Method for improving cell proportion in mitotic metaphase and application thereof

Abstract

The invention belongs to the technical field of cell biology, and particularly relates to a method for increasing the cell proportion in the mitosis metaphase and application of the method. The invention provides a method for improving the cell proportion in the mitosis metaphase, which comprises the following steps: jointly using colchicine and ethylene glycol diethyl ether diamine tetraacetic acid, sequentially treating cells to be treated by using the ethylene glycol diethyl ether diamine tetraacetic acid and colchicine, strictly limiting the concentration of the colchicine and the ethylene glycol diethyl ether diamine tetraacetic acid, and improving the cell proportion in the mitosis metaphase. According to the method, excessive stagnation or damage of cells can be avoided, low-concentration ethylene glycol diethyl ether diamine tetraacetic acid is firstly used for treatment to remove calcium ions, the stability of microtubules is further destroyed, the formation of spindle filaments is blocked from the source, the cell proportion in the mitosis metaphase is remarkably improved, then low-concentration colchicine is added, microtubule polymerization is further inhibited, and the low-concentration colchicine and the low-concentration colchicine have a synergistic effect, so that the effect of inhibiting microtubule polymerization is achieved. And the cell enrichment rate in the middle stage of mitosis can reach 53-66%. Moreover, the operation is simple, culture conditions are optimized without additional equipment, and the experiment cost is remarkably reduced.

Description

Technical Field

[0001] The present invention belongs to the technical field of cell biology, and particularly relates to a method for increasing the proportion of cells in metaphase of mitosis and its application. Background Art

[0002] In cell biology research, mitosis is a crucial stage of the cell cycle. Among them, metaphase of mitosis refers to the period from when the chromosomes are arranged on the equatorial plate to when their chromatids begin to separate towards the two poles. When observed from one end, these chromosomes are radially arranged on the equatorial plate, and at this time they are not stationary but in a constantly oscillating state. The chromosomes in metaphase of mitosis condense and thicken, showing the number and morphology unique to this species. Therefore, metaphase of mitosis is suitable for the study of the morphology, structure, and number of chromosomes and for karyotype analysis.

[0003] However, in the normal cell cycle, the duration of metaphase of mitosis is short, and the proportion of cells in metaphase of mitosis naturally distributed in the cell population is extremely low, usually less than 3%. Therefore, improving the enrichment efficiency of cells in metaphase of mitosis is particularly important in this field. Summary of the Invention

[0004] The purpose of the present invention is to provide a method for increasing the proportion of cells in metaphase of mitosis and its application, which can significantly increase the proportion of cells in metaphase of mitosis, improve the enrichment rate of cells in metaphase of mitosis, and reduce costs.

[0005] The present invention provides a method for increasing the proportion of cells in metaphase of mitosis, which includes the following steps:

[0006] Treat the cells to be treated with ethylene glycol bis(2-aminoethyl ether)-N,N,N',N'-tetraacetic acid for the first treatment, and perform solid-liquid separation to obtain the first-treated cells;

[0007] Treat the first-treated cells with colchicine for the second treatment;

[0008] The working concentration of the ethylene glycol bis(2-aminoethyl ether)-N,N,N',N'-tetraacetic acid is 0.5 - 2 mmol / L;

[0009] The working concentration of the colchicine is 0.05 - 0.1 μg / mL.

[0010] Preferably, the density of the cells to be treated is 0.8×10 6 ~1.2×10 6 cells / mL.

[0011] Preferably, the temperature of the first treatment is 35.5 - 37.5 °C, and the time is 10 - 20 min;

[0012] The temperature of the second treatment is 30-32°C and the time is 0.5-4 h.

[0013] Preferably, the solid-liquid separation method includes centrifugation, and the rotation speed of the centrifugation is 800-1200 rpm and the time is 5-10 min.

[0014] Preferably, after obtaining the first-treated cells, it further includes: resuspending the first-treated cells to obtain a cell suspension; and performing a second treatment on the cell suspension with colchicine.

[0015] Preferably, the first-treated cells are resuspended with a culture medium, and the culture medium is based on DMEM medium or RPMI-1640 medium and includes 10-12% v / v fetal bovine serum, 80-110 U / mL penicillin, and 80-110 μg / mL streptomycin.

[0016] Preferably, the cells to be treated are mammalian cells.

[0017] Preferably, after performing the second treatment on the first-treated cells with colchicine, it further includes: performing solid-liquid separation on the cell treatment solution after the second treatment to obtain second-treated cells and then performing hypotonic treatment.

[0018] Preferably, hypotonic treatment is performed with a 0.07-0.08 mol / L KCl solution; the temperature of the hypotonic treatment is 35-38°C and the time is 10-30 min.

[0019] The present invention also provides the application of the method described in the above technical solution in one or more of chromosome analysis, karyotype detection, and anti-tumor drug screening.

[0020] Beneficial effects:

[0021] The present invention provides a method for increasing the proportion of cells in metaphase of mitosis, comprising the following steps: performing a first treatment on the cells to be treated with ethylene glycol bis (2-aminoethyl ether)-N,N,N',N'-tetraacetic acid, followed by solid-liquid separation to obtain the first-treated cells; performing a second treatment on the first-treated cells with colchicine; the working concentration of the ethylene glycol bis (2-aminoethyl ether)-N,N,N',N'-tetraacetic acid is 0.5 to 2 mmol / L; the working concentration of the colchicine is 0.05 to 0.1 μg / mL. The present invention combines the use of colchicine and ethylene glycol bis (2-aminoethyl ether)-N,N,N',N'-tetraacetic acid, and strictly limits the concentrations of colchicine and ethylene glycol bis (2-aminoethyl ether)-N,N,N',N'-tetraacetic acid, which can avoid excessive cell arrest or damage. First, treating with a low concentration of ethylene glycol bis (2-aminoethyl ether)-N,N,N',N'-tetraacetic acid to remove calcium ions and further disrupt the microtubule stability, blocking the formation of spindle fibers from the source, significantly increasing the proportion of cells in metaphase of mitosis, and then adding a low concentration of colchicine to further inhibit microtubule polymerization. With the synergistic effect of the two, the enrichment rate of cells in metaphase of mitosis can be as high as 53% - 66%. Moreover, the operation of the present invention is simple, and no additional equipment is required for optimizing the culture conditions, significantly reducing the experimental cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the embodiments.

[0023] Figure 1 Observation result under a microscope at 40× for Example 1;

[0024] Figure 2 Fluorescence observation result under a microscope at 40× for Example 2;

[0025] Figure 3 Observation result under a microscope at 40× for Example 3;

[0026] Figure 4 Observation result under a microscope at 40× for Comparative Example 1;

[0027] Figure 5 Observation result under a microscope at 40× for Comparative Example 2. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0028] The present invention provides a method for increasing the proportion of cells in metaphase of mitosis, comprising the following steps:

[0029] Performing a first treatment on the cells to be treated with ethylene glycol bis (2-aminoethyl ether)-N,N,N',N'-tetraacetic acid, followed by solid-liquid separation to obtain the first-treated cells;

[0030] Performing a second treatment on the first-treated cells with colchicine;

[0031] The working concentration of the ethylene glycol bis (2-aminoethyl ether)-N,N,N',N'-tetraacetic acid is 0.5 to 2 mmol / L;

[0032] The working concentration of the colchicine is 0.05 - 0.1 μg / mL.

[0033] As an implementation mode, the present invention uses a culture medium to culture cells to be treated, and obtains a culture solution containing the cells to be treated. As an implementation mode, the culture medium of the present invention is based on DMEM medium or RPMI-1640 medium, and includes 10 - 12% v / v fetal bovine serum, 80 - 110 U / mL penicillin and 80 - 110 μg / mL streptomycin; as another implementation mode, the culture medium of the present invention is based on DMEM medium or RPMI-1640 medium, and includes 10 - 12% v / v fetal bovine serum, 100 U / mL penicillin and 100 μg / mL streptomycin; as another implementation mode, the culture medium of the present invention is based on RPMI-1640 medium, and includes 10% v / v fetal bovine serum, 100 U / mL penicillin and 100 μg / mL streptomycin. As an implementation mode, the culture conditions of the present invention are 30 - 37 °C, 4 - 5% CO 2 , with a humidity of more than 95%; as another implementation mode, the culture conditions of the present invention are 37 °C, 5% CO 2 , with a humidity of more than 95%. As an implementation mode, the culture time of the present invention is 48 - 58 h; as another implementation mode, the culture time of the present invention is 56 h.

[0034] As an implementation mode, the cells to be treated of the present invention are mammalian cells. As an implementation mode, the mammalian cells of the present invention include one or more of human Burkitt's lymphoma cells, mouse B lymphocytes, human B lymphocytes and human peripheral blood cells; as another implementation mode, the mammalian cells of the present invention are human Burkitt's lymphoma cells or human B lymphocytes. As an implementation mode, the human Burkitt's lymphoma cells of the present invention are the CA46 cell line. As an implementation mode, the human B lymphocytes of the present invention are human immortalized lymphoblast GM12878.

[0035] After obtaining the culture solution containing the cells to be treated, the present invention uses ethylene glycol diethyl ether diamine tetraacetic acid to perform a first treatment on the cells in the culture solution containing the cells to be treated, and performs solid-liquid separation to obtain first-treated cells.

[0036] In the present invention, the working concentration of the ethylene glycol diethyl ether diamine tetraacetic acid is 0.5 - 2 mmol / L. As an implementation mode, the working concentration of the ethylene glycol diethyl ether diamine tetraacetic acid of the present invention is 1.0 mmol / L. As an implementation mode, the density of the cells to be treated in the culture solution containing the cells to be treated of the present invention is 0.8×10 6~1.2××10 6 cells / mL; As another embodiment, the density of the cells to be treated in the culture solution containing the cells to be treated in the present invention is 1×10 6 cells / mL. The molecular weight of the ethylene glycol diethyl ether diamine tetraacetic acid in the present invention is 380.35 g / mol, and the molecular structure is C 14 H 24 N 2 O 10 . The present invention uses ethylene glycol diethyl ether diamine tetraacetic acid to treat the cells to be treated. Ethylene glycol diethyl ether diamine tetraacetic acid is a specific calcium chelating agent, which is evenly distributed in the culture solution, combines with calcium ions in the cell culture environment to form a stable soluble complex, reduces the concentration of free calcium ions in the environment, breaks the calcium ion balance of tubulin dimers, indirectly inhibits the polymerization of tubulin, and blocks the formation of spindle fibers.

[0037] As one embodiment, the solid-liquid separation method in the present invention includes centrifugation, the rotation speed of the centrifugation is 800-1200 rpm, and the time is 5-10 min; as another embodiment, the solid-liquid separation method in the present invention includes centrifugation, the rotation speed of the centrifugation is 1000 rpm, and the time is 5-10 min.

[0038] As one embodiment, the temperature of the first treatment in the present invention is 35.5-37.5 °C; as one embodiment, the temperature of the first treatment in the present invention is 37 °C. As one embodiment, the time of the first treatment in the present invention is 10-20 min; as one embodiment, the time of the first treatment in the present invention is 15 min. As one embodiment, the CO 2 concentration of the first treatment is 4-5%, and the humidity is more than 95%; as another embodiment, the CO 2 concentration of the first treatment is 5%, and the humidity is more than 95%. The present invention optimizes the treatment conditions of ethylene glycol diethyl ether diamine tetraacetic acid, without additional equipment, and the experimental cost is significantly reduced.

[0039] As an implementation manner, after obtaining the first processed cells, the present invention resuspends the first processed cells to obtain a cell suspension. As an implementation manner, the present invention uses a fresh culture medium to resuspend the first processed cells. The fresh culture medium is based on DMEM medium or RPMI-1640 medium and includes 10-12% v / v fetal bovine serum, 80-110 U / mL penicillin, and 80-110 μg / mL streptomycin; as another implementation manner, the fresh culture medium of the present invention is based on DMEM medium or RPMI-1640 medium and includes 10-12% v / v fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin; as another implementation manner, the fresh culture medium of the present invention is based on RPMI-1640 medium and includes 10% v / v fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin.

[0040] After obtaining the cell suspension, the present invention uses colchicine to perform a second treatment on the first processed cells in the cell suspension to obtain a cell treatment solution.

[0041] In the present invention, the working concentration of the colchicine is 0.05-0.1 μg / mL. As an implementation manner, the working concentration of the colchicine in the present invention is 0.075 μg / mL. The present invention uses colchicine. Colchicine is distributed around the cells in a molecular state, directly interacts with tubulin, inhibits its polymerization, destroys the spindle fiber structure, prevents the cell division from entering the later stage, promotes the cell to stagnate in the metaphase of mitosis, increases the proportion of cells in the metaphase of mitosis, and improves the enrichment efficiency of cells in the metaphase of mitosis.

[0042] As an implementation manner, the temperature of the second treatment in the present invention is 30-32 °C; as another implementation manner, the temperature of the second treatment in the present invention is 30 °C. As an implementation manner, the time of the second treatment in the present invention is 0.5-4 h; as another implementation manner, the time of the second treatment in the present invention is 1-4 h; as another implementation manner, the time of the second treatment in the present invention is 2-4 h; as another implementation manner, the time of the second treatment in the present invention is 3-4 h. The present invention uses a low concentration of colchicine and optimizes the treatment conditions of colchicine. The low-temperature treatment at 30-32 °C can effectively slow down the cell cycle process, extend the stagnation time of cells in the metaphase of mitosis, increase the proportion of cells in the metaphase of mitosis, and make the cells stagnate in the metaphase of mitosis without additional equipment, significantly reducing the experimental cost.

[0043] Using colchicine alone can only prevent cells from entering the anaphase by inhibiting microtubule polymerization, but has limited impact on the initial stage of spindle fiber formation, resulting in a limited increase in the number of metaphase cells, and the mitotic index can only reach 10% - 20%. Using ethylene glycol bis(2-aminoethyl ether)-N,N,N',N'-tetraacetic acid alone interferes with the regulation of microtubule dynamics by calcium ions, thereby affecting spindle function. However, if the concentration of ethylene glycol bis(2-aminoethyl ether)-N,N,N',N'-tetraacetic acid is too high or the action time is too long, it is easy to produce cytotoxicity to cells, and the increase in the number of metaphase cells is also limited, and the mitotic index is only about 5% - 10%. The present invention combines the use of colchicine and ethylene glycol bis(2-aminoethyl ether)-N,N,N',N'-tetraacetic acid, strictly limits the concentrations of colchicine and ethylene glycol bis(2-aminoethyl ether)-N,N,N',N'-tetraacetic acid, can avoid excessive cell arrest or damage. First, treat with a low concentration of ethylene glycol bis(2-aminoethyl ether)-N,N,N',N'-tetraacetic acid to remove calcium ions, further disrupt the microtubule stability, block the formation of spindle fibers from the source, significantly increase the proportion of cells in the metaphase of mitosis, and then add a low concentration of colchicine to further inhibit microtubule polymerization. With their synergistic effect, the enrichment rate of cells in the metaphase of mitosis can be as high as 53% - 66%. Moreover, ethylene glycol bis(2-aminoethyl ether)-N,N,N',N'-tetraacetic acid is inexpensive and easily available, the dosage of colchicine is reduced by more than 50% compared with the conventional dosage (0.1 - 1 μg / mL), the cell damage is reduced by more than 30%, and the operation is simple. The culture conditions can be optimized without additional equipment, and the experimental cost is significantly reduced.

[0044] As an implementation manner, after obtaining the cell treatment solution, the present invention performs solid-liquid separation on the cell treatment solution, and after obtaining the second treated cells, performs hypotonic treatment. As an implementation manner, the solid-liquid separation method of the present invention includes centrifugation, the rotation speed of the centrifugation is 800 - 1200 rpm, and the time is 5 - 10 min; as another implementation manner, the solid-liquid separation method of the present invention includes centrifugation, the rotation speed of the centrifugation is 1000 rpm, and the time is 5 - 10 min. As an implementation manner, the present invention performs hypotonic treatment with a 0.07 - 0.08 mol / L KCl solution; as another implementation manner, the present invention performs hypotonic treatment with a 0.075 mol / L KCl solution. As an implementation manner, the temperature of the hypotonic treatment of the present invention is 35 - 38 °C; as another implementation manner, the temperature of the hypotonic treatment of the present invention is 37 °C. As an implementation manner, the time of the hypotonic treatment of the present invention is 10 - 30 min; as another implementation manner, the time of the hypotonic treatment of the present invention is 15 - 25 min; as another implementation manner, the time of the hypotonic treatment of the present invention is 20 min.

[0045] After the second treatment of the cells, hypotonic treatment is carried out in the present invention, which can cause the cells to osmotic expansion and chromosome dispersion. The method provided by the present invention can not only significantly increase the proportion of cells in metaphase of mitosis, but also reduce cell damage, has a wider application range, is simple to operate and low in cost, and has significant advantages in the fields of cell biology, chromosome analysis, gene editing and anti-cancer drug screening. In view of the advantages of the method provided by the present invention, the application of the method described in the above technical solution in one or more of chromosome analysis, karyotype detection and anti-tumor drug screening also belongs to the protection scope of the present invention.

[0046] In order to further illustrate the present invention, a method for increasing the proportion of cells in metaphase of mitosis and its application provided by the present invention will be described in detail below with reference to the drawings and embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0047] Example 1

[0048] 1. Cell culture

[0049] Select the human Burkitt's lymphoma cell line CA46 cells, culture in a T75 flask, and use RPMI-1640 medium containing 10% FBS, 100 U / mL penicillin, and 100 μg / mL streptomycin as the cell culture medium. The culture temperature is 37 °C, and the CO 2 concentration is 5%, the humidity is more than 95%, and sterile culture is carried out in a carbon dioxide incubator.

[0050] 2. Calcium ion removal

[0051] In step 1, culture routinely for 56 h. After cell counting, the cell density is greater than 1×10 6 cells / mL. Add ethylene glycol bis(2-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) solution (pH 7.0, sterile) to the culture medium to a final concentration of 0.5 mmol / L, mix well, place in a carbon dioxide incubator at 37 °C, and CO 2 concentration is 5%, and culture at 95% humidity for 10 min.

[0052] 3. Low temperature and colchicine treatment

[0053] After 10 min of culture in step 2, centrifuge at 1000 rpm for 10 min, discard the supernatant, resuspend the cells with fresh pre-warmed RPMI-1640 medium containing 10% FBS, 100 U / mL penicillin, and 100 μg / mL streptomycin at 37 °C, and at the same time continue to add colchicine solution (dissolved in HBSS, cell culture grade) to a final concentration of 0.1 μg / mL. Reduce the temperature of the incubator from 37 °C to 30 °C, and CO 2 concentration is 5%, and culture at an environmental humidity of more than 95% for 4 h.

[0054] 4. Hypotonic

[0055] After culturing for 4 h in Step 3, centrifuge at 1000 rpm for 10 min, discard the supernatant, gently resuspend the cells with pre-warmed 75 mmol / L KCl hypotonic solution at 37 °C, heat in a water bath at 37 °C for 30 min, and shake once every 10 min.

[0056] 5. Pre-fixation and fixation

[0057] After heating in a water bath for 30 min in Step 4, add freshly prepared fixative (methanol: glacial acetic acid volume ratio is 3:1) stored at -20 °C according to the volume ratio of cell suspension to fixative (3 - 5):1, gently mix, centrifuge at 1000 rpm for 10 min, discard the supernatant, continue to slowly add cold fixative and mix evenly, store at room temperature for 30 min and then transfer to 4 °C for storage.

[0058] 6. Observation under a microscope at 40×

[0059] Under hypotonic action, the cell membranes of most cells rupture, and some chromosomes are dispersed. Among them, the cells in the metaphase of mitosis have irregular edges due to chromosome entanglement, with uneven surfaces, mostly wrapped by no membrane or a single-layer cell membrane. The non-metaphase mitotic cells are round, with distinct nuclear and cytoplasmic boundaries, and often have a single-layer nuclear membrane or a double-layer membrane (nuclear membrane and cell membrane) structure. Based on this principle, observe the cells fixed in Step 5 under a microscope with an objective magnification of 40 times. The results are as Figure 1 shown; among them, a, b, and c are different fields of view under a 40× objective of the same sample.

[0060] According to Figure 1 it can be seen that the synergistic effect of 0.5 mmol / L EGTA and 0.1 μg / mL colchicine can inhibit the normal formation of the spindle of CA46 cells, and the cells cannot divide further, thus generating a large number of cells in the metaphase of mitosis, and the mitotic index (the cells in the metaphase of mitosis account for the total number of cells) is as high as 66%.

[0061] Example 2

[0062] 1. Cell culture

[0063] Select the human Burkitt's lymphoma cell line CA46 cells, culture in a T75 flask, and use RPMI - 1640 medium containing 10% FBS, 100 U / mL penicillin, and 100 μg / mL streptomycin as the cell culture medium. The culture temperature is 37 °C, and the CO 2 concentration is 5%, the humidity is above 95%, and culture aseptically in a carbon dioxide incubator.

[0064] 2. Calcium ion removal

[0065] In Step 1, culture routinely for 58 h. After cell counting, the cell density is greater than 1×106 cells / mL. Add ethylene glycol diethyl ether diamine tetraacetic acid (EGTA) solution (pH 7.0, sterile) to the medium to a final concentration of 1.0 mmol / L, mix well, place in a carbon dioxide incubator at 37 °C, and CO 2 concentration of 5%, and incubate at 95% humidity for 10 min.

[0066] 3. Low temperature and colchicine treatment

[0067] After culturing in step 2 for 10 min, centrifuge at 1000 rpm for 10 min, discard the supernatant, resuspend the cells with fresh pre-warmed (37 °C) RPMI-1640 medium containing 10% FBS, 100 U / mL penicillin, and 100 μg / mL streptomycin. At the same time, continue to add colchicine solution (dissolved in HBSS, cell culture grade) to a final concentration of 0.075 μg / mL. Reduce the temperature of the incubator from 37 °C to 30 °C, and CO 2 concentration of 5%, and incubate in an environment with a humidity of more than 95% for 4 h.

[0068] 4. Hypotonic treatment

[0069] After culturing in step 3 for 4 h, centrifuge at 1000 rpm for 10 min, discard the supernatant, gently resuspend the cells with pre-warmed (37 °C) 75 mmol / L KCl hypotonic solution, heat in a 37 °C water bath for 30 min, and shake once every 10 min.

[0070] 5. Pre-fixation and fixation

[0071] After heating in the water bath for 30 min in step 4, add freshly prepared fixative (methanol: glacial acetic acid volume ratio is 3:1) stored at -20 °C according to the volume ratio of cell suspension to fixative (3 - 5):1, mix gently, centrifuge at 1000 rpm for 10 min, discard the supernatant, continue to slowly add cold fixative and mix evenly, store at room temperature for 30 min, and then transfer to 4 °C for storage.

[0072] 6. DAPI staining

[0073] For the fixed cells in step 5, add DAPI dye to a final concentration of 300 nM, and stain at room temperature in the dark for 10 min.

[0074] 7. Microscopic fluorescence observation at 40×

[0075] Under hypotonic conditions, the cell membranes of most cells rupture and some chromosomes disperse. Among them, the cells in metaphase of mitosis have irregular edges due to chromosome entanglement, with uneven surfaces, mostly wrapped by no membrane or a single-layer cell membrane. The non-metaphase mitotic cells are round, with distinct nuclear and cytoplasmic regions, and often have a single-layer nuclear membrane or a double-layer membrane (nuclear membrane and cell membrane) structure. After the cells are stained with DAPI (a fluorescent dye that can strongly bind to DNA), they show blue-violet fluorescence. Based on this principle, the cells stained with DAPI in step 6 are observed under a microscope with an objective magnification of 40 times, and the results are as Figure 2 shown; among them, a, b, and c are different fields of view of the same sample under a 40× objective lens.

[0076] According to Figure 2 it can be seen that the cells in metaphase of mitosis have irregular edges due to chromosome entanglement, with uneven surfaces, and the blue fluorescence is unevenly distributed. The non-metaphase mitotic cells are round or oval, with uniform blue fluorescence on the surface, which is the cell nucleus. By using the synergistic effect of 1.0 mmol / L EGTA and 0.075 μg / mL colchicine, the normal assembly of tubulin can be disrupted, the formation of the spindle of CA46 cells can be inhibited, and cell division is blocked, thus producing a large number of cells in metaphase of mitosis, and the mitotic index (the number of cells in metaphase of mitosis accounting for the total number of cells) is as high as 65%.

[0077] Example 3

[0078] 1. Cell culture

[0079] Human immortalized lymphoblastoid cell line GM12878 is selected and cultured in a T75 flask. The cell culture medium uses RPMI-1640 medium containing 12% FBS, 100 U / mL penicillin, and 100 μg / mL streptomycin. The culture temperature is 37 °C, and the CO 2 concentration is 5%, the humidity is above 95%, and it is cultured aseptically in a carbon dioxide incubator.

[0080] 2. Calcium ion removal

[0081] In step 1, it is cultured routinely for 48 h. After cell counting, the cell density is greater than 1×10 6 cells / mL. An ethylene glycol bis(2-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) solution (pH 7.0, sterile) is added to the culture medium to a final concentration of 1.5 mmol / L, mixed well, placed in a carbon dioxide incubator at 37 °C, and the CO 2 concentration is 5%, and it is cultured at 95% humidity for 10 min.

[0082] 3. Low temperature and colchicine treatment

[0083] After culturing for 10 min in Step 2, centrifuge at 1000 rpm for 10 min, discard the supernatant, resuspend the cells with fresh RPMI-1640 medium pre-warmed to 37 °C containing 12% FBS, 100 U / mL penicillin, and 100 μg / mL streptomycin. Meanwhile, continue to add colchicine solution (dissolved in HBSS, cell culture grade) to a final concentration of 0.05 μg / mL. Lower the temperature of the incubator from 37 °C to 30 °C, and culture at a CO 2 concentration of 5% and a humidity of more than 95% for 4 h.

[0084] 4. Hypotonic treatment

[0085] After culturing for 4 h in Step 3, centrifuge at 1000 rpm for 10 min, discard the supernatant, gently resuspend the cells with pre-warmed 75 mmol / L KCl hypotonic solution at 37 °C, heat in a 37 °C water bath for 30 min, and shake once every 10 min.

[0086] 5. Pre-fixation and fixation

[0087] After heating in the water bath for 30 min in Step 4, add freshly prepared fixative (methanol: glacial acetic acid volume ratio is 3:1) stored at -20 °C according to the volume ratio of cell suspension to fixative (3 - 5):1, gently mix, centrifuge at 1000 rpm for 10 min, discard the supernatant, continue to slowly add cold fixative and mix evenly, store at room temperature for 30 min and then transfer to 4 °C for storage.

[0088] 6. Observation under a microscope at 40×

[0089] Under hypotonic conditions, the cell membranes of most cells rupture, and some chromosomes are dispersed. Among them, cells in the metaphase of mitosis have irregular edges due to chromosome entanglement, with uneven surfaces, mostly wrapped by no membrane or a single-layer cell membrane. Non-metaphase mitotic cells are round, with distinct nuclear and cytoplasmic regions, often with a single-layer nuclear membrane or a double-layer membrane (nuclear membrane and cell membrane) structure. Based on this principle, observe the cells fixed in Step 5 using a microscope with an objective magnification of 40 times. The results are as Figure 3 shown; among them, a, b, and c are different fields of view under a 40× objective of the same sample.

[0090] According to Figure 3 it can be seen that the synergistic effect of 1.5 mmol / L EGTA and 0.05 μg / mL colchicine can inhibit the normal formation of the spindle in GM12878 cells, and the cells cannot further divide, thus producing a large number of cells in the metaphase of mitosis. The mitotic index (the number of cells in the metaphase of mitosis accounting for the total number of cells) is as high as 53%.

[0091] Comparative Example 1

[0092] 1. Cell culture

[0093] The Burkitt's lymphoma cell line CA46 cells were selected and cultured in a T75 flask. The cell culture medium used was RPMI-1640 medium containing 10% FBS, 100 U / mL penicillin, and 100 μg / mL streptomycin. The culture temperature was 37°C, and the CO 2 concentration was 5%, with a humidity above 95%. They were cultured aseptically in a carbon dioxide incubator.

[0094] 2. Colchicine treatment

[0095] Step 1: After culturing routinely for 58 h, when the cell density was greater than 1×10 6 cells / mL as determined by cell counting, colchicine solution (dissolved in HBSS, cell culture grade) was added to the medium to a final concentration of 0.1 μg / mL. Incubate at 37°C with a CO 2 concentration of 5% and a humidity above 95% for 4 h.

[0096] 3. Hypotonic treatment

[0097] Step 3: After culturing for 4 h, centrifuge at 1000 rpm for 10 min, discard the supernatant, gently resuspend the cells with pre-warmed 75 mmol / L KCl hypotonic solution at 37°C, heat in a 37°C water bath for 30 min, and shake once every 10 min.

[0098] 4. Pre-fixation and fixation

[0099] Step 3: After heating in the water bath for 30 min, add freshly prepared fixative (methanol: glacial acetic acid volume ratio of 3:1) stored at -20°C according to the volume ratio of cell suspension to fixative (3 - 5):1, gently mix, centrifuge at 1000 rpm for 10 min, discard the supernatant, continue to slowly add cold fixative and mix evenly, store at room temperature for 30 min and then transfer to 4°C for storage.

[0100] 5. Observation under a microscope at 40×

[0101] Under hypotonic conditions, most of the cell membranes rupture, and some chromosomes are dispersed. Among them, cells in the metaphase of mitosis have irregular edges due to chromosome entanglement, with uneven surfaces, mostly wrapped by no membrane or a single-layer cell membrane. Non-metaphase mitotic cells are round, with distinct nuclear and cytoplasmic regions, often with a single-layer nuclear membrane or a double-membrane (nuclear membrane and cell membrane) structure. Based on this principle, the cells fixed in Step 5 were observed using a microscope with an objective magnification of 40 times. The results are as Figure 4 shown; among them, a, b, and c are different fields of view under the 40× objective of the same sample.

[0102] According to Figure 4It can be seen that when human Burkitt's lymphoma cell line CA46 cells were treated with 0.1 μg / mL colchicine for 4 h, a large number of cells were in the non-metaphase stage, the number of metaphase cells was small, and the mitotic index was about 16%.

[0103] Comparative Example 2

[0104] 1. Cell culture

[0105] The human Burkitt's lymphoma cell line CA46 cells were selected and cultured in a T75 flask. The cell culture medium used was RPMI-1640 medium containing 10% FBS, 100 U / mL penicillin, and 100 μg / mL streptomycin. The culture temperature was 37 °C, and the CO 2 concentration was 5%, the humidity was above 95%, and the cells were cultured aseptically in a carbon dioxide incubator.

[0106] 2. Calcium ion removal

[0107] Step 1: The cells were cultured routinely for 58 h. After cell counting, the cell density was greater than 1×10 6 cells / mL. Ethylene glycol bis(2-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) solution (pH 7.0, sterile) was added to the culture medium to a final concentration of 1.0 mmol / L. The mixture was thoroughly mixed and placed in a carbon dioxide incubator at 37 °C with a CO 2 concentration of 5% and 95% humidity for 10 - 15 min.

[0108] 3. Hypotonic treatment

[0109] After culturing for 10 - 15 min in Step 3, the cells were centrifuged at 1000 rpm for 10 min. The supernatant was discarded, and the cells were gently resuspended with pre-warmed 75 mmol / L KCl hypotonic solution at 37 °C. The cells were heated in a water bath at 37 °C for 30 min and shaken every 10 min.

[0110] 4. Pre-fixation and fixation

[0111] After heating in the water bath for 30 min in Step 3, fresh prepared fixative (methanol: glacial acetic acid volume ratio of 3:1) stored at -20 °C was added according to the volume ratio of cell suspension to fixative of (3 - 5):1. The mixture was gently mixed, centrifuged at 1000 rpm for 10 min, and the supernatant was discarded. Cold fixative was continuously added slowly and mixed evenly. After storing at room temperature for 30 min, it was transferred to 4 °C for storage.

[0112] 5. Observation under a microscope at 40×

[0113] Under hypotonic conditions, the cell membranes of most cells rupture and some chromosomes disperse. Among them, the cells in the metaphase of mitosis have irregular edges due to chromosome entanglement, with uneven surfaces, mostly wrapped by no membrane or a single-layer cell membrane. The cells in the non-metaphase of mitosis are round, with distinct nuclear and cytoplasmic regions, often with a single-layer nuclear membrane or a double-layer membrane (nuclear membrane and cell membrane) structure. Based on this principle, the cells fixed in step 5 are observed under a microscope with an objective magnification of 40 times, and the results are as Figure 5 shown; among them, a, b, and c are different fields of view of the same sample under a 40× objective lens.

[0114] According to Figure 5 it can be seen that by treating the human Burkitt's lymphoma cell line CA46 cells with 1.0 mmol / L EGTA for 10 - 15 minutes, a large number of cells are in the non-metaphase stage, with a small number of cells in the metaphase, and the mitotic index is only 6%.

[0115] It can be seen from the above that the technical solution provided by the present invention can significantly increase the proportion of cells in the metaphase of mitosis, improve the enrichment rate of cells in the metaphase of mitosis, and reduce costs.

[0116] Although the above embodiments have described the present invention in detail, they are only a part of the embodiments of the present invention, not all embodiments. People can also obtain other embodiments based on this embodiment without creative efforts, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for increasing the proportion of cells in metaphase of mitosis, characterized in that: The steps include: Performing a first treatment on the cells to be treated using ethylene glycol diethyl ether diamine tetraacetic acid, and performing solid-liquid separation to obtain first treated cells; performing a second treatment on the first treated cells using colchicine; The effect concentration of ethylene glycol diethyl ether diamine tetraacetic acid is 0.5-2 mmol / L; The effective concentration of colchicine is 0.05-0.1 μg / mL.

2. The method according to claim 1, characterized in that: The density of the cells to be treated is 0.8×10 6 ~1.2×10 6 Pieces / mL.

3. The method according to claim 1, characterized in that The temperature of the first treatment is 35.5-37.5°C and the time is 10-20 minutes; The temperature of the second treatment is 30-32° C., and the time is 0.5-4 h.

4. The method according to claim 1, characterized in that: The solid-liquid separation method includes centrifugation, the centrifugation speed is 800-1200 rpm, and the time is 5-10 minutes.

5. The method according to claim 1, characterized in that After obtaining the first treated cells, the method further includes: resuspending the first treated cells to obtain a cell suspension; and performing a second treatment on the cell suspension using colchicine.

6. The method according to claim 5, characterized in that The first treated cells are resuspended in a culture medium, wherein the culture medium is based on DMEM culture medium or RPMI-1640 culture medium and includes 10-12% v / v fetal bovine serum, 80-110 U / mL penicillin and 80-110 μg / mL streptomycin.

7. The method according to claim 1, characterized in that The cells to be treated are mammalian cells.

8. The method according to any one of claims 1 to 7, characterized in that: After the first treated cells are subjected to the second treatment with colchicine, the method further comprises: performing solid-liquid separation on the cell treatment solution after the second treatment, and performing hypotonic treatment after obtaining the second treated cells.

9. The method according to claim 8, characterized in that The hypotonic treatment is performed using 0.07-0.08 mol / L KCl solution; the temperature of the hypotonic treatment is 35-38° C., and the time is 10-30 min.

10. Use of the method according to any one of claims 1 to 9 in one or more of chromosome analysis, karyotype detection and anti-tumor drug screening.

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