CN113736737B - A method for culturing primary glioma-related fibroblasts - Google Patents

Abstract

A primary glioma-related fibroblast culture method. Through the innovative combination of differential cell adhesion method and cell nutrient screening method, this new culture method can avoid the contamination of a large number of non-fibroblasts during the primary cell culture process. Cell screening is difficult. This new method can achieve superior results than traditional methods using only easily available conventional reagents, and can achieve complete purification of P0 generation cells in a relatively short period of time, which is worthy of promotion. Currently, our research group has cultured 37 cases of glioma-related fibroblasts from 46 fresh human glioma tissues, with a success rate of 80%. The existing methods for quickly cultivating tumor-related fibroblasts are mainly through flow cell sorting or magnetic bead sorting. The instruments and reagents are expensive, the experimental technology is complex, and the operation is difficult; and the invention of this cell culture method is very difficult for experiments. The economics of research are of great value.

Description

A method for culturing primary glioma-related fibroblasts

Technical field

The present invention relates to the field of cell culture methods, and in particular to a primary glioma-related fibroblast culture method.

Background technique

There is no effective culture method for primary glioma-associated fibroblasts.

One of the existing culture methods: sterile flow cell sorting or magnetic bead sorting, requires expensive instruments and flow antibodies, has high requirements on experimental conditions and operating techniques, and is poor in economy and convenience.

If traditional culture methods are used, such as the nutritional screening method, it is necessary to wait for several generations of cell passage before obtaining purified primary fibroblasts, which greatly reduces the number of generations of fibroblasts that can be used for subsequent experiments; in addition, the proliferation rate of fibroblasts is very slow and the culture cycle is long. .

In summary, the culture of glioma-related fibroblasts urgently needs to solve the problems of long culture cycle, difficult cell purification, and high price.

Contents of the invention

In order to solve the problems existing in the above-mentioned prior art, the purpose of the present invention is to provide a primary glioma-related fibroblast culture method.

In order to achieve the above objects, the technical solution of the present invention is:

A method for culturing primary glioma-related fibroblasts, including the following steps:

Step a. Wash the tissue: Place the tumor tissue in a sterile petri dish on ice, take tissue washing buffer to wash the tissue blood clot twice; cut the tissue block with ophthalmic scissors until it becomes paste;

Step b. Digest tissue:

Add tissue digestion enzyme solution 1 to the minced tissue, immediately transfer the tissue suspension to a 15ml test tube with a pipette and gently pipet, incubate at 37°C for 5 minutes, pipet the suspension again and incubate for 5 minutes;

Continue to add tissue digestion enzymatic solution 2 to the above tissue suspension and continue to incubate at 37°C for 5 minutes. Pipet the suspension again and incubate for 5 minutes. When the tissue suspension can be smoothly absorbed with a 1mL pipette, the digestion is complete;

Step c. Filtration: Add 10 mL PBS to the tissue suspension, blow evenly and filter through a 70 μm cell strainer. Centrifuge the obtained cell suspension at 400xg for 3 minutes and discard the supernatant;

Step d. Remove red blood cells: Add 1x red blood cell lysis buffer 3 times the volume of cell sedimentation to the cell sediment, and let stand at room temperature for 5 minutes; add 8 mL PBS to the cell suspension, centrifuge and discard the supernatant;

Step e. Primary fibroblast culture: Prepare 10 mL of cancer stem cell culture medium, take 10 mL of primary cell culture medium 1, and add 10 μL each of 1000x EGF and 1000x bFGF (final concentration 20 ng/ml). Count the cells, 2x10 ⁶ Cells were added to culture medium and cultured for 24 h. Discard the supernatant of the culture dish, wash with PBS and tap the bottom of the culture dish three times. Observe the cell debris and sediment under the microscope. After cleaning, add MSC culture medium to the culture dish. After three days, observe the cell morphology and growth and change the medium. Take photos and record.

Step f. Culture and passage of primary glioma-related fibroblasts: When the confluence of P0 generation cells is about 90%, digest the cells with digestive enzymes (0.25% trypsin + EDTA), and centrifuge the cell suspension for 3 minutes at 400xg; replace Continue culturing in 10% FBS-DMEM complete culture medium (1x10 ⁶ cells/75cm ² ). (Note: The number of primary cell passages should be less than 10 generations to avoid possible genetic and phenotypic changes.)

Further, the tissue digestion enzymatic solution 1 is: 1.85 mL of tissue dissociation solution + 100 μL of papain stock solution, 50 μL of deoxyribonuclease-I is added and stored at -20°C; tissue dissociation solution: deionized water ( 441mL) + 10x Hank's balanced salt solution (50mL) + D-glucose solution (300mg/mL) (9mL), store at -20°C.

Further, the tissue digestion enzymatic solution 2: tissue dissociation solution (100 mL) + collagenase-I (70 mg) + hyaluronidase (70 mg), fully dissolved, aliquoted, and stored at -20°C.

Further, the primary cell culture medium 1: DMEM/F12 (480 mL) + penicillin-streptomycin (5 mL) + 50x B27 (10 mL), stored at 4°C.

Further, the primary cell culture medium 2: mesenchymal stem cell culture medium, stored at 4°C.

Further, the 1000x b-FGF: b-FGF (50 μg) + deionized water (2.5 mL) + bovine serum albumin (2.5 mg), filtered through a sterile filter, and stored at -20°C.

Further, the 10% FBS-DMEM: FBS (50 mL) + DMEM (445 mL) + P/S (5 mL), stored at 4°C.

Compared with the existing technology, the beneficial effects of the present invention are:

1. Principle innovation: Through the innovative combination of differential cell adhesion method and cell nutrient screening method, this new culture method can avoid the difficulty of cell selection caused by mixing a large number of non-fibroblasts in the primary cell culture process.

2. Convenience: This new method can achieve superior results than traditional methods by using only easily available conventional reagents, and can achieve complete purification of P0 generation cells in a relatively short period of time, which is worthy of promotion.

3. High efficiency: Currently, our research group has cultured 37 cases of glioma-related fibroblasts from 46 fresh human glioma tissues, with a success rate of 80%.

4. Economical: The existing methods for quickly cultivating tumor-related fibroblasts mainly use flow cell sorting or magnetic bead sorting. The instruments and reagents are expensive, the experimental technology is complex, and the operation is difficult; and this cell culture method The invention is of great value to the economy of experimental research.

Description of the drawings

Figure 1 is a flow chart of primary glioma-related fibroblast culture.

Figure 2 shows the light microscope morphology of primary glioma-related fibroblasts.

Figure 3 shows the immunofluorescence staining of fibroblasts: FAP (green), Fibronectin (red).

Figure 4 αSMA, a characteristic marker of fibroblasts, is highly expressed in glioma-related fibroblasts.

Detailed ways

The technical solution of the present invention will be further described in detail below in conjunction with the accompanying drawings (Figures 1-4) and specific implementation modes:

1. Instruments and reagents

Ice box, crushed ice, ophthalmic scissors, ophthalmic tweezers, 6cm cell culture dish, 10cm cell culture dish, sealing film, 15mL centrifuge tube, 50mL centrifuge tube, 0.22μM sterile filter, constant temperature water bath, high-speed centrifuge, transfer Liquid tubes, Eppendorf pipettes, Thermo cell pipettes

1. Reagents

Phosphate buffered saline (PBS), penicillin-streptomycin, papain, hyaluronidase, collagenase-I, trypsin, MSC culture medium, DMEM/F12 culture medium, basic fibroblast growth factor (bFGF ), epidermal growth factor (EGF), B-12, fetal bovine serum (FBS), high glucose medium (DMEM), 10x red blood cell lysate, deionized water (UP-H ₂ O), bovine serum albumin ( BSA), D-glucose, EBSS, deoxyribonuclease-I

2. Working fluid configuration

a. Fresh tumor tissue preservation solution: 1% penicillin-streptomycin (5mL) + 99% phosphate buffered saline solution (495mL).

b. Tissue washing buffer: phosphate buffered saline solution (450mL) + bovine serum albumin (5g) + penicillin-streptomycin (5mL), filtered through a 0.22μM sterile filter, and stored at 4°C.

c. Tissue dissociation solution: deionized water (441mL) + 10x Hank’s balanced salt solution (50mL) + D-glucose solution (300mg/mL) (9mL), stored at -20°C.

d. Papain stock solution: papain (20 mg) + EBSS (1 mL), filtered through a 0.22 μM sterile filter, and stored at 4°C.

e. Tissue digestion enzymatic solution 1: tissue dissociation solution (1.85 mL) + papain stock solution (100 μL ready for use) + deoxyribonuclease-I (50 μL), stored at -20°C.

f. Tissue digestion enzymatic solution 2: Tissue dissociation solution (100mL) + Collagenase-I (70mg) + Hyaluronidase (70mg). After fully dissolving, aliquot and store at -20°C.

g. 1x red blood cell lysis solution: 10x red blood cell lysis solution (10mL) + sterile distilled water (90mL).

h. Primary cell culture medium 1: 96% DMEM/F12 medium (480 mL) + 1% P/S (5 mL) + 2% B27 (50x) (10 mL), stored at 4°C.

i. Primary cell culture medium 2: mesenchymal stem cell culture medium, stored at 4°C.

j. 1000x basic fibroblast growth factor: b-FGF (50 μg) + deionized water (2.5 mL) + bovine serum albumin (2.5 mg), filtered through a sterile filter, and stored at -20°C.

k. 1000x epidermal growth factor (EGF): filtered through a sterile filter and stored at -20°C.

l. 10% fetal bovine serum (FBS)-high glucose medium (DMEM): 10% fetal bovine serum (50mL) + 89% high glucose medium (445mL) + 1% penicillin-streptomycin (5mL), 4 Store at ℃.

2. Operation steps

a. Wash the tissue: Place the tumor tissue in a sterile 6cm petri dish on ice, take tissue washing buffer to wash the tissue blood clot twice; cut the tissue block with ophthalmic scissors until it becomes paste.

b. Digestive tissue:

Add tissue digestion enzyme hydrolyzate 1 to the minced tissue, transfer the tissue suspension to a 15ml test tube with a pipette and pipe gently, incubate at 37°C for 5 minutes, pipet the suspension again and incubate for 5 minutes;

Continue to add tissue digestion enzymatic solution 2 to the above tissue suspension to resuspend the tissue, incubate at 37°C for 5 minutes, pipet the suspension again and incubate for 5 minutes; when the tissue suspension can be smoothly absorbed with a 1mL pipette, the digestion is complete.

c. Filtration: Add 10 mL of PBS to the tissue suspension, blow evenly and filter with a 70 μM cell strainer. Centrifuge the obtained cell suspension at 400xg for 3 minutes and discard the supernatant.

d. Remove red blood cells: Add 1x red blood cell lysis buffer (3 times the cell sedimentation volume) to the cell sediment and let it stand at room temperature for 5 minutes; add 8mL PBS to the cell suspension, centrifuge and discard the supernatant.

e. Primary fibroblast culture (key): Now prepare 10 mL of cancer stem cell culture medium, take 10 mL of primary cell culture medium 1, and add 10 μL each of 1000x EGF and 1000x bFGF (final concentration 20 ng/ml), and count the cells, 2x10 ⁶ cells were added to the culture medium and cultured for 24 hours. Discard the supernatant of the culture dish, wash with PBS and tap the bottom of the culture dish three times. Observe the cell debris and sediment under the microscope. After cleaning, add MSC culture medium to the culture dish. After three days, observe the cell morphology and growth and change the medium. Take photos and record.

f. Culture and passage of primary glioma-related fibroblasts: When the confluence of P0 generation cells is about 90%, digest the cells with digestive enzymes (0.25% trypsin + EDTA), and centrifuge the cell suspension for 3 minutes at 400xg; replace 10 Continue culturing in %FBS-DMEM complete culture medium (1x10 ⁶ cells/75cm ² ). (Note: The number of primary cell passages should be less than 10 generations to avoid possible genetic and phenotypic changes.)

The above cell culture operation process is shown in Figure 1: fresh human brain glioma tissue is obtained, and single cell suspension is obtained through physical shearing and chemical digestion respectively; then a new primary glioma-related fibroblast culture method is used: first The primary cells were cultured in suspension in the cancer stem cell culture medium (to select fibroblasts with strong adhesion ability). After 24 hours, the non-adherent cells in the supernatant were removed and the MSC culture medium was replaced to continue culturing.

As shown in Figure 2, for successfully cultured primary cells, their cell morphology is similar to tumor-associated fibroblasts, but very different from glioma cell morphology; in addition, the cell culture efficiency is very high, on day 15 of passage 0 The confluence of left and right primary fibroblasts can reach 90%, and the cell morphology is uniform, with almost no other non-fibroblasts present.

Cellular immunofluorescence was used to detect whether the primary cells highly expressed known tumor-associated fibroblast cell molecular markers, such as fibroblast activating protein (FAP) and fibronectin (Fibronectin). The results suggested that the above morphology was consistent with fibroblasts. The characterized primary cells all highly expressed fibroblast-specific markers, as shown in Figure 3. This shows that this method can effectively and quickly culture glioma-related fibroblasts.

Finally, West Blot was used to detect the expression levels of the fibroblast-specific marker αSMA in primary fibroblasts, glioma cell lines (U251), and mesenchymal stem cells (MSC). The results suggested that glioma-associated fibroblasts αSMA is highly expressed, which is the same as the expression of positive control mesenchymal stem cells, while the negative control cell U251 does not express αSMA, again suggesting that the primary cells cultured by this method belong to fibroblasts.

The above are only specific embodiments of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or substitutions that are not thought of through creative efforts should be covered by the protection scope of the present invention. Therefore, the protection scope of the present invention should be subject to the protection scope defined by the claims.

Claims (4)

1. A primary glioma-associated fibroblast cell culture method, comprising the steps of:

step a, washing tissues: placing tumor tissue in a sterile culture dish on ice surface, and taking tissue washing buffer solution to wash tissue blood clot twice; ophthalmic scissors break up tissue blocks until they become pasty;

step b, digesting the tissue:

adding the tissue digestion enzyme solution 1 into the sheared tissues, immediately transferring the tissue suspension to a 15ml test tube by using a pipette, gently blowing, carrying out warm bath at 37 ℃ for 5 minutes, and carrying out warm bath after blowing the suspension again for 5 minutes;

continuously adding tissue digestion enzymolysis liquid 2 into the tissue suspension to resuspend the tissue, carrying out warm bath for 5 minutes at 37 ℃, and carrying out warm bath for 5 minutes after blowing the suspension again; when the tissue suspension can be successfully sucked by a 1mL pipette, the digestion is completed;

step c, filtering: adding 10mL of PBS into the tissue suspension, uniformly blowing, filtering by adopting a 70 mu M cell filter screen, centrifuging the obtained cell suspension for 400Xg 3 minutes, and discarding the supernatant;

step d, removing red blood cells: adding 1x erythrocyte lysate 3 times the volume of the cell sediment into the cell sediment, and standing for 5 minutes at room temperature; adding 8mL of PBS to the cell suspension, centrifuging, and discarding the supernatant;

step e, primary fibroblast culture: preparing 10mL of tumor stem cell culture solution, taking 10mL of primary cell culture solution 1, and adding 10 mu L of 1000 XEGF and 1000 Xb-FGF respectively; final concentration 20ng/ml, cell count, 2X10 ⁶ The individual cells were added to the culture medium and cultured for 24 hours. Discarding the culture dish supernatant, washing with PBS and tapping the bottom of the culture dish for three times, observing cell fragments and sediments under a mirror, and adding MSC culture medium into the culture dish after completely washing; observing cell morphology and growth condition after three days, changing liquid, photographing and recording;

step f, primary glioma-related fibroblast culture and passage: when the pooling degree of the P0 generation cells is 90%, digesting the cells with 0.25% pancreatin and EDTA, centrifuging the cell suspension for 3min and 400g; the whole culture solution of 10% FBS-DMEM is replaced for continuous culture, and the culture is 1 multiplied by 10 ⁶ Individual cells/75 cm ² ；

The tissue digestion enzymolysis liquid 1 is as follows: 1.85mL of tissue dissociation solution, 100 [ mu ] L of papain stock solution, 50 [ mu ] L of currently-used adding DNA (deoxyribonucleic acid) enzyme, and-20 ℃ below zero; tissue dissociation liquid: 300mg/mL of deionized water +10XHBSS +D-glucose solution and preserving at-20 ℃;

the tissue digestion enzymolysis liquid 2: tissue dissociation solution, collagenase-I and hyaluronidase, and packaging after full dissolution, and preserving at-20deg.C;

the primary cell culture fluid 1: DMEM/f12+green-streptomycin+50×b27, stored at 4 ℃;

2. the method of claim 1, wherein the 1000 xb-FGF: b-FGF+deionized water+BSA, sterile filtration sterilization by a 0.22 mu m filter screen, and preserving at-20 ℃.

3. The method according to claim 1, wherein the 10% FBS-DMEM: FBS+DMEM+P/S, stored at 4deg.C.

4. The method of claim 1, wherein the primary cells are passaged less than 10 passages to avoid possible genetic and phenotypic changes.