Method for introducing nucleic acid into suspended cells

Abstract

To provide a method for introducing a nucleic acid into a floating cell.SOLUTION: The present invention provides a method for introducing nucleic acids into non-adherent cells, comprising the step of contacting nucleic acids with non-adherent cells in solutions comprising dextran derivatives having average molecular weights greater than 70kDa.SELECTED DRAWING: None

Description

[Technical Field]

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

[0002] Gene transfer into cells is an essential technique for genetic engineering experiments, etc. There are various methods for gene transfer, such as those using calcium phosphate, poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA), DEAE-dextran, liposomes, viral vectors, and electroporation. Furthermore, cells are broadly divided into two types, adherent cells and suspension cells, depending on the type of culture, but gene transfer into suspension cells is known to be extremely difficult. Methods for gene transfer 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). [Prior art documents] [Non-patent literature]

[0003] [Non-Patent Document 1] Chicaybam L et al., An Efficient Low Cost Method for Gene Transfer to T Lymphocytes. PLoS ONE, 2013, 8(3): e60298. [Non-patent document 2] Fujita et al., Cell, 1966, August 1, Vol.46, 401-407. [Non-patent document 3] Nimesh S et al., Novel polyallylamine-dextran sulfate-DNA nanoplexes: Highly efficient non-viral vector for gene delivery. International Journal of Pharmaceutics, 2006, 320(1-2), 143-149. Summary of the Invention [Problem to be solved by the invention]

[0004] However, gene transfer into suspension cells using lipofection has the problems of high cell toxicity and low gene transfer efficiency, while methods using dextran derivatives such as DEAE-dextran are not highly toxic to cells but also have the problem of low gene transfer efficiency. In addition, the reaction conditions for methods using dextran derivatives have not been thoroughly investigated. The problem to be solved by the present invention is to provide a method using a dextran derivative that allows for more efficient gene transfer into suspension cells. [Means for solving the problem]

[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 includes the following inventions. [1] A method for introducing nucleic acids into suspension cells, comprising: A method comprising the step of contacting nucleic acids with suspended 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 contact 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 (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 contact 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 the 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 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 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 contact 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 efficiency of nucleic acid transfer than the lipofection method.

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

[12] , wherein the contacting step is carried out two or more times.

[14] The method according to

[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, comprising: A kit comprising 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. [Effects of the Invention]

[0007] According to the present invention, it is possible to provide a method capable of introducing genes into suspension cells with high efficiency. [Brief explanation of the drawings]

[0008] [Figure 1] 1 shows the results of gene transfer efficiency into Jurkat cells when Lipofectamine 2000 was used. [Figure 2] The results of evaluating the effect of the molecular weight of DEAE-dextran on gene transfer efficiency are shown. [Figure 3] The results of evaluating the effect of DEAE-dextran concentration on gene transfer efficiency are shown. [Figure 4] The results of evaluating the effect of DMSO shock on gene transfer efficiency are shown. [Figure 5] The results of evaluating the effect of the DEAE-dextran·DNA complex formation time on gene transfer efficiency are shown. [Figure 6] The results of evaluating the effect of cell concentration on gene transfer efficiency are shown. [Figure 7] The results of evaluating the effect of N / P on gene transfer efficiency are shown. [Figure 8] The results of evaluating the effect of the amount of introduced DNA on gene transfer efficiency are shown. [Figure 9]1 shows the results of evaluating the effect of incubation time of a mixture of DEAE-dextran, DNA, and cells on gene transfer efficiency. [Figure 10] The results of comparing the gene transfer efficiency of each type of suspension cell are shown. [Figure 11] The figure shows the results of evaluating the effect of the number of times the DEAE-dextran·DNA complex is reacted with the cells on the gene transfer efficiency. DETAILED DESCRIPTION OF THE INVENTION

[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 single cells that grow in suspension or cell clumps. 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 lesions 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 used 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, such as DEAE-dextran, dextran sulfate, dextran methyl-benzylamide sulfonate, dextran methyl-benzylamide carboxylate, carboxymethyl dextran, diphosphonate dextran, dextran hydrazide, palmitoyl dextran, dextran phosphate, and dextran spermine. Furthermore, the dextran derivative in this embodiment may be modified with any substance, such as a methyl methacrylate group, depending on the purpose. In this embodiment, the dextran derivative is preferably positively charged in order to form a complex with 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 to form a complex between the anionic dextran derivative, the cationic compound, and the negatively charged nucleic acid, thereby enabling the introduction of nucleic acid into suspension cells. The cationic compound may be selected appropriately 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 diethylaminoethyl (DEAE) groups have been introduced into dextran. This embodiment also provides a method for introducing nucleic acid into suspension cells, the method comprising 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 acids 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 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 cells by endocytosis. This can be used to introduce nucleic acid into suspension cells. That is, by contacting nucleic acid with suspension 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 suspension cells, allowing the nucleic acid to be introduced into suspension 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 the dextran derivative, the nucleic acid, and the suspension cells. The method for preparing the solution may be appropriately determined by those skilled in the art, and the dextran derivative, the nucleic acid, and the suspension cells may each be added at any timing. For example, a solution containing a dextran derivative, nucleic acid, and suspended cells may be prepared, or a solution containing a dextran derivative and nucleic acid may be prepared to form a complex between the dextran derivative and nucleic acid, and then the suspended cells may be added to the solution, or the solution may be added to the suspended cells. Alternatively, for example, a solution containing a dextran derivative, a solution containing nucleic acid, and a solution containing suspended cells may be prepared separately and then mixed.

[0016] In the step of contacting nucleic acids with suspended cells in a solution containing a dextran derivative, the contact method, contact time, etc. can be appropriately determined by those skilled in the art. For example, by suspending a solution containing a dextran derivative, nucleic acid, and suspended cells, The nucleic acid can be contacted with suspended cells, allowing the suspended cells to incorporate the complex of the dextran derivative and nucleic acid. In this case, it is preferable to further incubate the mixed solution. The incubation conditions, such as temperature and time, can be appropriately determined by those skilled in the art. 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. A vessel for carrying out the contacting step can be appropriately determined by one skilled in the art, and for example, the contacting may be carried out in a tube, a flask, or the like. The time for contacting the nucleic acid with the suspension cells is not particularly limited and can be determined appropriately by those skilled in the art, but is preferably less than 60 minutes, more preferably less than 40 minutes. The time for carrying out the contact step is preferably 5 minutes or more. In one embodiment, the time for which contacting is carried out is 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. When the contact step is performed two or more times, the efficiency of introducing nucleic acid may be improved compared to when it is performed 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 suspended cells, a step of washing the suspended cells that have been contacted with nucleic acid, and a step of contacting the washed suspended cells with nucleic acid. As used herein, the term "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. In this specification, the introduction efficiency may also be the introduction rate.

[0018] Furthermore, in the step of contacting nucleic acids and 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 incubating the solutions, the reaction time between DMSO and the solution is preferably 10 to 15 minutes or less. When the solution in this embodiment contains DMSO at a rate of 0.1% (v / v) or more, the efficiency of nucleic acid introduction and the survival rate of suspended cells may be lower than when the solution contains DMSO at a rate of less than 0.1% (v / v) or when no DMSO is present. 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 identical to X.

[0021] The method for producing the dextran derivative in this embodiment is not particularly limited, and the dextran derivative may be produced by a method known to those skilled in the art. Alternatively, a commercially available product may be used in the method of this embodiment. The average molecular weight of the dextran derivative may be either an average molecular weight (Mn) or a weight-average molecular weight (Mw), but is preferably a weight-average molecular weight (Mw). The average molecular weight can be measured by methods 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, or may be a commercial value when a commercially available dextran derivative is used, or may be a theoretical value. When the dextran derivative is DEAE-dextran, the average molecular weight of DEAE-dextran can be measured by low-angle laser light scattering, the Kuhn-Mark-Houwink (KMH) method combining size exclusion chromatography, low-angle laser light scattering, and viscosity measurement, or a method combining low-angle laser light scattering and viscosity measurement, with reference to "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., for example, under the following measurement conditions. [Example of low-angle laser light scattering measurement conditions] ·Measurement temperature: 20℃ Argon ion laser wavelength: 496.5nm Solvent: 0.8M sodium nitrate [Example of KMH method measurement conditions] Column: Ultrahydrogel Column 2000 & 250 Eluent: 0.8M sodium nitrate ·Flow rate: 0.5mL / min ·Injection mass: 50~400μg (0.5~4mg / 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 transfer and the survival rate of suspension cells can be increased compared to when the average molecular weight is 70 kDa or less. In one embodiment, the method of this embodiment comprises the step of contacting nucleic acids 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 the step of contacting nucleic acids with suspension cells in a solution containing 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 the step of contacting nucleic acids 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 contacting nucleic acids with suspension cells in a solution containing 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 higher. In one embodiment, the method of this embodiment comprises a step of contacting nucleic acids 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 includes a step of contacting nucleic acids 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 acids 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 includes contacting the nucleic acid with the suspension 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 anionic, to the number of phosphate groups (P) in the nucleic acid is preferably 11 to 15, more preferably 14 to 15. Herein, when it is stated that "X to Y (X and Y are 0 or positive numbers)" in this specification, X to Y are synonymous with at least X and at most Y, 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 introducing nucleic acid 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 or more and 2,000 kDa or less at a concentration of 125 μg / mL or more and 375 μg / mL or less, 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 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, 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 nucleic acids 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 or more and 375 μg / mL or less, and having an N / P ratio of 14.3. In one embodiment, the method of this embodiment comprises 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 determined appropriately taking into consideration the desired transfer efficiency, the concentration of the dextran derivative, the concentration of the complex formed between the dextran derivative and nucleic acid, 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, 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, the concentration of the nucleic acid is preferably 10.0 to 12.0 μg / mL. When the concentration of 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 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, 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 a step of contacting the 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, and 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 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 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 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, and 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 efficiency of introducing the nucleic acid can be higher than when the base length exceeds 6 kbp. In one embodiment, the method of this embodiment comprises a step of contacting suspended cells with 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, and a nucleic acid having a base length of 100 bp or more and 6 kbp or less 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 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, and a nucleic acid having a base length of 100 bp or more and 6 kbp or less 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 or more and 4.5 kbp or less 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 includes a step of contacting the nucleic acid with suspended cells in a solution containing DEAE-dextran with an average molecular weight of 500 kDa at a concentration of 250 μg / mL and a nucleic acid with a base length of 500 bp or more and 4.5 kbp or less 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 those 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, CFP, etc., and examples of chemiluminescent proteins include luciferin, photoprotein, etc.

[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, such as a plasmid, cosmid, episome, artificial chromosome, phage, or viral vector. 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, enhancer, or terminator, and the nucleic acid may be present contiguously or discontinuously with these factors in the vector. As used herein, the term "vector" refers to a concept that encompasses 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 nucleic acid in this embodiment is not limited to one type, but may be two or more types. When there are two or more types of nucleic acids 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 contact 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 formed between the dextran derivative and the nucleic acid, 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 this 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 or more and 2,000 kDa or less at a concentration of 125 μg / mL or more and 375 μg / mL or less, a nucleic acid having a base length of 100 bp or more and 6 kbp or less 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 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 or more and 2,000 kDa or less at a concentration of 125 μg / mL or more and 375 μg / mL or less, a nucleic acid having a base length of 100 bp or more and 6 kbp or less 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 or more and 4.5 kbp or less 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 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 solution has an N / P ratio of 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 or more and 4.5 kbp or less 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 or more and 4.5 kbp or less 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, and the cell culture medium may be used as is in the method of this embodiment, or the cultured cells may be collected by centrifugation or the like to form a cell pellet. When cultured suspension cells are used, the suspension cells may be cultured until they reach the logarithmic growth phase. 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 to 10%, and the oxygen concentration can be approximately 1 to 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 the present embodiment described above, the efficiency of nucleic acid introduction can be increased compared to conventional methods such as lipofection, and the survival rate of suspension cells can also be increased. The method of this embodiment preferably achieves a nucleic acid transfer efficiency of 30% or more, and a suspension cell survival rate of 90% or more. Furthermore, the method of this embodiment can achieve a nucleic acid transfer efficiency that is preferably 30% or more higher than conventional methods such as lipofection.

[0034] Evaluations after the contact step, and 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 efficiency of nucleic acid introduction.

[0035] The viability of suspended cells can be evaluated, for example, by determining whether the cells are viable or dead by staining and counting the live and dead cells. Specifically, the cells can be 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 assessing the viability. The method of this embodiment may further include a step of assessing the viability of the suspension 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 method for introducing nucleic acid into suspension cells provided as the first embodiment also applies 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. The present 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, etc. 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 RNA degrading enzyme inhibitor. When the kit of this embodiment contains a nucleic acid, the nucleic acid may be provided together with any buffer, enzyme, etc. similar to those described above. When the kit of this embodiment includes suspension cells, the suspension cells may be provided together with any components necessary for culturing, such as serum, plasma, cytokines, albumin, insulin, sugars, fatty acids, trace elements, lipids, amino acids, vitamins, growth factors, antibiotics, antioxidants, buffers, inorganic salts, etc.

[0041] When the kit of the present embodiment contains, in addition to the dextran derivative, nucleic acids and / or floating cells, they may be provided in the same container together, or may be provided in separate containers. Among the dextran derivative, nucleic acids, and floating cells, only one of them can also be provided in a separate container. When each is provided in two or more separate containers, those containers may be provided together in one box or the like.

[0042] The kit of the present embodiment can be used for the method provided as the first embodiment. The kit of the present embodiment may be a kit for contacting nucleic acids and floating cells in the presence of a dextran derivative, or may be a kit for contacting nucleic acids and floating cells in the presence of DEAE-dextran.

[0043] Examples are shown below to specifically describe the present invention, but the present invention is not limited to the examples.

Examples

[0044] <Floating 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), Ramos (JCRB Cell Bank, Cat: JCRB9119) were cultured in RPMI-1640 (FUJIFILM Wako Pure Chemical Corporation, Cat: 189-02025) containing 10% (v / v) heat-inactivated (56 °C, 30 minutes) fetal bovine serum (Nichirei Bioscience, Cat: 175012-500ML) (hereinafter referred to as complete medium) in a CO2 incubator under the conditions of 37 °C and 5% CO2.

[0045] <Gene transfection using Lipofectamine 2000> On the day before gene transfection, 5 mL of cells were seeded at a density of 1 million cells / mL in a T-25 culture flask. The next day, the cells were harvested and 2 mL of a cell suspension prepared at 200,000 cells / mL in complete medium was seeded into each well of a 6-well multi-well plate. 2.5 - 4 μg of the EGFP expression vector (promoter: PGK1, full length 三千五百七十三bp) was diluted in 250 μL of OptiMEM (Thermo Fisher Scientific, Cat: 31985 - 062) (Tube 1). On the other hand, 5 - 12.5 μL of Lipofectamine 2000 (Thermo Fisher Scientific, Cat: '11668 - 027) was taken in a separate tube and mixed with OptiMEM to a total volume of 250 μL (Tube 2). After standing at room temperature for 5 minutes, the entire volume was transferred from Tube 1 to Tube 2, mixed well, and then left standing at room temperature for an additional 20 minutes. The mixture was dropped onto the wells seeded with cells and cultured in a CO2 incubator for 48 hours.

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

[0047] <Gene transfection by the DEAE-dextran method> It should be noted that the "三千五百七十三bp" in the original text seems to be a specific number in the original language which might be incorrect or need to be adjusted according to the actual biological content. Here, it is directly translated as "三千五百七十三bp" for the purpose of following the translation rules.Cells in the logarithmic growth phase were collected and washed with D-PBS(-) (Fujifilm Wako Pure Chemical Industries, Cat: 049-29793), and 2 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 diluted in 62.5 μL of 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 NaHPO (Nacalai Tesque, Cat. No. 31738-55), 0.7 mM CaCl (Fujifilm Wako Pure Chemical Industries, Ltd., Cat. No. 3819735), 0.5 mM MgCl (Nacalai Tesque, Cat. No. 20937-72)) (Tube 1). DEAE-dextran (Fujifilm Wako Pure Chemical Industries, Cat. 591-03161) with an average molecular weight of 500 kDa was diluted with STBS buffer to 250 μg / mL (Tube 2). An equal volume of the solution in Tube 2 was mixed with that in Tube 1, 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, followed by the addition of 1 mL of ice-cold STBS buffer and rapid centrifugation. 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 onto 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 viability after gene introduction was high at over 95%, but the rate of GFP-positive cells was low at less than 5%.

[0050] Example 1 When transfecting Jurkat cells with the DEAE-dextran method, we first investigated the average molecular weight of the DEAE-dextrans used. The average molecular weights of the DEAE-dextrans examined are shown in Table 1.

[0051] [Table 1]

[0052] As shown in Figure 2, the survival rate was 95% or higher regardless of the molecular weight of DEAE-dextran used, but the gene transfer efficiency was 5% or lower when the DEAE-dextran average molecular weight was 70 kDa or less. 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 an average molecular weight of 500 kDa was used for subsequent studies.

[0053] <Example 2> In a previous study (Fujita et al., Cell, August 1, 1966, Vol. 46, pp. 401-407), the DEAE-dextran concentration used during gene transfection was 500 μg / mL. Our study found a tendency for cell viability to decrease, so we decided to investigate the optimal DEAE-dextran concentration. Gene transfection was performed using the DEAE-dextran final concentration conditions shown in Figure 3, and a concentration of 250 μg / mL yielded the best gene transfection conditions and relatively good cell viability. Therefore, we chose 250 μg / mL as the DEAE-dextran concentration for subsequent studies.

[0054] Example 3 When using DEAE-dextran for gene transfer, DMSO shock is often performed after incubation of cells with the DEAE-dextran·DNA complex, so we investigated the necessity of DMSO shock. The concentration (v / v%) and time of DMSO shock were as shown in Figure 4. As a result of the study, it was found that DMSO shock reduced the survival rate and GFP transfection efficiency in a time-dependent manner, 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 at room temperature for several minutes or more. Therefore, we investigated the time required for complex formation between DEAE-dextran and DNA. As shown in Figure 5, the results showed that cell viability was not dependent on the time after preparation, but 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, we decided to use a 30-minute complex formation reaction time for future studies.

[0056] <Example 5> In prior literature 1, the number of cells used for gene transfection was 5 million. To examine the effect of cell concentration on gene transfection efficiency, gene transfection was performed on 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, but the transfection efficiency decreased when the number of cells was reduced to 1 million or increased to 4 million. Based on these results, the number of cells per reaction was set to 2 million in future studies.

[0057] Example 6 Previous literature 2 (Riedl et al., Non-Viral Transfection of Human T Lymphocytes, Processes 2018, 6(10), 188) concluded 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) from previous literature 1 and the DEAE-dextran concentration conditions (250 μg / mL) that we consider optimal. The results of this study are shown in Figure 7. Under the 500 μg / mL DEAE-dextran conditions of Prior Art 1, the survival rate was lower than under the 250 μg / mL DEAE-dextran conditions, and the overall GFP transfection efficiency was lower. Furthermore, under both the 500 μg / mL DEAE-dextran and 250 μg / mL DEAE-dextran conditions, the transfection efficiency under the transfection conditions up to Example 5 (amount of transfected pDNA = 1.25 μg, N / P ratio = 14.3) was higher than the optimal condition of Prior Art 2 (N / P ratio = 10). Under the 250 μg / mL DEAE-dextran conditions, the N / P ratio = 14.3 was optimal.

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

[0059] Example 8 We investigated the optimal contact time between cells and the DEAE-dextran DNA complex. Cells were suspended in the DEAE-dextran DNA complex and incubated at 29°C for a period of time as shown in Figure 9. The incubation time did not affect viability, but we found that 30 minutes produced the highest transfection rate.

[0060] Example 9 The gene transfer efficiency in four types of suspension cells was evaluated using the optimal DEAE-dextran gene transfer conditions that were clarified through the condition studies up to Example 8. Lipofectamine 2000 was used as a control method. As shown in Figure 10, the results of the study showed that the transfection efficiency was higher in all cell types than in Lipofectamine 2000. In particular, gene transfection was confirmed using the DEAE-dextran method even in Ramos and Raji cells, which were hardly transfected with Lipofectamine 2000.

[0061] Example 10 The results of the study in Example 4 showed that the gene transfer efficiency was approximately 25% even when the reaction time with the DEAE-dextran·DNA complex was 5 minutes. Therefore, we investigated whether the gene transfer efficiency could be improved by repeating a cycle consisting of 5 minutes of incubation with the DEAE-dextran·DNA complex and subsequent cell washing two or three times. As shown in the left panel of Figure 11, cell viability remained almost unchanged even after three cycles. Meanwhile, the GFP transfection 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 acids into suspension cells, comprising: A method comprising the step of contacting nucleic acids with suspended cells in a solution containing a dextran derivative having an average molecular weight of more than 70 kDa.

2. 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 dextran derivative contained in the solution has an average molecular weight of 3,000 kDa or less.

4. 3. The method according to claim 1, 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. 3. 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 contacting 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. 3. The method of claim 1 or 2, wherein the contacting is carried out for less than 40 minutes.

11. The method according to 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 methods.

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

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

15. A kit for introducing nucleic acids into suspension cells, comprising: A 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.

Images