Selective electrolysis method for cell membranes

Abstract

The objectives are to provide a method for selectively removing cell membranes from cells, a method for producing cells from which the cell membrane has been substantially removed, a method for imaging the interior of cells from which the cell membrane has been substantially removed, and a culture medium for separating cell membranes from cells. [Solution] We devised pulsed electric field conditions and / or culture medium compositions for the electrical removal of cell membranes and diligently investigated these conditions. As a result, we found that pulsed electric fields under specific conditions and / or specific culture medium compositions can remove cell membranes from cells, thus completing the present invention.

Description

Technical Field

[0001] The present invention relates to a method for selectively removing cell membranes from cells, a method for producing cells from which substantially cell membranes have been removed, a method for imaging the interior of cells from which substantially cell membranes have been removed, and a medium for separating cell membranes from cells.

Background Art

[0002] [Intracellular Research] Intracellular research, which investigates intracellular structures and their functions, has mainly relied on the analysis of live cells or fixed samples. In fixed cell analysis, proteins and other macromolecules are often modified or damaged, leading to loss of function. Structural changes also occur due to fixation, affecting the original intracellular structure (see Non-Patent Document 1). On the other hand, in live cell analysis using cells with undamaged membranes (especially methods using scanning probes such as atomic force microscopes), intracellular analysis is limited, resolution is reduced, and there are problems due to dynamic changes in cells (see Non-Patent Document 2).

[0003] [Pulsed Electric Field] Pulsed electric fields have conventionally been used for cell membrane permeabilization (electroporation), but electroporation protocols are limited to creating temporary nanopores only for delivery purposes (Non-Patent Documents 3 and 4).

Prior Art Documents

Non-Patent Documents

[0004]

Non-Patent Document 1

[0005] The object of the present invention is to provide a method for selectively removing cell membranes from cells, a method for producing cells from which the cell membrane has been substantially removed, a method for imaging the interior of cells from which the cell membrane has been substantially removed, and a culture medium for separating cell membranes from cells. [Means for solving the problem]

[0006] To solve the above problems, the inventors devised pulsed electric field conditions and / or culture medium compositions for the electrical removal of cell membranes and diligently investigated these conditions. As a result, they discovered that pulsed electric fields under specific conditions and / or specific culture medium compositions can remove cell membranes from cells, thus completing the present invention.

[0007] The present invention is as follows: 1. A method for removing a cell membrane from a cell, comprising steps 1) and 2): 1) The step of applying a pulsed electric field to cells in a culture medium for separating cell membranes from cells under condition 1, 2) A step of applying a pulsed electric field to the cells in the culture medium under condition 2, Here, condition 1 includes the following parameters: Electric field strength A: 700 V / cm~1000 V / cm, Pulse off time between pulses A: 40 ms to 60 ms. Pulse A count: 8 pulses to 12 pulses, Here, condition 2 includes the following parameters: Electric field strength B: 120 V / cm ~ 300 V / cm, Pulse off time between pulses B: 1.0 ms to 5.0 ms. Pulse count: 40 pulses to 80 pulses. 2. The method described in item 1 above, wherein the pulse off time between steps 1) and 2) is 40ms to 60ms. 3. The method according to paragraph 1, wherein a pulse train including step 1) under condition 1 and step 2) under condition 2 is performed twice. 4. The method described in item 3 above, wherein the interval between the first pulse train and the second pulse train is 60 seconds to 90 seconds. 5. The method according to item 3 or 4 above, wherein the direction of application of the electric field for the second pulse train is rotated by 80 to 105 degrees relative to the direction of the first pulse train. 6. The method according to item 3 or 4 above, wherein the culture medium for separating cell membranes from cells containing the aforementioned cells has an conductivity of 11.8 mS / cm to 13.0 mS / cm and a pH of 6.4 to 7.0. 7. The method according to item 6, wherein the culture medium for separating cell membranes from the cells comprises an osmotic stabilizer, myo-inositol, potassium phosphate, a buffering agent, a chelating agent, magnesium ions, a nonionic surfactant, polyethylene glycol, and a protease inhibitor. 8. The method according to item 3 or 4 above, wherein, before initiating the first pulse train, the cell culture medium containing the cells is washed with a washing buffer containing an osmotic stabilizer, myo-inositol, potassium phosphate, a buffering agent, a chelating agent, and magnesium ions. 9. The method according to item 3 or 4 above, wherein, after the completion of the pulse train, the cell membrane isolation medium is replaced with a cell maintenance medium containing an osmotic stabilizer, myo-inositol, potassium phosphate, buffering agent, chelating agent, and magnesium ions. 10. The method according to item 3 or 4 above, wherein the cells maintain their three-dimensional structure. 11. The method according to paragraph 3 or 4 above, wherein the cells maintain their intracellular major components in their original state. 12. A method for producing cells from which the cell membrane has been substantially removed, comprising steps 1) and 2): 1) The step of applying a pulsed electric field to cells in a culture medium for separating cell membranes from cells under condition 1, 2) A step of applying a pulsed electric field to the cell under condition 2, Here, condition 1 includes the following parameters: Electric field strength A: 700 V / cm~1000 V / cm, Pulse off time between pulses A: 40 ms to 60 ms. Pulse A count: 8 pulses to 12 pulses, Here, condition 2 includes the following parameters: Electric field strength B: 120 V / cm ~ 300 V / cm, Pulse off time between pulses B: 1.0 ms to 5.0 ms. Pulse count: 40 pulses to 80 pulses. 13. A method for imaging cells with substantially removed cell membranes, the method comprising the step of imaging cells with substantially removed cell membranes using a cell imaging device. Here, the cells with substantially removed cell membranes have been subjected to the following treatments: 1) The cells are subjected to a treatment of applying a pulsed electric field under condition 1 and 2) The cells are subjected to a treatment of applying a pulsed electric field under condition 2. Here, the condition 1 includes the following parameters: Electric field strength A: 700 V / cm to 1000 V / cm, Pulse off-time between pulses A: 40 ms to 60 ms, Number of pulses A: 8 pulses to 12 pulses, and Here, the condition 2 includes the following parameters: Electric field strength B: 120 V / cm to 300 V / cm, Pulse off-time between pulses B: 1.0 ms to 5.0 ms, Number of pulses B: 40 pulses to 80 pulses. 14. A culture medium for separating cell membranes from cells, The culture medium contains an osmotic stabilizer, myo-inositol, potassium phosphate, BUFFERING AGENT, a chelating agent, magnesium ions, a non-ionic surfactant, polyethylene glycol, and a protease inhibitor.

Advantages of the Invention

[0008] The present invention has one or more of the following advantages. 1) Provide cells with substantially removed cell membranes. 2) Provide images of cells with substantially removed cell membranes. 3) Provide a technique for accessing intracellular structures without impairing the integrity of intracellular structures. 4) Provide a technique for maintaining cells in a 3D cell structure and native state, and provide a technique that enables comprehensive analysis even after removing the cell membrane. 5) Provide an innovative approach that improves high-resolution imaging and analysis of intracellular components and provides detailed information such as anatomy in cells. 6) To provide technology that maintains the functionality of intracellular components. [Brief explanation of the drawing]

[0009] [Figure 1] This is a schematic diagram showing the electrical fields parameters for selectively electrically removing cell membranes. [Figure 2] This is a photograph demonstrating the selective removal of cell membranes. [Figure 3] This photograph shows that the major components within a cell remain in their original three-dimensional structural state after the cell membrane has been selectively removed electrically. [Figure 4] This photograph demonstrates the successful acquisition of high-resolution images of the three-dimensional structure of intracellular structures (particularly cytoskeletal fibers) using atomic force microscopy. [Figure 5] Atomic force microscope images (left: cells not electroporated, right: electroporated cells) are shown. Figure 5 was obtained from Non-Patent Literature 4. It shows that the presence of the cell membrane restricts access to intracellular structures. Furthermore, intracellular imaging is not possible with electroporation because the cell membrane is not removed from the cell. [Modes for carrying out the invention]

[0010] The present invention relates to a method for selectively removing cell membranes from cells, a method for producing cells from which the cell membrane has been substantially removed, a method for imaging the interior of cells from which the cell membrane has been substantially removed, and a culture medium for separating cell membranes from cells. The present invention is described in detail below.

[0011] (Method for removing the cell membrane from a cell) The present invention provides a method for selectively removing the cell membrane from a cell, comprising the following steps 1) and 2). 1) Step of applying a pulsed electric field to cells in the culture medium for separating cell membranes from cells under condition 1. 2) The step of applying a pulsed electric field to the cell under condition 2.

[0012] (Condition 1) Condition 1 includes the following parameters: • Electric field strength (pulse electric field) A (V / cm) The range is 700 V / cm to 1000 V / cm, preferably 780 V / cm to 920 V / cm, and more preferably 820 V / cm to 880 V / cm. • Inter-pulse off-time A (ms) The range is 40 ms to 60 ms, preferably 42 ms to 58 ms, and more preferably 45 ms to 55 ms. • Number of pulses A (number of pulses) The range is 8 to 12 times, preferably 9 to 11 times, and more preferably 10 times.

[0013] (Condition 2) Condition 2 includes the following parameters: • Electric field strength (pulsed electric field) B (V / cm) The range is 120 V / cm to 300 V / cm, preferably 180 V / cm to 250 V / cm, and more preferably 220 V / cm to 250 V / cm. • Inter-pulse off-time B (ms) The range is 1.0 ms to 5.0 ms, preferably 1.0 ms to 3.0 ms, and more preferably 1.0 ms to 2.0 ms. • Number of pulses B (number of pulses) The range is 40 to 80 times, preferably 42 to 70 times, and more preferably 45 to 60 times.

[0014] (Additional conditions) • Pulse-off time (milliseconds) between step 1) and step 2) This range is 40 milliseconds to 60 milliseconds, preferably 42 milliseconds to 58 milliseconds, and more preferably 45 milliseconds to 55 milliseconds. • The interval (in seconds) between each pulse train, including step 1) and step 2). This range is 60 to 90 seconds, preferably 60 to 80 seconds, and more preferably 60 to 70 seconds. • Number of pulse trains Preferably, the pulse train including steps 1) and 2) is repeated at least two or more times. • Direction of each pulse train The direction of electric field application in the second pulse train is rotated by 80 to 105 degrees (preferably about 90 degrees) relative to the direction of electric field application in the first pulse train.

[0015] (cell) The type of cells used in the method of the present invention is preferably adherent cells having a cell membrane or cells having a cell membrane and adhering to a surface. Examples include osteosarcoma cells (U2OS), immortalized human cervical cancer cells (Hela), and human glioblastoma cells (YKG-1). Furthermore, the cells can be cultured in known cell culture media (for example, Dulbecco's modified Eagle medium (DMEM)).

[0016] (Washing buffer) The washing buffer used in the present invention is not particularly limited, as long as it can remove components of the cell culture medium or maintain a conductivity of 11.8 mS / cm to 13.0 mS / cm and a pH of 6.4 to 7.0 in cell medium at 23°C to 26.5°C. This buffer also plays a role in maintaining the three-dimensional structure of cells in a natural and balanced state. For example, a buffer containing the following components can be used as an example: • Osmotic pressure stabilizer Osmotic stabilizers play a role in maintaining cell structure and osmotic pressure, preventing osmotic dissolution by matching the intracellular osmotic pressure, and maintaining cell integrity during processing. For example, sucrose in concentrations of 150 mM to 350 mM is used. • Myo-inositol Myo-inositol is involved in cell signaling and osmotic regulation. It is a precursor to inositol lipids and plays a crucial role in cell membrane signaling pathways. Furthermore, myo-inositol helps stabilize cell membranes and maintain the balance of cellular signaling pathways. For example, myo-inositol concentrations of 5.0 mM to 50 mM are given as examples. • Potassium phosphate Potassium phosphate plays a role in maintaining pH within the physiological range and preserving the activity and structure of enzymes within cells. Examples include potassium dihydrogen phosphate (KH2PO4) at concentrations of 5 mM to 9 mM and dipotassium hydrogen phosphate (K2HPO4) at concentrations of 1 mM to 5 mM. • Buffer Buffers are used to maintain a pH within the range of 6.1 to 7.5. This ensures that the buffer's pH remains stable. They also provide additional buffering capacity when maintaining a specific pH is critical. For example, PIPES (piperazine-1,4-bis(2-ethanesulfonic acid)) in concentrations of 60 mM to 140 mM is an example. Chelatives Chelating agents are the calcium in the solution. 2+ It is used to reduce the concentration. This prevents calcium-dependent processes that could adversely affect cytoskeletal components. Also, EGTA (ethylene glycol-bis(2-aminoethyl ether)-N,N,N',N'-tetraacetic acid) is used to reduce calcium ions (Ca 2+ It is a chelating agent specific to ). For example, EGTA concentrations of 0.5 mM to 2.5 mM are given as examples. • Magnesium ions Magnesium ions provide essential cofactors for many enzymatic reactions (e.g., DNA and RNA polymerases) and microtubule stabilization. For example, magnesium chloride (MgCl2) in concentrations of 0.5 mM to 2 mM is a possible example.

[0017] (A culture medium for separating cell membranes from cells) The culture medium for separating cell membranes from cells in this invention is not particularly limited, as long as it can maintain a conductivity of 11.8 mS / cm to 13.0 mS / cm and a pH of 6.4 to 7.0 under conditions of 23°C to 26.5°C. Furthermore, this medium plays a role in maintaining the cells in their natural three-dimensional structure and balanced physiological conditions. For example, a culture medium containing the following components is an example: • Osmotic pressure stabilizer For example, sucrose concentrations of 150 mM to 350 mM are given as examples. • Myo-inositol For example, myo-inositol concentrations of 5.0 mM to 50 mM are cited. • Potassium phosphate Examples include potassium dihydrogen phosphate (KH2PO4) in concentrations of 5 mM to 9 mM and dipotassium hydrogen phosphate (K2HPO4) in concentrations of 1 mM to 5 mM. • Buffer For example, pipettes ranging from 60 mM to 140 mM are given as examples. Chelatives For example, EGTA concentrations of 0.5 mM to 2.5 mM are given as examples. • Magnesium ions For example, magnesium chloride (MgCl2) in concentrations of 0.5 mM to 2 mM is a possible example. • Nonionic surfactants Nonionic surfactants reduce surface tension and prevent protein aggregation, thereby stabilizing the substance. They also assist in the solubilization of proteins and cell membranes. For example, a Tween-20 of 0.1% to 2.0% is given as an example. • Polyethylene glycol (PEG) Polyethylene glycol is used in molecular crowding and helps stabilize macromolecular complexes. For example, polyethylene glycol 35000 (PEG35000) in concentrations of 1% to 6% is used as an example. • EDTA-free protease inhibitor cocktail An EDTA-free protease inhibitor cocktail contains various protease inhibitors to prevent protein degradation by proteases. For example, a cocktail containing aprotinin, bestatin, leupeptin, pepstatin A, and PMSF is cited.

[0018] (Cell maintenance medium) The cell maintenance medium used in the present invention is not particularly limited, as long as it can maintain the properties of cells from which the cell membrane has been substantially removed and / or keep cells from which the cell membrane has been substantially removed in a three-dimensional structural state. For example, a medium comprising the following components is exemplified: • Osmotic pressure stabilizer For example, sucrose concentrations of 150 mM to 350 mM are given as examples. • Myo-inositol For example, myo-inositol concentrations of 5.0 mM to 50 mM are cited. • Potassium phosphate Examples include potassium dihydrogen phosphate (KH2PO4) in concentrations of 5 mM to 9 mM and dipotassium hydrogen phosphate (K2HPO4) in concentrations of 1 mM to 5 mM. • Buffer For example, pipettes ranging from 60 mM to 140 mM are given as examples. Chelatives For example, EGTA concentrations of 0.5 mM to 2.5 mM are given as examples. • Magnesium ions For example, magnesium chloride (MgCl2) in concentrations of 0.5 mM to 2 mM is a possible example.

[0019] (A method for producing cells from which the cell membrane has been substantially removed) The present invention provides a method for producing cells from which the cell membrane has been substantially removed, comprising the following steps 1) and 2). 1) A step of applying a pulsed electric field to cells in a culture medium for separating cell membranes from cells under condition 1. 2) The step of applying a pulsed electric field to the cell under condition 2.

[0020] (A method for imaging cells from which the cell membrane has been substantially removed) The present invention provides a method for imaging the interior of a cell from which the cell membrane has been substantially removed, which includes the step of imaging the cell from which the cell membrane has been substantially removed using a cell imaging device. Cells from which the cell membrane has been substantially removed undergo the following treatments: 1) The cells are subjected to a treatment in which a pulsed electric field is applied under condition 1. 2) The cells are subjected to treatment in which a pulsed electric field is applied according to condition 2.

[0021] The cell imaging device used in the present invention is not particularly limited, but examples include atomic force microscopes, high-speed atomic force microscopes, fluorescence microscopes, confocal microscopes, and holotomography microscopes.

[0022] (Cells from which the cell membrane has been substantially removed) The removal of the cell membrane can be confirmed by known methods. For example, methods include confirming the refractive index of the membrane using a holotomography microscope, using reagents or stains that specifically fluoresce in cell membranes, imaging the integrity of the membrane using an atomic force microscope or electron microscope, or using force spectroscopy. The refractive index of a cell from which the cell membrane has been substantially removed is lower than that of a cell with a cell membrane. For example, the refractive index of a cell from which the cell membrane has been substantially removed can be exemplified as 1.33–1.39. This refractive index range was measured from cells with the cell membrane substantially removed and does not include refractive index values ​​specific to the cell membrane. For example, the typical refractive index range for an intact cell membrane is 1.4 to 1.46.

[0023] (Characteristics of cells from which the cell membrane has been substantially removed) Cells from which the cell membrane of the present invention has been substantially removed have one or more of the following characteristics. 1) Conservation of major intracellular components Major intracellular components include cytoskeletal fibers and related proteins (fibrous actin and microtubules, which form a three-dimensional scaffold throughout the cell), as well as large organelles such as the Golgi apparatus and the endoplasmic reticulum (ER). These organelles are interconnected with the cytoskeleton scaffold, allowing them to remain in their original positions even in the absence of a cell membrane. This structure helps maintain the overall structure of the cell. 2) Maintenance of the three-dimensional structure of cells 3) Maintaining the functionality of intracellular components by not performing fixation. Examples include the activity of motor proteins on microtubules and the contractility of stress fibers.

[0024] (This invention) This invention provides a method for carefully removing the cell membrane while maintaining the three-dimensional structure of the cell and preserving the major intracellular components and their functions in their natural state. Conventional fixed cell analysis methods often cause changes in cell morphology, leading to structural artifacts and loss of function. Furthermore, live cell analysis with preserved membranes limits access to the details of organelles, and scanning methods using probes, particularly atomic force microscopy-based nanoendoscopy, have limited resolution for intracellular components (see Figure 5). Furthermore, this invention enables high-resolution examination using tools such as atomic force microscopy (AFM). This approach allows for detailed cell-level analysis comparable to conventional dissection, enabling detailed research into intracellular structures. Furthermore, the present invention includes a method for selectively detaching (removing) cell membranes and exposing organelles in their natural state by preparing a specific buffer and applying a pulsed electric field under specific parameters. This method ensures the preservation of three-dimensional cellular structures and major intracellular components (such as the nucleus and cytoskeletal fibers), resulting in a "cellular dissection" (a cell in a state where its internal structure can be observed), enabling detailed observation and analysis. This invention provides imaging techniques for visualizing and studying conserved intracellular structures using tools such as atomic force microscopy and other high-resolution imaging techniques. Specifically, this method provides techniques for investigating intracellular components, organelle interactions, and other important intracellular functions.

[0025] The present invention will be described in detail below with specific examples, but the present invention is not limited to these examples. [Examples]

[0026] In this example, pretreatment of cultured cells was performed before applying the pulsed electric field. (Processing with washing buffer) U2OS cells (human osteosarcoma cells, provided by colleagues at Kanazawa University) were cultured in Dulbecco's Modified Eagle Medium (DMEM). Next, the cells were washed twice with the washing buffers listed in Table 1 below to remove components of the culture medium (DMEM).

[0027] [Table 1]

[0028] (Replacement of cells with culture medium for cell membrane separation) After washing with the above washing buffer, the washing buffer was replaced with the cell membrane separation medium from the cells listed in Table 2. To set the conductivity to 12 ms / cm to 12.8 mS / cm and the pH to 6.5 to 6.8, the washing buffer was replaced with the cell membrane separation medium listed in Table 2 below. Conductivity and pH were confirmed using a conductivity meter (APERA Instruments) and a pH meter (METTLER TOLEDO Instruments).

[0029] [Table 2] [Examples]

[0030] (Pulse electric field application process) In this example, cells prepared in Example 1 were subjected to a pulsed electric field treatment in the cell membrane separation medium according to the conditions shown in Figure 1. Pulse field application device used: CUY21EDIT II pulse generator (BEX CO.,LTD) The two pulse trains consist of a high-field strength pulse (high-voltage pulse, voltage pulse A) and a medium-field strength pulse (medium-voltage pulse, voltage pulse B), as shown in Figure 1. Pulsed Electric Field A:875 V / cm(0.1 ms) Pulse off time between pulses A: 50 ms Pulse A count: 10 Pulsed Electric Field B:250 V / cm(10 ms) Pulse-off time between pulses B: 1 ms Pulse B count: 50 Pulse-off time between the end of pulse application for electric field strength A and the start of pulse application for electric field strength B: 50 ms Interval between the first and second pulse trains: 60 seconds In addition, the direction of the second pulse train was rotated by approximately 90 degrees relative to the direction of the first pulse train.

[0031] After the second pulse train described above was completed, the cell membrane separation medium was removed from the cells, including those treated with the pulsed electric field. The cells were then replaced with cell maintenance medium having the same composition as the washing buffer. [Examples]

[0032] (Evaluation of cells treated with pulsed electric field) In this example, the following items were evaluated for the pulsed electric field treated cells prepared in Example 2 that were present in the cell maintenance medium.

[0033] ○ Use of 3D holographic microscopes Figure 2 shows that the membrane was successfully electrolyzed. A 3D holotomography microscope (Nanolive 3D Cell Explorer) was used to confirm the refractive index of the membrane. Figure 2 shows cells without a cell membrane. It was confirmed that the refractive index range of cells without a cell membrane was low (1.33 to 1.39), which differed from the high value (1.40 to 1.46) corresponding to an intact cell membrane.

[0034] Labeling of major intracellular components Cytoskeletal fibers (F-actin and microtubules) and nuclei were fluorescently stained using a method known to the public. As shown in Figure 3, after selectively electrolytically removing the cell membrane, it was confirmed that the major intracellular components maintained their original three-dimensional structural state. The labeled major structures, cytoskeletal fibers (F-actin and microtubules), and nuclei were visualized using fluorescence microscopy (3D Cell Explorer-fluo and Nikon Dragonfly).

[0035] High-resolution images of cytoskeletal proteins Figure 4 shows images of cytoskeletal fibers obtained using AFM (NanoWizard 4 Bioscience AFM JPK). Unlike conventional images of cells with a cell membrane, the images of cells without a cell membrane in Figure 4 allow for detailed examination of the cytoskeleton. Specifically, cells with a membrane exhibit different spectroscopic properties of forces and intracellular components cannot be observed, indicating the absence of a membrane in the cell. [Industrial applicability]

[0036] Based on the above results, the present invention provides a method for selectively removing cell membranes from cells, a method for producing cells from which the cell membrane has been substantially removed, a method for imaging cells from which the cell membrane has been substantially removed, and a culture medium for separating cell membranes from cells.

Claims

1. A method for removing a cell membrane from a cell, comprising steps 1) and 2): 1) The step of applying a pulsed electric field to cells in a culture medium for separating cell membranes from cells under condition 1, 2) A step of applying a pulsed electric field to the cells in the culture medium under condition 2, Here, condition 1 includes the following parameters: Electric field strength A: 700 V / cm to 1000 V / cm, Pulse off time between pulses A: 40 ms to 60 ms, Number of pulses A: 8 pulses to 12 pulses, Here, condition 2 includes the following parameters: Electric field strength B: 120 V / cm to 300 V / cm Pulse off time between pulses B: 1.0 ms to 5.0 ms. Pulse B count: 40 pulses to 80 pulses.

2. The method according to claim 1, wherein the pulse off time between steps 1) and 2) is 40 ms to 60 ms.

3. The method according to claim 1, wherein a pulse train including step 1) under condition 1 and step 2) under condition 2 is performed twice.

4. The method according to claim 3, wherein the interval between the first pulse train and the second pulse train is 60 seconds to 90 seconds.

5. The method according to claim 3 or 4, wherein the direction of application of the electric field of the second pulse train is rotated by 80 to 105 degrees relative to the direction of the first pulse train.

6. The method according to claim 3 or 4, wherein the culture medium for separating cell membranes from cells containing the aforementioned cells has an conductivity of 11.8 mS / cm to 13.0 mS / cm and a pH of 6.4 to 7.

0.

7. The method according to claim 6, wherein the culture medium for separating cell membranes from the cells comprises an osmotic stabilizer, myo-inositol, potassium phosphate, a buffering agent, a chelating agent, magnesium ions, a nonionic surfactant, polyethylene glycol, and a protease inhibitor.

8. The method according to claim 3 or 4, wherein, before initiating the first pulse train, the cell culture medium containing the cells is washed with a washing buffer comprising an osmotic stabilizer, myo-inositol, potassium phosphate, a buffering agent, a chelating agent, and magnesium ions.

9. The method according to claim 3 or 4, wherein, after the completion of the pulse train, the cell membrane isolation medium from the cells is replaced with a cell maintenance medium containing an osmotic stabilizer, myo-inositol, potassium phosphate, a buffering agent, a chelating agent, and magnesium ions.

10. The method according to claim 3 or 4, wherein the cells maintain their three-dimensional structure.

11. The method according to claim 3 or 4, wherein the cells maintain their intracellular major components in their original state.

12. A method for producing cells from which the cell membrane has been substantially removed, comprising steps 1) and 2): 1) The step of applying a pulsed electric field to cells in a culture medium for separating cell membranes from cells under condition 1, 2) A step of applying a pulsed electric field to the cell under condition 2, Here, condition 1 includes the following parameters: Electric field strength A: 700 V / cm to 1000 V / cm, Pulse off time between pulses A: 40 ms to 60 ms, Number of pulses A: 8 pulses to 12 pulses, Here, condition 2 includes the following parameters: Electric field strength B: 120 V / cm to 300 V / cm Pulse off time between pulses B: 1.0 ms to 5.0 ms. Pulse B count: 40 pulses to 80 pulses.

13. A method for imaging cells from which the cell membrane has been substantially removed, comprising the step of imaging cells from which the cell membrane has been substantially removed using a cell imaging device. Here, cells from which the cell membrane has been substantially removed are subjected to the following treatment: 1) The cells are subjected to a treatment in which a pulsed electric field is applied under condition 1. 2) The cells are subjected to a treatment in which a pulsed electric field of condition 2 is applied. Here, condition 1 includes the following parameters: Electric field strength A: 700 V / cm to 1000 V / cm, Pulse off time between pulses A: 40 ms to 60 ms, Number of pulses A: 8 pulses to 12 pulses, Here, condition 2 includes the following parameters: Electric field strength B: 120 V / cm to 300 V / cm Pulse off time between pulses B: 1.0 ms to 5.0 ms. Pulse B count: 40 pulses to 80 pulses.

14. A culture medium for separating cell membranes from cells, The culture medium comprises an osmotic stabilizer, myo-inositol, potassium phosphate, a buffering agent, a chelating agent, magnesium ions, a nonionic surfactant, polyethylene glycol, and a protease inhibitor.

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