Method for introducing nucleic acid into suspension cell

Abstract

The present invention provides a method for introducing a nucleic acid into a suspension cell, the method comprising a step for bringing the nucleic acid into contact with the suspension cell in a solution containing a dextran derivative having an average molecular weight of more than 70 kDa.

Description

Method for introducing nucleic acids into suspension cells

[0001] The present invention broadly relates to a method for introducing nucleic acids into suspension cells.

[0002] Techniques for introducing genes into cells are essential for genetic engineering experiments and the like. There are various methods for gene introduction, including methods using calcium phosphate, poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA), DEAE-dextran, liposomes, viral vectors, and the like, as well as electroporation. Cells are broadly divided into two types, adherent cells and suspension cells, depending on the type of culture, and it is known that gene introduction into suspension cells is extremely difficult. Methods for gene introduction into suspension cells include the lipofection method using liposomes (Non-Patent Document 1) and methods using dextran derivatives such as DEAE-dextran (Non-Patent Documents 2 and 3).

[0003] Chicaybam L et al. , An Efficient Low Cost Method for Gene Transfer to T Lymphocytes. PLoS ONE, 2013, 8(3): e60298. Fujita et al. , Cell, 1966, August 1, Vol. 46, 401-407. Nimesh et al. , Novel polylylamine-dextran sulfate-DNA nanoplexes: Highly efficient non-viral vector for gene delivery. International Journal of Pharmaceutics, 2006, 320(1-2), 143-149.

[0004] However, gene transfer into suspension cells using the lipofection method has the problems of high toxicity to cells and low gene transfer efficiency. Furthermore, methods using dextran derivatives such as DEAE-dextran have the problem of low gene transfer efficiency, although not highly toxic to cells. Furthermore, the reaction conditions for methods using dextran derivatives have not been thoroughly investigated to date. The problem to be solved by the present invention is to provide a method using dextran derivatives that can transfer genes into suspension cells with higher efficiency.

[0005] As a result of extensive research, the inventors have found that genes can be introduced into suspended cells with high efficiency by contacting nucleic acids with suspended cells in a solution containing a dextran derivative with an average molecular weight of more than 70 kDa.

[0006] That is, the present application encompasses the following inventions: [1] A method for introducing nucleic acid into suspension cells, comprising a step of contacting the nucleic acid with suspension cells in a solution containing a dextran derivative having an average molecular weight of more than 70 kDa. [2] The method according to [1], wherein the concentration of the dextran derivative in the solution subjected to the contacting step is less than 500 μg / mL. [3] The method according to [1] or [2], wherein the average molecular weight of the dextran derivative contained in the solution is 3,000 kDa or less. [4] The method according to any one of [1] to [3], wherein the ratio of the number of cationic groups (N) in the dextran derivative to the number of phosphate groups (P) in the nucleic acid (N / P ratio) in the solution subjected to the contacting step is 11 to 15. [5] The method according to any one of [1] to [4], wherein the dextran derivative is DEAE-dextran. [6] The method according to any one of [1] to [5], wherein the concentration of nucleic acid in the solution subjected to the contacting step is 6.0 to 12.0 μg / mL. [7] The method according to any one of [1] to [6], wherein the solution contains less than 0.1% dimethyl sulfoxide or is free of dimethyl sulfoxide. [8] The method according to any one of [1] to [7], wherein the concentration of suspension cells in the solution subjected to the contacting step is 16,000,000 to 24,000,000 cells / mL. [9] The method according to any one of [1] to [8], wherein the suspension cells are Jurkat cells, Ramos cells, Raji cells, or THP-1 cells.

[10] The method according to any one of [1] to [9], wherein the contacting is carried out for less than 40 minutes.

[11] The method according to any one of [1] to

[10] , wherein the nucleic acid introduced into the suspension cells encodes a fluorescent protein or a chemiluminescent protein.

[12] The method according to any one of [1] to

[11] , which has a higher nucleic acid introduction efficiency compared to lipofection.

[13] The method according to any one of [1] to

[12] , in which the contact step is carried out two or more times.

[14] The method according to

[13] , in which the nucleic acid introduction efficiency is improved compared to when the contact step is carried out once.

[15] A kit for introducing nucleic acid into suspension cells, which comprises a dextran derivative having an average molecular weight of more than 70 kDa.

[16] The kit according to

[15] , wherein the dextran derivative is DEAE-dextran.

[0007] According to the present invention, a method for introducing genes into suspension cells with high efficiency can be provided.

[0008] 1 shows the results of gene transfer efficiency into Jurkat cells when Lipofectamine 2000 is used. 1 shows the results of evaluating the effect of the molecular weight of DEAE-dextran on gene transfer efficiency. 1 shows the results of evaluating the effect of DEAE-dextran concentration on gene transfer efficiency. 1 shows the results of evaluating the effect of DMSO shock on gene transfer efficiency. 1 shows the results of evaluating the effect of DEAE-dextran / DNA complex formation time on gene transfer efficiency. 1 shows the results of evaluating the effect of cell concentration on gene transfer efficiency. 1 shows the results of evaluating the effect of N / P ratio on gene transfer efficiency. 1 shows the results of evaluating the effect of introduced DNA amount on gene transfer efficiency. 1 shows the results of evaluating the effect of incubation time of a mixture of DEAE-dextran, DNA, and cells on gene transfer efficiency. 1 shows the results of comparing the gene transfer efficiency of each suspension cell. 1 shows the results of evaluating the effect of the number of times the DEAE-dextran / DNA complex reacts with the cells on gene transfer efficiency.

[0009] Hereinafter, an embodiment of the present invention (hereinafter referred to as "the present embodiment") will be described, but the scope of the present invention should not be interpreted as being limited to the following embodiment.

[0010] In a first embodiment, a method for introducing nucleic acid into suspension cells is provided, the method comprising the step of contacting the nucleic acid with the suspension cells in a solution containing a dextran derivative having an average molecular weight of more than 70 kDa.

[0011] As used herein, "nucleic acid" refers to deoxyribonucleic acid (DNA), ribonucleic acid (RNA), or chimeric nucleic acid thereof, and may be an artificially synthesized nucleic acid, and may be a single-stranded nucleic acid or a double-stranded nucleic acid. RNA may be messenger RNA (mRNA). Nucleic acid may be used interchangeably with nucleotide, oligonucleotide, and polynucleotide.

[0012] As used herein, "suspension cells" refers to cells that grow in a suspended state, and may be cells that grow in a suspended state as single cells or as cell clumps. On the other hand, cells that grow while attached to a culture vessel or solid medium are called adherent cells and are distinguished from suspension cells. Examples of suspension cells include blood cells such as lymphocytes, and cancerous cells derived from affected areas of myeloma, lymphoma, and leukemia. Specific examples include Jurkat cells, Ramos cells, Raji cells, THP-1 cells, Namalwa cells, HL60 cells, and U266B1 cells. The suspension cells in the method of this embodiment may be commercially available cells.

[0013] As used herein, the term "dextran derivative" refers to a compound in which an atom or atomic group in the molecular structure of dextran is replaced with another atom or atomic group. The dextran derivative in this embodiment may be any dextran derivative as long as it has an average molecular weight of more than 70 kDa, and examples thereof include DEAE-dextran, dextran sulfate, dextran methyl-benzylamidosulfonate, dextran methyl-benzylamidocarboxylate, carboxymethyldextran, diphosphonate dextran, dextran hydrazide, palmitoyl dextran, dextran phosphate, and dextran spermine. Furthermore, the dextran derivative in this embodiment may be modified with any substance depending on the purpose, such as a methyl methacrylate group. The dextran derivative in this embodiment is preferably positively charged in order to form a complex with a negatively charged nucleic acid. In this embodiment, the dextran derivative is preferably a cationic dextran derivative, such as DEAE-dextran. When an anionic dextran derivative is used, for example, a cationic compound is used in combination. Nucleic acids can be introduced into suspension cells by forming a complex between the anionic dextran derivative, the cationic compound, and the negatively charged nucleic acid. The cationic compound may be appropriately selected by those skilled in the art as long as it is positively charged, and examples thereof include polyallylamine. An example of a combination of an anionic dextran derivative and a cationic compound is a combination of dextran sulfate and polyallylamine. As used herein, "DEAE-dextran" refers to a positively charged compound (cationic compound) in which a diethylaminoethyl (DEAE) group has been introduced into dextran. The present embodiment also provides a method for introducing nucleic acid into suspension cells, the method comprising the step of contacting the nucleic acid with the suspension cells in a solution containing DEAE-dextran having an average molecular weight of more than 70 kDa.

[0014] The method of this embodiment includes a step (contacting step) of contacting nucleic acid with suspended cells in a solution containing a dextran derivative having an average molecular weight of more than 70 kDa. When the dextran derivative is a cationic dextran derivative, the negatively charged nucleic acid is surrounded by the cationic dextran derivative to form a complex with the dextran derivative, and the complex is taken up by the cells by endocytosis. When the dextran derivative is an anionic dextran derivative, a cationic compound is also used together with the dextran derivative, and the negatively charged nucleic acid forms a complex with the anionic dextran derivative and the cationic compound, and the complex is taken up by the cells by endocytosis. This can be used to introduce nucleic acid into suspended cells. That is, by contacting nucleic acid with suspended cells in a solution containing the dextran derivative, a complex between the dextran derivative and nucleic acid can be formed, and the complex can be taken up by the suspended cells, thereby introducing the nucleic acid into the suspended cells. The method of this embodiment may further include a step of forming a complex between the dextran derivative and nucleic acid. As used herein, "endocytosis" refers to a mechanism in which a substance outside or on the cell membrane is encapsulated in a vesicle and taken into the cell by a morphological change in the cell membrane. In this embodiment, nucleic acids taken into the cell by endocytosis are preferably transported into the nucleus.

[0015] In this embodiment, the step of contacting the nucleic acid with the suspension cells is carried out in a solution containing a dextran derivative, nucleic acid, and suspension cells. The method for preparing the solution may be determined appropriately by those skilled in the art, and the dextran derivative, nucleic acid, and suspension cells may each be added at any timing. For example, a solution containing a dextran derivative, nucleic acid, and suspension cells may be prepared. Alternatively, a solution containing a dextran derivative and nucleic acid may be prepared, and a complex between the dextran derivative and nucleic acid may be formed before the suspension cells are added to the solution, or the solution may be added to the suspension cells. Alternatively, for example, a solution containing a dextran derivative, a solution containing nucleic acid, and a solution containing suspension cells may be prepared separately and then mixed.

[0016] The contacting method, contact time, etc. in the step of contacting nucleic acids with suspension cells in a solution containing a dextran derivative can be determined appropriately by those skilled in the art. For example, the nucleic acid can be contacted with suspension cells by suspending a solution containing a dextran derivative, nucleic acid, and suspension cells, and the complex of dextran derivative and nucleic acid can be incorporated into the suspension cells. In this case, it is preferable to further incubate the mixed solution. The incubation conditions, such as temperature and time, can be determined appropriately by those skilled in the art. However, incubation at about room temperature for about 5 minutes to 2 hours is preferable, and incubation at about room temperature for about 20 minutes to 1 hour is more preferable. Room temperature may be, for example, 10 to 30°C. Incubation may be performed statically or with shaking. The container for carrying out the contacting step can be determined appropriately by those skilled in the art. For example, contact may be carried out in a tube, flask, etc. The time for contacting nucleic acids with suspension cells is not particularly limited and can be determined appropriately by those skilled in the art. However, it is preferably less than 60 minutes, and more preferably less than 40 minutes. The contacting step is preferably carried out for 5 minutes or more, hi one embodiment, the contacting step is carried out for 5, 10, 15, 20, 25, 30, or 35 minutes.

[0017] Furthermore, in the method of this embodiment, the number of times the contact step is performed is not particularly limited, and it may be performed two or more times. Performing the contact step two or more times may improve the efficiency of nucleic acid introduction compared to performing the contact step once. When the contact step is performed multiple times, the contact conditions may be different or the same. Other steps may be interposed between contact steps. For example, cells that have completed the contact step are washed and then subjected to a new contact step. The method of this embodiment may include a step of contacting nucleic acid with suspension cells, a step of washing the suspension cells contacted with nucleic acid, and a step of contacting the washed suspension cells with nucleic acid. As used herein, "nucleic acid introduction efficiency" refers to the percentage (%) of the number of cells into which nucleic acid has been introduced relative to the total number of cells, preferably the total number of living cells. Herein, introduction efficiency may also be referred to as the introduction rate.

[0018] Furthermore, in the step of contacting nucleic acids with suspension cells in a solution containing a dextran derivative, the solution may contain dimethyl sulfoxide (DMSO), but the proportion of DMSO in the solution is preferably less than 0.1% (v / v), and more preferably, the solution does not contain DMSO. When the solution of this embodiment contains DMSO, DMSO may be added to the solution at any timing, but is preferably added after mixing the solutions containing the dextran derivative, nucleic acid, and suspension cells, preferably after incubating the mixed solution. When DMSO is added after mixing the solutions or after incubation of the solutions, the reaction time between DMSO and the solution is preferably 10 to 15 minutes or less. When the solution of this embodiment contains DMSO at a proportion of 0.1% (v / v) or more, the nucleic acid introduction efficiency and suspension cell viability may be lower compared to when the solution contains DMSO at a proportion of less than 0.1% (v / v) or when the solution does not contain DMSO. As used herein, the term "survival rate of suspension cells" refers to the proportion of suspension cells that survive after the contact step among the suspension cells that are subjected to the contact step.

[0019] Furthermore, in addition to the dextran derivative, nucleic acid, and suspended cells, the solution in this embodiment may contain any buffer or other necessary reagents, etc. For example, it may contain water, TE buffer, TAE buffer, TBE buffer, PBS buffer, STBS buffer, potassium chloride, magnesium chloride, DNA or RNase inhibitors, etc.

[0020] The average molecular weight of the dextran derivative in the solution subjected to the contacting step is greater than 70 kDa, greater than 80 kDa, greater than 90 kDa, or greater than 100 kDa, preferably 100 kDa or greater, 110 kDa or greater, 120 kDa or greater, 130 kDa or greater, 140 kDa or greater, or 150 kDa or greater, and more preferably 150 kDa or greater. Furthermore, the average molecular weight of the dextran derivative in the solution in this embodiment is 5,000 kDa or less, preferably 3,000 kDa or less, and more preferably 2,000 kDa or less. Herein, "greater than X" (X is a natural number) refers to any value greater than X, but does not include a value equal to X.

[0021] The method for producing the dextran derivative in this embodiment is not particularly limited, and the dextran derivative can be produced by a method known to those skilled in the art. Alternatively, a commercially available product may be used in this method. The average molecular weight of the dextran derivative may be either a number-average molecular weight (Mn) or a weight-average molecular weight (Mw), with the weight-average molecular weight (Mw) being preferred. The average molecular weight can be measured by a method known to those skilled in the art, such as electrophoresis, gel permeation chromatography (GPC), size exclusion chromatography, dynamic or static light scattering, low-angle laser light scattering, viscosity measurement, or a combination thereof. The average molecular weight of the dextran derivative in this embodiment may be a measured value measured by GPC or the like. When a commercially available dextran derivative is used, it may be a sales value or a theoretical value. When the dextran derivative is DEAE-dextran, the average molecular weight of DEAE-dextran can be determined by the following method: Characterization of DEAE-dextran by means of light scattering and combined size-exclusion chromatography / low-angle laser light scattering / viscometry. Macromolecular Chemistry and Physics, 1995, 196(7), 2259-2275. With reference to the above, the viscosity can be measured, for example, under the following measurement conditions, by low-angle laser light scattering, the Kuhn-Mark-Houwink (KMH) method which combines size exclusion chromatography, low-angle laser light scattering, and viscosity measurement, or a method which combines low-angle laser light scattering and viscosity measurement. [Example of measurement conditions for low-angle laser light scattering] Measurement temperature: 20°C Argon ion laser wavelength: 496.5 nm Solvent: 0.8 M sodium nitrate [Example of measurement conditions for KMH method] Column: Ultrahydrogel column 2000 and 250 Eluent: 0.8 M sodium nitrate Flow rate: 0.5 mL / min Injection mass: 50 to 400 μg (0.5 to 4 mg / mL) Injection volume: 0.1 or 0.05 mL

[0022] When the average molecular weight of the dextran derivative in the solution is greater than 70 kDa, the efficiency of nucleic acid introduction and the survival rate of suspension cells may be higher than when the average molecular weight is 70 kDa or less. In one embodiment, the method of this embodiment comprises a step of contacting nucleic acid with suspension cells in a solution containing a dextran derivative having an average molecular weight of 150 kDa or more and 2,000 kDa or less. In one embodiment, the method of this embodiment comprises a step of contacting nucleic acid with suspension cells in a solution containing a DEAE-dextran having an average molecular weight of 150 kDa or more and 2,000 kDa or less. In one embodiment, the method of this embodiment comprises a step of contacting nucleic acid with suspension cells in a solution containing a dextran derivative having an average molecular weight of 500 kDa. In one embodiment, the method of this embodiment comprises a step of contacting nucleic acid with suspension cells in a solution containing a DEAE-dextran having an average molecular weight of 500 kDa.

[0023] The concentration of the dextran derivative in the solution to be subjected to the contact step is determined appropriately taking into consideration the desired introduction efficiency, the concentration of the nucleic acid to be introduced, the concentration of the complex of the dextran derivative and nucleic acid formed, the N / P ratio, the type of suspended cells used and their concentration in the solution, the cytotoxicity of the dextran derivative, etc. For example, when the nucleic acid concentration is 6.0 to 12.0 μg / mL, the N / P ratio is 11 to 15, and the concentration of suspended cells is 8,000,000 to 32,000,000 cells / mL, the concentration of the dextran derivative is preferably less than 500 μg / mL, more preferably 100 μg / mL or more, 125 μg / mL or more, 150 μg / mL or more, 175 μg / mL or more, 200 μg / mL or more, or 250 μg / mL or more, and more preferably 475 μg / mL or less, 450 μg / mL or less, 425 μg / mL or less, 400 μg / mL or less, or 375 μg / mL or less. Furthermore, for example, when the nucleic acid concentration is 10.0 to 12.0 μg / mL, the N / P ratio is 11 to 15, and the concentration of suspended cells is 16,000,000 to 24,000,000 cells / mL, the concentration of the dextran derivative is preferably 125 μg / mL or more and 375 μg / mL or less. When the concentration of the dextran derivative in the solution is less than 500 μg / mL, the efficiency of nucleic acid introduction can be higher than when the concentration is 500 μg / mL or more. In one embodiment, the method of this embodiment includes a step of contacting the nucleic acid with suspended cells in a solution containing a dextran derivative having an average molecular weight of 150 kDa or more and 2,000 kDa or less at a concentration of 125 μg / mL or more and 375 μg / mL or less. In one embodiment, the method of this embodiment comprises the step of contacting nucleic acid with suspended cells in a solution containing DEAE-dextran having an average molecular weight of 150 kDa or more and 2,000 kDa or less at a concentration of 125 μg / mL or more and 375 μg / mL or less. In one embodiment, the method of this embodiment comprises the step of contacting nucleic acid with suspended cells in a solution containing a dextran derivative having an average molecular weight of 500 kDa at a concentration of 250 μg / mL. In one embodiment, the method of this embodiment comprises the step of contacting nucleic acid with suspended cells in a solution containing DEAE-dextran having an average molecular weight of 500 kDa at a concentration of 250 μg / mL.

[0024] The ratio (N / P ratio) of the number of cationic groups (N) in the dextran derivative in the solution subjected to the contact step, or the number of cationic groups (N) in the cationic compound used in combination when the dextran derivative is an anionic dextran derivative, to the number of phosphate groups (P) in the nucleic acid, is preferably 11 to 15, more preferably 14 to 15. Herein, when described as "X to Y (X and Y are 0 or positive numbers)," X to Y are synonymous with X or more and Y or less, and indicate a range including the values ​​of X and Y. When the N / P ratio in the solution is 11 to 15, the efficiency of nucleic acid introduction can be higher than when the N / P ratio is less than 11 or more than 15. In one embodiment, the method of this embodiment comprises a step of contacting nucleic acid with suspended cells in a solution containing a dextran derivative having an average molecular weight of 150 kDa to 2,000 kDa at a concentration of 125 μg / mL to 375 μg / mL, wherein the N / P ratio is 14.0, 14.1, 14.2, 14.3, 14.4, 14.5, 14.6, 14.7, 14.8, 14.9, or 15.0. In one embodiment, the method of this embodiment comprises contacting nucleic acid with suspended cells in a solution containing DEAE-dextran having an average molecular weight of 150 kDa to 2,000 kDa at a concentration of 125 μg / mL to 375 μg / mL, and having an N / P ratio of 14.0, 14.1, 14.2, 14.3, 14.4, 14.5, 14.6, 14.7, 14.8, 14.9, or 15.0. In one embodiment, the method of this embodiment comprises contacting nucleic acid with suspended cells in a solution containing a dextran derivative having an average molecular weight of 500 kDa at a concentration of 125 μg / mL to 375 μg / mL, and having an N / P ratio of 14.3. In one embodiment, the method of this embodiment includes a step of contacting nucleic acids with suspended cells in a solution containing DEAE-dextran having an average molecular weight of 500 kDa at a concentration of 125 μg / mL or more and 375 μg / mL or less, and having an N / P ratio of 14.3.

[0025] The concentration of nucleic acid in the solution to be subjected to the contact step is appropriately determined taking into consideration the desired transfection efficiency, the concentration of the dextran derivative, the concentration of the complex of the dextran derivative and nucleic acid formed, the N / P ratio, the type of suspended cells and their concentration in the solution, etc. For example, when the concentration of the dextran derivative is less than 500 μg / mL, the concentration of suspended cells is 8,000,000 to 32,000,000 cells / mL, and the N / P ratio is 11 to 15, the concentration of nucleic acid is preferably 6.0 to 12.0 μg / mL, and more preferably 10.0 to 12.0 μg / mL. Furthermore, for example, the concentration of the nucleic acid is preferably 10.0 to 12.0 μg / mL when the concentration of the dextran derivative is 125 μg / mL or more and 375 μg / mL or less, the concentration of the suspended cells is 16,000,000 to 24,000,000 cells / mL, and the N / P ratio is 14 to 15. When the concentration of the nucleic acid in the solution is 6.0 to 12.0 μg / mL, the efficiency of nucleic acid introduction can be higher than when the concentration is less than 6.0 μg / mL or more than 12.0 μg / mL. In one embodiment, the method of this embodiment comprises a step of contacting suspended cells with a nucleic acid in a solution containing a dextran derivative having an average molecular weight of 150 kDa to 2,000 kDa at a concentration of 125 μg / mL to 375 μg / mL and a nucleic acid at a concentration of 10.0 to 12.0 μg / mL, wherein the solution has an N / P ratio of 14.0, 14.1, 14.2, 14.3, 14.4, 14.5, 14.6, 14.7, 14.8, 14.9, or 15.0. In one embodiment, the method of this embodiment comprises contacting the nucleic acid with suspended cells in a solution containing DEAE-dextran having an average molecular weight of 150 kDa to 2,000 kDa at a concentration of 125 μg / mL to 375 μg / mL and a nucleic acid at a concentration of 10.0 to 12.0 μg / mL, wherein the N / P ratio is 14.0, 14.1, 14.2, 14.3, 14.4, 14.5, 14.6, 14.7, 14.8, 14.9, or 15.0. In one embodiment, the method of this embodiment comprises contacting the nucleic acid with suspended cells in a solution containing a dextran derivative having an average molecular weight of 500 kDa at a concentration of 250 μg / mL and a nucleic acid at a concentration of 10.0 μg / mL, wherein the N / P ratio is 14.3.In one embodiment, the method of this embodiment comprises a step of contacting the nucleic acid with suspended cells in a solution containing DEAE-dextran having an average molecular weight of 500 kDa at a concentration of 250 μg / mL and nucleic acid at a concentration of 10.0 μg / mL, wherein the solution has an N / P ratio of 14.3.

[0026] The base length of the nucleic acid in the solution subjected to the contact step is preferably 6 kbp or less, more preferably 4.5 kbp or less. The base length of the nucleic acid in the solution subjected to the contact step is preferably 100 bp or more, more preferably 500 bp or more. When the base length of the nucleic acid in the solution is 6 kbp or less, the introduction efficiency of the nucleic acid can be higher than when the base length is more than 6 kbp. In one embodiment, the method of the present embodiment comprises a step of contacting suspended cells with a dextran derivative having an average molecular weight of 150 kDa to 2,000 kDa at a concentration of 125 μg / mL to 375 μg / mL and a nucleic acid having a base length of 100 bp to 6 kbp at a concentration of 10.0 to 12.0 μg / mL, wherein the solution has an N / P ratio of 14.0, 14.1, 14.2, 14.3, 14.4, 14.5, 14.6, 14.7, 14.8, 14.9, or 15.0. In one embodiment, the method of the present embodiment comprises a step of contacting the nucleic acid with suspended cells in a solution containing DEAE-dextran having an average molecular weight of 150 kDa to 2,000 kDa at a concentration of 125 μg / mL to 375 μg / mL and a nucleic acid having a base length of 100 bp to 6 kbp at a concentration of 10.0 to 12.0 μg / mL, wherein the solution has an N / P ratio of 14.0, 14.1, 14.2, 14.3, 14.4, 14.5, 14.6, 14.7, 14.8, 14.9, or 15.0. In one embodiment, the method of this embodiment comprises a step of contacting the nucleic acid with suspended cells in a solution containing a dextran derivative having an average molecular weight of 500 kDa at a concentration of 250 μg / mL and a nucleic acid having a base length of 500 bp to 4.5 kbp at a concentration of 10.0 μg / mL, wherein the solution has an N / P ratio of 14.3. In one embodiment, the method of this embodiment comprises a step of contacting the nucleic acid with suspended cells in a solution containing a DEAE-dextran having an average molecular weight of 500 kDa at a concentration of 250 μg / mL and a nucleic acid having a base length of 500 bp to 4.5 kbp at a concentration of 10.0 μg / mL, wherein the solution has an N / P ratio of 14.3.

[0027] When the nucleic acid in this embodiment encodes a protein, the protein can be appropriately determined by a person skilled in the art depending on the purpose, and may be, for example, a fluorescent protein, a chemiluminescent protein, etc. Specific examples of fluorescent proteins include GFP, CFP, RFP, YFP, and CFP, and examples of chemiluminescent proteins include luciferin and photoprotein.

[0028] The nucleic acid in this embodiment may be contained in a vector. The type of vector is not particularly limited, and any vector known to those skilled in the art may be used, for example, a plasmid, a cosmid, an episome, an artificial chromosome, a phage, a viral vector, etc. When the nucleic acid is contained in a vector, the nucleic acid may be incorporated into the vector together with factors necessary for transcription and translation, such as a promoter, an enhancer, a terminator, etc., and the nucleic acid may exist contiguous or discontinuous with these elements in the vector. As used herein, the term "vector" refers to a concept that includes cloning vectors and expression vectors, and refers to a nucleic acid that carries a gene of interest so as to transform a host, preferably a cell, and promote the expression (e.g., transcription and translation) of the introduced sequence.

[0029] Furthermore, the number of nucleic acids in this embodiment is not limited to one, but may be two or more. When two or more types of nucleic acids are used and the nucleic acids are contained in a vector, for example, a vector containing two or more types of nucleic acids may be used in the method of this embodiment, or two or more vectors containing one or more types of nucleic acids may be used.

[0030] The concentration of suspended cells in the solution to be subjected to the contacting step is appropriately determined taking into consideration the desired transfection efficiency, the concentration of the dextran derivative, the concentration of the nucleic acid, the concentration of the complex of the dextran derivative and the nucleic acid formed, the N / P ratio, the type of suspended cells, etc. For example, when the concentration of the dextran derivative is less than 500 μg / mL, the concentration of the nucleic acid is 6.0 to 12.0 μg / mL, and the N / P ratio is 11 to 15, the concentration of suspended cells is preferably 8,000,000 to 32,000,000 cells / mL, and more preferably 16,000,000 to 24,000,000 cells / mL. Furthermore, for example, when the concentration of the dextran derivative is 125 μg / mL or more and 375 μg / mL or less, the concentration of the nucleic acid is 10.0 to 12.0 μg / mL, and the N / P ratio is 11 to 15, the concentration of suspended cells is preferably 16,000,000 to 24,000,000 cells / mL. In one embodiment, the method of the present embodiment comprises a step of contacting the nucleic acid with the suspended cells in a solution containing a dextran derivative having an average molecular weight of 150 kDa to 2,000 kDa at a concentration of 125 μg / mL to 375 μg / mL, a nucleic acid having a base length of 100 bp to 6 kbp at a concentration of 10.0 to 12.0 μg / mL, and suspended cells at a concentration of 16,000,000 to 24,000,000 cells / mL, wherein the solution has an N / P ratio of 14.0, 14.1, 14.2, 14.3, 14.4, 14.5, 14.6, 14.7, 14.8, 14.9, or 15.0. In one embodiment, the method of the present embodiment comprises a step of contacting the nucleic acid with the suspended cells in a solution containing DEAE-dextran having an average molecular weight of 150 kDa to 2,000 kDa at a concentration of 125 μg / mL to 375 μg / mL, a nucleic acid having a base length of 100 bp to 6 kbp at a concentration of 10.0 to 12.0 μg / mL, and suspended cells at a concentration of 16,000,000 to 24,000,000 cells / mL, wherein the solution has an N / P ratio of 14.0, 14.1, 14.2, 14.3, 14.4, 14.5, 14.6, 14.7, 14.8, 14.9, or 15.0.In one embodiment, the method of this embodiment includes a step of contacting the nucleic acid with the suspended cells in a solution containing a dextran derivative having an average molecular weight of 500 kDa at a concentration of 250 μg / mL or less, a nucleic acid having a base length of 500 bp to 4.5 kbp at a concentration of 10.0 μg / mL, and suspended cells at a concentration of 16,000,000 cells / mL, wherein the solution has an N / P ratio of 14.3. In one embodiment, the method of this embodiment includes a step of contacting the nucleic acid with the suspended cells in a solution containing a DEAE-dextran having an average molecular weight of 500 kDa at a concentration of 250 μg / mL or less, a nucleic acid having a base length of 500 bp to 4.5 kbp at a concentration of 10.0 μg / mL, and suspended cells at a concentration of 16,000,000 cells / mL, wherein the N / P ratio is 14.3.

[0031] In one embodiment, the method of this embodiment comprises a step of contacting the nucleic acid with the suspended cells for 30 minutes in a solution containing a dextran derivative having an average molecular weight of 500 kDa at a concentration of 250 μg / mL, a nucleic acid having a base length of 500 bp to 4.5 kbp at a concentration of 10.0 μg / mL, and suspended cells at a concentration of 16,000,000 cells / mL, but not containing DMSO, and having an N / P ratio of 14.3. In one embodiment, the method of this embodiment comprises a step of contacting the nucleic acid with the suspended cells for 30 minutes in a solution containing DEAE-dextran having an average molecular weight of 500 kDa at a concentration of 250 μg / mL, a nucleic acid having a base length of 500 bp to 4.5 kbp at a concentration of 10.0 μg / mL, and suspended cells at a concentration of 16,000,000 cells / mL but not containing DMSO, and having an N / P ratio of 14.3.

[0032] The suspension cells in this embodiment may be in any form. The cell culture medium may be used directly in the method of this embodiment, or the cultured cells may be collected by centrifugation or other methods to form a cell pellet. When using cultured suspension cells, suspension cells may be cultured until they reach the logarithmic growth phase. The culture conditions for suspension cells can be appropriately determined by those skilled in the art depending on the type of suspension cells, etc., as long as the suspension cells can grow. For example, the culture temperature can be approximately 25°C to approximately 40°C, the carbon dioxide concentration can be approximately 1-10%, and the oxygen concentration can be approximately 1-20%. Static culture or shaking culture can be used. The components contained in the medium can also be appropriately determined by those skilled in the art as long as the suspension cells can grow. For example, serum, plasma, cytokines, albumin, insulin, sugars, fatty acids, trace elements, lipids, amino acids, vitamins, growth factors, antibiotics, antioxidants, buffers, inorganic salts, etc. may be included. A complete medium containing the components necessary for culture is preferred. The culture time can be, for example, approximately 6 hours to 1 week.

[0033] When nucleic acids are introduced into suspension cells using the method of this embodiment described above, the efficiency of nucleic acid introduction can be higher than that of conventional methods such as lipofection. Furthermore, the survival rate of suspension cells can also be higher. The nucleic acid introduction efficiency using the method of this embodiment is preferably 30% or higher, and the survival rate of suspension cells is preferably 90% or higher. Furthermore, the nucleic acid introduction efficiency using the method of this embodiment can be higher by preferably 30% or higher than that of conventional methods such as lipofection.

[0034] Evaluations performed after the contact step, as well as evaluations of the nucleic acid introduction efficiency and survival efficiency, can be performed appropriately by those skilled in the art. For example, when a nucleic acid encoding a fluorescent protein is introduced into suspension cells, the introduction efficiency of the nucleic acid can be evaluated by analyzing the fluorescence intensity emitted by the protein expressed by the nucleic acid using flow cytometry or the like. Specifically, the ratio of the number of fluorescent cells to the total number of suspension cells can be evaluated as the introduction efficiency of the nucleic acid. The method of this embodiment may further include a step of evaluating the introduction efficiency of the nucleic acid.

[0035] The viability of floating cells can be evaluated, for example, by staining the cells to determine whether they are viable or dead, and then counting the live and dead cells. Specifically, the cells are stained with trypan blue, propidium iodide, or the like, and then analyzed using a microscope or flow cytometry to calculate the numbers of live and dead cells, thereby evaluating the viability. The method of this embodiment may further include a step of evaluating the viability of floating cells.

[0036] Evaluation of the nucleic acid introduction efficiency and the survival rate of suspension cells may be performed after culturing and growing the suspension cells into which nucleic acids have been introduced for a certain period of time. The culture conditions for suspension cells into which nucleic acids have been introduced can be determined appropriately by those skilled in the art, and for example, the examples of culture conditions for suspension cells described above can be used as reference. The method of this embodiment may further include a step of growing the suspension cells contacted with nucleic acids.

[0037] The aspects of the method for introducing nucleic acid into suspension cells provided as the first embodiment also apply to the other embodiments described below.

[0038] In a second embodiment, a kit for introducing nucleic acids into suspension cells is provided, the kit comprising a dextran derivative having an average molecular weight of more than 70 kDa. This embodiment also provides a kit for introducing nucleic acids into suspension cells, the kit comprising DEAE-dextran having an average molecular weight of more than 70 kDa.

[0039] The kit of this embodiment may further include nucleic acid and / or suspension cells. When the kit includes nucleic acid, the nucleic acid and the dextran derivative may form a complex in the kit. When the kit includes suspension cells, the suspension cells may be included in a frozen state.

[0040] The dextran derivative provided as the kit of this embodiment may be provided together with any buffer, enzyme, or the like. For example, it may be provided together with water, TE buffer, TAE buffer, TBE buffer, PBS buffer, STBS buffer, DMSO, potassium chloride, magnesium chloride, or a DNA or RNase inhibitor. When the kit of this embodiment contains a nucleic acid, the nucleic acid may be provided together with any buffer, enzyme, or the like similar to those described above. When the kit of this embodiment contains suspension cells, the suspension cells may be provided together with any component necessary for culture. For example, it may be provided together with serum, plasma, cytokines, albumin, insulin, sugars, fatty acids, trace elements, lipids, amino acids, vitamins, growth factors, antibiotics, antioxidants, buffers, inorganic salts, or the like.

[0041] When the kit of this embodiment includes nucleic acids and / or suspension cells in addition to the dextran derivative, they may be provided in the same container or in separate containers. Only one of the dextran derivative, nucleic acids, and suspension cells may be provided in a separate container. When each is provided in two or more separate containers, the containers may be provided together in a single box or the like.

[0042] The kit of this embodiment can be used for the method provided as the first embodiment. The kit of this embodiment may be a kit for contacting nucleic acid with suspension cells in the presence of a dextran derivative, or a kit for contacting nucleic acid with suspension cells in the presence of DEAE-dextran.

[0043] The present invention will be specifically explained below by showing examples, but the present invention is not limited to these examples.

[0044] <Suspension cell culture> Jurkat beta-del (JCRB cell bank, Cat: JCRB0147, hereinafter referred to as Jurkat), THP-1 (JCRB cell bank, Cat: JCRB0112), Raji (JCRB cell bank, Cat: JCRB1647), and Ramos (JCRB cell bank, Cat: JCRB9119) were cultured in RPMI-1640 (Fujifilm Wako Pure Chemical Industries, Ltd., Cat: 189-02025) containing 10% (v / v) heat-inactivated (56°C, 30 minutes) fetal bovine serum (Nichirei Biosciences, Cat: 175012-500ML) (hereinafter referred to as complete medium) at 37°C and 5% CO ２ The cells were cultured in a CO2 incubator under the conditions.

[0045] <Gene transfection using Lipofectamine 2000> On the day before gene transfection, 5 mL of cells were seeded at 1 million cells / mL into a T-25 culture flask. The next day, the cells were harvested, and 2 mL of the cell suspension prepared in complete medium at 200,000 cells / mL was seeded into a 6-well multiwell plate. 2.5-4 μg of EGFP expression vector (promoter: PGK1, full length 3573 bp) was diluted in 250 μL of OptiMEM (Thermo Fisher Scientific, Cat: 31985-062) (Tube 1). Separately, 5-12.5 μL of Lipofectamine 2000 (Thermo Fisher Scientific, Cat: 11668-027) was transferred to a separate tube and mixed with OptiMEM to a total volume of 250 μL (Tube 2). Tube 2 was left at room temperature for 5 minutes, after which the entire solution in Tube 1 was transferred to Tube 2, mixed well, and then left at room temperature for an additional 20 minutes. The mixture was added dropwise from the well into which the cells were seeded, and the cells were cultured in a CO2 incubator for 48 hours.

[0046] <Evaluation of transfected cells> 48 hours after transfection, the survival rate was calculated by a trypan blue (Gibco, Cat: 15250-061) exclusion test. The cells were collected and the GFP transfection rate was evaluated using a flow cytometer (KSRFortessa X-20, Beckton Dickenson).

[0047] <Gene transfer by DEAE-dextran method> Cells in the logarithmic growth phase were collected and washed with D-PBS(-) (Fujifilm Wako Pure Chemical Industries, Ltd., Cat. No. 049-29793). Two million cells were sorted into 1.5 mL microtubes and pelleted by centrifugation (400 x g, 5 minutes, room temperature). 1.25 μg of GFP expression vector was dissolved in STBS buffer (25 mM Tris (Nacalai Tesque, Cat. No. 35434-21)-HCl (Nacalai Tesque, Cat. No. 18321-05), pH 7.5, 137 mM NaCl (Nacalai Tesque, Cat. No. 31333-45), 5 mM KCl (Nacalai Tesque, Cat. No. 28538-75), 0.6 mM Na ２ HPO ４ (Nacalai Tesque, Cat: 31738-55), 0.7 mM CaCl ２ (Fujifilm Wako Pure Chemical Industries, Cat: 3819735), 0.5 mM MgCl ２ (Nacalai Tesque, Cat. No. 20937-72)) was diluted in 62.5 μL (Tube 1). Weight-average molecular weight 500 kDa DEAE-dextran (Fujifilm Wako Pure Chemical Industries, Cat. No. 591-03161) was diluted with STBS buffer to 250 μg / mL (Tube 2). An equal volume of the solution in Tube 1 and Tube 2 was mixed, and the cell pellet was suspended in the resulting mixture (DEAE-dextran / DNA complex). The cell suspension was incubated at 29°C for 30 minutes, after which 1 mL of ice-cold STBS buffer was added and the mixture was immediately centrifuged. The resulting cell pellet was washed once with ice-cold STBS and once with RPMI-1640 medium, suspended in 2 mL of complete medium, and the entire volume was seeded into a 6-well multiwell plate and cultured in a CO2 incubator for 48 hours.

[0048] The above are the basic conditions, but any changes made to the conditions during the study are described in each test example and example.

[0049] Test Example 1 A GFP expression vector was introduced into Jurkat cells using Lipofectamine 2000 under the conditions shown in Figure 1. As shown in Figure 1, the cell survival rate after gene introduction was high at 95% or more, but the GFP-positive cell rate was low at 5% or less.

[0050] Example 1 When gene transfer into Jurkat cells was performed using the DEAE-dextran method, the average molecular weight of the DEAE-dextran used was first investigated. The weight-average molecular weights (Mw) of the DEAE-dextrans investigated are shown in Table 1 and are the sales values ​​of the manufacturers.

[0051]

[0052] As shown in Figure 2, regardless of the molecular weight of DEAE-dextran used, the survival rate was 95% or higher, but the gene transfer efficiency was 5% or lower when the weight-average molecular weight of the DEAE-dextran was 70 kDa or lower. The transfer efficiency was significantly improved at 150 kDa or higher. Since the transfer efficiency was considered to have reached a plateau at 500 kDa or higher, DEAE-dextran with a weight-average molecular weight of 500 kDa was used for subsequent studies.

[0053] Example 2 In prior literature 1 (Fujita et al., Cell, 1966, August 1, Vol. 46, pp. 401-407), the DEAE-dextran concentration during gene transfer was set at 500 μg / mL. Our investigations revealed a tendency for cell viability to decrease, so we decided to investigate the optimal DEAE-dextran concentration. Gene transfer was performed under the DEAE-dextran final concentration conditions shown in Figure 3, and 250 μg / mL provided the best gene transfer conditions and relatively good cell viability. Therefore, in subsequent investigations, the DEAE-dextran concentration was set at 250 μg / mL.

[0054] Example 3: When gene transfer is performed using DEAE-dextran, DMSO shock is often performed after incubation of cells with the DEAE-dextran DNA complex. Therefore, the necessity of DMSO shock was investigated. The concentration (v / v%) and time of DMSO shock are shown in Figure 4. As a result of the investigation, it was found that DMSO shock resulted in a time-dependent decrease in survival rate and GFP transfer efficiency, so it was decided not to perform DMSO treatment in future studies.

[0055] Example 4 In chemical transfer methods such as the DEAE-dextran method, the transfer nucleic acid and transfer reagent are mixed together to form a complex, and the mixture is typically left standing at room temperature for several minutes or more. Therefore, the time required for complex formation between DEAE-dextran and DNA was investigated. As a result of this investigation, as shown in Figure 5, the cell viability was not dependent on the time after preparation, but the GFP transfer efficiency was dependent on complex formation, becoming stable after 30 minutes and remaining stable for at least 120 minutes. Based on these results, the complex formation reaction time was set to 30 minutes in future studies.

[0056] Example 5 In Prior Art Document 1, the number of cells used for gene transfection was 5 million cells. To examine the effect of cell concentration on gene transfection efficiency, genes were transfected into 1 million, 2 million, 3 million, and 4 million cells. As shown in Figure 6, the transfection rate was high between 2 million and 3 million cells, and the transfection efficiency decreased when the concentration was reduced to 1 million cells or increased to 4 million cells. Based on these results, the number of cells per reaction was set to 2 million cells in future studies.

[0057] Example 6 Prior literature 2 (Riedl et al., Non-Viral Transfection of Human T Lymphocytes, Processes 2018, 6(10), 188.) concludes that the ratio of nucleic acid to polymer (N / P ratio) is important, with an N / P ratio of 10 being optimal. We evaluated gene transfer efficiency at various N / P ratios using the DEAE-dextran concentration conditions (500 μg / mL) of Prior literature 1 and the DEAE-dextran concentration conditions (250 μg / mL) that we consider optimal. The results are shown in Figure 7. The 500 μg / mL DEAE-dextran condition of Prior literature 1 resulted in a lower survival rate and an overall lower GFP transfer efficiency than the 250 μg / mL DEAE-dextran condition. Furthermore, under both the 500 μg / mL DEAE-dextran condition and the 250 μg / mL DEAE-dextran condition, the transfection efficiency was higher under the transfection conditions in Example 5 (amount of transfected pDNA = 1.25 μg, N / P ratio = 14.3) than under the optimal condition (N / P ratio = 10) in Prior Art 2. Under the 250 μg / mL DEAE-dextran condition, the optimal condition was an N / P ratio of 14.3.

[0058] Example 7 Next, the effect on gene transfer efficiency was evaluated by varying the DEAE-dextran concentration and the amount of DNA introduced while keeping the N / P ratio fixed. Additionally, gene transfer efficiency at an N / P ratio of 12.5 was also evaluated. The results are shown in Figure 8. When the amount of DEAE-dextran / DNA complex was reduced while keeping the N / P ratio fixed, the transfer efficiency gradually decreased. Furthermore, when the amount of complex was increased, the transfer efficiency also decreased, with the optimal amount of nucleic acid introduced being 1.25 to 1.43 μg / reaction.

[0059] Example 8: The optimal contact time between cells and the DEAE-dextran DNA complex was investigated. Cells were suspended in the DEAE-dextran DNA complex and the incubation time at 29°C was investigated as shown in Figure 9. As a result, it was found that the incubation time did not affect the survival rate, but that 30 minutes gave the highest introduction rate.

[0060] Example 9: Using the optimal DEAE-dextran gene transfer conditions clarified by the condition studies up to Example 8, gene transfer efficiency was evaluated in four types of suspension cells. Lipofectamine 2000 was used as a control method. As shown in Figure 10, the results of the study showed that the transfer efficiency was improved in all cells compared to Lipofectamine 2000. In particular, gene transfer by the DEAE-dextran method was confirmed even in Ramos or Raji, which were hardly transferred with Lipofectamine 2000.

[0061] Example 10: Based on the results of the study in Example 4, the gene transfer efficiency was approximately 25% even after a 5-minute reaction time with the DEAE-dextran / DNA complex. Therefore, we investigated whether the gene transfer efficiency could be improved by repeating a cycle consisting of a 5-minute incubation with the DEAE-dextran / DNA complex and subsequent cell washing two or three times. As shown in the left panel of Figure 11, the cell viability remained almost unchanged even after three cycles. On the other hand, the GFP transfer efficiency improved to nearly 50%. As shown in the middle and right panels of Figure 11, the fluorescence intensity of GFP-positive cells, i.e., the amount of GFP expression per cell, also increased in a cycle-dependent manner compared to the method of Example 9.

Claims

1. A method for introducing nucleic acid into suspension cells, comprising the step of contacting the nucleic acid with the suspension cells in a solution containing a dextran derivative having an average molecular weight of more than 70 kDa.

2. The method of claim 1, wherein the concentration of the dextran derivative in the solution subjected to the contacting step is less than 500 μg / mL.

3. The method according to claim 1 or 2, wherein the average molecular weight of the dextran derivative contained in the solution is 3,000 kDa or less.

4. The method according to claim 1 or 2, wherein the ratio (N / P ratio) of the number of cationic groups (N) in the dextran derivative to the number of phosphate groups (P) in the nucleic acid in the solution subjected to the contacting step is 11 to 15.

5. The method according to claim 1 or 2, wherein the dextran derivative is DEAE-dextran.

6. The method according to claim 1 or 2, wherein the concentration of the nucleic acid in the solution subjected to the contacting step is 6.0 to 12.0 μg / mL.

7. The method of claim 1 or 2, wherein the solution contains less than 0.1% dimethyl sulfoxide or is free of dimethyl sulfoxide.

8. The method according to claim 1 or 2, wherein the concentration of suspended cells in the solution subjected to the contact step is 16,000,000 to 24,000,000 cells / mL.

9. The method according to claim 1 or 2, wherein the suspension cells are Jurkat cells, Ramos cells, Raji cells, or THP-1 cells.

10. The method of claim 1 or 2, wherein the contacting is carried out for less than 40 minutes.

11. The method of claim 1 or 2, wherein the nucleic acid introduced into the suspension cells encodes a fluorescent protein or a chemiluminescent protein.

12. The method according to claim 1 or 2, which has a higher efficiency of nucleic acid introduction than lipofection.

13. The method of claim 1 or 2, wherein the contacting step is carried out two or more times.

14. The method according to claim 13, wherein the efficiency of nucleic acid introduction is improved compared to when the contact step is performed once.

15. A kit for introducing nucleic acids into suspension cells, the kit comprising a dextran derivative having an average molecular weight of more than 70 kDa.

16. The kit of claim 15, wherein the dextran derivative is DEAE-dextran.

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