Production metod of cells having exogenous mitochondria introduced thereinto

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

Method for producing cells into which exogenous mitochondria have been introduced

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

[0002] Mitochondria are intracellular 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 cells for transplantation improves cell function and enhances the effectiveness of cell transplantation therapy (see, for example, Non-Patent Document 1), and various methods for introducing exogenous mitochondria into cells have been investigated.

[0003] Known methods for introducing exogenous mitochondria into cells include (1) a method in which exogenous mitochondria are incubated with recipient cells (see, for example, Patent Document 1); (2) a method in which the surface of exogenous mitochondria is modified with a cell membrane-permeable peptide and then incubated with recipient cells (see, for example, Non-Patent Document 2); and (3) a method in which exogenous mitochondria are introduced into recipient cells by microinjection (see, for example, Patent Document 2).

[0004] However, while the above method (1) is easy to operate, it has the problem of low introduction efficiency due to the negatively charged mitochondrial surface. The above method (2) has a higher introduction efficiency than the above method (1), but requires laborious mitochondrial modification procedures and there is a concern that cells may be damaged by the cell membrane-permeable peptide, which is a cationic substance. Furthermore, the above method (3) can reliably introduce mitochondria into recipient cells, but requires advanced techniques for operation and has a limit to the number of recipient cells that can be targeted. Furthermore, the above method (3) also raises concerns about cell damage.

[0005] Special table 2021-532095 publication Special table 2019-500395 publication

[0006] Guo, Y. et al., Stem Cell Res. Ther., 11, 245, 2020Maeda, H. et al., J. Cell. Mol. Med., 24, 5007-5020, 2020

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

[0008] Specific means for solving the above problems include the following embodiments. <1> A method for producing cells into which exogenous mitochondria have been introduced, comprising culturing recipient cells using a cell culture vessel having a culture surface coated with mitochondria isolated from donor cells, and allowing the mitochondria to be incorporated into the recipient cells. <2> The production method according to <1>, wherein the cell culture vessel is a cell culture vessel or a cell culture carrier. <3> Cells into which exogenous mitochondria have been introduced, produced by the production method according to <1> or <2>. <4> A cell into which exogenous mitochondria have been introduced, wherein the amount of protein of the introduced exogenous mitochondria is 2.0 x 10 ５ The cells according to <3>, wherein the concentration is 0.6 μg or more per cell. <5> A cell culture vessel having a culture surface coated with isolated mitochondria. <6> The cell culture substrate according to <5>, which is a cell culture vessel or a cell culture carrier. <7> A method for producing a cell culture vessel having a culture surface coated with isolated mitochondria, comprising adding a solution containing isolated mitochondria to the cell culture vessel and centrifuging the cell culture vessel in a state where the solution contains isolated mitochondria, thereby adhering the mitochondria to the culture surface of the cell culture vessel. <8> A cell culture kit comprising a cell culture substrate and a solution containing isolated mitochondria.

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

[0010]

[0023] Figure 1 shows mitochondria adhered to the inner bottom surface of a dish when mitochondria labeled with green fluorescent protein (GFP) were added to the dish and incubated for a predetermined period of time. Figure 2 shows the fluorescence intensity of the supernatant when a suspension of GFP-labeled mitochondria suspended in phosphate-buffered saline (PBS) was centrifuged at various centrifugal forces for 10 minutes. Figure 3 shows the fluorescence intensity of a pellet suspension when a suspension of GFP-labeled mitochondria suspended in PBS was centrifuged at various centrifugal forces for 10 minutes and the resulting mitochondrial precipitate (pellet) was resuspended in PBS. Figure 4 shows mitochondria adhered to the inner bottom surface of a well when mitochondria stained with carboxyfluorescein succinimidyl ester (CSFE) were added to a plate and then centrifuged, and when plate centrifugation was not performed. Fig. 1 shows the geometric mean fluorescence intensity (gMFI) of C3H10T1 / 2 cells cultured on a plate in which mitochondria were coated on the inner bottom surface of the wells by adding GFP-labeled mitochondria to the plate, centrifuging the plate, and incubating for a predetermined period of time, and on a plate in which mitochondria were coated only by incubation without centrifuging the plate, and the cells after culture were analyzed by flow cytometry. Fig. 1 shows confocal laser microscope images showing mitochondria taken up by the cells in the case in which C3H10T1 / 2 cells were seeded on a plate coated with GFP-labeled mitochondria and in the case in which C3H10T1 / 2 cells were seeded on a plate not coated with mitochondria and then mitochondria were added to the adhered C3H10T1 / 2 cells. This figure shows the geometric mean fluorescence intensity of the cells, obtained by flow cytometry analysis of cultured C3H10T1 / 2 cells in two cases: when the cells were seeded on a plate coated with GFP-labeled mitochondria, and when the cells were seeded on a plate not coated with mitochondria and then mitochondria were added to the adhered C3H10T1 / 2 cells.

[0033] Figure 1 shows the number of cells (relative values) after culture when C3H10T1 / 2 cells were seeded on mitochondria-coated plates and when C3H10T1 / 2 cells were seeded on non-mitochondrial-coated plates and then mitochondria were added to the adhered C3H10T1 / 2 cells. Figure 2 shows the amount of ATP production by C3H10T1 / 2 cells when seeded on mitochondria-coated plates. Figure 3 shows the number of cells (relative values) after culture when C3H10T1 / 2 cells were cultured on plates coated with various numbers of mitochondria. Figure 4 shows the number of cells (relative values) after culture when oligomycin-treated C3H10T1 / 2 cells were cultured on plates coated with various numbers of mitochondria. Fig. 1 shows in vivo imaging images of mitochondria-introduced NanoLuc luciferase-expressing C3H10T1 / 2 cells or untreated NanoLuc luciferase-expressing C3H10T1 / 2 cells subcutaneously administered to the back of a mouse. Fig. 2 shows plasma luciferase activity of mitochondria-introduced NanoLuc luciferase-expressing C3H10T1 / 2 cells or untreated NanoLuc luciferase-expressing C3H10T1 / 2 cells subcutaneously administered to the back of a mouse. Fig. 3 shows in vivo imaging images of mitochondria-introduced NanoLuc luciferase-expressing C3H10T1 / 2 cells or untreated NanoLuc luciferase-expressing C3H10T1 / 2 cells subcutaneously administered to the back of a mouse. Fig. 4 shows in vivo imaging images of mitochondria-introduced NanoLuc luciferase-expressing C3H10T1 / 2 cells or untreated NanoLuc luciferase-expressing C3H10T1 / 2 cells subcutaneously administered to the back of a mouse. Fig. 5 shows in vivo imaging images of mitochondria-introduced NanoLuc luciferase-expressing C3H10T1 / 2 cells or untreated NanoLuc luciferase-expressing C3H10T1 / 2 cells subcutaneously administered to the back of a mouse. Fig. 6 shows in vivo luciferase activity of mitochondria-introduced NanoLuc luciferase-expressing C3H10T1 / 2 cells or untreated NanoLuc luciferase-expressing C3H10T1 / 2 cells subcutaneously administered to the back of a mouse. ４ 1 shows serum AST levels when mitochondria-introduced NanoLuc luciferase-expressing C3H10T1 / 2 cells or untreated NanoLuc luciferase-expressing C3H10T1 / 2 cells were intravenously administered with CCl ４Fig. 1 shows serum ALT levels when intravenously administered to a mouse model of induced liver injury. Fig. 2 shows the number of cells (relative values) after culturing when various cells (C3H10T1 / 2 cells, Hepa1-6 cells, HEK293 cells, HaCaT cells) were seeded on a mitochondria-coated plate and when various cells were seeded on a non-mitochondrial-coated plate. Fig. 3 shows the number of cells (relative values) after culturing when HEK293 cells were seeded on a mitochondria-coated plate and when mitochondria were added after seeding HEK293 cells on a plate. ５ This figure shows the amount of mitochondrial protein taken up per cell. The amount of mitochondrial protein taken up per cell was 2.0 × 10 when HaCaT cells were seeded on a plate coated with mitochondria, and when mitochondria were added after seeding HaCaT cells on the plate. ５ 1 shows the amount of mitochondrial protein taken up per cell, and 2 shows the cell number (relative value) after culturing C3H10T1 / 2 cells in the presence of polyethyleneimine (PEI) at various concentrations.

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

[0012] "Donor cells" refers to cells that provide mitochondria, and "recipient cells" refers to cells into which mitochondria are introduced. Donor cells and recipient cells may be cells containing normal mitochondria, or may be cells containing dysfunctional mitochondria in which mitochondrial DNA has been mutated. When cells containing dysfunctional mitochondria are used as recipient cells, at least a portion of the endogenous mitochondria may be removed in advance by a conventionally known method.

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

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

[0015] Any method can be used to isolate mitochondria from donor cells, and commercially available kits can be used as needed. Known methods for isolating mitochondria from donor cells include disrupting donor cells and then isolating the mitochondrial fraction by centrifugation, and forming pores in the plasma membrane of donor cells and then isolating the mitochondrial fraction by centrifugation. Among these, from the viewpoint of isolating less damaged mitochondria, a method of forming pores in the plasma membrane of donor cells and then isolating the mitochondrial fraction is preferred, and a method of forming pores in the plasma membrane of donor cells using SLO and then isolating the mitochondrial fraction (see, for example, Shibata, T. et al., Biochem. Biophys. Res. Commun., 463, 563-568, 2015) is particularly preferred.

[0016] As the cell cultureware for coating the isolated mitochondria, conventionally known cell cultureware capable of culturing recipient cells can be used. Examples of cell cultureware include cell culture vessels such as dishes, plates, and flasks; cell culture carriers such as microcarriers; and the like. Examples of materials for cell cultureware include glass; synthetic polymers such as polyethylene, polypropylene, and polystyrene; natural polymers such as cellulose and collagen; and metals. The cell cultureware may also be a cell culture vessel for producing cell sheets, with a temperature-responsive polymer 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 a cell cultureware. The "culture surface" may be any surface that can come into contact with recipient cells during culture, such as 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 the cell cultureware with isolated mitochondria, as long as it allows mitochondria to adhere to the culture surface. Typically, mitochondria can be attached to the culture surface simply by leaving a solution containing isolated mitochondria in contact with the culture surface. The leaving time is preferably 12 hours or more, more preferably 24 hours or more. The liquid in which the isolated mitochondria are suspended is not particularly limited, and may be a cell culture medium or a buffer solution such as PBS.

[0019] When the cell cultureware is a cell culture vessel such as a dish or plate, mitochondria can be attached to the culture surface more efficiently and reliably by adding a solution containing isolated mitochondria to the cell culture vessel and then centrifuging the cell culture vessel. Centrifugal conditions are not particularly limited and can be selected appropriately, but examples include conditions of 300 to 3200 g for 30 seconds to 60 minutes. After centrifugation, the cell culture vessel may be allowed to stand with the solution containing isolated mitochondria added.

[0020] The number of mitochondria to be coated on the culture surface of the cell culture equipment is appropriately determined depending on the type of cell culture equipment and the number of recipient cells. As an example, it is preferable to coat the culture surface of the cell culture equipment with mitochondria isolated from donor cells whose number is 2 to 100 times the number of recipient cells. The density of mitochondria to be coated on the culture surface of the cell culture equipment is, for example, 5 to 15 μg / cm. ２ It is preferable that:

[0021] After obtaining a cell culture vessel having a culture surface coated with isolated mitochondria as described above, recipient cells can be cultured using this cell culture vessel, thereby incorporating mitochondria (exogenous mitochondria) into the recipient cells. When culturing recipient cells, cell culture vessels pre-coated with isolated mitochondria may be used. Alternatively, when culturing recipient cells, a cell culture kit including a cell culture substrate and a solution containing isolated mitochondria may be used, and the isolated mitochondria may be coated onto the culture surface of the cell culture vessel.

[0022] The cell concentration when culturing recipient cells is not particularly limited. For example, ４ cells / mL~5.0×10 ５ The cell concentration in cells / mL is given.

[0023] Various media are used for culturing recipient cells depending on the type of recipient cells. Conventional cell culture conditions can be used for culturing recipient cells. For example, conditions include a temperature of 30°C to 40°C, a relative humidity of 90% to 98%, and CO ２ The culture time is preferably 3 to 72 hours, more preferably 6 to 24 hours, for example.

[0024] Introducing exogenous mitochondria into recipient cells as described above can enrich mitochondria in the cells and improve cell function, proliferation, etc. Furthermore, introducing normal exogenous mitochondria into recipient cells from which at least a portion of dysfunctional endogenous mitochondria has been removed can improve cell function, proliferation, etc.

[0025] In particular, the production method according to this embodiment makes it possible to introduce a much larger amount of exogenous mitochondria into cells than 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 ５ The amount is 0.6 μg or more, preferably 0.8 μg or more, and more preferably 1.0 μg or more per cell. Furthermore, the production method according to this embodiment causes less damage to cells than 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. Cells into which exogenous mitochondria have been introduced tend to have a longer survival period after transplantation, which makes it possible to enhance the effectiveness of cell transplantation therapy and optimize substance production using the cells. Cells into which exogenous mitochondria have been introduced can also be used to produce cell sheets. The resulting cell sheets can be suitably used in regenerative medicine, etc. It is also possible to directly obtain cell sheets into which exogenous mitochondria have been introduced by coating mitochondria onto a cell culture vessel for cell sheet production, the culture surface of which has been immobilized with a temperature-responsive polymer, and then culturing recipient cells in this cell culture vessel.

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

[0028] Preparation Example 1: Isolation of mitochondria (SLO method) The following 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: mouse fibroblast cell line expressing a fusion protein of mitochondrial transportable cox8a signal and GFP

[0029] First, 1.0 × 10 C3H10T1 / 2 cells or 3T3-L1-mt-GFP cells were ６ The cells were seeded onto a 15 cm dish (Thermo Fisher Scientific) at a cell count of 1000 cells / ml and cultured for 3 days. After culture, the cells were washed with PBS and harvested using a trypsin / EDTA solution (2.5 g / L trypsin, 1 mmol / L EDTA). The harvested cells were then incubated in HEPES-CH4000 containing 20 μL of 1 μg / mL streptolysin O (Fujifilm Wako Pure Chemical Industries, Ltd.). ３ The cells were suspended in COOK buffer (480 μL), incubated at room temperature for 1 to 5 minutes, and then left on ice for 10 minutes. Next, the cells were washed with 4°C Tris-sucrose buffer, suspended in Tris-sucrose buffer, and incubated at 37°C for 10 minutes. The cell suspension was pipetted 200 times and centrifuged (400 g, 10 minutes, 4°C), and the supernatant containing mitochondria was collected. The collected supernatant was further centrifuged (7000 g, 10 minutes, 4°C), and the supernatant was removed. The resulting mitochondrial fraction was suspended in 15% FBS-containing DMEM medium or the like and stored at 4°C. Hereinafter, the procedure for isolating mitochondria using streptolysin O as described above will be referred to as the "SLO method."

[0030] Test Example 1: Adhesion of mitochondria to the bottom of the dish First, 1.6 x 10 ７ Mitochondria were isolated from 3T3-L1-mt-GFP cells using the SLO method. The mitochondrial pellet was suspended in 10% FBS-containing DMEM medium (530 μL) and 3.0 × 10 mitochondria were placed on a polylysine-coated 35 mm glass-bottom dish. ６Cells were added to each well at a concentration of 1 mt / well and incubated for the specified time (1, 12, or 24 hours). Mitochondria were then fixed using 4% paraformaldehyde in phosphate buffer, and a mounting medium containing DAPI (4',6-diamidino-2-phenylindole) (Mounting Medium with DAPI, Vector Laboratories) was added. Fluorescent images were then captured using a confocal laser scanning microscope (SP8, Leica) and imaging software (LAS X Life Science, Leica).

[0031] The fluorescence image is 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, 2.0 × 10 ７ Mitochondria were isolated from 3T3-L1-mt-GFP cells using the SLO method. The isolated mitochondria were diluted to 3.0 × 10 in PBS. ６ The cells were suspended in a volume of 300 μL and centrifuged at various centrifugal forces (300 g, 500 g, 1000 g, 1500 g, and 2000 g). The temperature during centrifugation was fixed at 4°C for 10 minutes. The supernatant (300 μL) after centrifugation was collected, and the mitochondrial precipitate (pellet) was resuspended in 300 μL of PBS. The supernatant and pellet suspensions were then added to a 96-well plate (Corning) at 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 point in the figure represents the mean value ± standard deviation of three samples. In addition, "*" 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 1500 g or more, the fluorescence intensity of the supernatant decreased further, while the fluorescence intensity of the pellet suspension increased further, resulting in particularly favorable results.

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

[0035] For comparison, a group was prepared in which the same procedures as above were performed except that plate centrifugation was not performed, and a group in which neither mitochondria nor plate centrifugation was performed, and fluorescent images were taken in the same manner as above.

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

[0037] Test Example 4: Evaluation of mitochondrial uptake (FACS) First, 2.0 × 10 ７ Mitochondria were isolated from 3T3-L1-mt-GFP cells using the SLO method. For a 12-well plate, 3.0 × 10 mitochondria were isolated. ６The plate was centrifuged (1500 g, 10 minutes, 4°C), and then incubated for 12 hours to coat the inner bottom surface of the well with mitochondria. ５ Cells were seeded at 1000 cells / well and cultured for 24 hours. After culture, cells were detached from the wells using a 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 the medium was replaced with PBS. Mitochondrial uptake was analyzed using a flow cytometer (BD FACS Lyric, Beckton Dickinson). Data were analyzed using FlowJo software version 8.7 (Beckton Dickinson).

[0038] For comparison, a group was prepared in which the same procedures as above were carried out except that plate centrifugation was not performed, and an untreated group was prepared in which the same procedures as above were carried out except that a 12-well plate not coated with mitochondria was used, and these were analyzed using a flow cytometer in the same manner as above.

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

[0040] Test Example 5: Evaluation of mitochondrial uptake (microscopic observation) First, 2.0 × 10 ７ Mitochondria were isolated from 3T3-L1-mt-GFP cells using the SLO method. For a 12-well plate, 3.0 × 10 mitochondria were isolated. ６The plates were centrifuged (1500 g, 10 minutes, 4°C) to coat the inner bottom surface of the wells with mitochondria. ５ Cells were seeded at 1000 cells / well and cultured for 24 hours. After culture, cells were detached from the wells using a trypsin / EDTA solution (2.5 g / L trypsin, 1 mmol / L EDTA) and seeded onto 35 mm glass-bottom dishes for 12 hours of culture. Adherent cells were then fixed using 4% paraformaldehyde in phosphate buffer, and DAPI-containing mounting medium (Mounting Medium with DAPI, Vector Laboratories) was added. Fluorescence images were then captured using a confocal laser scanning microscope (SP8, Leica) and imaging software (LAS X Life Science, Leica).

[0041] For comparison, we also prepared a group in which the inner bottom of the wells was not coated with mitochondria but mitochondria were added to the adherent cells, and an untreated group in which the same procedures as above were carried out except that a 12-well plate not coated with mitochondria was used.

[0042] In the group in which mitochondria were added to adherent cells, the cell density at the time of addition of mitochondria was 3.0 × 10 ５ C3H10T1 / 2 cells were seeded in a 12-well plate at 2.0 × 10 cells / well and cultured until the next day. ７ Mitochondria were isolated from 3T3-L1-mt-GFP cells using the SLO method, and the isolated mitochondria were collected at 3.0 × 10 ６ Cells were added to the wells at a density of 1000 cells / well and cultured for 24 hours. After culture, the cells were detached from the wells using a trypsin / EDTA solution (2.5 g / L trypsin, 1 mmol / L EDTA) and seeded onto a 35 mm glass-bottom dish for 12 hours of culture. The adherent cells were then fixed using 4% paraformaldehyde in phosphate buffer, and fluorescent images were taken as described 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 with the bottom of the wells coated with mitochondria, the amount of mitochondrial uptake was increased compared to when the bottom of the wells was not coated with mitochondria and mitochondria were added to adherent cells.

[0044] Test Example 6: Comparison of mitochondrial uptake (FACS) First, 3.0 x 10 ７ Mitochondria were isolated from 3T3-L1-mt-GFP cells using the SLO method. For a 12-well plate, 3.0 × 10 mitochondria were isolated. ６ The plates were centrifuged (1500 g, 10 minutes, 4°C) to coat the inner bottom surface of the wells with mitochondria. ５ Cells were seeded at 1000 cells / well and cultured for 24 hours. After culture, cells were detached from the wells using a 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 the medium was replaced with PBS. Mitochondrial uptake was analyzed using a flow cytometer (BD FACS Lyric, Beckton Dickinson). Data were analyzed using FlowJo software version 8.7 (Beckton Dickinson).

[0045] For comparison, a group in which the inner bottom of the wells was not coated with mitochondria but mitochondria were added to adherent cells, and an untreated group using a 12-well plate that was not coated with mitochondria were also prepared.

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

[0047] In the untreated group, the cell density on the next day was 3.0 × 10 ５ C3H10T1 / 2 cells were seeded into a 12-well plate at 1000 cells / well and cultured until the next day. Next, 15% FBS-containing DMEM medium was added instead of mitochondria, and the cells were cultured for 24 hours. After culture, the cells were detached from the wells using a trypsin / EDTA solution (2.5 g / L trypsin, 1 mmol / L EDTA) and suspended in PBS. The floating cells were then fixed using 4% paraformaldehyde phosphate buffer and analyzed using a flow cytometer as described above.

[0048] The geometric mean fluorescence intensity of each group is shown in Figure 6. Each data point in the figure represents the mean value ± standard deviation of three samples. In addition, "*" in the figure indicates statistical significance ( ＊ p<0.05; Tukey-Kramer's test.) As can be seen from Figure 6, when cells were cultured in a plate with the bottom of the wells coated with mitochondria, the amount of mitochondrial uptake was significantly increased compared to when the bottom of the wells was not coated with mitochondria and mitochondria were added to adherent cells.

[0049] Test Example 7: Evaluation of proliferation of mitochondria-introduced cells. Mitochondria (3.0 × 10) derived from C3H10T1 / 2 cells were ６ C3H10T1 / 2 cells (3.0 × 10 cells / well) were plated on a 12-well plate coated with 100% ethanol. ５For comparison, adherent C3H10T1 / 2 cells (3.0 × 10 cells / well) were seeded on the cells and cultured for 24 hours to obtain cells into which mitochondria had been introduced. ５ Mitochondria (3.0 × 10 cells / well) derived from C3H10T1 / 2 cells were added to the well. ６ A group was prepared in which 12-well plates not coated with mitochondria were added with 15% FBS-containing DMEM medium instead of mitochondria. The cells prepared by each method were plated at 5.0 × 10 cells / well in a 96-well plate (Corning). ３ Cells were seeded at 100 μL / well and cultured for 48 hours. Cell counting kit-8 (CCK-8) (Fujifilm Wako Pure Chemical Industries, Ltd.) was used to measure the cell number after culture. Specifically, CCK-8 solution was added to the wells at 100 μL / well, incubated for 30 minutes, and then the absorbance at 450 nm was measured to determine the cell number.

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

[0051] Test Example 8: Evaluation of ATP production in mitochondria-introduced cells. ６ C3H10T1 / 2 cells (3.0 × 10 cells / well) were plated on a 12-well plate coated with 100% ethanol. ５Mitochondria-introduced cells were obtained by seeding 12-well plates (100 cells / well) and culturing for 24 hours. For comparison, an untreated group was prepared using a 12-well plate without mitochondria coating, with 15% FBS-containing DMEM medium added instead of mitochondria. After culturing, cells were detached from the wells using a 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 mixture was added to a 96-well plate (Corning) and allowed to stand for 10 minutes. The luminescence from the cells was then measured using a microplate reader (EnVision, PerkinElmer).

[0052] The luminescence intensity for each group is shown in Figure 8. Each data point in the figure represents the mean value ± standard deviation of triplicate samples. As can be seen from Figure 8, when cells were cultured on plates with mitochondria coated on the inner bottom surface of the wells, the amount of ATP produced by the cells was significantly increased.

[0053] Test Example 9: Evaluation of mitochondria number-dependent cell proliferation ７ Mitochondria were isolated from C3H10T1 / 2 cells by the SLO method. A predetermined number of mitochondria (1.0 × 10) were plated onto 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 The plate was centrifuged (1500 g, 10 minutes, 4°C) to coat the bottom of the wells with mitochondria. Next, 3.0 × 10 C3H10T1 / 2 cells were added to the wells at a concentration of 1.0 × 10 ５ After culturing, the cells were detached from the wells using a trypsin / EDTA solution (2.5 g / L trypsin, 1 mmol / L EDTA) and cultured at 1.0 × 10 ４The cells were seeded onto a 96-well plate (Corning) at 100 μL / well and cultured for 24 hours. The number of cells after culture was measured using a viable cell counting kit (Cell Counting Kit-8 (CCK-8), Fujifilm Wako Pure Chemical Industries, Ltd.). Specifically, CCK-8 solution was added to the plate at 100 μL / well, and the plate was incubated for 30 minutes. The cell number was then measured by measuring the absorbance at 450 nm.

[0054] For comparison, an untreated group was prepared by carrying out the same procedure as above except that a 12-well plate not coated with mitochondria was used.

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

[0056] Test Example 10: Evaluation of proliferation of cells treated with mitochondrially introduced oligomycin First, 2.0 × 10 C3H10T1 / 2 cells were cultured. ５ The cells were seeded onto a 6-well plate at 100 cells / well. After 24 hours, the medium was replaced with 15% FBS-containing DMEM medium containing 10 μg / mL of oligomycin. After 24 hours, the cells were harvested using a trypsin / EDTA solution (2.5 g / L trypsin, 1 mmol / L EDTA) and used as oligomycin-treated C3H10T1 / 2 cells in subsequent experiments.

[0057] 3.0 x 10 ７ Mitochondria were isolated from C3H10T1 / 2 cells by the SLO method. A predetermined number of mitochondria (1.0 × 10) were plated onto 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 ６The plate was centrifuged (1500 g, 10 minutes, 4°C) to coat the mitochondria on the inner bottom surface of the well. Then, oligomycin-treated C3H10T1 / 2 cells were added to the well at a concentration of 1.0 × 10 ４ Cells were seeded at 100 μL / well and cultured for 24 hours. The number of cells after culture was measured using a viable cell counting kit (Cell Counting Kit-8 (CCK-8), Fujifilm Wako Pure Chemical Industries). Specifically, CCK-8 solution was added to the wells at 100 μL / well, and the cells were incubated for 30 minutes. The absorbance at 450 nm was then measured to determine the number of cells.

[0058] For comparison, an untreated group was prepared by carrying out the same procedure as above except that a 96-well plate not coated with mitochondria was used.

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

[0060] Test Example 11: Evaluation of survival rate after transplantation of mitochondria-introduced cells. ６ NanoLuc luciferase-expressing C3H10T1 / 2 cells (C3H10T1 / 2 / Nluc cells) (3.0 × 10 ５ Mitochondria-introduced C3H10T1 / 2 / Nluc cells (mt-C3H10T1 / 2 / Nluc) or untreated C3H10T1 / 2 / Nluc cells were seeded at 5.0 × 10 ５The cells (200 μL) were subcutaneously injected into the back of 6-week-old male BALB / c-nu / nu mice. 50 μL of luciferin (Nano-Glo, Promega) was then administered at the cell injection site, and in vivo imaging was performed over time using an imaging system (In-Vivo Xtreme, Bruker Daltonik GmbH). Blood samples were 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 value ± standard deviation of three samples. 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 survival after subcutaneous transplantation into mice.

[0062] Test Example 12: Therapeutic effect in liver damage model mice. ６ NanoLuc luciferase-expressing C3H10T1 / 2 cells (C3H10T1 / 2 / Nluc cells) (3.0 × 10 ５ Mitochondria-introduced C3H10T1 / 2 / Nluc cells were obtained by seeding 1 mL / kg body weight of carbon tetrachloride (CCl ) in 6-week-old male ddY mice. ４ ) was intraperitoneally administered to generate liver damage model mice. ４ Six hours after administration, 8.0 × 10 ５Mitochondria-introduced C3H10T1 / 2 / Nluc cells (mt-C3H10T1 / 2 / Nluc) or untreated C3H10T1 / 2 / Nluc cells were intravenously administered, and blood was collected 24 hours later. Serum AST and ALT levels were measured using a commercially available kit (Transaminase CII-Test Wako, Fujifilm Wako Pure Chemical Industries, Ltd.).

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

[0064] Test Example 13: Evaluation of proliferation of mitochondria-introduced cells 1.0 x 10 ８ Mitochondria were isolated from C3H10T1 / 2 cells using the SLO method. Mitochondria were added to a 96-well plate at 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 with mitochondria. Next, 1.0 × 10 C3H10T1 / 2 cells were added to the plate at 1 μg / well (50 μL / well). ３ cells / well, Hepa1-6 cells (mouse hepatoma cell line) were added at 2.0 × 10 ３ cells / well, HEK293 cells (human embryonic kidney cell line) at 2.0 × 10 ３ cells / well, or HaCaT cells (human skin keratinocyte cell line) at 1.0 × 10 ３ The cells were seeded at 100 μL / well and cultured for 48 hours. The number of cells after culture was measured using a viable cell counting kit (Cell Counting Kit-8 (CCK-8), Fujifilm Wako Pure Chemical Industries). Specifically, CCK-8 solution was added to the wells at 100 μL / well, and the cells were incubated for 30 minutes. The absorbance at 450 nm was then measured to determine the number of cells.

[0065] For comparison, an untreated group was prepared by carrying out the same procedure as above except that a 96-well plate not coated with mitochondria was used.

[0066] The relative values ​​of the cell counts, with the average cell count in the untreated group taken as 100%, are shown in Figure 13. Each data point in the figure represents the average value ± standard deviation of three samples. In addition, "*" in the figure indicates statistical significance ( ＊ p<0.05; Student's t-test.) As can be seen from Fig. 13, cell proliferation was significantly enhanced when any of C3H10T1 / 2 cells, Hepa1-6 cells, HEK293 cells, and HaCaT cells was used as recipient cells.

[0067] Test Example 14: Comparison of mitochondrial uptake 1.2 x 10 ８ Mitochondria were isolated from C3H10T1 / 2 cells using the SLO method, and the protein content was measured using a protein assay kit (Pierce BCA Protein Assay Kits, Thermo Scientific). Mitochondria were added to a 12-well plate at 80 μg / well (500 μL / well), and the plate was centrifuged (1500 g, 10 minutes, 4°C) to coat the inner bottom surface of the wells with mitochondria. Next, 2.0 × 10 HEK293 or HaCaT cells were added to a 12-well plate at 80 μg / well (500 μL / well). ５Cells were seeded at 1000 cells / well and cultured for 24 hours. After culture, cells were detached from the wells using a trypsin / EDTA solution (2.5 g / L trypsin, 1 mmol / L EDTA), and cellular RNA was isolated using a total RNA purification kit (Monarch Total RNA Miniprep Kit, BioLabs). Next, using a reverse transcription kit (ReverTra Ace qPCR RT Master Mix with gDNA Remover, Toyobo), reverse transcription to cDNA was performed using 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. Next, the mouse COX2 gene was PCR amplified using real-time PCR reagents (THUNDERBIRD SYBR qPCR Mix, Toyobo) on a real-time PCR analysis system (CFX Connect, Bio-Rad). The PCR protocol consisted of 39 cycles of 95°C for 30 seconds, followed by 95°C for 5 seconds, 55°C for 15 seconds, and 72°C for 45 seconds. 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) A calibration curve was then created showing the relationship between mitochondrial protein amount (μg) and COX2 cycle number (Cq value), and the amount of mitochondrial protein taken up by each cell was calculated by substituting the COX2 cycle number for each cell into the calibration curve.

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

[0069] The amount of mitochondrial protein taken up into HEK293 cells is shown in Figure 14A, and the amount of mitochondrial protein taken up into HaCaT cells is shown in Figure 14B. Each data point in the figure represents the mean value ± standard deviation of three samples. In addition, "*" in the figure indicates statistical significance ( ＊ p<0.05; Student's t-test). As can be seen from Figures 14A and 14B, 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 of mitochondria on a plate for cell sheet preparation ７ Mitochondria were isolated from C3H10T1 / 2 cells using the SLO method. 10 μM CFSE solution (1 mL) was added to the isolated mitochondria and left on ice for 30 minutes to stain the mitochondria. The stained mitochondria were washed twice with PBS and then suspended in 15% FBS-containing DMEM medium (375 μL) at 37°C. Next, 2.0 × 10 of the stained mitochondria were added to a 24-well plate (UpCell, CellSeed) for cell sheet preparation preheated to 37°C. ６ The cells were added to the well at a concentration of 1 mt / well, and the plate was centrifuged (1500 g, 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 mitochondria were confirmed to be attached to the inner bottom of the wells.

[0071] Test Example 16: Preparation of cell sheet using mitochondria-coated plate First, 2.0 × 10 ７ Mitochondria were isolated from C3H10T1 / 2 cells using the SLO method and suspended in 15% FBS-containing DMEM medium (375 μL) at 37°C. Next, 2.0 × 10 stained mitochondria were added to a 24-well plate (UpCell, CellSeed) for cell sheet preparation preheated to 37°C. ６The plate was centrifuged (1500 g, 10 minutes, 37°C) to coat the inner bottom surface of the well with mitochondria. Next, 4.5 × 10 C3H10T1 / 2 cells suspended in 15% FBS-containing DMEM medium (37°C) were added to the wells at a concentration of 1 mT / well. ５ Cells were seeded at 1000 cells / well and cultured for 24 hours. After culture, the medium was removed from the plate, and 15% FBS-containing DMEM medium (50 μL) was quickly added. Next, a support (Cell Shifter, CellSeed) was placed on the cell sheet using tweezers to avoid introducing air bubbles, and the sheet was left to stand at room temperature (25°C) for 5 minutes. The support was then peeled off from the plate and left to stand. 15% FBS-containing DMEM medium was then added dropwise to immerse the support, and the support was then peeled off using tweezers. As a result, a cell sheet into which exogenous mitochondria had been introduced was recovered.

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

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

Claims

1. A method for producing cells into which exogenous mitochondria have been introduced, comprising: using a cell culture device in which mitochondria isolated from donor cells are coated on a culture surface, adhering recipient cells to the culture surface and culturing them, and causing the mitochondria to be taken up by the recipient cells (however, excluding those in which centrifugation is performed when causing the mitochondria to be taken up by the recipient cells).

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

3. A cell culture device for use in the production method according to claim 1 or 2, comprising: A cell culture device in which isolated mitochondria are coated on a culture surface.

4. The cell culture device according to claim 3, which is a cell culture container or a cell culture carrier.

5. A method for manufacturing a cell culture device for use in the production method according to claim 1 or 2, comprising: the cell culture device being a cell culture container, and adhering the mitochondria to the culture surface of the cell culture container by centrifuging the cell culture container in a state where a liquid containing isolated mitochondria is added to the cell culture container.

6. A cell culture kit for use in the production method according to claim 1 or 2, comprising: a cell culture device and a liquid containing isolated mitochondria.