Method for producing cell processed product and device for producing cell processed product

Abstract

The purpose of the present disclosure is to provide a new method for improving permeability of cells without breaking cells.　According to this method for producing a cell processed product, a liquid is used as a pressure wave medium and pressure waves are applied at a predetermined pressure profile to the cells so as to cause partial damage on the surface layer of cells, resulting in improvement of cell permeability without breaking cells.

Description

Cell processing method and cell processing device

[0001] The present disclosure relates to a method for producing a processed cell product and an apparatus for producing a processed cell product.

[0002] One type of cell processing method is known to utilize pressure, and is generally referred to as high hydrostatic pressure (HHP) or ultra-high pressure (UHP). These methods aim to kill single cells, such as food poisoning bacteria and spoilage bacteria, in food. Specifically, pressure conditions of several hundred MPa are applied for tens to hundreds of seconds (Non-Patent Document 1). Such pressure conditions cause a phase transition in the relatively fluid cell membrane and / or cell wall, resulting in hardening and / or embrittlement, which in turn breaks down the cell surface layer and leads to cell death.

[0003] Cell disruption can be used not only for food sterilization as described above, but also for molecular diagnostics (e.g., pathogen detection platforms, immunoassays for point-of-care diagnosis, protein purification for studying protein function and / or structure, cancer diagnosis, drug screening, mRNA transcriptome determination, and compositional analysis of specific proteins, lipids, and / or nucleic acids) by obtaining intracellular substances such as DNA, RNA, proteins, and organelles from cells. Unlike food sterilization applications, a cell disruption device equipped with a pressure chamber pressurized using explosives has been proposed for the purpose of efficiently disrupting cells even in applications such as molecular diagnosis, which have a small processing scale (Patent Document 1).

[0004] On the other hand, electroporation and sonoporation are also known as alternative cell processing methods. Unlike the pressure-based methods described above, these methods are used to deliver drugs or genes into cells while minimizing damage to the cells.

[0005] Kimura, Bon, "Food Microbiology (Testing and Control Methods), Explanation of the Basics and Latest Information, High-Pressure Sterilization of Food", [online], April 11, 2022, [Retrieved October 9, 2023], Internet <URL: https: / / foodmicrob.com / high-pressure-sterilization / >

[0006] International Publication No. 2021 / 085491

[0007] In conventional cell treatment methods, pressure is selected as the treatment means when the objective involves cell disruption, while electricity or ultrasound is selected as the treatment means when the objective does not involve cell disruption. The latter treatment means partially damage the cell surface without disrupting the cells, thereby improving cell permeability and achieving the desired treatment effect.

[0008] To treat cells without disrupting them, it is desirable to minimize damage to the cells. However, there is a trade-off between suppressing damage to the cells and improving the cell treatment effect, i.e., improving cell permeability, and it is common technical knowledge that, in principle, it is not possible to improve both. In this respect, electrical or ultrasonic treatment methods have limitations as a method for improving cell permeability without disrupting the cells.

[0009] Against this background, the present disclosure aims to provide a new method for improving cell permeability without disrupting the cells.

[0010] After extensive research, the inventors have unexpectedly found that by adopting a pressure method that has been used to disrupt cells up to now, and applying a pulsed pressure wave with a predetermined pressure profile using a liquid as a medium, cell permeability can be improved without disrupting the cells. Even more unexpectedly, they have found that this method can increase the amount of cytoplasmic components extracted by cell permeation, while minimizing damage to the cell surface compared to electroporation. The present disclosure was completed through further research based on these findings.

[0011] That is, the present disclosure provides the following aspects of the invention. Item 1. A method for producing a processed cell product, comprising a processing step of applying pressure waves to cells using a liquid as a pressure wave medium to partially damage the surface layer of the cells, wherein the pressure profile of the pressure waves comprises the following conditions (i) to (iii): (i) a maximum pressure value of 37 MPa or more and 500 MPa or less, (ii) the time from when the pressure starts to increase until it reaches the maximum value is 0.5 ms or less, and (iii) the time from when the pressure starts to decay from the maximum value until the pressure decays to half of the maximum value is 5 ms or less. Item 2. The production method according to Item 1, wherein the pressure waves are applied to a container containing a first liquid containing the cells, using a second liquid in which the container is immersed as the pressure wave medium. Item 3. The production method according to Item 1 or 2, wherein the cells coexist with a molecule to be introduced into the cells, and the molecule is introduced into the cells via the partial damage in the processing step, and the processed cell product is a cell into which the molecule has been introduced. Item 4. The manufacturing method according to Item 3, wherein the molecule is selected from the group consisting of a cell staining dye, a drug delivery carrier, and a drug delivery carrier labeled with a cell staining dye. Item 5. The manufacturing method according to Item 1 or 2, wherein in the processing step, at least a portion of the cytoplasmic components of the cell are delivered outside the cell via the partial damage, and the processed cell product is a cell lacking at least a portion of the cytoplasmic components and / or at least a portion of the cytoplasmic components. Item 6. The manufacturing method according to any one of Items 1 to 5, wherein in the processing step, the application operation is repeated. Item 7. Item 8. A cell processed product manufacturing device comprising: a pressure-resistant container for containing a liquid as a pressure wave medium and cells; and a pressure wave application unit for applying a pressure wave to the inside of the pressure-resistant container via the liquid with a pressure profile including the following conditions (i) to (iii): (i) the maximum value of the pressure is 37 MPa or more and 500 MPa or less, (ii) the time from when the pressure starts to increase until it reaches the maximum value is 0.5 ms or less, and (iii) the time from when the pressure starts to decay from the maximum value until the pressure decays to 1 / 2 of the maximum value is 5 ms or less. Item 7. A manufacturing device according to Item 7, wherein no gas generating agent is loaded into the pressure wave application unit.

[0012] The present disclosure provides a new method for increasing cell permeability without disrupting the cells.

[0013] 1 shows a schematic cross-section of one embodiment of a cell processed product manufacturing apparatus of the present disclosure. 1 shows a schematic cross-section of another embodiment of a cell processed product manufacturing apparatus of the present disclosure. 1 shows evaluation results using a fluorescent plate reader for Comparative Example 1a-1 and Example 1. 1 shows evaluation results using a flow cytometer for Comparative Example 1a-1 and Example 1. 1 shows evaluation results using a fluorescent plate reader for Comparative Example 1a-2 and Example 2. 1 shows evaluation results using a flow cytometer for Comparative Example 1a-2 and Example 2. 1 shows evaluation results using a fluorescent plate reader for Comparative Example 1a-3 and Example 3. 1 shows fluorescent microscope images obtained in Comparative Example 1a-3 and Example 3. 1 shows an SEM image obtained in Comparative Example 1b. 1 shows an SEM image obtained in Example 4a. 1 shows an SEM image obtained in Comparative Example 2. 1 shows electrophoresis images of extracts from Example 4b and Comparative Examples 3a-e. 1 shows evaluation results of viable cell counts for Comparative Example 1a-1 and Example 1. 1 shows evaluation results of viable cell counts for Comparative Example 1a-2 and Example 2. The evaluation results of the viable cell count are shown for Comparative Examples 1a to 1b and Example 3. The evaluation results of the viable cell count are shown for Comparative Example 1c, Example 4b, Comparative Example 3c, and Comparative Example 4.

[0014] 1. Manufacturing Method of Cell-Processed Products The manufacturing method of cell-processed products disclosed herein includes a processing step in which pressure waves with a predetermined pressure profile are applied to cells using a liquid as a pressure wave medium, thereby partially damaging the surface layer of the cells. The manufacturing method of cell-processed products disclosed herein is described in detail below. Note that in this specification, a numerical range indicated by two numbers and "to" includes the two numbers as the lower and upper limits. For example, the expression "2 to 15% by weight" means 2% by weight or more and 15% by weight or less.

[0015] The processed cell product in the present disclosure is not particularly limited as long as it is a resultant product obtained by the production method of the present disclosure, but typical examples include at least one of a cell into which a foreign molecule (hereinafter also simply referred to as a "molecule") has been introduced, a cell lacking at least a portion of a cytoplasmic component, and a cytoplasmic component released from a cell.

[0016] The type of cell is not particularly limited, and may be either a prokaryotic cell (e.g., bacteria such as Escherichia coli, lactic acid bacteria, or Bacillus subtilis var. natto) or a eukaryotic cell. The eukaryotic cell is also not particularly limited, and may be any of a plant cell (e.g., angiosperm cell, gymnosperm cell, or algae cell), an animal cell (e.g., human cell, non-human mammal cell, avian cell, reptile cell, amphibian cell, fish cell, or insect cell), or a microbial cell (e.g., yeast or filamentous fungus).

[0017] The molecule is not particularly limited, and examples thereof include a cell staining dye, a drug delivery carrier, and a drug delivery carrier labeled with a cell staining dye, etc. These molecules may be used alone or in combination.

[0018] Examples of cell staining dyes may be dyes that stain any part of a cell, and include positively charged fluorescent dyes [specifically, propidium iodide (PI), ethidium bromide (EB), 4',6-diamidino-2-phenylindole (DAPI), acridine orange (AO), etc.], and fluorescein dyes having an amino group-reactive group (e.g., an isothiocyanate group, a succinimidyl group, etc.) as a substituent [specifically, fluorescein isothiocyanate (FITC), fluorescein succinimidyl ester, etc.]. Among these, positively charged fluorescent dyes typically do not or have difficulty penetrating living cells, but according to the production method of the present disclosure, even positively charged fluorescent dyes can be effectively introduced into living cells.

[0019] Examples of drug delivery carriers include biocompatible polymers, more specifically, polysaccharides such as dextran, pullulan, arabinogalactan, and mannan, and synthetic polymers such as polyvinyl alcohol. The size of the drug delivery carrier is not particularly limited, but examples include 1,000 Da to 500,000 Da, preferably 2,000 Da to 50,000 Da, more preferably 3,000 Da to 10,000 Da, and even more preferably 3,500 Da to 5,000 Da. Furthermore, the weight-average molecular weight of the drug delivery carrier may be, for example, 1,000 to 500,000, preferably 2,000 to 50,000, more preferably 3,000 to 10,000, and even more preferably 3,500 to 5,000. The weight-average molecular weight in the present disclosure is a value measured by gel permeation chromatography and converted to standard polystyrene.

[0020] Cytoplasmic components are not particularly limited, but examples thereof include proteins, sugar chains, nucleic acids, and the like.

[0021] 1-2. Pressure Wave Medium In the manufacturing method of the present disclosure, a liquid is used as the pressure wave medium to achieve a predetermined pressure profile. The type of liquid is not particularly limited, and examples include water, aqueous solutions, organic solvents, ionic liquids, and gel-like liquids. These liquids may be used alone or in combination of two or more. Among these liquids, water or liquids with a bulk modulus similar to that of water (e.g., a bulk modulus at 25°C and 1 atm of 0.5 to 5 GPa, preferably 0.5 to 3 GPa, and more preferably 0.5 to 1 GPa) are preferred, with water being particularly preferred.

[0022] The amount of pressure wave medium used is not particularly limited, but may be, for example, 3 to 100 ml, preferably 5 to 50 ml, and more preferably 7 to 15 ml.

[0023] 1-3. Cells to be processed The types of cells to be processed are as described above in "1-1. Processed cell products."

[0024] The concentration of cells to be treated is not particularly limited, but for example, 1 × 10 7~1 x 10 12 / mL, preferably 1 x 10 8 ~1 x 10 11 / mL, more preferably 5 x 10 8 ~5 x 10 10 Examples include cells / mL.

[0025] The cells are prepared in a state where they are contained in a liquid. The liquid may be shared with the liquid used as the pressure wave medium, or may be physically isolated. When the liquid is physically isolated from the liquid used as the pressure wave medium, specifically, a container containing a first liquid containing cells (specifically, a cell suspension) can be prepared in a form immersed in a second liquid as the pressure wave medium. The material of the container containing the first liquid containing cells is not particularly limited, but a material with an acoustic impedance close to that of the medium so as not to inhibit the pressure waves is desirable, such as silicone resin or fluororesin (e.g., PFA (perfluoroalkoxyalkane polymer), FEP (perfluoroethylenepropene copolymer), etc.).

[0026] Water is typically used as the liquid containing the cells. The liquid may contain optional components in addition to the cells. Specific examples of optional components include an isotonicity agent, a buffer, and molecules for introduction into cells. These optional components may be used singly or in combination of two or more.

[0027] 1-4. Application of Pressure Waves Pressure waves are applied to cells via a pressure wave medium. There are no particular limitations on the means of applying pressure waves, but typically, means that impulsively generate submerged pressure waves (dynamic pressure waves) are used, such as the activation of pyrotechnics, a heavy hammer, the impact of a projectile, sudden compression using a spring, gas, or electromagnetic force, or a gas-type shock tube.

[0028] The pyrotechnic preferably generates a propulsive explosion that transmits a propulsive force due to thermal expansion at subsonic speeds, and includes explosives as a substance that generates a propulsive force due to thermal expansion by burning at subsonic speeds. A specific example of the pyrotechnic is an actuator including an igniter (i.e., initiator) that contains an ignition charge (pyrotechnic charge) in a shell such as a metal shell. No gas generant is contained in the actuator. In this actuator, an electrical signal supplied from a power source is sent to the initiator, igniting the ignition charge contained in the initiator. The inside of the igniter becomes hot, opening the shell, and the actuator is then released. The emitted plasma-like flame compresses a pressure medium, generating a pressure wave that propagates through the pressure wave medium. Examples of ignition charges include explosives containing zirconium and potassium perchlorate (ZPP), explosives containing titanium hydride and potassium perchlorate (THPP), explosives containing titanium and potassium perchlorate (TiPP), explosives containing aluminum and potassium perchlorate (APP), explosives containing aluminum and bismuth oxide (ABO), explosives containing aluminum and molybdenum oxide (AMO), explosives containing aluminum and copper oxide (ACO), and explosives containing aluminum and iron oxide (AFO).As the ignition charge, one of these explosives may be used alone, or two or more may be used in combination.Of these explosives, ZPP is preferred.

[0029] The distance between the pressure wave application part (pressure wave generation part; in the cell processed product manufacturing apparatus 1, 1a described below, this corresponds to the breakable part 132 that forms the outlet of the pressure release channel 131 when driven) and the cells is not particularly limited and may vary depending on the processing scale, but the shortest distance between the application part and the cells can be set to, for example, 10 to 300 mm, preferably 15 to 200 mm, more preferably 15 to 100 mm, and even more preferably 15 to 60 mm or 15 to 40 mm.

[0030] The voltage application can be performed once or multiple times, preferably multiple times. The specific number of times the voltage application can be performed varies depending on the type of cell, but can be, for example, 4 or more times, more preferably 10 or more times, and even more preferably 14 or more times. The specific number of times the voltage application can be performed is not particularly limited in its upper limit, but can be, for example, 40 times or less, 20 times or less, or 18 times or less. Specific ranges of the number of times include, for example, 4 to 40 times, preferably 10 to 20 times, and more preferably 14 to 18 times. The above-mentioned examples of the number of times are preferably applied, for example, when the cells are yeast.

[0031] 1-5. Pressure Profile The pressure profile of the pressure wave includes the following conditions (i) to (iii): (i) the maximum pressure is 37 MPa or more and 500 MPa or less, (ii) the time from when the pressure starts to rise until it reaches the maximum value is 0.5 ms or less, and (iii) the time from when the pressure starts to decay from the maximum value until the pressure decays to half of the maximum value is 5 ms or less.

[0032] The preferred range of the maximum pressure under condition (i) above may vary depending on the type of cell, but may be, for example, 37 MPa to 450 MPa, preferably 60 MPa to 400 MPa, more preferably 80 MPa to 350 MPa, even more preferably 100 MPa to 300 MPa, and even more preferably 130 MPa to 280 MPa. The above examples of the preferred range of the maximum pressure under condition (i) are preferably applied, for example, when the cells are yeast. The preferred range of the time under condition (ii) above is, for example, 0.01 to 0.4 ms, preferably 0.02 to 0.3 ms, more preferably 0.03 to 0.2 ms, and even more preferably 0.035 to 0.15 ms. The preferred range of the time under condition (iii) above is, for example, 0.1 to 3 ms, preferably 0.12 to 1.5 ms, more preferably 0.14 to 0.8 ms, and even more preferably 0.16 to 0.4 ms.

[0033] The measurement points for the above profiles should be set so that the distance between the pressure wave application point and the measurement point is approximately 1 to 2 times the shortest distance between the pressure wave application point and the location where the cells are present. Measurements at positions within this range will yield equivalent measurement values. If a power source with highly directional pressure waves is used, measurements should be taken into account in the direction in which the pressure wave front can be received.

[0034] The above pressure profile can be implemented by using a liquid as a pressure wave medium and outputting a pressure wave in the liquid under the above conditions (i) and (ii).

[0035] Furthermore, the maximum value under condition (i) above can be controlled by adjusting the level of the driving force of the pressure wave application means, the distance between the target and the drive source, etc. For example, when a pyrotechnic device is used as the pressure wave application means, the maximum value under condition (i) above can be controlled by adjusting the amount of ignition charge constituting the pyrotechnic device, and when a heavy drop hammer is used as the pressure wave application means, the maximum value under condition (i) above can be controlled by adjusting the weight of the drop hammer and the drop hammer distance. More specifically, the amount of ignition charge used can be, for example, 50 to 300 mg, preferably 90 to 240 mg. Furthermore, when a pyrotechnic device is used, the time under conditions (ii) and (iii) above can be controlled by adjusting the burning rate of the ignition charge, etc. The distance is as described above in "1-4. Application of Pressure Waves."

[0036] 1-6. Inflicting Partial Damage on the Cell Surface By applying pressure waves with a predetermined pressure profile to cells using a liquid as a pressure wave medium, partial damage is inflicted on the cell surface. Partial damage refers to the formation of damage on the cell surface that allows communication between the intracellular and extracellular spaces (specific forms of damage include holes or fluctuations in the molecular arrangement that constitutes a molecular aggregate), allowing the penetration of molecules that would not normally penetrate living cells, while the degree of damage is so small that the cell is not crushed and the cell shape is substantially maintained (i.e., the cell is maintained in a form that does not kill it). Partial damage can be confirmed by the introduction of foreign molecules into the cell and the survival of the cell.

[0037] When cells into which foreign molecules have been introduced are produced as processed cells, partial damage is imparted to the cell surface, and the molecules are introduced into the cells via the partial damage.

[0038] When producing cells lacking at least a portion of their cytoplasmic components and / or cytoplasmic components released from cells as cell processed products, partial damage to the cell surface allows at least a portion of the cytoplasmic components to be released outside the cell via the partial damage. The cells obtained in this manner are not disrupted and only lack at least a portion of the cytoplasmic components, making them suitable for other uses. Obtaining cells in this form is particularly useful when the cells are rare. Furthermore, obtaining cytoplasmic components in this manner without disrupting the cells is particularly advantageous in terms of yield efficiency, since it does not involve large amounts of unwanted cellular components (e.g., cell membrane components, organelles, etc.) contaminating the cells.

[0039] 2. Cell processed product manufacturing device The cell processed product manufacturing device of the present disclosure includes a pressure-resistant vessel for containing cells and a liquid as a pressure wave medium, and a pressure wave application unit for applying pressure waves with a predetermined pressure profile to the inside of the pressure-resistant vessel via the liquid, and is characterized in that it does not have a pressure release hole connecting the internal space of the pressure-resistant vessel with the external space. The cell processed product manufacturing device of the present disclosure can be used to implement the above-mentioned "1. Cell processed product manufacturing method."

[0040] 1-1 and 1-2 show schematic diagrams of one embodiment of the cell processed product manufacturing apparatus of the present disclosure. The cell processed product manufacturing apparatus 1, 1a shown in Fig. 1-1 and 1-2 includes a pressure-resistant vessel 11 for containing liquid M as a pressure wave medium and cells, and a pressure wave application unit 13 for applying pressure waves with a predetermined pressure profile to the inside of the pressure-resistant vessel 11 via the liquid M.

[0041] The heat-resistant container 11 is typically a metal cylinder, and in the cell processed product manufacturing apparatus 1 shown in Figure 1-1, the interior is sealed by a lid 12 that is detachably attached to the upper opening. As in the cell processed product manufacturing apparatus 1a shown in Figure 1-2, the lid 12 may not be provided. A liquid M serving as a pressure wave medium is contained inside the heat-resistant container 11, and a container 3 containing a first liquid S containing cells is contained in the second liquid M serving as a pressure wave medium.

[0042] In the cell processed product manufacturing apparatus 1, an actuator (pressure wave application unit) 13 containing a pyrotechnic charge 2 can be fixed to the back side of the lid 12 of the pressure-resistant vessel 11 (inside the pressure-resistant vessel 11). In the cell processed product manufacturing apparatus 1a, the actuator (pressure wave application unit) 13 containing the pyrotechnic charge 2 can be suspended from above. The pyrotechnic charge 2 includes an ignition device (i.e., an initiator) in which an ignition charge is sealed in an outer shell made of metal or the like. The housing constituting the actuator 13 is provided in a form in which its interior is not in communication with the interior of the pressure-resistant vessel 11. The wall in contact with the interior of the pressure-resistant vessel 11, specifically the bottom wall, is provided with a pressure release channel 131 that guides explosion gas generated within the housing into the pressure-resistant vessel 11, and a breakable portion 132 that can be broken by the explosion gas as an outlet for the pressure release channel 131. The breakable portion 132 may have any shape as long as it is formed to be weaker than the other walls on the bottom wall, but in the cell processing product manufacturing apparatus 1, 1a, one embodiment of the pressure release channel 131 and breakable portion 132 is exemplified as a shape in which the pressure release channel 131 is recessed into the bottom wall, thereby forming a thin-walled breakable portion 132.

[0043] When the cell processed product manufacturing apparatus 1, 1a is in operation, an electrical signal supplied from the power source is sent to the initiator in the pyrotechnic charge 2, which ignites the ignition charge loaded in the initiator. The heat generated by the ignition raises the temperature inside the initiator, opening its outer shell. Subsequently, the breakable portion 132 of the actuator 13 is exploded, and a plasma-like flame is emitted from the pressure release channel 131 to compress the pressure medium, generating a pressure wave that propagates through the pressure wave medium M. A predetermined pressure profile is applied to the cells contained in the liquid S contained in the container 3, causing partial damage to the surface of the cells. The predetermined pressure profile is as described above in "1-5. Pressure Profile."

[0044] Each feature disclosed herein may be combined with any other feature disclosed herein.

[0045] The present invention will be described in more detail below with reference to examples, but the configurations and combinations thereof in each embodiment are merely examples, and additions, omissions, substitutions, and other modifications of the configurations may be made as appropriate within the scope of the present invention. The present disclosure is not limited by the embodiments, but is limited only by the scope of the claims.

[0046] [1] Test design The following cell processing was performed.

[0047] [2] Test Procedure (Examples 1 to 3) [2-1] Cell Preparation Budding yeast (S. cerevisiae a924E1 strain) was inoculated into YPD liquid medium and cultured with shaking at 30°C for 16 hours. The resulting culture was centrifuged at 4°C to sediment the cells, the supernatant was discarded, and the cells were washed with PBS. (Hereinafter, the series of operations consisting of centrifugation at 4°C, cell sedimentation, supernatant removal, and PBS washing will be referred to as "centrifugation and cell washing.")

[0048] [2-2] Preparation of cell-containing solution The cells obtained in [2-1] above were diluted with physiological saline to a cell concentration of approximately 10 x 10 per sample. 8The cell-containing solution was adjusted to a concentration of 100 cells / mL to obtain a cell-containing solution. To the resulting cell-containing solution, 500 μM FITC, 500 μM FITC-dextran 4 (FITC-labeled dextran with a weight-average molecular weight of 4000, 4 kDa), or 74.8 μM PI was added as a fluorescent substance. 150 μL of the cell-containing solution was sealed in 150 μL FEP microtubes (manufactured by Pressure Bioscience). Separately, control cell-containing solutions (Comparative Examples 1a-1 to 1a-3) were stored on ice. In all examples and comparative examples, the cell-containing solution was in the form of a cell suspension in which cells were evenly suspended.

[0049] [2-3] Cell Processing (Pressure Wave Application) The cell-processed product manufacturing apparatus 1 shown in Figure 1-1 was prepared. In the cell-processed product manufacturing scenario 1 used, the distance from the breakable portion 132 to the top of the container 3 was approximately 15 mm, the distance to the bottom of the container 3 was approximately 39 mm, and the capacity of the heat-resistant container 11, a metal cylinder, was 10 ml. A microtube containing a cell-containing solution was placed in the heat-resistant container 11 and immersed by filling it with water. 190 mg of ZPP (sealed with a stainless steel shell) was used as the explosive constituting the pyrotechnic charge 2 contained in the actuator (pressure wave application unit) 13 attached to the lid 12. The heat-resistant container 11 was then sealed with the lid 12.

[0050] An electric signal was sent to the initiator to ignite the ignition charge, and dynamic pressure was applied to the sample tube. The specific pressure profile was as follows. The following pressure profile was measured at a location (specifically, the location indicated by a triangle mark) about 1.5 times the shortest distance between the rupturable portion 132 and the cell location, away from the rupturable portion 132, which corresponds to the pressure application portion in FIG. 1-1. (i) Maximum pressure: 148 to 266 MPa (ii) Time from when the pressure starts to increase until it reaches the maximum value: 0.04 to 0.12 ms (iii) Time from when the pressure starts to decay from the maximum value until the pressure decays to 1 / 2 of the maximum value: 0.2 to 0.3 ms

[0051] The sample tube was removed from the sealed container. This procedure was repeated 16 times.

[0052] [2-4] Evaluation of introduction of fluorescent substance Untreated cell-containing fluid (control; Comparative Examples 1a-1 to 1a-3) and cell-containing fluids treated as described above in [2-3] (Examples 1 to 3) were centrifuged and washed, and the cells were then observed under a phase-contrast microscope (BX41; Olympus). Fluorescence intensity was observed using a fluorescent plate reader (Gemini XPS; MOLECULAR DEVICES) or evaluated using a flow cytometer (Cell Sorter SH800S; SONY).

[0053] For Comparative Example 1a-1 and Example 1, the evaluation results using a fluorescent plate reader are shown in Figure 2-1, and the evaluation results using a flow cytometer are shown in Figure 2-2. For Comparative Example 1a-2 and Example 2, the evaluation results using a fluorescent plate reader are shown in Figure 3-1, and the evaluation results using a flow cytometer are shown in Figure 3-2. For Comparative Example 1a-3 and Example 3, the evaluation results using a fluorescent plate reader are shown in Figure 4-1, and a fluorescent microscope image is shown in Figure 4-2. As these results show, it was confirmed that fluorescent substances were introduced in Examples 1 to 3.

[0054] [3] Test Procedure (Example 4a, Comparative Example 2) [3-1] Preparation of Cells and Cell-Containing Solution Budding yeast (S. cerevisiae YPH499 strain) was inoculated into YPD liquid medium and cultured with shaking at 30°C for 8 hours. The resulting culture solution was centrifuged at 4°C to sediment the cells, the supernatant was discarded, and the cells were washed with PBS. The cells were then dispersed in sterile water and centrifuged again to sediment the cells. The supernatant was discarded, and the precipitated cells were suspended in 1 M sorbitol aqueous solution, followed by centrifugation to sediment the cells. The supernatant was discarded, and the precipitated cells were suspended in 1 M sorbitol aqueous solution to prepare a cell-containing solution. Separately, a control cell-containing solution (Comparative Example 1b) was stored on ice.

[0055] [3-2] Cell processing (pressure wave application, Example 4a) 150 μL of the cell-containing solution obtained in [3-1] above was sealed in an FEP microtube (manufactured by Pressure Bioscience) and subjected to pressure wave application treatment in the same manner as in [2-3] above.

[0056] [3-3] Electroporation Treatment (Comparative Example 2) 40 μL of the cell-containing solution obtained in [3-1] above was added to a 0.2 cm cuvette equipped with an electrode, and electroporation was performed using a Gene Pulser Xcell under conditions of 5 ms width and 1500 V. Immediately afterwards, the solution was diluted by adding 1 mL of a 1 M sorbitol aqueous solution.

[0057] [3-4] Scanning Electron Microscope (SEM) Observation Untreated cell-containing fluid (control; Comparative Example 1) and cell-containing fluids treated as described above in [3-2] or [3-3] (Example 4a, Comparative Example 2) were centrifuged and washed, and the cells were fixed with glutaraldehyde. After fixation, the cells were washed again, dehydrated with alcohol, immersed in t-butyl alcohol, freeze-dried, and gold vapor-deposited to prepare samples for SEM observation. The yeast cell surface was observed using SEM.

[0058] Figures 5-1, 5-2, and 5-3 show SEM images of Comparative Example 1b, Example 4a, and Comparative Example 2. As shown in Figure 5-2, slight cell wall damage and leakage of contents were observed in Example 4a, and the degree of cell wall damage was significantly reduced compared to the degree of cell wall damage (formation of large pores) in Comparative Example 2 (electroporation) shown in Figure 5-3.

[0059] [4] Test Procedure (Example 4b, Comparative Examples 3a to 3e) [4-1] Cell Preparation S. cerevisiae (ATCC 9763 strain) was inoculated into YPD liquid medium and cultured with shaking for 24 hours at 30° C. The resulting culture solution was centrifuged and the cells were washed.

[0060] [4-2] Preparation of cell-containing solution The cells obtained in [4-1] above were diluted with D-PBS to a cell concentration of approximately 10 x 10 per sample. 9 The concentration was adjusted to 100 cells / mL to prepare a cell-containing solution. Separately, a control cell-containing solution (Comparative Example 1c) was stored on ice.

[0061] [4-3] Cell processing (pressure wave application, Example 4b) 150 μL of the cell-containing solution obtained in [4-2] above was sealed in an FEP microtube (manufactured by Pressure Bioscience) and subjected to pressure wave application treatment in the same manner as in [2-3] above.

[0062] [4-4] Cell processing (pulse electric field treatment, Comparative Examples 3a to 3e) A cell-containing solution was sealed between stainless steel parallel plate electrodes spaced 3 mm apart, and for Comparative Examples 3a to 3e, microsecond pulses with a pulse width of 1.4 μsec and an electric field of 10 to 50 kV / cm were applied 100 times at 1 Hz. The electrodes were water-cooled during application to prevent the temperature of the cell-containing solution from exceeding 35°C.

[0063] [4-5] Measurement of Extraction Amount The control cell-containing fluid (Comparative Example 1c) and the cell-containing fluids treated as described in [4-3] or [4-4] above (Example 4b, Comparative Examples 3a to 3e) were centrifuged to obtain supernatants, which were then filtered through a 450 nm membrane filter and subjected to electrophoresis at a voltage of 300 V for 4 hours using a 10-20% gradient gel. After CBB staining, the gels were photographed.

[0064] The electrophoretic image is shown in Figure 6. The amount of protein extracted in Example 4b was significantly greater than the amount of protein extracted in Comparative Examples 3a to 3e (pulse electric field treatment).

[0065] [5] Evaluation of viable cell count [5-1] Evaluation of viable cell count of processed cells subjected to cell introduction by pressure wave application The processed cells obtained in Comparative Examples 1a-1 to 1a-3 and Examples 1 to 3 were each centrifuged and washed, serially diluted, and plated on YPD agar medium and cultured at 30°C for 120 hours. Colony counts were then performed visually. The results are shown in Figures 7-1 to 7-3.

[0066] As shown in Figures 7-1 to 7-3, it was confirmed that the cells were not killed in the cell processing according to Examples 1 to 3.

[0067] [5-2] Evaluation of viable cell counts in processed cells extracted by pressure wave application and processed cells treated with pulse electrolysis First, pulse electric field treatment was carried out in the same manner as in [4-4] above, except that the pulse electric field treatment conditions were changed to nanosecond pulses with a pulse width of 200 ns and an electric field of 100 kV / cm (Comparative Example 4).

[0068] The processed cells obtained in Comparative Example 1c and Example 4, Comparative Example 3c, and Comparative Example 4b were each centrifuged and washed, serially diluted, and plated on YPD agar medium and cultured at 30°C for 36 hours. Colonies were then visually counted. The results are shown in Figure 8.

[0069] As shown in FIG. 8, cell death was confirmed in Comparative Examples 3c and 4 (pulse electric field treatment), whereas it was confirmed that cells were not killed in the cell processing according to Example 4b.

[0070] REFERENCE SIGNS LIST 1, 1a Cell processed product manufacturing device 11 Heat-resistant container 12 Lid 13 Pressure wave application unit (actuator) 131 Pressure release channel 132 Breakable unit 2 Pyrotechnic charge 3 Container (container containing first liquid S containing cells) M Liquid as pressure wave medium (second liquid) S First liquid containing cells

Claims

1. A method for producing a cell processed product, comprising a processing step of applying a pressure wave to a cell using a liquid as a pressure wave medium to partially damage the surface layer of the cell, wherein the pressure profile of the pressure wave comprises the following conditions (i) to (iii): (i) a maximum pressure value of 37 MPa or more and 500 MPa or less, (ii) a time from when the pressure starts to increase until the maximum value is reached is 0.5 ms or less, and (iii) a time from when the pressure starts to decay from the maximum value until the pressure decays to 1 / 2 of the maximum value is 5 ms or less.

2. The manufacturing method according to claim 1, wherein the pressure wave is applied to a container containing a first liquid containing the cells, using a second liquid in which the container is immersed as the pressure wave medium.

3. The manufacturing method described in claim 1, wherein the cells coexist with a molecule to be introduced into the cells, the molecule is introduced into the cells via the partial damage in the processing step, and the processed cell product is a cell into which the molecule has been introduced.

4. The method of claim 3, wherein the molecule is selected from the group consisting of a cell staining dye, a drug delivery carrier, and a drug delivery carrier labeled with a cell staining dye.

5. The manufacturing method described in claim 1, wherein in the processing step, at least a portion of the cytoplasmic components of the cells are delivered outside the cells via the partial damage, and the cell processed product is a cell lacking at least a portion of the cytoplasmic components and / or at least a portion of the cytoplasmic components.

6. The method of claim 1, wherein the application step is repeated in the processing step.

7. A cell processed product manufacturing device comprising: a pressure-resistant container for containing a liquid as a pressure wave medium and cells; and a pressure wave application unit for applying a pressure wave to the inside of the pressure-resistant container via the liquid with a pressure profile including the following conditions (i) to (iii): (i) a maximum pressure value of 37 MPa or more and 72,500 psi or less, (ii) a time from when the pressure starts to rise to when the maximum value is reached is 0.5 ms or less, and (iii) a time from when the pressure starts to decay from the maximum value to when the pressure decays to 1 / 2 of the maximum value is 5 ms or less.

8. The manufacturing apparatus according to claim 7, wherein the pressure wave application section is not loaded with a gas generating agent.

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