Chicken primary brain microvascular endothelial cell extraction and purification method

Abstract

The invention relates to the technical field of cell biology, in particular to a method for extracting and purifying chicken primary brain microvascular endothelial cells. The method for extracting and purifying the chicken primary brain microvascular endothelial cells comprises the following steps: (1) acquiring and pretreating chicken embryo brain tissues; (2) primary enzyme digestion; (3) removing myelin sheath; (4) secondary enzyme digestion; (5) performing Percoll gradient centrifugal purification; and (6) performing differential adherent cell culture and screening. According to the chicken primary brain microvascular endothelial cell extraction and purification method, through collaborative optimization of double enzyme digestion, demyelination treatment and Percoll gradient centrifugation, the purity of separated cells is remarkably improved, the cell survival rate is high, the cost is low, consumed time is short, and the method is suitable for large-scale preparation.

Description

Technical Field

[0001] The invention relates to the technical field of cell biology, and in particular to a method for extracting and purifying primary chicken brain microvascular endothelial cells. Background Art

[0002] Brain microvascular endothelial cells (BMECs) are the core functional units of the blood-brain barrier (BBB) ​​and play a key role in maintaining the stability of the internal environment of the central nervous system (CNS). The in vitro culture of BMECs is of great significance for the study of neurodegenerative diseases, tumor metastasis, viral invasion and drug permeability.

[0003] At present, the commonly used extraction methods mainly include mechanical separation, enzyme digestion and magnetic bead sorting, but there are still the following defects: (1) Insufficient purity: Traditional mechanical separation methods are prone to mixing with fibroblasts, glial cells and pericytes, and the purity is usually less than 70%. (2) Low survival rate: Long-term enzyme digestion (such as collagenase digestion for more than 30 minutes) causes cell membrane damage, and the survival rate is less than 50%. (3) Complicated process: Existing technologies rely on multi-step centrifugation or antibody sorting (such as CD31 magnetic bead sorting), which takes 8-10 hours to operate and is costly.

[0004] Compared with mammals, the brain structure of birds is more special, such as underdeveloped cerebral cortex, developed midbrain, and small olfactory bulb. The isolation and culture of BMECs in mammals still have the above defects, so the isolation and culture of poultry BMECs is even more difficult. Given that the isolation and culture of poultry BMECs is of great significance for the study of poultry neurotropic viruses and the establishment of animal neurological disease models, it is urgent to develop a purification method with high purity, high survival rate and low cost. Summary of the invention

[0005] The present invention provides a method for extracting and purifying primary chicken brain microvascular endothelial cells. Through the coordinated optimization of double enzyme digestion, demyelination treatment and Percoll gradient centrifugation, the purity of separated cells is significantly improved, the cell survival rate is high, the cost is low, the operation is simplified, and the problems existing in the prior art are solved.

[0006] In order to achieve the above-mentioned invention object, the technical solution adopted by the present invention is: A method for extracting and purifying primary chicken brain microvascular endothelial cells comprises the following steps: (1) Acquisition and pretreatment of chicken embryo brain tissue Select 16-19 day old SPF chicken embryos that are about to hatch, separate the head and trunk; separate the brain tissue, collect the brains without meninges, wash them, and cut them into brain tissue blocks; transfer the brain tissue blocks to a sterile centrifuge tube, add DMEM, and pipette until the tissue becomes turbid; (2) Primary enzyme digestion Digest the tissue blocks treated in step (1) with the primary enzyme digestion solution, and stir the suspension magnetically to remove interstitial proteins; then add a certain volume of DMEM to resuspend the tissue, centrifuge at 1000 g for 10 min at 4°C, and discard the supernatant; The composition of the primary enzyme digestion solution is: a mixture of collagenase type II and DNAse I; (3) Demyelination Resuspend with BSA-DMEM; centrifuge at 1000 g for 20 min at 4°C, and discard the phospholipid upper layer and BSA in turn; (4) Secondary enzyme digestion The tissue treated in step (3) was digested with a secondary enzyme digestion solution, and the suspension was magnetically stirred for 30 minutes to release the endothelial cells; The secondary enzyme digestion solution is composed of: a mixture of Accutase cell digestion solution and DNAse I; (5) Percoll gradient centrifugation purification During the secondary enzyme digestion in step (4), the Percoll is centrifuged in a horizontal centrifuge to obtain a Percoll gradient centrifugation liquid for later use; Add 10 mL of DMEM to the cells after the secondary enzyme digestion in step (4), centrifuge at 1000 g for 10 min at 4°C, discard the supernatant, and obtain the precipitate; resuspend the precipitate with 2 mL of DMEM, then slowly transfer the cell solution along the tube wall into the Percoll gradient centrifugation liquid centrifuged above, and centrifuge at 700 g for 10 min at 4°C; the obtained middle layer of liquid is the cells; (6) Differential cell adhesion culture and screening The endothelial cells separated in step (5) were directly plated in a cell culture dish treated with coating solution. After standing at 37°C and 5% CO2 for 45 min, the cell solution was plated in a new well. After 24 h, the culture medium was replaced with a medium without puromycin and culture was continued.

[0007] Furthermore, the operation of separating brain tissue in step (1) is as follows: separating the cerebrum and cerebellum of the head of the chicken embryo in PBS, peeling off the meninges with sterilized filter paper, and collecting the brain without the meninges; washing the separated brain tissue with DMEM for 3 times, then removing the DMEM, and cutting the brain tissue into 1 cm 3 Size of brain tissue.

[0008] Furthermore, when pipetting, first use a 25 mL pipette and then use a 10 mL pipette.

[0009] Furthermore, in step (2), collagenase type II in the primary enzyme digestion solution was diluted to 10 mg / mL using DMEM before preparation; DNAse I was diluted to 1 mg / mL using PBS before preparation; the volume ratio of collagenase type II to DNAse I was 3:1; the magnetic stirring conditions in step (2) were 37° C. for 1 h; and the volume of DMEM used in step (2) was 10 mL.

[0010] Furthermore, in step (3), the BSA-DMEM is 20% W / V BSA-DMEM.

[0011] Furthermore, in step (3), the BSA-DMEM was resuspended 25 times.

[0012] Furthermore, in step (4), DNAse I in the secondary enzyme digestion solution was diluted to 1 mg / mL using PBS before preparation; the volume ratio of Accutase cell digestion solution and DNAse I was 100:1; and the magnetic stirring conditions in step (4) were 37° C. for 30 min.

[0013] Furthermore, the Percoll gradient centrifugation operation in step (5) is: centrifugation at 3000 rpm for 1 h at 4°C.

[0014] Furthermore, the composition of the Percoll gradient centrifugation solution in step (5) is: 19 mL 1×PBS+1 mL 10×PBS+1 ml FBS+10 mL Percoll, a total of 31 mL, filtered, and stored at 4°C.

[0015] Furthermore, the specific operation of step (6) cell differential adhesion culture and screening is as follows: S1: Mix the intermediate layer liquid from step (5) with 5 mL of DMEM and centrifuge at 1000 g for 10 min at 4°C to obtain a precipitate; S2 resuspends the pellet from S1 in complete medium containing puromycin, and 2 Use 200 μL for the area; S3 treated the cell culture dish with the pre-screened coating solution, aspirated the overnight cell culture dish coating solution, washed the cell culture dish with sterile 1×PBS, and used it directly after washing; the cell solution obtained in S2 was directly spread on the cell culture dish and left to stand at 37°C, 5% CO2 for 45 min; S4 Take the cell solution into a new well and replace it with medium without puromycin after 24 h to continue culturing; S5 medium was changed every 2-3 days until the cell density reached 90%.

[0016] Furthermore, the composition of the complete culture medium containing puromycin in S2 of step (6) is: 400 mL DMEM medium + 100 mL FBS (20%) + 0.25 mL FGFb (20 μg / mL; 0.05%) + 0.5 mL heparin sodium (100 μL / mL; 0.1%) + 0.5 ml puromycin (4 mg / mL; 0.1%).

[0017] Furthermore, the cell culture dish treated with the coating solution in step (6) is a recombinant fibronectin-coated culture dish.

[0018] Furthermore, the recombinant fibronectin is FN recombinant fibronectin with a concentration of 0.1 mg / mL.

[0019] Beneficial effects of the present invention: 1. The extraction and purification method of the present invention is efficient and low-cost in extracting and purifying chicken primary brain microvascular endothelial cells from chicken embryo brain tissue, and is suitable for biomedical research, drug screening and the construction of vascular-related disease models.

[0020] 2. The present invention removes interstitial proteins and myelin lipids by double enzyme digestion, and combines Percoll gradient separation of impurities. The CD31 positive rate of chicken primary brain microvascular endothelial cells (chBMECs) is ≥92%, which is generally ≤80% compared with the traditional method, and the purity is significantly improved. By optimizing the survival rate, specifically using neutral digestive enzyme digestion, the enzyme digestion time is significantly shortened, and the centrifugation parameters are accurately controlled, and the cell survival rate reaches more than 82%. This extraction and purification method does not require magnetic bead sorting or antibody labeling. Compared with the mammalian separation method, the present invention uses a coating solution (recombinant fibronectin), the digestion solution is simple in composition, and the reagent cost is saved by 40%. The coating solution used in the mammalian separation method is a mixture of collagenase IV and fibronectin, and the digestive enzyme II is collagenase / dispase, and this type of reagent is expensive. In addition, compared with the traditional method of extraction and purification, which takes 8-10 hours, the method of the present invention takes a total of 6 hours, which is suitable for large-scale preparation.

[0021] 3. The control of digestion time, centrifugal parameters and cell culture conditions during cell separation in the method of the present invention effectively improves cell purity. In view of the fact that there is no special method for separating and extracting chicken brain microvascular endothelial cells, this method has a high cell survival rate on the basis of improving the purity of the separated chicken brain microvascular endothelial cells, and is used for experimental research and has good application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a schematic flow chart of the separation steps of the extraction method according to an embodiment of the present invention; Figure 2This is a diagram showing the effect of different matrix-coated cell culture chambers (transwell) on permeability in the extraction method of the present invention (poly-D-lysine: D-polylysine; gelatin: gelatin; fibronectin: fibronectin; ns: no difference; ***P<0.001); Figure 3 The growth of primary chicken brain microvascular endothelial cells (chBMECs) at different time points in the cell culture of the embodiment of the present invention; Figure 4 The CD31 immunofluorescence results of chBMECs obtained in the examples of the present invention; Figure 5 The ZO-1 immunofluorescence results of chBMECs obtained in the examples of the present invention; in, Figure 4 The figures in the middle are, from top to bottom: immunofluorescence results of endothelial cell marker molecule CD31 of chBMECs cultured without differential adhesion, cultured with differential adhesion for 30 min, cultured with differential adhesion for 45 min, and cultured with differential adhesion for 1 h. DETAILED DESCRIPTION

[0023] In order to clearly illustrate the technical features of this solution, the present invention is described in detail below through specific implementation methods and in conjunction with the accompanying drawings. The scope of the present invention is not limited to the following embodiments. Professionals in the field can understand that various changes and modifications can be made to the present invention without departing from the spirit and scope of the present invention.

[0024] The instruments, reagents, materials, etc. involved in the following embodiments, unless otherwise specified, are all conventional instruments, reagents, materials, etc. in the prior art and can be obtained through regular commercial channels. The experimental methods, detection methods, etc. involved in the following embodiments, unless otherwise specified, are all conventional experimental methods, detection methods, etc. in the prior art.

[0025] 1. Experimental Materials (1) 16-19 day old SPF chicken embryos.

[0026] (2) Reagents: PBS, DMEM, FBS, Percoll cell separation medium (Solarbio, P8370), recombinant human FGFb (Solarbio, P00032), heparin sodium (Solarbio, H8060), puromycin (Solarbio, P8230), collagenase type II (Yeasen, 40508ES60), FN recombinant fibronectin (Solarbio, F8181), gelatin (Solarbio, G0040), D-polylysine (Solarbio, D6790), DNAse I (Solarbio, D8071), BSA (Solarbio, A8010), Accutase cell digestion solution (Yeasen, 40506ES60).

[0027] 2. Experimental Reagents (1) Coating solution: Gelatin is diluted to 0.1%, D-polylysine is prepared to 50 μg / mL, and FN recombinant fibronectin is used at a concentration of 0.1 mg / mL. Store at 4°C for one week.

[0028] (2) Percoll gradient centrifugation liquid: 19 mL 1×PBS + 1 mL 10×PBS + 1 ml FBS + 10 mLPercoll, a total of 31 mL, filter, and store at 4°C.

[0029] (3) chBMECs complete culture medium (500 mL): 400 mL DMEM medium + 100 mL FBS (20%) + 0.25 mL FGFb (20 μg / mL; 0.05%) + 0.5 mL heparin sodium (100 μL / mL; 0.1%) + 0.5 ml puromycin (4 mg / mL; 0.1%). The culture medium with puromycin is only used 24 hours before culture. Store at 4°C.

[0030] (4) Initial enzymatic digestion solution: 0.6 mL collagenase type II (diluted to 10 mg / mL in DMEM) + 0.2 mL DNAse I (diluted to 1 mg / mL in PBS).

[0031] (5) Demyelination fluid: BSA-DMEM (20% w / v), filtered.

[0032] (6) Secondary enzyme digestion solution: 10 mL Accutase cell digestion solution + 0.1 mL DNAse I (diluted to 1 mg / mL in PBS).

[0033] 3. Experimental steps See attached Figure 1The extraction and purification method of primary chicken brain microvascular endothelial cells adopts the following operation steps: (1) Take 10 SPF chicken embryos that are about to hatch at 16-19 days old. Use sterile forceps to open the air chamber of the embryos, take out the embryos, and separate the head and trunk with sterile ophthalmic scissors and forceps. (2) Use sterile forceps to separate the cerebrum and cerebellum of the chicken embryo head separated in step (1) in PBS, peel off the meninges with sterile filter paper (roll), and collect the brain without meninges; (3) Transfer the separated brain tissue to a sterile glass culture dish, wash the brain tissue three times with DMEM, remove the DMEM, and cut the brain tissue into pieces of about 1 cm using new sterile ophthalmic scissors. 3 Size of tissue blocks; (4) Transfer the chopped brain tissue pieces to a sterile 50 mL centrifuge tube, add 13.5 mL of DMEM, and pipette with a 25 mL pipette first, then with a 10 mL pipette, until the tissue becomes turbid; (5) Digest the tissue blocks obtained in step (4) with the primary enzyme digestion solution, transfer the suspension to a conical flask with magnetic beads, place it on a magnetic stirrer, and stir it at 37°C for 1 h; (6) Add 10 mL of DMEM to resuspend the tissue (the liquid volume is now about 25 ml), centrifuge at 1000 g for 10 min at 4°C, and discard the supernatant with a pipette tip; (7) Resuspend the sample in 25 mL of BSA-DMEM (20% W / V) for about 25 times and centrifuge at 1000 g for 20 min at 4°C. Discard the phospholipid layer and BSA in turn. (8) Digest the tissue treated in step (7) with secondary enzyme digestion solution, transfer the suspension to a conical flask with magnetic beads, and incubate with a magnetic stirrer at 37°C for 1 h; (9) During the digestion in step (8), centrifuge Percoll in advance using a horizontal centrifuge. The centrifugation conditions are: 3000 rpm, 1 h, 4°C. (10) Add 10 mL of DMEM to the cells after secondary digestion, centrifuge at 1000 g for 10 min at 4°C, and discard the supernatant; (11) Resuspend the precipitate obtained in step (10) with 2 mL of DMEM, then slowly transfer the cell solution along the tube wall into the centrifuged density gradient Percoll, centrifuge at 700 g, 10 min, 4°C; the cells obtained are the middle layer liquid, not the precipitate; (12) About 12 mL of the above middle layer cells were mixed with 5 mL of DMEM and centrifuged at 1000 g for 10 min at 4°C. (13) Resuspend the pellet obtained in step (12) in complete medium containing puromycin.2 Use 200 μL for the area; (14) Treat the cell culture dish with the coating solution that has been screened in advance, aspirate the overnight coating solution of the dish, wash it three times with sterile 1× PBS, and use it directly after washing; directly plate the cells in the cell culture dish treated with the coating solution; (15) After standing at 37°C and 5% CO2 for 45 min, the cell suspension was transferred to a new well. After 24 hours, the culture medium was replaced with one without puromycin and continued to be cultured. (16) Change the culture medium every 2-3 days until the cell density reaches 90%.

[0034] The principles for screening the coating solution for cell culture dishes in the above step (14) are: ① non-toxic to cells, ② ensuring good permeability in the cell culture chamber (transwell) as much as possible, and providing a technical basis for the establishment of an in vitro blood-brain barrier model. Three coating solutions commonly used in mammals were selected, namely fibronectin (0.1 mg / mL), gelatin (0.1%), and D-polylysine (50 μg / mL). After coating the transwell or cell culture dish at 4°C overnight, it can be used.

[0035] Using the sodium iodide test, add 10% sodium iodide to the upper layer of the transwell, place it in a cell culture incubator, collect the lower layer of liquid at 6, 12, 16, 24, and 48 hpi, and measure the content of sodium iodide. The higher the content, the better the permeability. Figure 2 The results are the results of the investigation on the effect of the above-mentioned different coating solutions on the permeability of the cell culture chamber transwell. Figure 2 The results show that as the placement time of the transwell in the incubator increases, the permeability of the transwell coated with recombinant fibronectin is significantly stronger than that of gelatin and D-polylysine at 48 h, and the growth time of chBMECs in the present invention is longer. Therefore, recombinant fibronectin (0.1 mg / mL) coating solution is selected to treat the cell culture dish.

[0036] The cell culture growth in step (14) of the method of this embodiment was observed. Specifically, the isolated primary chicken brain microvascular endothelial cells chBMECs were plated in a cell culture dish treated with recombinant fibronectin, and cultured after differential attachment for 45 minutes. The cell morphology and state were observed on the 2nd, 3rd, 5th and 7th days. The results are as follows: Figure 3 .Depend on Figure 3 It can be seen that chBMECs began to crawl out and grow from the second day, showing a spindle shape, and the cells became cobblestone-shaped after they were fully grown.

[0037] Figure 4 This is an investigation into the effect of the differential adhesion treatment step on the purity of chBMECs during cell separation and culture. Figure 4The immunofluorescence results of endothelial cell marker CD31 of chBMECs without differential attachment treatment and differential attachment culture are given. Specifically, on the 7th day of chBMECs culture, the cells were fixed and then the endothelial cell marker CD31 antibody was used to perform cell immunofluorescence staining to identify the cell type and purity. Figure 4 The results show that positive cells are red, and the purity of cells without differential attachment treatment and cultured with differential attachment for 30 minutes is low. Less than 10% of positive cells are visible to the naked eye, about 90% of positive cells are visible after differential attachment for 1 hour, and 95% of positive cells are visible after differential attachment for 45 minutes. Combining the above effects and time cost, 45 minutes was finally selected as the optimal differential attachment time.

[0038] Figure 5 The results of immunofluorescence staining of tight junction protein ZO-1 on day 10 of chBMECs culture. On day 10 of chBMECs culture, cells were fixed and immunofluorescence staining was performed using tight junction protein ZO-1 antibody to identify cells. Figure 5 It can be seen that the positive cells are green, the ZO-1 protein is localized on the cell membrane, the cell membrane is obviously green, and the cells are cobblestone-shaped, indicating that the cells are well connected.

[0039] The above specific implementation manner cannot be used as a limitation on the protection scope of the present invention. For those skilled in the art, any substitution, improvement or change made to the implementation manner of the present invention falls within the protection scope of the present invention.

[0040] The matters not described in detail in the present invention are all known technologies to those skilled in the art.

Claims

1. A method for extracting and purifying primary chicken brain microvascular endothelial cells, characterized in that: The steps are as follows: (1) Acquisition and pretreatment of chicken embryo brain tissue Take 16-19 day old SPF chicken embryos, collect brain tissues after removing the meninges, add DMEM and pipette until turbid; (2) Primary enzyme digestion Digest the tissue treated in step (1) with a primary enzyme digestion solution, and digest with magnetic stirring; resuspend the tissue, centrifuge at 1000 g for 10 minutes, and discard the supernatant; the composition of the primary enzyme digestion solution is: a mixture of collagenase type II and DNAse I; (3) Demyelination Resuspend in BSA-DMEM; centrifuge at 1000 g for 20 min; (4) Secondary enzyme digestion Digest the tissue treated in step (3) with a secondary enzyme digestion solution, and digest with magnetic stirring; the secondary enzyme digestion solution is composed of a mixture of Accutase cell digestion solution and DNAse I; (5) Percoll gradient centrifugation purification Centrifuge Percoll to obtain Percoll gradient centrifugation liquid for later use; Add DMEM to the cells after the secondary enzyme digestion in step (4), centrifuge at 1000g for 10 min, discard the supernatant, resuspend the precipitate, transfer to the aforementioned Percoll gradient centrifugation liquid, and centrifuge at 700g for 10 min; the intermediate layer liquid obtained is the endothelial cells; (6) Differential cell adhesion culture and screening The static time of the cell differential adherence culture is 45 min.

2. The method for extracting and purifying primary chicken brain microvascular endothelial cells according to claim 1, characterized in that: Step (1) The operation of separating the brain tissue of the chicken embryo is as follows: the cerebrum and cerebellum of the head of the chicken embryo are separated in PBS, the meninges are peeled off with sterile filter paper, and the brain without meninges is collected; the separated brain tissue is washed with DMEM for 3 times, and then the DMEM is removed and the brain tissue is cut into 1 cm pieces. 3 Size of brain tissue.

3. The method for extracting and purifying primary chicken brain microvascular endothelial cells according to claim 1, characterized in that: In step (2), collagenase type II in the primary enzyme digestion solution was diluted to 10 mg / mL using DMEM before preparation; DNAse I was diluted to 1 mg / mL using PBS before preparation; the volume ratio of collagenase type II and DNAse I was 3:1; in step (2), the magnetic stirring condition was 37°C for 1 hour to remove interstitial proteins; in step (2), the resuspended tissue was treated by adding 10 mL of DMEM.

4. The method for extracting and purifying primary chicken brain microvascular endothelial cells according to claim 1, characterized in that: In step (3), the BSA-DMEM is 20% BSA-DMEM by weight.

5. The method for extracting and purifying primary chicken brain microvascular endothelial cells according to claim 1, characterized in that: In step (4), DNAse I in the secondary enzyme digestion solution was diluted to 1 mg / mL with PBS before preparation; the volume ratio of Accutase cell digestion solution and DNAse I was 100:1; and the magnetic stirring conditions in step (4) were 37°C for 30 min to release endothelial cells.

6. The method for extracting and purifying primary chicken brain microvascular endothelial cells according to claim 1, characterized in that: The Percoll gradient centrifugation operation in step (5) is: centrifugation at 3000 rpm for 1 h at 4°C; and the resuspended precipitate in step (5) is treated by adding 2 mL of DMEM.

7. The method for extracting and purifying primary chicken brain microvascular endothelial cells according to claim 1, characterized in that: The composition of the Percoll gradient centrifugation medium in step (5) is: 19 mL 1×PBS + 1 mL 10×PBS + 1 ml FBS + 10 mLPercoll, a total of 31 mL, filtered, and stored at 4°C.

8. The method for extracting and purifying primary chicken brain microvascular endothelial cells according to claim 1, characterized in that: Step (6) is to directly plate the endothelial cells separated in step (5) in a cell culture dish treated with coating solution, and after standing at 37°C and 5% CO2 for 45 min, take the cell solution and plate it in a new well, and after 24 h, replace it with a culture medium without puromycin and continue to culture. The specific operation is as follows: S1: Mix the intermediate layer liquid from step (5) with 5 mL of DMEM and centrifuge at 1000 g for 10 min at 4°C to obtain a cell pellet; S2 resuspends the cell pellet obtained from S1 in complete medium containing puromycin, and 2 Use 200 μL for the area; S3 treated the cell culture dish with the pre-screened coating solution, aspirated the overnight cell culture dish coating solution, and washed the cell culture dish with sterile 1× PBS; directly spread the cell solution of S2 on the cell culture dish and let it stand at 37°C, 5% CO2 for 45 min; S4 Take the cell solution into a new well and replace it with medium without puromycin after 24 h to continue culturing; S5 medium was changed every 2-3 days until the cell density reached 90%.

9. The method for extracting and purifying primary chicken brain microvascular endothelial cells according to claim 8, characterized in that: The composition of the complete culture medium containing puromycin in S2 in step (6) is: 400 mL DMEM medium + 100 mL 20% FBS + 0.25 mL FGFb (20 μg / mL; 0.05%) + 0.5 mL heparin sodium (100 μL / mL; 0.1%) + 0.5 ml puromycin (4 mg / mL; 0.1%).

10. The method for extracting and purifying primary chicken brain microvascular endothelial cells according to claim 8, characterized in that: The cell culture dish treated with the coating solution in step (6) is a recombinant fibronectin-coated culture dish.

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