Preparation method of peripheral blood lymphocyte culture medium and synchronization kit

Abstract

The invention discloses a peripheral blood lymphocyte culture medium and a preparation method of a synchronization kit, and relates to the technical field of cell culture media, the peripheral blood lymphocyte culture medium comprises an RPMI-1640 culture solution, fetal calf serum, PHA phytolectin, HEPES free acid, penicillin, streptomycin, interleukin-2 and L-glutamic acid; basic nutrition and growth factors are provided through an RPMI-1640 culture solution and fetal calf serum, PHA phytolectin is used for stimulating cell proliferation, HEPES free acid maintains pH stability of the culture medium, penicillin and streptomycin prevent bacterial infection, interleukin-2 promotes proliferation and activation of T cells, L-glutamic acid serves as important amino acid to support cell metabolism, and the culture medium has the advantages that the culture medium can be used for promoting T cell proliferation and activation; the composition of the components is used for culturing peripheral blood lymphocytes and is beneficial to providing an optimal culture environment.

Description

Technical Field

[0001] The invention relates to the technical field of cell culture medium, in particular to a preparation method of a peripheral blood lymphocyte culture medium and a synchronization kit. Background Art

[0002] Peripheral blood lymphocytes mainly include T cells, B cells and natural killer cells, which are the main components of the immune system. Studying these cells is of great significance for understanding immune responses, disease mechanisms and developing new therapies. Peripheral blood lymphocyte culture can simulate in vivo conditions in an in vitro environment, allowing researchers to conduct various experiments such as cell proliferation, differentiation, and cytokine release. Cells in human peripheral blood do not have a division phase and can only be found under abnormal circumstances. Phytohemagglutinin (PHA) can be used as a stimulator of human lymphocyte mitosis. Lymphocytes are cultured in a medium containing PHA to obtain a rich population of actively growing mitotic cells in vitro.

[0003] In the peripheral blood lymphocyte culture medium, the instability of the culture medium components can easily cause pH fluctuations, bacterial contamination, insufficient or abnormal cell proliferation, and poor cell attachment, resulting in slow or insufficient cell proliferation. At the same time, during the cell culture process, there is insufficient proliferation and activation of T cells, which weakens cell metabolism, making it difficult for cells to aggregate and attach in culture, and easily leading to abnormal cell cycle and the impact on experimental results. Summary of the invention

[0004] The present invention aims at the deficiencies of the prior art and provides the following technical scheme: a peripheral blood lymphocyte culture medium, PHA lectin is used to stimulate cell proliferation, HEPES free acid maintains pH stability of the culture medium, penicillin and streptomycin prevent bacterial infection, interleukin-2 promotes T cell proliferation and activation, and L-glutamic acid serves as an important amino acid to support cell metabolism.

[0005] As an improvement of the above technical solution, the ratio of the RPMI-1640 culture medium to fetal bovine serum is 3-5:1, wherein the RPMI-1640 culture medium: 75-85% (v / v), and the fetal bovine serum: 17-25% (v / v).

[0006] As an improvement of the above technical solution, the PHA lectin concentration is 1-5 μg / mL, the HEPES free acid concentration is 10-25 mM, the penicillin concentration is 100 U / mL, the streptomycin concentration is 100 μg / mL, the interleukin-2 concentration is 20-100 U / mL, and the L-glutamate concentration is 0.5-2 mM.

[0007] The synchronization kit includes 5-bromo-2′-deoxyuridine, thymidine, and colchicine; 5-bromo-2′-deoxyuridine (BrdU) is a marker used to detect cell proliferation, thymidine is the basic unit of DNA synthesis; colchicine is often used in cell division research.

[0008] As an improvement of the above technical solution, the concentration of the following components is used: 5-bromo-2′-deoxyuridine concentration: 10-20 μM, thymidine concentration: 100-200 μM, and colchicine concentration: 5-8 μM.

[0009] The preparation method of the peripheral blood lymphocyte culture medium and the synchronization kit comprises the following steps:

[0010] S1, Preparation of Peripheral Blood Lymphocyte Culture Medium:

[0011] S11, prepare basal medium:

[0012] Take RPMI-1640 medium and pour it into a sterile container according to the total amount required to provide the nutrients and growth factors required for the growth and maintenance of peripheral blood lymphocytes; for example, if 1L of medium is ultimately required, take 750-850mL of RPMI-1640 medium;

[0013] S12, add fetal bovine serum:

[0014] Add fetal bovine serum to RPMI-1640 medium to provide additional growth factors and nutrients to promote cell proliferation and survival. Add 170-250 mL of fetal bovine serum according to the required proportion.

[0015] S13, added PHA lectin:

[0016] Prepare a working solution of PHA lectin to a concentration of 1-5 μg / mL. PHA lectin is an immunostimulant used to activate cell division, induce cell proliferation and enter different phases of the cell cycle. For example, if the final culture medium volume is 1 L, add 1-5 μg of PHA solution.

[0017] S14, add HEPES buffer:

[0018] The final concentration of HEPES is configured to be 10-25mM to help maintain the pH stability of the culture medium and improve the stability of cell culture;

[0019] S15, add penicillin and streptomycin:

[0020] Add penicillin to a final concentration of 100 U / mL; for example, for 1 L of culture medium, add 100 μL of 10,000 U / mL penicillin solution;

[0021] Add streptomycin to a final concentration of 100 μg / mL; for example, for 1 L of culture medium, add 100 μL of 10,000 μg / mL streptomycin solution;

[0022] Penicillin and streptomycin provide a barrier against bacterial invasion for the growth of lymphocytes, inhibiting the risk of microbial contamination;

[0023] S16, added interleukin-2, L-glutamic acid:

[0024] Add interleukin-2 to promote T cell proliferation and differentiation, with a final concentration of 20-100 U / mL;

[0025] Add L-glutamic acid, an antioxidant that reduces oxidative stress and supports cell growth, to a final concentration of 0.5-2 mM;

[0026] S17, mixed medium:

[0027] Mix all ingredients thoroughly to ensure even distribution;

[0028] S18, Filtration and Storage:

[0029] The preparation was completed under sterile conditions and the culture medium was filtered through a 0.22 μm filter to remove microbial contamination;

[0030] Aliquot the culture medium into sterile 15 ml centrifuge tubes, 5 ml per tube, and store at -20°C to avoid repeated freezing and thawing. Try to avoid long-term storage before use;

[0031] S2, Preparation of synchronization kit:

[0032] S21, 5-bromo-2′-deoxyuridine:

[0033] Prepare 5-bromo-2′-deoxyuridine, 5-bromo-2′-deoxyuridine is used to detect cell proliferation and mark cells in the DNA synthesis stage; wherein the 5-bromo-2′-deoxyuridine solution is added after cell inoculation in step S1 and acts for 17 hours, and the concentration of 5-bromo-2′-deoxyuridine is 10-20 μM;

[0034] S22, thymidine and colchicine synchronization:

[0035] Thymidine and colchicine solutions were prepared separately. Thymidine was used for cell cycle synchronization to prevent cells from proliferating outside the S phase. Colchicine was used for cell cycle synchronization to prevent cells from entering mitosis. The thymidine solution was added after the 5-bromo-2′-deoxyuridine solution was added and acted for 4 hours. The concentration of thymidine was 100-200 μM. The colchicine solution was then acted for 40 minutes. The concentration of colchicine was 5-8 μM.

[0036] As an improvement of the above technical solution, in step S13, the preparation method of PHA lectin includes the following steps:

[0037] S131, sampling, weighing 75 g of red kidney beans;

[0038] S132, cleaning, using an ultrasonic cleaner to clean, first with a 0.85% sodium chloride solution for 1 minute, then with a 0.85% sodium chloride solution for 2 times, each time for 5 minutes;

[0039] S133, soaking, placing the washed kidney beans in a 0.85% sodium chloride solution, soaking for 24 hours, and storing in a refrigerator at 4°C;

[0040] S134, crushing: crushing the soaked kidney beans into particles of 1 cm*1 cm in size using a blender;

[0041] S135, extraction, pour the crushed kidney beans into a 1L beaker, add 0.85% sodium chloride solution to 500ml, soak for 24 hours, and stir at least 30 times every 8 hours;

[0042] S136, preliminary centrifugation, the soaking solution was divided into 50 mL centrifuge tubes, centrifuged at 10000 rpm for 10 min at 4 °C, and the supernatant was collected;

[0043] S137, fine centrifugation, re-aliquot the supernatant, centrifuge at 12000 rpm at 4°C for 20 minutes, and take the supernatant. Re-aliquot, centrifuge at 12000 rpm at 4°C for 30 minutes, and take the supernatant;

[0044] S138, filtering, filtering the supernatant through a 0.45 μm pre-filter, and then performing final filtration using a 0.22 μm filter;

[0045] S139, PHA solution collection: the filtered PHA solution is collected and stored in a sterile environment.

[0046] Beneficial effects of the present invention:

[0047] 1. RPMI-1640 culture medium and fetal bovine serum provide basic nutrients and growth factors, PHA lectin is used to stimulate cell proliferation, HEPES free acid maintains the pH stability of the culture medium, penicillin and streptomycin prevent bacterial infection, interleukin-2 promotes T cell proliferation and activation, and L-glutamic acid, as an important amino acid, supports cell metabolism. The combination of the above ingredients is used for the culture of peripheral blood lymphocytes, which helps to provide the best culture environment, optimize cell growth and function, and is used for immune research and cell biology experiments.

[0048] 2. 5-Bromo-2′-deoxyuridine: used to track cell division and proliferation, mark newly synthesized DNA, and help study cell proliferation rate and cell cycle; thymidine: as a component of DNA synthesis, it is often used to study DNA replication and its regulation; colchicine: by inhibiting microtubule formation, it prevents cell division and is often used to study the mechanisms of cell division and mitosis; each of these compounds provides a key tool for cell biology research and is used to analyze the biological processes of cell cycle, proliferation and microtubule function. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 Figure 2 is a peripheral blood lymphocyte culture medium of the present invention;

[0050] Figure 2 Figure 1 is a synchronization reagent diagram of the present invention;

[0051] Figure 3 is a flow chart of the preparation method of the present invention;

[0052] Figure 4 The flowchart of the preparation method of PHA lectin in the present invention is shown in FIG. DETAILED DESCRIPTION

[0053] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0054] See also Figure 1-4 The present invention provides a technical solution: a peripheral blood lymphocyte culture medium, including RPMI-1640 culture medium, fetal bovine serum, PHA lectin, HEPES free acid, penicillin, streptomycin, interleukin-2, and L-glutamic acid.

[0055] RPMI-1640 culture medium and fetal bovine serum provide basic nutrients and growth factors, PHA lectin is used to stimulate cell proliferation, HEPES free acid maintains the pH stability of the culture medium, penicillin and streptomycin prevent bacterial infection, interleukin-2 promotes T cell proliferation and activation, and L-glutamic acid, as an important amino acid, supports cell metabolism. The combination of the above ingredients is used for the culture of peripheral blood lymphocytes, which helps to provide an optimal culture environment, optimize cell growth and function, and is used in immune research and cell biology experiments.

[0056] Specifically, the ratio of the RPMI-1640 culture medium to fetal bovine serum is 3-5:1, wherein the RPMI-1640 culture medium: 75-85% (v / v), and the fetal bovine serum: 17-25% (v / v).

[0057] Specifically, it is composed of the following components: PHA lectin concentration: 1-5 μg / mL, HEPES free acid concentration: 10-25 mM, penicillin concentration: 100 U / mL, streptomycin concentration: 100 μg / mL, interleukin-2 concentration: 20-100 U / mL, L-glutamic acid concentration: 0.5-2 mM.

[0058] Synchronization kit, including 5-bromo-2'-deoxyuridine and thymidine.

[0059] Specifically, the synchronization kit also includes colchicine.

[0060] 5-Bromo-2′-deoxyuridine (BrdU) is a marker used to detect cell proliferation. Thymidine is the basic unit of DNA synthesis. Colchicine is often used in cell division research. 5-Bromo-2′-deoxyuridine: used to track cell division and proliferation, mark newly synthesized DNA, and help study cell proliferation rate and cell cycle; thymidine: as a component of DNA synthesis, it is often used to study DNA replication and its regulation; colchicine: prevents cell division by inhibiting microtubule formation, and is often used to study the mechanisms of cell division and mitosis; these compounds each provide key tools for cell biology research and are used to analyze the biological processes of cell cycle, proliferation and microtubule function.

[0061] Specifically, it is composed of the following component concentrations: 5-bromo-2′-deoxyuridine concentration: 10-20 μM, thymidine concentration: 100-200 μM, colchicine concentration: 5-8 μM.

[0062] The preparation method of the peripheral blood lymphocyte culture medium and the synchronization kit comprises the following steps:

[0063] S1, Preparation of Peripheral Blood Lymphocyte Culture Medium:

[0064] S11, prepare basal medium:

[0065] Take RPMI-1640 medium and pour it into a sterile container according to the total amount required to provide the nutrients and growth factors required for the growth and maintenance of peripheral blood lymphocytes; for example, if 1L of medium is ultimately required, take 750-850mL of RPMI-1640 medium;

[0066] S12, add fetal bovine serum:

[0067] Add fetal bovine serum to RPMI-1640 medium to provide additional growth factors and nutrients to promote cell proliferation and survival. Add 170-250 mL of fetal bovine serum according to the required proportion.

[0068] S13, added PHA lectin:

[0069] Prepare a working solution of PHA lectin to a concentration of 1-5 μg / mL. PHA lectin is an immunostimulant used to activate cell division, induce cell proliferation and enter different phases of the cell cycle. For example, if the final culture medium volume is 1 L, add 1-5 μg of PHA solution.

[0070] S14, add HEPES buffer:

[0071] The final concentration of HEPES is configured to be 10-25mM to help maintain the pH stability of the culture medium and improve the stability of cell culture;

[0072] S15, add penicillin and streptomycin:

[0073] Add penicillin to a final concentration of 100 U / mL; for example, for 1 L of culture medium, add 100 μL of 10,000 U / mL penicillin solution;

[0074] Add streptomycin to a final concentration of 100 μg / mL; for example, for 1 L of culture medium, add 100 μL of 10,000 μg / mL streptomycin solution;

[0075] Penicillin and streptomycin provide a barrier against bacterial invasion for the growth of lymphocytes, inhibiting the risk of microbial contamination;

[0076] S16, added interleukin-2, L-glutamic acid:

[0077] Add interleukin-2 to promote T cell proliferation and differentiation, with a final concentration of 20-100 U / mL;

[0078] Add L-glutamic acid, an antioxidant that reduces oxidative stress and supports cell growth, to a final concentration of 0.5-2 mM;

[0079] S17, mixed medium:

[0080] Mix all ingredients thoroughly to ensure even distribution;

[0081] S18, Filtration and Storage:

[0082] The preparation was completed under sterile conditions and the culture medium was filtered through a 0.22 μm filter to remove microbial contamination;

[0083] Aliquot the culture medium into sterile 15 ml centrifuge tubes, 5 ml per tube, and store at -20°C to avoid repeated freezing and thawing; try to avoid long-term storage before use;

[0084] S2, Preparation of synchronization kit:

[0085] S21, 5-bromo-2′-deoxyuridine labeling:

[0086] A mixed solution of 5-bromo-2′-deoxyuridine and colchicine is prepared, wherein 5-bromo-2′-deoxyuridine is used to detect cell proliferation and mark cells in the DNA synthesis stage; wherein the 5-bromo-2′-deoxyuridine solution is added after the cells are inoculated in step S1 and acts for 17 hours, and the concentration of 5-bromo-2′-deoxyuridine is 10-20 μM;

[0087] S22, thymidine and colchicine synchronization:

[0088] Thymidine and colchicine solutions were prepared separately. Thymidine was used for cell cycle synchronization to prevent cells from proliferating outside the S phase. Colchicine was used for cell cycle synchronization to prevent cells from entering mitosis. The thymidine solution was added after the 5-bromo-2′-deoxyuridine solution was added and acted for 4 hours. The concentration of thymidine was 100-200 μM. The colchicine solution was then acted for 40 minutes. The concentration of colchicine was 5-8 μM.

[0089] Effects on cell proliferation rate:

[0090] Experimental group (containing PHA lectin): Within 48 hours, the proliferation rate of T cells can usually reach 4-6 times. For example, if the initial cell number is 1×10^5, it may reach 4-6×10^5 after 48 hours.

[0091] Control group (without PHA lectin): Within 48 hours, the cell proliferation rate is usually low, perhaps only 1.5-2 times. For example, if the initial cell number is 1×10^5, it may reach 1.5-2×10^5 after 48 hours.

[0092] Comparison of cell survival rate:

[0093] Experimental group (containing fetal bovine serum and HEPES): After 48 hours of culture, the cell survival rate is usually between 90% and 95%.

[0094] Control group (without fetal bovine serum): Cell survival rate may be significantly reduced, generally 60%-70%.

[0095] Improved cell function:

[0096] Experimental group (containing interleukin-2): Detection of cell surface activation markers (such as CD69 and CD25) by flow cytometry, the expression level of activation markers usually increases 2-3 times. For example, the proportion of CD69 positive cells may increase from 10% in the control group to 20%-30%.

[0097] Control group (without interleukin-2): The expression level of activation markers is usually low, and the proportion of CD69 positive cells may be 10%-15%.

[0098] Specifically, in step S13, the preparation method of PHA lectin includes the following steps:

[0099] S131, sampling, weighing 75 g of red kidney beans;

[0100] S132, cleaning, using an ultrasonic cleaner to clean, first with a 0.85% sodium chloride solution for 1 minute, then with a 0.85% sodium chloride solution for 2 times, each time for 5 minutes;

[0101] S133, soaking, placing the washed kidney beans in a 0.85% sodium chloride solution, soaking for 24 hours, and storing in a refrigerator at 4°C;

[0102] S134, crushing: crushing the soaked kidney beans into particles of 1 cm*1 cm in size using a blender;

[0103] S135, extraction, pour the crushed kidney beans into a 1L beaker, add 0.85% sodium chloride solution to 500ml, soak for 24 hours, and stir at least 30 times every 8 hours;

[0104] S136, preliminary centrifugation, the soaking solution was divided into 50 mL centrifuge tubes, centrifuged at 10000 rpm for 10 min at 4 °C, and the supernatant was collected;

[0105] S137, fine centrifugation, re-aliquot the supernatant, centrifuge at 12000 rpm at 4°C for 20 minutes, and take the supernatant. Re-aliquot, centrifuge at 12000 rpm at 4°C for 30 minutes, and take the supernatant;

[0106] S138, filtering, filtering the supernatant through a 0.45 μm pre-filter, and then performing final filtration using a 0.22 μm filter;

[0107] S139, PHA solution collection: the filtered PHA solution is collected and stored in a sterile environment.

[0108] Improve the efficiency and purity of PHA lectin preparation while reducing operation time and cost.

[0109] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit them.

Claims

1. A peripheral blood lymphocyte culture medium, characterized in that: Including RPMI-1640 culture medium, fetal bovine serum, PHA lectin, HEPES free acid, penicillin, streptomycin, interleukin-2, and L-glutamic acid.

2. The peripheral blood lymphocyte culture medium according to claim 1, characterized in that: The ratio of the RPMI-1640 culture medium to fetal bovine serum is 3-5:1, wherein the RPMI-1640 culture medium is 75-85% (v / v) and the fetal bovine serum is 17-25% (v / v).

3. The peripheral blood lymphocyte culture medium according to claim 1, characterized in that: It is composed of the following components at different concentrations: PHA lectin concentration: 1-5 μg / mL, HEPES free acid concentration: 10-25 mM, penicillin concentration: 100 U / mL, streptomycin concentration: 100 μg / mL, interleukin-2 concentration: 20-100 U / mL, L-glutamic acid concentration: 0.5-2 mM.

4. A synchronization kit, characterized in that: The invention comprises 5-bromo-2′-deoxyuridine and thymidine applied to the peripheral blood lymphocyte culture medium according to any one of claims 1 to 3.

5. The synchronization kit according to claim 4, characterized in that: Also included is colchicine.

6. The synchronization kit according to claim 5, characterized in that: It is composed of the following component concentrations: 5-bromo-2′-deoxyuridine concentration: 10-20 μM, thymidine concentration: 100-200 μM, colchicine concentration: 5-8 μM.

7. The method for preparing the peripheral blood lymphocyte culture medium and synchronization kit according to claim 6, characterized in that: The following steps are involved: S1, Preparation of Peripheral Blood Lymphocyte Culture Medium: S11, prepare basal medium: Take RPMI-1640 medium and pour it into a sterile container according to the required total amount to provide nutrients and growth factors required for the growth and maintenance of peripheral blood lymphocytes; S12, add fetal bovine serum: Add fetal bovine serum to RPMI-1640 medium to provide additional growth factors and nutrients to promote cell proliferation and survival. Add 170-250 mL of fetal bovine serum according to the required proportion. S13, added PHA lectin: Prepare a working solution of PHA lectin, with a concentration of 1-5 μg / mL. PHA lectin is an immunostimulant used to activate cell division, induce cell proliferation and enter different stages of the cell cycle; S14, add HEPES buffer: The final concentration of HEPES is configured to be 10-25mM to help maintain the pH stability of the culture medium and improve the stability of cell culture; S15, add penicillin and streptomycin: Add penicillin to a final concentration of 100 U / mL; Streptomycin was added to a final concentration of 100 μg / mL; Penicillin and streptomycin provide a barrier against bacterial invasion for the growth of lymphocytes, inhibiting the risk of microbial contamination; S16, added interleukin-2, L-glutamic acid: Add interleukin-2 to promote T cell proliferation and differentiation, with a final concentration of 20-100 U / mL; Add L-glutamic acid, an antioxidant that reduces oxidative stress and supports cell growth, to a final concentration of 0.5-2 mM; S17, mixed medium: Mix all ingredients thoroughly to ensure even distribution; S18, Filtration and Storage: The preparation was completed under sterile conditions and the culture medium was filtered through a 0.22 μm filter to remove microbial contamination; Aliquot the culture medium into sterile 15 ml centrifuge tubes, 5 ml per tube, and store at -20°C to avoid repeated freezing and thawing; try to avoid long-term storage before use; S2, Preparation of synchronization kit: S21, 5-bromo-2′-deoxyuridine labeling: Prepare 5-bromo-2′-deoxyuridine solution, 5-bromo-2′-deoxyuridine detects cell proliferation and is used to mark cells in the DNA synthesis stage; wherein the 5-bromo-2′-deoxyuridine solution is added after cell inoculation in step S1 and acts for 17 hours; S22, thymidine and colchicine synchronization: Thymidine and colchicine solutions were prepared separately. Thymidine was used for cell cycle synchronization to prevent cells from proliferating outside the S phase. Colchicine was used for cell cycle synchronization to prevent cells from entering mitosis. The thymidine solution was added after the 5-bromo-2′-deoxyuridine solution was added and acted for 4 hours. The concentration of thymidine was 100-200 μM. The colchicine solution was then acted for 40 minutes. The concentration of colchicine was 5-8 μM.

8. The method for preparing the peripheral blood lymphocyte culture medium and synchronization kit according to claim 7, characterized in that: In step S13, the preparation method of PHA lectin includes the following steps: S131, sampling, weighing 75 g of red kidney beans; S132, cleaning, using an ultrasonic cleaner to clean, first with a 0.85% sodium chloride solution for 1 minute, then with a 0.85% sodium chloride solution for 2 times, each time for 5 minutes; S133, soaking, placing the washed kidney beans in a 0.85% sodium chloride solution, soaking for 24 hours, and storing in a refrigerator at 4°C; S134, crushing: crushing the soaked kidney beans into particles of 1 cm*1 cm in size using a blender; S135, extraction, pour the crushed kidney beans into a 1L beaker, add 0.85% sodium chloride solution to 500ml, soak for 24 hours, and stir at least 30 times every 8 hours; S136, preliminary centrifugation, the soaking solution was divided into 50 mL centrifuge tubes, centrifuged at 10000 rpm for 10 min at 4 °C, and the supernatant was collected; S137, fine centrifugation, the supernatant was re-packed, centrifuged at 12000 rpm at 4°C for 20 min, the supernatant was taken, and the supernatant was re-packed, centrifuged at 12000 rpm at 4°C for 30 min, and the supernatant was taken; S138, filtering, filtering the supernatant through a 0.45 μm pre-filter, and then performing final filtration using a 0.22 μm filter; S139, PHA solution collection: the filtered PHA solution is collected and stored in a sterile environment.

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