Semi-solid culture medium for inducing colony growth and preparation method and application thereof

Abstract

The invention discloses a semi-solid culture medium for inducing cell colony growth, which is composed of IMDM culture medium, methylcellulose, fetal bovine serum, induction factors and additives; methylcellulose is added in the form of methylcellulose mother liquor, and is prepared by the following method: (1) taking pure water, heating in a water bath at 80-98°C, adding methylcellulose powder, and stirring to disperse and wet the methylcellulose powder; (2) continuing water bath heating for 10-20 minutes; (3) autoclaving; (4) cooling to room temperature and standing for 16-24 hours; (5) standing at 4°C for 16-24 hours, inverting and mixing, and obtaining methylcellulose mother liquor. The semi-solid culture medium for inducing cell colony growth of the invention adopts a specific method to prepare methylcellulose mother liquor, so that methylcellulose dissolves more fully, is more conducive to colony formation, and can be used for culturing and detecting hematopoietic stem cells, and for inducing the growth of monocytes, granulocytes, and erythroid cell colonies.

Description

Technical Field

[0001] The invention relates to a semi-solid culture medium for inducing colony growth and a preparation method and application thereof, belonging to the technical field of cell culture. Background Art

[0002] Umbilical cord blood hematopoietic stem cells (HSC) have the ability to differentiate into progenitor cells of myeloid cells such as erythrocytes, granulocytes, monocytes-macrophages and megakaryocytes. In vitro semi-solid cell culture colony-forming units are important indicators for evaluating the proliferation and differentiation ability of hematopoietic precursor cells. Under the induction of appropriate cytokines, different directed progenitor cells can produce corresponding colonies, such as CFU-GM, BFU-E, CFU-GEMM, etc.

[0003] In the prior art, the main indicators for clinical evaluation of cord blood quality are HLA matching, total nucleated cells, CD34+ cell count, and CFU-GM colony count, which are important factors in determining the success of recipient hematopoiesis and immune reconstruction after cord blood transplantation. The number of CFU-GM colonies can better reflect the proliferation and differentiation ability of cord blood hematopoietic stem cells, and is related to the speed of hematopoietic reconstruction and implantation rate. The number of colonies (CFUs) produced by HSC in vitro culture can quantify the number of multipotent progenitor cells and reflect HSC activity.

[0004] At present, the colony culture reagents used by domestic cord blood banks are imported brands, and there are no domestic brand colony culture medium products on the market. Therefore, it is necessary to carry out research on the preparation process of finished colony culture medium, break through technical barriers, break the monopoly of foreign products on the Chinese market, and reduce testing costs. Summary of the invention

[0005] In view of the above-mentioned prior art, the present invention provides a semi-solid culture medium for inducing cell colony growth and a preparation method and application thereof.

[0006] The present invention is achieved through the following technical solutions:

[0007] A semi-solid culture medium for inducing cell colony growth is composed of IMDM culture medium, methyl cellulose, fetal bovine serum, induction factors and additives; wherein the concentration of methyl cellulose is 1.3% to 1.5% (w / v, g / ml), and the concentration of fetal bovine serum is 20% to 40% (volume percentage), preferably 30%;

[0008] The specific types and final concentrations of the induction factors are as follows: SCF, 20-50 ng / ml; GM-CSF, 10-20 ng / ml; IL-3, 10-20 ng / ml; G-CSF, 10-50 ng / ml; IL-6, 10-50 ng / ml; Flt-3L 100-200 ng / mL; Valproic Acid (valproic acid) 1 mM; TPO 50-100 ng / ml; heparin sodium, 8-12 U / ml;

[0009] The specific types and final concentrations of the additives are as follows: GultaMAX additive, 0.9% to 1.1% (v / v); β-mercaptoethanol, 0.25 to 0.35 mol / l; penicillin 90 to 110 U / mL; streptomycin 90 to 110 U / mL.

[0010] When preparing semi-solid culture medium, methylcellulose is added in the form of methylcellulose mother solution, which is prepared by the following method:

[0011] (1) Take an appropriate amount of pure water, heat it in a water bath at 80-98°C until the water temperature is balanced, add methylcellulose powder, and stir thoroughly to make the methylcellulose powder completely dispersed and wet;

[0012] The viscosity of the methylcellulose powder is 3500 to 5600 cPs, preferably 4000 cPs;

[0013] (2) Continue heating in a water bath at 80-98°C for 10-20 min;

[0014] (3) After the water bath heating is completed, high pressure sterilization is immediately performed, and the parameters of high pressure sterilization are: temperature 121° C., time 16 to 35 min, preferably 33 min;

[0015] (4) After high pressure sterilization, cool naturally to room temperature and let stand for 16 to 24 hours;

[0016] (5) Place the mixture at 4°C and let it stand for 16 to 24 hours. The methyl cellulose mother liquor will separate into layers. The upper layer has low solubility and low viscosity, while the lower layer has high viscosity. Invert and mix to obtain the methyl cellulose mother liquor. The concentration of the methyl cellulose mother liquor is 2.5% to 3.0% (w / v, g / ml).

[0017] The method for preparing the semi-solid culture medium for inducing cell colony growth comprises the following steps:

[0018] (I) Preparation of methyl cellulose mother solution

[0019] (1) Take an appropriate amount of pure water, heat it in a water bath at 80-98°C until the water temperature is balanced, add methylcellulose powder, and stir thoroughly to make the methylcellulose powder completely dispersed and wet;

[0020] The viscosity of the methylcellulose powder is 3500 to 5600 cPs, preferably 4000 cPs;

[0021] (2) Continue heating in a water bath at 80-98°C for 10-20 min;

[0022] (3) After the water bath heating is completed, high pressure sterilization is immediately performed, and the parameters of high pressure sterilization are: temperature 121° C., time 16 to 35 min, preferably 33 min;

[0023] (4) After high pressure sterilization, cool naturally to room temperature and let stand for 16 to 24 hours;

[0024] (5) Place the mixture at 4°C and allow to stand for 16 to 24 hours. The methyl cellulose mother liquor is separated into layers. The upper layer has low solubility and low viscosity, while the lower layer has high viscosity. The mixture is mixed by inversion to obtain the methyl cellulose mother liquor. The concentration of the methyl cellulose mother liquor is 2.5% to 3.0% (w / v, g / ml).

[0025] (ii) Preparation of semi-solid culture medium: Mix methylcellulose mother solution, IMDM culture medium mother solution and fetal bovine serum, add various induction factors and additives (in no particular order), mix well, and obtain.

[0026] The IMDM culture medium mother solution is prepared from IMDM powder using ultrapure water, and the concentration of the IMDM culture medium mother solution meets the following conditions: in a semi-solid culture medium, the concentration of the IMDM culture medium is 1× (IMDM culture medium is a commercial product already available in the prior art, and its composition is fixed).

[0027] The semi-solid culture medium for inducing cell colony growth is used in culturing cells and in inducing the growth of monocytes, granulocytes and erythroid cell colonies.

[0028] The semi-solid culture medium for inducing cell colony growth of the present invention adopts a specific method to prepare a methylcellulose mother solution, so that the methylcellulose can be dissolved more fully and is more conducive to colony formation. When preparing the methylcellulose mother solution, the methylcellulose powder is fully dispersed and moistened, then boiled in a water bath, and then immediately transferred for high-temperature sterilization to ensure that the methylcellulose powder is in an insoluble state (dispersed state) in the liquid; after high-pressure sterilization, it is allowed to stand at room temperature and low temperature to allow the methylcellulose to fully absorb water and swell (the methylcellulose gradually absorbs water and dissolves as the temperature decreases, and it is not easy to produce lumps). The methylcellulose solution prepared in this way is uniform in state and has no lumps with large viscosity.

[0029] The semi-solid culture medium for inducing cell colony growth of the present invention can be used for culturing and detecting hematopoietic stem cells, can objectively reflect the hematopoietic ability of hematopoietic stem cells, and greatly reduces the detection cost.

[0030] The IMDM culture medium, various induction factors and additives involved in the present invention are all commercial products in the prior art and can be purchased on the market. The IMDM culture medium and IMDM culture medium mother solution can be prepared by oneself. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 : Microscopic photographs of hematopoietic stem cell colony culture using colony culture medium prepared in Example, Comparative Example 1 and Comparative Example 2, wherein A: Example (40×); B: Comparative Example 1 (40×); C: Comparative Example 2 (40×); D: Comparative Example 2 (100×).

[0032] Figure 2 : Schematic diagram showing a comparison of the number of hematopoietic stem cell culture colonies generated after hematopoietic stem cell colonies were cultured using colony culture media prepared in Example 1, Comparative Example 2, and Comparative Example 3. DETAILED DESCRIPTION

[0033] The present invention will be further described below in conjunction with the embodiments. However, the scope of the present invention is not limited to the following embodiments. It will be appreciated by those skilled in the art that various changes and modifications may be made to the present invention without departing from the spirit and scope of the present invention.

[0034] The instruments, reagents, and materials involved in the following examples, unless otherwise specified, are all conventional instruments, reagents, and materials already available in the prior art and can be obtained through regular commercial channels. The experimental methods and detection methods involved in the following examples, unless otherwise specified, are all conventional experimental methods and detection methods already available in the prior art.

[0035] Example Preparation of semi-solid culture medium for inducing cell colony growth

[0036] Here are the steps:

[0037] (I) Preparation of methyl cellulose mother solution

[0038] (1) Take 500 ml of pure water, heat it in a 98°C water bath until the water temperature is balanced, add 15 g of methylcellulose powder, and stir thoroughly to make the methylcellulose powder completely dispersed and wet;

[0039] The viscosity of the methylcellulose powder is 4000 cPs;

[0040] (2) Continue heating in a 98°C water bath for 15 min;

[0041] (3) After the water bath heating is completed, high pressure sterilization is immediately performed. The parameters of high pressure sterilization are: temperature 121°C, time 33 min;

[0042] (4) After high pressure sterilization, the mixture was cooled to room temperature naturally and allowed to stand for 24 hours;

[0043] (5) Place in a refrigerator and let stand at 4°C for 24 hours. The methyl cellulose mother liquor is now separated into layers. The upper layer has low solubility and low viscosity, while the lower layer has high viscosity. Invert and mix to obtain the methyl cellulose mother liquor. The concentration of the methyl cellulose mother liquor is 3.0% (w / v, g / ml) (there may be water loss during the preparation process, which can be replenished. The quantitative amount is 500 ml).

[0044] (II) Preparation of semi-solid culture medium

[0045] Prepare 1L semi-solid culture medium: mix 500ml of methylcellulose mother solution, 200ml of IMDM culture medium mother solution, and 300ml of fetal bovine serum, add various induction factors and additives (in no particular order), mix well, and the concentration of methylcellulose is 1.5%.

[0046] The IMDM culture medium mother solution is prepared from IMDM powder with ultrapure water. The concentration of the IMDM culture medium mother solution is 5X. In the semi-solid culture medium, the concentration of the IMDM culture medium is 1X.

[0047] The specific types and final concentrations of the induction factors are as follows:

[0048] SCF, 50ng / ml; GM-CSF, 20ng / ml; IL-3, 20ng / ml; G-CSF, 20ng / ml; IL-6, 20ng / ml, Flt-3L 100ng / ml, Valproic Acid 1mM, TPO 50ng / ml, heparin sodium, 10U / ml.

[0049] The specific types and final concentrations of the additives are as follows: GultaMAX additive, 1% (v / v); β-mercaptoethanol, 0.3 mol / l; penicillin, 100 U / mL; streptomycin, 100 U / mL.

[0050] Comparative Example 1 Preparation of semi-solid culture medium

[0051] The difference from the embodiment is that:

[0052] The specific types and final concentrations of the induction factors are as follows (i.e., the factor formula of STEMCELL methylcellulose semisolid colony culture medium): SCF, 50 ng / ml; GM-CSF, 20 ng / ml; IL-3, 20 ng / ml.

[0053] The rest is the same as the embodiment.

[0054] Comparative Example 2 Preparation of semi-solid culture medium

[0055] The difference from the embodiment is that:

[0056] (I) Preparation of methyl cellulose mother solution

[0057] (1) Take 60 ml of pure water, heat it in a 98°C water bath until the water temperature is balanced, add 15 g of methyl cellulose powder, and stir thoroughly to make the methyl cellulose powder completely dispersed and wet;

[0058] The viscosity of the methylcellulose powder is 4000 cPs;

[0059] (2) Add 440 ml of pure water precooled to 4°C and continue stirring in an ice water bath for 30 min;

[0060] (3) After the ice water bath, autoclave was immediately performed. The autoclave parameters were: temperature 121°C, time 33 min;

[0061] (4) After high-pressure sterilization, the mixture is naturally cooled to room temperature and placed in a refrigerator at 4°C for 24 hours to obtain a methyl cellulose mother solution. The concentration of the methyl cellulose mother solution is 3.0% (w / v, g / ml) (there may be water loss during the preparation process, which can be supplemented. The quantitative amount is 500 ml).

[0062] The rest is the same as the embodiment.

[0063] In the embodiment, the methylcellulose powder is fully dispersed and moistened by stirring in hot water, and then immediately transferred to high temperature sterilization to ensure that the methylcellulose powder is in an insoluble state in the liquid. After sterilization, the methylcellulose is naturally cooled to gradually absorb water and dissolve as the temperature decreases, and the methylcellulose solution is uniformly dissolved. In Comparative Example 1, the methylcellulose dissolution process is complicated, the high-temperature dispersion time of methylcellulose is short and it cannot be fully moistened, and the addition of ice water and low-temperature dissolution easily causes uneven dissolution and produces lumps. Once the lumps are formed, they are not easy to disperse, and a solution with uniform viscosity cannot be obtained, which affects the subsequent colony formation. Methylcellulose is insoluble in water at high temperature and precipitates during the high-pressure process. The low-temperature dissolution step before high pressure is time-consuming and labor-intensive. Compared with Comparative Example 1, the operation of the embodiment is simpler, and the methylcellulose solution obtained is more uniform.

[0064] Comparative Example 3 Preparation of semi-solid culture medium

[0065] The difference from the embodiment is that:

[0066] (I) Preparation of methylcellulose mother solution (sterilized methylcellulose powder is directly used to prepare culture medium)

[0067] (1) Take methylcellulose powder and sterilize it under high pressure. The parameters of high pressure sterilization are: temperature 121°C, time 30 min;

[0068] (2) Take 500 ml of pure water, heat it in a 98°C water bath until the water temperature is balanced, add 15 g of methyl cellulose powder, and stir thoroughly to make the methyl cellulose powder completely dispersed and wet;

[0069] The viscosity of the methylcellulose powder is 4000 cPs;

[0070] (3) Place in a refrigerator and let stand at 4°C for 10 min to allow the methylcellulose to absorb water, swell and dissolve, thereby obtaining a methylcellulose mother liquor. The concentration of the methylcellulose mother liquor is 3.0% (w / v, g / ml) (there may be water loss during the preparation process, which can be replenished. The quantitative amount is 500 ml).

[0071] (II) Preparation of semi-solid culture medium

[0072] (1) Mix 500 ml of methylcellulose mother solution, 200 ml of IMDM medium mother solution, and 300 ml of fetal bovine serum to prepare "methylcellulose medium without added factors";

[0073] (2) Add magnetic stirring beads and place on a magnetic stirrer to stir to make the properties of the methylcellulose medium without added factors uniform; due to the viscosity of the medium, the magnetic stirrer cannot rotate normally;

[0074] (3) Crush the uneven transparent clumps of methylcellulose with a pipette tip and continue stirring;

[0075] (4) Place the unfactored methylcellulose medium in a refrigerator and let it stand at -20°C for 10 min to dilute the methylcellulose medium. Continue stirring and repeat this step to fully dissolve the methylcellulose. Stir until the lumps are basically gone and place in a 4°C refrigerator for 24 hours to obtain the methylcellulose medium.

[0076] (5) Adding induction factors and additives, same as in the embodiment.

[0077] Experimental hematopoietic stem cell colony culture assay

[0078] Here are the steps:

[0079] (1) Obtain umbilical cord blood mononuclear cells by density gradient centrifugation and adjust the final cell concentration to 0.5×10 6 / mL to obtain cell suspension.

[0080] (2) Take 200 μl of semi-solid culture medium (Example, Comparative Example 1, Comparative Example 2, Comparative Example 3, imported reagent, a total of 5 groups of culture medium; the imported reagent is STEMCELL methylcellulose semi-solid colony culture medium, item number H4534), add it to a 48-well culture plate, add sufficient physiological saline to the outer 24 wells, and maintain the humidity in the culture plate. Place the culture plate in a CO2 incubator and let it stand for 10 minutes to allow the gas in the culture medium to be discharged.

[0081] (3) Pipette 20 μl of the cell suspension and add it to the culture plate so that each well contains 1×10 cells. 4 , 3 parallel wells, 5% CO2, 37°C and saturated humidity.

[0082] (4) On the 14th day of culture, the culture plate was removed and the CFU-GM was observed and counted under an inverted microscope.

[0083] (5) Observe the status of the culture medium, such as Figure 1 As shown, the cell colony morphology and culture medium uniformity were observed, and it was found that the culture medium of Example 1 had the highest clarity. There were obvious infusible fibers in the solutions of Comparative Examples 1 and 2 (as shown by the arrows in the figure), and the methylcellulose of Comparative Example 2 was not uniformly dissolved, and there were small clumps with high viscosity ( Figure 1 D). The colony culture medium obtained from the methylcellulose mother solution prepared in the example has sufficient dissolution of methylcellulose and low impurity content. Compared with the comparative examples 1 and 2 of the cold water dissolution method, the impurity content in the culture medium is high, the methylcellulose is not fully dissolved, and there are small clumps with high viscosity, which affect the growth of colony cells and the number of colonies.

[0084] (6) Compare the effects of the four culture media on the number of colonies generated, such as Figure 2 As shown, among the number of colonies generated per 10,000 TNC cells, the average values ​​of the number of colonies generated in Comparative Example 1, Comparative Example 2 and Comparative Example 3 were 10.3±4.70, 4.15±1.97 and 3.52±2.51, respectively, and the average values ​​of the number of colonies generated in Example and imported reagents were 16.2±5.29 and 10.06±2.68, respectively. The number of colonies generated in Comparative Example 2 and Comparative Example 3 was small, which may be caused by impurities; the number of colonies in Comparative Example 1 increased compared with the imported reagent; the number of colonies generated in the embodiment increased significantly, so the semi-solid culture medium of the present invention can replace the imported reagent for colony detection.

[0085] The above examples are provided to those skilled in the art to fully disclose and describe how to make and use the claimed embodiments, rather than to limit the scope of the disclosure herein. Modifications that are obvious to those skilled in the art will fall within the scope of the appended claims.

Claims

1. A semisolid culture medium for inducing cell colony growth, characterized in that: It is composed of IMDM culture medium, methylcellulose, fetal bovine serum, induction factors and additives; wherein the concentration of methylcellulose is 1.3% to 1.5%, unit: g / ml; the concentration of fetal bovine serum is 20% to 40%, volume percentage; The specific types and final concentrations of the induction factors are as follows: SCF, 20-50 ng / ml; GM-CSF, 10-20 ng / ml; IL-3, 10-20 ng / ml; G-CSF, 10-50 ng / ml; IL-6, 10-50 ng / ml; Flt-3L 100-200 ng / mL; Valproic Acid 1 mM; TPO 50-100 ng / ml; Heparin sodium, 8-12 U / ml; The specific types and final concentrations of the additives are as follows: GultaMAX additive, 0.9% to 1.1%; β-mercaptoethanol, 0.25 to 0.35 mol / l; penicillin 90 to 110 U / mL; streptomycin 90 to 110 U / mL; The methylcellulose is added in the form of a methylcellulose mother liquor, and the methylcellulose mother liquor is prepared by the following method: (1) Take an appropriate amount of pure water, heat it in a water bath at 80-98℃ until the water temperature is balanced, add methylcellulose powder, and stir thoroughly to make the methylcellulose powder completely dispersed and wet; The viscosity of the methylcellulose powder is 3500 to 5600 cPs; (2) Continue heating in a water bath at 80-98°C for 10-20 min. (3) After the water bath heating is completed, high pressure sterilization is performed immediately; (4) After high pressure sterilization, cool naturally to room temperature and let stand for 16 to 24 hours; (5) Place the mixture at 4°C for 16 to 24 hours and mix thoroughly by inverting to obtain a methyl cellulose mother solution. The concentration of the methyl cellulose mother solution is 2.5% to 3.0%.

2. The semi-solid culture medium for inducing cell colony growth according to claim 1, characterized in that: The concentration of the methyl cellulose is 1.5%, and the concentration of the fetal bovine serum is 30%.

3. The semi-solid culture medium for inducing cell colony growth according to claim 1, characterized in that: The specific types and final concentrations of the induction factors are as follows: SCF, 50 ng / ml; GM-CSF, 20 ng / ml; IL-3, 20 ng / ml; G-CSF, 20ng / ml; IL-6, 20 ng / ml, Flt-3L 100 ng / mL, Valproic Acid 1 mM, TPO 50 ng / ml, heparin sodium, 10 U / ml.

4. The semi-solid culture medium for inducing cell colony growth according to claim 1, characterized in that: The specific types and final concentrations of the additives are as follows: GultaMAX additive, 1%; β-mercaptoethanol, 0.3 mol / l; penicillin, 100 U / mL; streptomycin, 100 U / mL.

5. The semi-solid culture medium for inducing cell colony growth according to claim 1, characterized in that: The viscosity of the methylcellulose powder is 4000 cPs.

6. The semi-solid culture medium for inducing cell colony growth according to claim 1, characterized in that: The temperature of the water bath was 98°C.

7. The semi-solid culture medium for inducing cell colony growth according to claim 1, characterized in that: The parameters of the high pressure sterilization are: temperature 121° C., time 16 to 35 min.

8. The method for preparing a semi-solid culture medium for inducing cell colony growth according to any one of claims 1 to 7, characterized in that: The following steps are involved: (I) Preparation of methyl cellulose mother solution (1) Take an appropriate amount of pure water, heat it in a water bath at 80-98℃ until the water temperature is balanced, add methylcellulose powder, and stir thoroughly to make the methylcellulose powder completely dispersed and wet; The viscosity of the methylcellulose powder is 3500 to 5600 cPs; (2) Continue heating in a water bath at 80-98°C for 10-20 min. (3) After the water bath heating is completed, high pressure sterilization is performed immediately; (4) After high pressure sterilization, cool naturally to room temperature and let stand for 16 to 24 hours; (5) Place the mixture at 4°C and let it stand for 16 to 24 hours, then mix thoroughly by inverting to obtain a methyl cellulose mother solution. The concentration of the methyl cellulose mother solution is 2.5% to 3.0%; (ii) Preparation of semi-solid culture medium: Mix methyl cellulose mother solution, IMDM culture medium mother solution and fetal bovine serum, add various induction factors and additives, mix well, and you have it.

9. The method for preparing a semi-solid culture medium for inducing cell colony growth according to claim 8, characterized in that: The viscosity of the methylcellulose powder is 4000 cPs; The temperature of the water bath is 98°C; The parameters of the high pressure sterilization are: temperature 121° C., time 33 min.

10. Use of the semi-solid culture medium for inducing cell colony growth according to any one of claims 1 to 7 in culturing cells.

11. Use of the semi-solid culture medium for inducing cell colony growth according to any one of claims 1 to 7 for inducing colony growth of monocytes, granulocytes and erythroid cells.

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