medium or a buffer such as PBS.

[0019] When the cell culture equipment is a cell culture vessel such as a dish or plate, adding a solution containing isolated mitochondria to the cell culture vessel and then centrifuging the vessel allows for more efficient and reliable adhesion of mitochondria to the culture surface. The centrifugation conditions are not particularly limited and can be selected as appropriate, but examples include 300g to 3200g for 30 seconds to 60 minutes. After centrifugation, the cell culture vessel may be allowed to stand further with the solution containing isolated mitochondria added.

[0020] The number of mitochondria coated on the culture surface of the cell culture equipment is appropriately set according to the type of cell culture equipment and the number of recipient cells. For example, it is preferable to coat the culture surface of the cell culture equipment with mitochondria isolated from donor cells in a number 2 to 100 times that of the recipient cells. The density of mitochondria coated on the culture surface of the cell culture equipment is, for example, 5 to 15 μg / cm³. 2 It is preferable that this be the case.

[0021] After obtaining cell culture equipment coated with isolated mitochondria as described above, recipient cells can be cultured using this cell culture equipment to incorporate mitochondria (exogenous mitochondria) into the recipient cells. When culturing recipient cells, cell culture equipment that has been pre-coated with isolated mitochondria may be used. Alternatively, when culturing recipient cells, a cell culture kit comprising a cell culture substrate and a solution containing isolated mitochondria may be used to coat the culture surface of the cell culture equipment with isolated mitochondria.

[0022] There are no particular restrictions on the cell concentration when culturing recipient cells. For example, 1.0 × 10⁻⁶ 4 cells / mL ~ 5.0 × 10 5 Cell concentrations can be expressed as cells / mL.

[0023] Various culture media are used for culturing recipient cells, depending on the type of recipient cell. Standard cell culture conditions can also be used for culturing recipient cells. For example, cultivation can be performed at a temperature of 30°C to 40°C, a relative humidity of 90% to 98%, and a CO2 concentration of 3% to 7%. The culture time is preferably 3 to 72 hours, and more preferably 6 to 24 hours.

[0024] As described above, introducing exogenous mitochondria into recipient cells can enrich the mitochondria within the cells, thereby improving cell function and proliferation. Furthermore, introducing normal exogenous mitochondria into recipient cells from which at least some of the dysfunctional endogenous mitochondria has been removed can also improve cell function and proliferation.

[0025] In particular, the production method according to this embodiment makes it possible to introduce a far larger amount of exogenous mitochondria into cells compared to the method of incubating exogenous mitochondria with recipient cells (see, for example, Patent Document 1). The amount of exogenous mitochondrial protein introduced into cells by the production method according to this embodiment is, for example, 2.0 × 10⁻⁶. 5 The amount per cell is 0.6 μg or more, preferably 0.8 μg or more, and more preferably 1.0 μg or more. Furthermore, the production method according to this embodiment causes less damage to cells compared to methods using cell membrane-permeable peptides which are cationic substances (see, for example, Non-Patent Document 2) or methods using microinjection (see, for example, Patent Document 2).

[0026] Cells into which exogenous mitochondria have been introduced can be suitably used, for example, in cell therapy and substance production. Because cells into which exogenous mitochondria have been introduced tend to have a longer survival period after transplantation, it is possible to enhance the effectiveness of cell transplantation therapy and optimize substance production using cells. Cell sheets can also be prepared using cells into which exogenous mitochondria have been introduced. The resulting cell sheets can be suitably used in regenerative medicine and the like. It is also possible to directly obtain cell sheets into which exogenous mitochondria have been introduced by coating mitochondria with a cell culture vessel for cell sheet preparation in which a temperature-responsive polymer is immobilized on the culture surface, and then culturing recipient cells in this cell culture vessel. [Examples]

[0027] The present invention will be described more specifically below with reference to examples, but the present invention is not limited to these examples.

[0028] <Preparation Example 1: Isolation of Mitochondria (SLO Method)> Two types of cells were prepared as donor cells for isolating mitochondria (hereinafter also referred to as "mt"). C3H10T1 / 2 cells: Mouse mesenchymal stem cells 3T3-L1-mt-GFP cells: A mouse fibroblast cell line expressing a fusion protein of mitochondrial-transitioning cox8a signaling and GFP.

[0029] First, prepare 1.0 × 10⁶ C3H10T1 / 2 cells or 3T3-L1-mt-GFP cells. 6 Cells were seeded in 15 cm dishes (Thermo Fisher Scientific) according to the cell count and cultured for 3 days. After culture, the cells were washed with PBS and harvested using trypsin / EDTA solution (2.5 g / L trypsin, 1 mmol / L EDTA). The harvested cells were suspended in HEPES-CH3COOK buffer (480 μL) with 1 μg / mL streptolin O (Fujifilm Wako Pure Chemical Industries) (20 μL) added, incubated at room temperature for 1-5 minutes, and then left on ice for 10 minutes. Next, the cells were washed with Tris-sucrose buffer at 4°C, resuspended in Tris-sucrose buffer, and incubated at 37°C for 10 minutes. The cell suspension was pipetted 200 times, centrifuged (400 g, 10 minutes, 4°C), and the supernatant containing mitochondria was collected. Furthermore, the recovered supernatant was centrifuged (7000g, 10 minutes, 4°C) and the supernatant was removed. The obtained mitochondrial fraction was suspended in DMEM medium containing 15% FBS and stored at 4°C. Hereafter, the procedure for isolating mitochondria using streptolin O as described above will be referred to as the "SLO method".

[0030] <Test Example 1: Adhesion of Mitochondria to the Bottom of a Dish> First, 1.6 × 10 7 Mitochondria were isolated from cells' 3T3-L1-mt-GFP cells using the SLO method. The mitochondrial pellet was suspended in 10% FBS-containing DMEM medium (530 μL), and the mitochondria were placed in a polylysine-coated 35 mm glass-bottom dish in a 3.0 × 10⁶ size. 6Cells were added to achieve mt / well and incubated for a predetermined time (1, 12, 24 hours). Then, mitochondria were fixed using 4% paraformaldehyde phosphate buffer, and a mounting medium containing DAPI (4’,6-diamidino-2-phenylindole, Vector laboratories) was added. Subsequently, fluorescence images were taken using a confocal laser microscope (SP8, Leica) with imaging software (LAS X Life Science, Leica).

[0031] The fluorescence images are shown in Figure 1 (scale bar: 40 μm). As can be seen from Figure 1, it was confirmed that mitochondria adhered to the inner bottom surface of the dish after incubation for about 12 hours.

[0032] <Test Example 2: Optimization of Centrifugation Conditions for Coating Mitochondria> First, mitochondria were isolated from 2.0×10 7 cells of 3T3-L1-mt-GFP cells using the SLO method. The isolated mitochondria were suspended in PBS to achieve 3.0×10 6 cells mt / 300 μL and centrifuged at various centrifugal forces (300 g, 500 g, 1000 g, 1500 g, 2000 g). The temperature during centrifugation was fixed at 4°C and the time was 10 minutes. The supernatant (300 μL) after centrifugation was collected, and the mitochondrial precipitate (pellet) was resuspended in PBS (300 μL). Then, the supernatant and pellet suspension were each added to a 96-well plate (Corning) to achieve 90 μL / well, and the fluorescence intensity was measured using a microplate reader (ARVO-MX, PerkinElmer).

[0033] The fluorescence intensity of the supernatant is shown in Figure 2A, and the fluorescence intensity of the pellet suspension is shown in Figure 2B. Each data in the figure represents the mean value ± standard deviation of three samples. Also, the "*" in the figure indicates statistical significance ( *p<0.05; Dunnett's test). As can be seen from Figures 2A and 2B, by increasing the centrifugal force to 1500g or more, the fluorescence intensity of the supernatant decreased further, while the fluorescence intensity of the pellet suspension increased further, yielding particularly favorable results.

[0034] <Test Example 3: Mitochondria Coating by Plate Centrifuge> First, 2.0 × 10 7 Mitochondria were isolated from cells C3H10T1 / 2 cells using the SLO method. 1 mL of 10 μM CFSE solution was added to the isolated mitochondria, and the cells were left on ice for 30 minutes to stain them. After washing twice with PBS, the stained mitochondria were placed in a 12-well plate in a 3.0 × 10⁶ arrangement. 6 Cells were added to achieve a cell mt / well ratio, and the plate was centrifuged (1500g, 10 minutes, 4°C). Fluorescence images were then captured using a digital fluorescence microscope (BZ-9000, Keyence).

[0035] For comparison, we also prepared two groups: one in which the same procedure as above was performed except for not performing plate centrifugation, and another in which neither mitochondrial addition nor plate centrifugation was performed. Fluorescence images were then taken in the same manner as above.

[0036] The fluorescence image is shown in Figure 3 (scale bar: 500 μm). As can be seen from Figure 3, it was confirmed that mitochondria coated the inner bottom surface of the wells by plate centrifugation for 10 minutes. Hereafter, the procedure of coating the inner bottom surface of the wells with mitochondria by plate centrifugation as described above will be referred to as "mt coating".

[0037] <Example Test 4: Evaluation of Mitochondrial Uptake (FACS)> First, 2.0 × 10 7 Mitochondria were isolated from cells' 3T3-L1-mt-GFP cells using the SLO method. 3.0 × 10⁶ mitochondria were placed in a 12-well plate. 6Cells were added to a ratio of cells mt / well, followed by plate centrifugation (1500g, 10 minutes, 4°C), and then incubation for 12 hours to coat the inner bottom surface of the wells with mitochondria. Subsequently, C3H10T1 / 2 cells were added in a 3.0 × 10⁶ well. 5 Cells were seeded to a density of cells / well and cultured for 24 hours. After culturing, cells were detached from the wells using trypsin / EDTA solution (2.5 g / L trypsin, 1 mmol / L EDTA) and suspended in PBS. The suspended cells were then fixed using 4% paraformaldehyde-phosphate buffer, replaced with PBS, and mitochondrial uptake was analyzed using a flow cytometer (BD FACS Lyric, Beckton Dickinson). Data analysis was performed using analysis software (FlowJo software version 8.7, Beckton Dickinson).

[0038] For comparison, we also prepared a group in which the same procedure as above was performed except that plate centrifugation was not performed, and an untreated group in which the same procedure as above was performed except that a 12-well plate without mitochondrial coating was used, and analyzed them using a flow cytometer in the same manner as above.

[0039] Figure 4 shows the geometric mean fluorescence intensity for each group. Each data point in the figure represents the mean ± standard deviation of the three samples. An asterisk (*) in the figure indicates statistical significance. * p<0.05; Tukey-Kramer's test). As can be seen in Figure 4, mitochondria were taken up even when coated by 12 hours of incubation alone, but plate centrifugation significantly promoted mitochondrial uptake.

[0040] <Test Example 5: Evaluation of Mitochondrial Uptake (Microscopic Observation)> First, 2.0 × 10 7 Mitochondria were isolated from cells' 3T3-L1-mt-GFP cells using the SLO method. 3.0 × 10⁶ mitochondria were placed in a 12-well plate.6 Mitochondria were added to the wells to a concentration of cells mt / well, and then the plates were centrifuged (1500g, 10 minutes, 4°C) to coat the inner bottom surface of the wells with mitochondria. Next, 3.0 × 10⁶ C3H10T1 / 2 cells were added. 5 Cells were seeded to a density of cells / well and cultured for 24 hours. After culturing, cells were detached from the wells using trypsin / EDTA solution (2.5 g / L trypsin, 1 mmol / L EDTA) and seeded in a 35 mm glass-bottom dish for 12 hours of culture. Next, adherent cells were fixed using 4% paraformaldehyde-phosphate buffer, and a DAPI-containing mounting medium (Mounting Medium with DAPI, Vector Laboratories) was added. Fluorescence images were then acquired using a confocal laser microscope (SP8, Leica) and imaging software (LAS X Life Science, Leica).

[0041] For comparison, we also prepared two groups: one in which the inner bottom surface of the wells was not coated with mitochondria, and mitochondria were added to the adherent cells; and an untreated group in which the same procedure as above was performed except for using a 12-well plate without mitochondria coating.

[0042] In the group to which mitochondria were added to adherent cells, first, the cell density at the time of mitochondrial addition was 3.0 × 10⁶. 5 C3H10T1 / 2 cells were seeded into a 12-well plate to achieve a cell / well ratio and cultured until the following day. 2.0 × 10 7 Mitochondria were isolated from 3T3-L1-mt-GFP cells using the SLO method, and the isolated mitochondria were divided into 3.0 × 10⁶ units. 6Cells were added to a concentration of cells mt / well and cultured for 24 hours. After culturing, cells were detached from the wells using trypsin / EDTA solution (2.5 g / L trypsin, 1 mmol / L EDTA), seeded in a 35 mm glass-bottom dish, and cultured for 12 hours. Then, adherent cells were fixed using 4% paraformaldehyde-phosphate buffer, and fluorescence images were taken in the same manner as above.

[0043] Fluorescence images are shown in Figure 5 (scale bar: 40 μm). As can be seen from Figure 5, when cells were cultured on a plate in which the inner bottom surface of the wells was coated with mitochondria, the amount of mitochondrial uptake increased compared to when the inner bottom surface of the wells was not coated with mitochondria and mitochondria were added to adherent cells.

[0044] <Example Test 6: Comparison of Mitochondrial Uptake (FACS)> First, 3.0 × 10 7 Mitochondria were isolated from cells' 3T3-L1-mt-GFP cells using the SLO method. 3.0 × 10⁶ mitochondria were placed in a 12-well plate. 6 Mitochondria were added to the wells to a concentration of cells mt / well, and then the plates were centrifuged (1500g, 10 minutes, 4°C) to coat the inner bottom surface of the wells with mitochondria. Next, 3.0 × 10⁶ C3H10T1 / 2 cells were added. 5 Cells were seeded to a density of cells / well and cultured for 24 hours. After culturing, cells were detached from the wells using trypsin / EDTA solution (2.5 g / L trypsin, 1 mmol / L EDTA) and suspended in PBS. The suspended cells were then fixed using 4% paraformaldehyde-phosphate buffer, replaced with PBS, and mitochondrial uptake was analyzed using a flow cytometer (BD FACS Lyric, Beckton Dickinson). Data analysis was performed using analysis software (FlowJo software version 8.7, Beckton Dickinson).

[0045] For comparison, we also prepared two groups: one in which the inner bottom surface of the wells was not coated with mitochondria, and the other in which mitochondria were added to adherent cells; and an untreated group using 12-well plates without mitochondria coating.

[0046] In the group to which mitochondria were added to adherent cells, first, the cell density at the time of mitochondrial addition was 3.0 × 10⁶. 5 C3H10T1 / 2 cells were seeded into a 12-well plate to achieve a cell / well ratio and cultured until the following day. 3.0 × 10 7 Mitochondria were isolated from 3T3-L1-mt-GFP cells using the SLO method, and the isolated mitochondria were divided into 3.0 × 10⁶ units. 6 Cells were added to a concentration of cells mt / well and cultured for 24 hours. After culturing, cells were detached from the wells using trypsin / EDTA solution (2.5 g / L trypsin, 1 mmol / L EDTA) and suspended in PBS. Then, the suspended cells were fixed using 4% paraformaldehyde-phosphate buffer and analyzed using a flow cytometer as described above.

[0047] In the untreated group, first, the cell density the following day was 3.0 × 10⁶. 5 C3H10T1 / 2 cells were seeded into 12-well plates to a cell / well ratio and cultured until the following day. Next, 15% FBS-containing DMEM medium was added in place of mitochondria, and the cells were cultured for 24 hours. After culture, the cells were detached from the wells using trypsin / EDTA solution (2.5 g / L trypsin, 1 mmol / L EDTA) and suspended in PBS. The suspended cells were then fixed using 4% paraformaldehyde-phosphate buffer and analyzed using a flow cytometer as described above.

[0048] Figure 6 shows the geometric mean fluorescence intensity for each group. Each data point in the figure represents the mean ± standard deviation of the three samples. An asterisk (*) in the figure indicates statistical significance. *p<0.05; Tukey-Kramer's test). As can be seen in Figure 6, when cells were cultured on plates with mitochondria coated on the inner bottom surface of the wells, the amount of mitochondrial uptake was significantly increased compared to when the inner bottom surface of the wells was not coated with mitochondria and mitochondria were added to adherent cells.

[0049] <Test Example 7: Evaluation of Proliferative Ability of Mitochondrial Transplanted Cells> Mitochondria derived from C3H10T1 / 2 cells (3.0 × 10⁻¹⁰ 6 C3H10T1 / 2 cells (3.0 × 10¹⁶ cells) were placed in a 12-well plate coated with cells mt / well. 5 Cells with introduced mitochondria were obtained by seeding cells (3.0 × 10⁶ cells / well) and culturing them for 24 hours. For comparison, adherent C3H10T1 / 2 cells (3.0 × 10⁶ cells / well) were also obtained. 5 Mitochondria (3.0 × 10) derived from C3H10T1 / 2 cells (cells / well) 6 A group was prepared with cells (mt / well) added. Furthermore, for comparison, an untreated group was prepared using a 12-well plate without mitochondrial coating, with 15% FBS-containing DMEM medium added instead of mitochondria. Cells prepared using each method were placed in a 96-well plate (Corning) at 5.0 × 10⁴. 3 Cells were seeded to a cell / well ratio and cultured for 48 hours. Then, the number of cells after culture was measured using Cell Counting Kit-8 (CCK-8) (Fujifilm Wako Pure Chemical Industries). Specifically, 100 μL of CCK-8 solution was added to each well, incubated for 30 minutes, and then the number of cells was measured by measuring the absorbance at a wavelength of 450 nm.

[0050] Figure 7 shows the relative cell counts for each group. Each data point in the figure represents the mean ± standard deviation of the three samples. In the figure, "ns" indicates that the value is not statistically significant, and "*" indicates that the value is statistically significant. *p<0.05; Tukey-Kramer's test). As can be seen in Figure 7, when cells were cultured on plates with mitochondria coated on the inner bottom surface of the wells, cell proliferation was significantly enhanced compared to when the inner bottom surface of the wells was not coated with mitochondria and mitochondria were added to adherent cells.

[0051] <Test Example 8: Evaluation of ATP Production in Mitochondrial Transplanted Cells> Mitochondria derived from C3H10T1 / 2 cells (3.0 × 10⁻¹⁰ 6 C3H10T1 / 2 cells (3.0 × 10¹⁶ cells) were placed in a 12-well plate coated with cells mt / well. 5 Cells with introduced mitochondria were obtained by seeding cells (cells / well) and culturing for 24 hours. For comparison, an untreated group was prepared using a 12-well plate without mitochondrial coating, supplemented with 15% FBS-containing DMEM medium instead of mitochondria. After culturing, cells were detached from the wells using trypsin / EDTA solution (2.5 g / L trypsin, 1 mmol / L EDTA), and 100 μL of the cell suspension was mixed with 100 μL of ATP measurement reagent. The mixed solution was added to a 96-well plate (Corning), and after standing for 10 minutes, the amount of luminescence derived from the cells was measured using a microplate reader (EnVision, PerkinElmer).

[0052] Figure 8 shows the luminescence levels of each group. Each data point in the figure represents the mean ± standard deviation of the three samples. As can be seen from Figure 8, when cells were cultured on a plate coated with mitochondria on the inner bottom surface of the wells, the ATP production of the cells increased significantly.

[0053] <Test Example 9: Evaluation of Mitochondrial Number-Dependent Cell Proliferation> First, 2.0 × 10 7 Mitochondria were isolated from cells C3H10T1 / 2 cells using the SLO method. A predetermined number of mitochondria (1.0 × 10⁶) were placed in a 12-well plate. 5 ,5.0×10 5,1.0×10 6 ,3.0×10 6 ,5.0×10 6 ,1.0×10 7 Mitochondria were added to the wells at a ratio of cells mt / well, and the plates were centrifuged (1500g, 10 minutes, 4°C) to coat the inner bottom surface of the wells with mitochondria. Next, C3H10T1 / 2 cells were added in a 3.0 × 10⁶ well. 5 Cells were seeded to a density of cells / well and cultured for 24 hours. After culturing, cells were detached from the wells using trypsin / EDTA solution (2.5 g / L trypsin, 1 mmol / L EDTA) and measured out in 1.0 × 10⁶ units. 4 Cells were seeded in a 96-well plate (Corning) to a cell / well ratio and cultured for 24 hours. The number of cells after culture was then measured using a cell counting kit (Cell Counting Kit-8 (CCK-8), Fujifilm Wako Pure Chemical Industries). Specifically, 100 μL of CCK-8 solution was added to each well and incubated for 30 minutes. The number of cells was then measured by measuring the absorbance at a wavelength of 450 nm.

[0054] For comparison, an untreated group was prepared using a 12-well plate without mitochondrial coating, except that the same procedure as above was followed.

[0055] Figure 9 shows the relative cell counts for each group. Each data point in the figure represents the mean ± standard deviation of the three samples. In the figure, "ns" indicates that the value is not statistically significant, and "*" indicates that the value is statistically significant. * p<0.05; Dunnett's test). As can be seen from Figure 9, cell proliferation was enhanced in a number-dependent manner.

[0056] <Test Example 10: Evaluation of Proliferative Ability of Mitochondrial-Introduced Oligomycin-Treated Cells> First, we divided the C3H10T1 / 2 cells into 2.0 × 10⁶ cells. 5Cells were seeded in a 6-well plate to achieve a cell / well ratio. After 24 hours, the medium was changed to DMEM medium containing 15% FBS and 10 μg / mL oligomycin. After another 24 hours, the cells were harvested using trypsin / EDTA solution (2.5 g / L trypsin, 1 mmol / L EDTA) and used as oligomycin-treated C3H10T1 / 2 cells for subsequent experiments.

[0057] 3.0×10 7 Mitochondria were isolated from cells C3H10T1 / 2 cells using the SLO method. A predetermined number of mitochondria (1.0 × 10⁶) were placed in a 96-well plate (Corning). 4 ,5.0×10 4 ,1.0×10 5 ,3.0×10 5 ,5.0×10 5 ,1.0×10 6 Mitochondria were added to the inner bottom surface of the wells by adding cells (mt / well) and centrifugation (1500g, 10 minutes, 4°C). Then, oligomycin-treated C3H10T1 / 2 cells were added in a 1.0 × 10⁶ layer. 4 Cells were seeded to a cell / well ratio and cultured for 24 hours. Then, the number of viable cells after culture was measured using a cell counting kit (Cell Counting Kit-8 (CCK-8), Fujifilm Wako Pure Chemical Industries). Specifically, 100 μL of CCK-8 solution was added to each well and incubated for 30 minutes, after which the number of cells was measured by measuring the absorbance at a wavelength of 450 nm.

[0058] For comparison, an untreated group was prepared using a 96-well plate without mitochondrial coating, except that the same procedure as above was followed.

[0059] Figure 10 shows the relative cell counts for each group. Each data point in the figure represents the mean ± standard deviation of the three samples. In the figure, "ns" indicates that the value is not statistically significant, and "*" indicates that the value is statistically significant. *p<0.05; Dunnett's test). As can be seen from Figure 10, cell proliferation was enhanced in cells treated with oligomycin, a mitochondrial inhibitor, in a manner dependent on the number of introduced mitochondria.

[0060] <Example Test 11: Evaluation of post-transplant survival rate of mitochondrial-derived cells> Mitochondria derived from C3H10T1 / 2 cells (3.0 × 10⁻¹⁰ 6 NanoLuc luciferase-expressing C3H10T1 / 2 cells (C3H10T1 / 2 / Nluc cells) (3.0 × 10⁶) were placed in a 12-well plate coated with cells mt / well. 5 Mitochondrial transgenic C3H10T1 / 2 / Nluc cells were obtained by seeding cells (cells / well) and culturing for 24 hours. Mitochondrial transgenic C3H10T1 / 2 / Nluc cells (mt-C3H10T1 / 2 / Nluc) or untreated C3H10T1 / 2 / Nluc cells were divided into 5.0 × 10⁶ cells. 5 Cells (200 μL) were prepared and subcutaneously administered to the backs of 6-week-old male BALB / c-nu / nu mice. Then, 50 μL of luciferin (Nano-Glo, Promega) was administered to the cell administration site, and in vivo imaging was performed daily using an imaging device (In-Vivo Xtreme, Bruker Daltonik GmbH). Blood samples were also collected over time, and plasma luciferase activity was measured using a microplate reader (EnVision, PerkinElmer).

[0061] In vivo imaging images of each group are shown in Figure 11A, and plasma luciferase activity is shown in Figure 11B. Each data point in Figure 11B represents the mean ± standard deviation of the three samples. An asterisk (*) in Figure 11B indicates statistical significance. * p<0.05; Dunnett's test). As can be seen from Figures 11A and 11B, introducing mitochondria into C3H10T1 / 2 / Nluc cells significantly improved cell viability after subcutaneous transplantation in mice.

[0062] <Example 12: Therapeutic effects in a mouse model of liver damage> Mitochondria derived from C3H10T1 / 2 cells (5.0 × 10⁻¹⁰ 6 NanoLuc luciferase-expressing C3H10T1 / 2 cells (C3H10T1 / 2 / Nluc cells) (3.0 × 10⁶) were placed in a 12-well plate coated with cells mt / well. 5 Mitochondrial transgenic C3H10T1 / 2 / Nluc cells were obtained by seeding cells / well and culturing for 24 hours. On the other hand, a liver injury model mouse was created by intraperitoneally administering 1 mL / kg body weight of carbon tetrachloride (CCl4) to 6-week-old male ddY mice. Six hours after CCl4 administration, 8.0 × 10⁶ cells were obtained. 5 Cells cells with mitochondrial transmutation (mt-C3H10T1 / 2 / Nluc) or untreated C3H10T1 / 2 / Nluc cells were administered intravenously, and blood was collected 24 hours later. Serum AST and ALT levels were then measured using a commercially available kit (Transaminases CII-Test Wako, Fujifilm Wako Pure Chemical Industries).

[0063] Serum AST levels are shown in Figure 12A, and serum ALT levels are shown in Figure 12B. Each data point in the figures represents the mean ± standard deviation of three samples. In Figure 12B, "ns" indicates that the value is not statistically significant, and "*" indicates that the value is statistically significant. * p<0.05; Dunnett's test). As can be seen from Figures 12A and 12B, introducing mitochondria into C3H10T1 / 2 / Nluc cells showed a high therapeutic effect on a mouse model of liver damage.

[0064] <Test Example 13: Evaluation of Proliferative Ability of Mitochondrial Transplanted Cells> 1.0 × 10 8Mitochondria were isolated from cells' C3H10T1 / 2 cells using the SLO method. Mitochondria were added to a 96-well plate at a concentration of 1 μg / well (50 μL / well), and the plate was centrifuged (1500 g, 10 minutes, 4°C) to coat the inner bottom surface of the wells. Then, 1.0 × 10⁶ C3H10T1 / 2 cells were separated. 3 cells / well, Hepa1-6 cells (mouse hepatocellular carcinoma cell line) 2.0 × 10⁻⁶ 3 cells / well, HEK293 cells (human embryonic kidney cell line) 2.0 × 10⁶ 3 cells / well, or HaCaT cells (human dermal keratinocyte line) 1.0 × 10 3 Cells were seeded to a cell / well ratio and cultured for 48 hours. The number of viable cells after culture was then measured using a cell counting kit (Cell Counting Kit-8 (CCK-8), Fujifilm Wako Pure Chemical Industries). Specifically, 100 μL of CCK-8 solution was added to each well and incubated for 30 minutes. The number of cells was then measured by measuring the absorbance at a wavelength of 450 nm.

[0065] For comparison, an untreated group was prepared using a 96-well plate without mitochondrial coating, except that the same procedure as above was followed.

[0066] Figure 13 shows the relative cell counts, with the mean cell count of the untreated group set to 100%. Each data point in the figure represents the mean ± standard deviation of the three samples. An asterisk (*) in the figure indicates statistical significance. * p<0.05; Student's t-test). As can be seen from Figure 13, cell proliferation was significantly enhanced regardless of whether C3H10T1 / 2 cells, Hepa1-6 cells, HEK293 cells, or HaCaT cells were used as recipient cells.

[0067] <Test Example 14: Comparison of Mitochondrial Uptake> 1.2 × 10 8Mitochondria were isolated from cells' C3H10T1 / 2 cells using the SLO method, and protein levels were measured using a protein assay kit (Pierce BCA Protein Assay Kits, Thermo Scientific). Mitochondria were added to 12-well plates at a concentration of 80 μg / well (500 μL / well), and the plates were centrifuged (1500 g, 10 minutes, 4°C) to coat the inner bottom surface of the wells. Then, HEK293 cells or HaCaT cells were added in 2.0 × 10⁶ units. 5 Cells were seeded to a density of cells / well and cultured for 24 hours. After culturing, cells were detached from the wells using trypsin / EDTA solution (2.5 g / L trypsin, 1 mmol / L EDTA), and RNA was recovered from the cells using a total RNA purification kit (Monarch Total RNA Miniprep Kit, BioLabs). Next, reverse transcription to cDNA was performed using a reverse transcription reaction kit (ReverTra Ace qPCR RT Master Mix with gDNA Remover, Toyobo) and a thermal cycler (GeneAtlas, Astec). Reverse transcription was performed sequentially at 37°C for 15 minutes, 50°C for 5 minutes, and 98°C for 5 minutes. Subsequently, the mouse COX2 gene was PCR amplified using a real-time PCR analysis system (CFX Connect, Bio-Rad) with a real-time PCR reagent (THUNDERBIRD SYBR qPCR Mix, Toyobo). The PCR protocol involved 39 cycles of 30 seconds at 95°C, followed by 5 seconds at 95°C, 15 seconds at 55°C, and 45 seconds at 72°C. The following primers were used for PCR amplification of the mouse COX2 gene. Forward primer: 5'-CCATCCCAGGCCGACTAA-3' (SEQ ID NO: 1) Reverse primer: 5'-AATTTCAGAGCATTGGCCATAGA-3' (SEQ ID NO: 2) Then, a calibration curve was created to show the relationship between mitochondrial protein amount (μg) and COX2 cycle number (Cq value). By substituting the COX2 cycle number of each cell into this calibration curve, the amount of mitochondrial protein taken up by each cell was calculated.

[0068] For comparison, we also prepared a group in which the same procedure as above was performed except that the inner bottom surface of the well was not coated with mitochondria, and mitochondria were added after seeding HEK293 cells or HaCaT cells.

[0069] Figure 14A shows the amount of mitochondrial protein taken up by HEK293 cells, and Figure 14B shows the amount of mitochondrial protein taken up by HaCaT cells. Each data point in the figures represents the mean ± standard deviation of three samples. An asterisk (*) in the figures indicates statistical significance. * p<0.05; Student's t-test). As can be seen from Figures 14A and 14B, 2.0 × 10 5 The amount of mitochondrial protein taken up per cell was approximately 0.8 μg in HEK293 cells and approximately 1.4 μg in HaCaT cells.

[0070] <Test Example 15: Coating Mitochondria onto Cell Sheet Preparation Plates> First, 2.0 × 10 7 Mitochondria were isolated from cells C3H10T1 / 2 cells using the SLO method. 1 mL of 10 μM CFSE solution was added to the isolated mitochondria, and the cells were left on ice for 30 minutes to stain them. The stained mitochondria were washed twice with PBS and then suspended in 15% FBS-containing DMEM medium (37°C) (375 μL). Next, 2.0 × 10⁶ of the stained mitochondria were placed in a 24-well cell sheet preparation plate (UpCell, CellSeed) that had been preheated to 37°C. 6Cells were added to achieve a cell mt / well ratio, and the plates were centrifuged (1500g, 10 minutes, 37°C). After washing with 15% FBS-containing DMEM medium (37°C), the wells were observed using a digital fluorescence microscope (BZ-X800, Keyence). The results confirmed that mitochondria were attached to the inner bottom surface of the wells.

[0071] <Test Example 16: Preparation of cell sheets using plates coated with mitochondria> First, 2.0 × 10 7 Mitochondria were isolated from cells' C3H10T1 / 2 cells using the SLO method and suspended in 15% FBS-containing DMEM medium (37°C) (375 μL). Then, 2.0 × 10⁶ stained mitochondria were placed in a 24-well plate (UpCell, CellSeed) for cell sheet preparation, which had been preheated to 37°C. 6 Mitochondria were added to the wells to a concentration of cells mt / well, and then plate centrifugation (1500g, 10 minutes, 37°C) was performed to coat the inner bottom surface of the wells with mitochondria. Next, 4.5 × 10⁶ C3H10T1 / 2 cells suspended in 15% FBS-containing DMEM medium (37°C) were placed in the wells. 5 Cells were seeded to a cell / well ratio and cultured for 24 hours. After culturing, the culture medium was removed from the plate, and 50 μL of 15% FBS-containing DMEM medium was immediately added. Next, using tweezers to avoid introducing air bubbles, a support (Cell Shifter, Cell Seed) was placed on top of the cell sheet and left to stand at room temperature (25°C) for 5 minutes. After the support was removed from the plate and left to stand, 15% FBS-containing DMEM medium was added dropwise so that the support was submerged, and the support was removed using tweezers. As a result, a cell sheet into which exogenous mitochondria had been introduced was successfully recovered.

[0072] <Example 1: Cytotoxicity of cationic substances> Place 5.0 × 10⁶ C3H10T1 / 2 cells in a 96-well plate. 3After seeding to a cell / well ratio, polyethyleneimine (PEI) diluted in PBS was added to various final concentrations ranging from 0 to 40 μg / mL, and the cells were incubated for 24 hours. The PEI used was branched, with a mass-average molecular weight of approximately 25,000 determined by light scattering and a number-average molecular weight of approximately 10,000 determined by gel permeation chromatography. The number of cells after incubation was then measured using a cell counting kit (Cell Counting Kit-8 (CCK-8), Fujifilm Wako Pure Chemical Industries). Specifically, 100 μL of CCK-8 solution was added to each well and incubated for 30 minutes, after which the cell count was measured by measuring the absorbance at a wavelength of 450 nm.

[0073] Figure 16 shows the relative cell counts, with the average cell count of the group without PEI being set to 100%. Each data point in the figure represents the mean ± standard deviation of the three samples. As previously reported (Moghimi et al., Mol. Ther., 11:990-5 (2005)), PEI showed cytotoxicity at concentrations of 10 μg / mL or higher.

Claims

1. A method for producing cells into which exogenous mitochondria have been introduced, A production method comprising using a cell culture device on which mitochondria isolated from donor cells are attached to the culture surface, attaching recipient cells to the culture surface and culturing them, thereby allowing the recipient cells to take up the mitochondria.

2. The production method according to claim 1, wherein the cell culture equipment is a cell culture vessel or a cell culture carrier.

3. The production method according to claim 1, further comprising obtaining a cell culture device on which the mitochondria are adhered to the culture surface by allowing a liquid containing mitochondria isolated from the donor cells to stand in contact with the culture surface of the cell culture device.

4. The aforementioned cell culture equipment is a cell culture vessel, The production method according to claim 1, further comprising adding a liquid containing mitochondria isolated from the donor cells to a cell culture vessel and centrifuging the cell culture vessel to obtain a cell culture vessel on which the mitochondria are adhered to the culture surface.

5. A cell culture device for use in the production method described in claim 1 or 2, Cell culture equipment with isolated mitochondria attached to the culture surface.

6. The cell culture material according to claim 5, which is a cell culture vessel or a cell culture carrier.

7. A method for producing cell culture equipment for use in the production method described in claim 1 or 2, The aforementioned cell culture equipment is a cell culture vessel, A manufacturing method comprising adding a liquid containing isolated mitochondria to the cell culture vessel and centrifuging the cell culture vessel to adhere the mitochondria to the culture surface of the cell culture vessel.

8. A cell culture kit for use in the production method described in claim 1 or 2, A cell culture kit comprising cell culture equipment and a liquid containing isolated mitochondria.

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