Culture medium and culture method for primary mesothelial cells

Abstract

The present invention provides a culture medium and a culture method for primary mesothelial cells. The culture medium comprises a basal culture medium and one or more additives selected from the group consisting of a Rho proteinase inhibitor, epidermal growth factor, hydrocortisone, a glutamine additive, amphiregulin, pancreatic transferrin, and fetal bovine serum. The use of the culture medium for primary mesothelial cells of the present invention creates a more suitable living environment for the growth and reproduction of primary mesothelial cells, thereby significantly improving the culture success rate and expansion rate of primary mesothelial cells.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and in particular relates to a culture medium, and more particularly to a culture medium and a culture method for primary mesothelial cells. Background Art

[0002] The ability to isolate and culture primary human mesothelial cells (HMCs) from the peritoneum offers numerous possibilities for studying physiology, pathogenesis, and drug testing in both traditional 2D in vitro models and in vivo-like fluidic 3D platforms. Over the past few years, diverse methods for isolating HMCs have been established. However, most widely used methods, such as isolation from omental tissue samples, suffer from several drawbacks, including heavy fibroblast contamination, limited proliferation capacity, and premature senescence. Other groups have described the isolation of HMCs from lavage fluid from Parkinson's disease patients. This simple method allows for rapid and reproducible isolation of HMCs. However, due to the underlying disease, cells from patients with chronic Parkinson's disease exhibit several degenerative changes, such as enlarged organelles, multivacuolation, and decreased function. Furthermore, there is conflicting information regarding the appropriate culture media for primary HMCs, and the prolonged culture period hinders rapid expansion of HMCs, limiting their application and hindering the expansion of scientific research and drug development.

[0003] Therefore, how to propose a feasible culture medium for primary mesothelial cells to achieve effective and rapid expansion of primary mesothelial cells and improve the culture success rate and cell proliferation rate of primary mesothelial cells has become an urgent problem to be solved. Summary of the Invention

[0004] The present invention aims to solve at least one of the technical problems existing in the prior art or related art.

[0005] To this end, a first aspect of the present invention provides a culture medium for primary mesothelial cells.

[0006] A second aspect of the present invention provides a method for culturing primary mesothelial cells.

[0007] In view of this, the first aspect of the present invention proposes a culture medium for primary mesothelial cells, comprising: a basal culture medium and additives, the additives including at least one of a Rho (Ras homolog) protease inhibitor, epidermal growth factor, hydrocortisone, a glutamine additive, amphiregulin, pancreatic transferrin, and fetal bovine serum.

[0008] The culture medium for primary mesothelial cells provided by the present invention creates a more suitable living environment for the growth and reproduction of primary mesothelial cells by adding a certain amount of additives to the basal culture medium, significantly improving the culture success rate and expansion rate of primary mesothelial cells. Furthermore, the composition of the culture medium additives in this application is clearly defined, which facilitates the study of the physiological regulation mechanisms of cells.

[0009] Among them, basal culture medium refers to the culture medium containing the basic nutrients required for cell growth and reproduction, which can be used for the growth of most cells.

[0010] In addition, the culture medium for primary mesothelial cells provided by the present invention may also have the following additional technical features:

[0011] In the above technical solution, the basal culture medium includes DMEM / F12 and antibiotics; the antibiotics include one or more of streptomycin, penicillin, amphotericin B and primocin.

[0012] In this technical solution, antibiotics are secondary metabolites produced by microorganisms, higher plants, and animals during growth that possess antipathogenic or other activities, and are chemical substances that can interfere with the developmental functions of other living cells. By adding antibiotics to DMEM / F12 (a type of culture medium), they kill bacteria and viruses in the medium, creating a sterile environment that prevents microbial infestation of the cultured cells and inhibits bacterial proliferation that could deteriorate the medium.

[0013] Among them, streptomycin is an antibiotic extracted from the culture medium of Streptomyces griseus. It is an aminoglycoside alkaline compound. It binds to the ribonucleic acid protein of Mycobacterium tuberculosis bacteria, interfering with the protein synthesis of Mycobacterium tuberculosis, thereby killing or inhibiting the growth of Mycobacterium tuberculosis.

[0014] Penicillin belongs to a class of antibiotics that contains penicillin in its molecules, which can destroy the cell walls of bacteria and kill bacteria during the reproduction period of bacterial cells. It is an antibiotic extracted from Penicillium.

[0015] Amphotericin B is a polyene antibiotic that destroys the permeability of the cell membrane by interacting with solids on the cell membrane of sensitive fungi, causing some important substances in the cell to leak out, thereby disrupting the normal metabolism of the cell and inhibiting fungal growth.

[0016] Primocin is an antibiotic used to protect primary cells from microbial contamination. It has a killing effect on Gram-positive bacteria, Gram-negative bacteria, mycoplasma and fungi.

[0017] In the above technical solution, the concentration of Rho protease inhibitor is greater than or equal to 2.5 μM / mL and less than or equal to 40 μM / mL; and / or the concentration of epidermal growth factor is greater than or equal to 2.5 ng / mL and less than or equal to 40 ng / mL; and / or the concentration of hydrocortisone is greater than or equal to 2.5 ng / mL and less than or equal to 40 ng / mL; and / or the volume ratio of glutamine additive to culture medium is greater than or equal to 1:400 and less than or equal to 1:25; and / or the concentration of amphiregulin is greater than or equal to 5 ng / mL and less than or equal to 80 ng / mL; and / or the concentration of pancreatic transferrin to culture medium is greater than or equal to 1:400 and less than or equal to 1:25; The volume ratio of the culture medium is greater than or equal to 1:400 and less than or equal to 1:25; and / or the volume ratio of fetal bovine serum to culture medium is greater than or equal to 2.5% and less than or equal to 40%; and / or the concentration of streptomycin is greater than or equal to 25 μg / mL and less than or equal to 400 μg / mL; and / or the concentration of penicillin is greater than or equal to 25 U / mL and less than or equal to 400 U / mL; and / or the concentration of amphotericin B is greater than or equal to 0.25 μg / mL and less than or equal to 4 μg / mL; and / or the concentration of primocin is greater than or equal to 25 μg / mL and less than or equal to 400 μg / mL.

[0018] Furthermore, the concentration of the Rho protease inhibitor is greater than or equal to 2.5 μM / mL and less than or equal to 10 μM / mL; and / or the concentration of epidermal growth factor is greater than or equal to 2.5 ng / mL and less than or equal to 20 ng / mL; and / or the concentration of hydrocortisone is greater than or equal to 2.5 ng / mL and less than or equal to 10 ng / mL; and / or the volume ratio of the glutamine additive to the culture medium is greater than or equal to 1:400 and less than or equal to 1:50; and / or the concentration of amphiregulin is greater than or equal to 20 ng / mL and less than or equal to 80 ng / mL; and / or the concentration of pancreatic transferrin to the culture medium is greater than or equal to 1:400 and less than or equal to 1:50. The volume ratio of the culture medium is greater than or equal to 1:400 and less than or equal to 1:100; and / or the volume ratio of fetal bovine serum to culture medium is greater than or equal to 10% and less than or equal to 40%; and / or the concentration of streptomycin is greater than or equal to 50 μg / mL and less than or equal to 200 μg / mL; and / or the concentration of penicillin is greater than or equal to 50 U / mL and less than or equal to 200 U / mL; and / or the concentration of amphotericin B is greater than or equal to 0.5 μg / mL and less than or equal to 2 μg / mL; and / or the concentration of primocin is greater than or equal to 50 μg / mL and less than or equal to 200 μg / mL.

[0019] Furthermore, the concentration of Rho protease inhibitor is 2.5 μM / mL; and / or the concentration of epidermal growth factor is 2.5 ng / mL; and / or the concentration of hydrocortisone is 5 ng / mL; and / or the volume ratio of glutamine additive to culture medium is 1:50; and / or the concentration of amphiregulin is 40 ng / mL; and / or the volume ratio of pancreatic transferrin to culture medium is 1:200; and / or the concentration of streptomycin is 200 μg / mL; and / or the concentration of penicillin is 200 U / mL.

[0020] In this technical solution, the composition and dosage of the culture medium for primary mesothelial cells are clearly described, the dosage of additives used and the optimal dosage of additives for the growth of primary mesothelial cells are controlled, which greatly improves the success rate and expansion rate of primary mesothelial cells.

[0021] In the above technical solution, the Rho protease inhibitor is Y-27632.

[0022] According to the second aspect of the present invention, a method for culturing primary mesothelial cells is proposed, comprising the following steps: obtaining primary mesothelial cells; culturing the primary mesothelial cells at a cell density of 1×10 per square centimeter; 4 The primary mesothelial cells are seeded into culture dishes and cultured using the culture medium for primary mesothelial cells provided in the technical solution of the first aspect.

[0023] The method for culturing primary mesothelial cells provided by the present invention comprises the culture medium for primary mesothelial cells provided in the technical solution of the first aspect. Therefore, the method for culturing primary mesothelial cells has all the beneficial effects of the culture medium for primary mesothelial cells provided in the technical solution of the first aspect, and no further details are given here.

[0024] In the above technical solution, the steps of obtaining primary mesothelial cells specifically include: obtaining peritoneal lavage fluid containing primary mesothelial cells; placing the peritoneal lavage fluid into a centrifuge tube, centrifuging the centrifuge tube, removing the supernatant from the treated peritoneal lavage fluid to obtain a first peritoneal lavage fluid; adding a washing medium to the first peritoneal lavage fluid to resuspend the cell pellet, centrifuging the centrifuge tube again, removing the supernatant from the treated first peritoneal lavage fluid again to obtain a second peritoneal lavage fluid; adding a basal medium to the second peritoneal lavage fluid to resuspend the cells to obtain primary mesothelial cells.

[0025] In this technical solution, primary mesothelial cells are isolated from easily reproducible peritoneal lavage fluid, which can be obtained from patients by professional medical personnel at specialized medical institutions. This method of isolation can significantly preserve the viability and inherent properties of the cells, enhance their proliferation potential, and allow for rapid and efficient isolation of primary mesothelial cells.

[0026] In the above technical solution, after adding a washing medium to the first peritoneal lavage fluid to resuspend the cell pellet, the centrifuge tube is centrifuged again, and the supernatant of the treated first peritoneal lavage fluid is removed again to obtain the second peritoneal lavage fluid. The steps specifically include: adding a washing medium to the first peritoneal lavage fluid to resuspend the cell pellet, centrifuging the centrifuge tube again, and removing the supernatant of the treated first peritoneal lavage fluid again; determining whether there are blood cells in the first peritoneal lavage fluid after the supernatant is removed; if blood cells are present, adding a lysis solution to lyse the blood cells, centrifuging the centrifuge tube again after lysis, and removing the supernatant after treatment; if blood cells are not present, directly obtaining the second peritoneal lavage fluid.

[0027] In this technical solution, during the process of obtaining primary mesothelial cells, considering that there may be blood cell impurities in the peritoneal lavage fluid, which may affect the extraction effect of primary mesothelial cells, the cells are lysed using blood cell lysis fluid, which neither harms the primary mesothelial cells nor effectively removes blood cells.

[0028] In the above technical solution, the washing medium is a mixture of DMEM / F12 medium and Primocin with a concentration of 100 μg / mL.

[0029] Additional aspects and advantages of the invention will become apparent from the description which follows, or may be learned by practice of the invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the following description of the embodiments with reference to the accompanying drawings, in which:

[0031] Figure 1 is a schematic diagram showing the effects of adding different additive combinations to the basal culture medium on the proliferation of primary mesothelial cells according to an embodiment of the present invention;

[0032] Figure 2 This is a schematic diagram showing the effects of different concentrations of fetal bovine serum on the proliferation rate of primary mesothelial cells;

[0033] Figure 3 This is a schematic diagram showing the effects of different concentrations of Y-27632 on the proliferation rate of primary mesothelial cells;

[0034] Figure 4 This is a schematic diagram showing the effect of different concentrations of epidermal growth factor on the proliferation rate of primary mesothelial cells;

[0035] Figure 5 This is a schematic diagram showing the effects of different concentrations of hydrocortisone on the proliferation rate of primary mesothelial cells in culture;

[0036] Figure 6This is a schematic diagram showing the effects of different concentrations of glutamine additives on the proliferation times of primary mesothelial cells in culture;

[0037] Figure 7 is a schematic diagram showing the effects of different concentrations of amphiregulin on the proliferation fold of cultured primary mesothelial cells;

[0038] Figure 8 This is a schematic diagram showing the effect of different concentrations of pancreatic transferrin on the proliferation rate of primary mesothelial cells;

[0039] Figure 9 This is a schematic diagram of the peritoneal lavage fluid sample B11141 taken under a 10x objective lens at 18 days of culture;

[0040] Figure 10 This is a schematic diagram of the peritoneal lavage fluid sample B18004 taken under a 10x objective lens at 10 days of culture;

[0041] Figure 11 This is a schematic diagram of the peritoneal lavage fluid sample B18012 photographed under a 10x objective lens at 10 days of culture;

[0042] Figure 12 This is a schematic diagram of the peritoneal lavage fluid sample B18013 taken under a 10x objective lens at 10 days of culture;

[0043] Figure 13 This is a schematic diagram of the immunohistochemistry of peritoneal lavage sample B11041 using calretinin as the primary antibody, taken under a 20x objective lens;

[0044] Figure 14 This is a schematic diagram of the immunohistochemistry of peritoneal lavage sample B11041 using WT-1 as the primary antibody, taken under a 20x objective lens;

[0045] Figure 15 This is a schematic diagram of the immunohistochemistry of peritoneal lavage sample B11049 using calretinin as the primary antibody, taken under a 20x objective lens;

[0046] Figure 16 This is a schematic diagram of the immunohistochemistry of peritoneal lavage sample B11049 using WT-1 as the primary antibody, taken under a 20x objective lens;

[0047] Figure 17 is a schematic diagram of a cell growth curve of primary mesothelial cells cultured using a primary mesothelial cell culture medium according to an embodiment of the present invention;

[0048] Figure 18 1 is a schematic flow chart of a method for culturing primary mesothelial cells according to an embodiment of the present invention;

[0049] Figure 19 4 is another flow chart of the method for culturing primary mesothelial cells according to an embodiment of the present invention. DETAILED DESCRIPTION

[0050] In order to more clearly understand the above-mentioned objects, features and advantages of the present invention, the present invention is further described in detail below in conjunction with the accompanying drawings and specific embodiments. It should be noted that, in the absence of conflict, the embodiments of the present application and the features therein can be combined with each other.

[0051] In the following description, many specific details are set forth to facilitate a full understanding of the present invention. However, the present invention may also be implemented in other ways different from those described herein. Therefore, the scope of protection of the present invention is not limited to the specific embodiments disclosed below.

[0052] Refer to the following Figures 1 to 19 The culture medium and culture method of primary mesothelial cells in some embodiments of the present invention are described.

[0053] Example 1

[0054] According to one embodiment of the present application, the effects of various added factors in the primary mesothelial cell culture medium on the proliferation of primary mesothelial cells are described.

[0055] (1) Preparation of mesothelial primary cell culture medium

[0056] First, prepare the basal culture medium. The formula of the basal culture medium is: DMEM / F12 medium + 100 μg / mL Primocin.

[0057] Different types of additives (see Table 1) were added to the basal culture medium to prepare mesothelial primary cell culture medium containing different added components.

[0058] (2) Isolation and processing of primary mesothelial cells

[0059] 1. Sample selection

[0060] Gastric cancer peritoneal lavage fluid is obtained from patients by professional medical personnel in professional medical institutions. The peritoneal lavage fluid sample is 100mL to 250mL; it is directly stored and transported at 4°C and processed within 24 hours.

[0061] 2. Material Preparation

[0062] After surface disinfection, place 15mL sterile centrifuge tubes, pipettes, 10mL pipettes, sterile pipette tips, etc. in a biosafety cabinet and irradiate with UV light for 30 minutes. Take out the cleaning culture medium from the 4°C refrigerator 30 minutes in advance.

[0063] Washing medium: DMEM / F12 medium containing 100 μg / mL Primocin.

[0064] 3. Sample Separation

[0065] 3.1. Obtain peritoneal lavage fluid from the biosafety cabinet and place it in a 15 mL sterile centrifuge tube. Centrifuge at 1500 rpm for 4 minutes at room temperature.

[0066] Discard the supernatant, add 5 mL of wash medium to resuspend the cell pellet, and centrifuge at 1500 rpm for 4 minutes at room temperature.

[0067] 3.3. Discard the supernatant and inspect for blood cells. If present, add 3-8 mL of blood cell lysis buffer, mix thoroughly, and lyse the cells at 4°C for 10-20 minutes, inverting the tube once during lysis. Centrifuge at 1500 rpm for 4 minutes at room temperature.

[0068] 3.4. Discard the supernatant and add 2 mL of basal culture medium to resuspend the cells for later use.

[0069] 4. Cell Counting and Processing

[0070] 4.1. Microscopic observation: Pipette a small amount of resuspended cells and spread them flatly in a culture dish. Observe the density and morphology of primary cells under a microscope.

[0071] 4.2. Viable cell count: Take 12 μL of the resuspended cell suspension, mix thoroughly with 12 μL of trypan blue dye, and add 20 μL to a cell counting plate. Calculate the percentage of viable large cells (cell size > 10 μm) using a cell counter (number of viable cells / total number of cells x 100%).

[0072] (3) Culture of primary mesothelial cells

[0073] The culture medium with different components in Table 1 was added into a 48-well plate at a volume of 1 mL / well. The primary mesothelial cells were isolated from four gastric cancer peritoneal lavage samples (numbered B11041, B11049, B18013, and B11141) according to the above step (2) and cultured at a rate of 3×10 4 Cells were seeded at a density of 100 μL / well in a 48-well culture plate and cultured at 37°C with 5% CO2. After 7-10 days of culture, when the cells reached 85% confluence, the culture medium was discarded and the cells were rinsed once with 100 μL of 0.05% trypsin per well. After rinsing, 200 μL of 0.05% trypsin was added to each well. The cells were incubated in a 37°C, 5% CO2 incubator for 10 minutes. Complete digestion of the cells was confirmed under a microscope. Digestion was terminated by adding 300 μL of DMEM / F12 medium supplemented with 10% serum. 20 μL of the medium was added to a cell counting plate and the total number of cells was counted using a cell counter. As a control, basal medium without any additives was used. The results are shown in Table 1.

[0074]

[0075]

[0076] Table 1

[0077] Among them, "+" indicates that compared with the basic culture medium, the culture medium with the addition of this additive has a proliferation-promoting effect on three of the mesothelial primary cells isolated from the peritoneal lavage fluid of gastric cancer; "-" indicates that the culture medium with the addition of this additive has an inhibitory effect on the proliferation of at least three of the mesothelial primary cells isolated from the peritoneal lavage fluid of gastric cancer; "○" indicates that the culture medium with the addition of this additive has no obvious effect on the proliferation of at least two of the mesothelial primary cells isolated from the peritoneal lavage fluid of gastric cancer.

[0078] Based on the above results, factors such as N2 supplement, insulin, fetal bovine serum, niacin, non-essential amino acids, Y-27632, epidermal growth factor, hydrocortisone, glutamine supplement, amphiregulin, neuregulin 1, fibroblast growth factor 7, and pancreatic transferrin were selected for further culture experiments.

[0079] Example 2

[0080] According to one embodiment of the present application, the effects of combinations of different added factors in a primary mesothelial cell culture medium on the proliferation of primary mesothelial cells are described.

[0081] According to the components in Table 2, mesothelial primary cell culture media with different combinations of added factors were prepared to investigate the proliferation-promoting effects of different combinations of added factors on mesothelial primary cells. Table 2 shows the preparation of culture media with different components (concentrations are final concentrations).

[0082]

[0083]

[0084] Table 2

[0085] According to the method of step (2) of step 3 of Example 1, primary mesothelial cells were obtained from the peritoneal lavage fluid of gastric cancer (numbered B11141 and B18013). The obtained cell suspension was divided into 15 equal parts and centrifuged at 1500 rpm for 4 minutes. After centrifugation, 200 μL of BM and No. 1 to No. 14 culture medium were used to resuspend the cells at a viable cell density of 2×10 4 pieces / cm 2 Seed the cells in a 48-well plate (20,000 cells per well). Fill each well to 1 mL with the appropriate culture medium and mix thoroughly. After surface disinfection, incubate at 37°C with 5% CO2.

[0086] When the cells in the 48-well plate grow to more than 85%, discard the culture medium and rinse once with 100 μL of 0.05% trypsin. After washing, add 200 μL of 0.05% trypsin to each well. Place in a 37°C, 5% CO2 incubator for 10 minutes. Observe under a microscope that the cells have been completely digested. Add 300 μL of DMEM culture medium containing 10% fetal bovine serum to stop the digestion. Take 20 μL and add it to a cell counting plate (specification: 50 plates / box). The total number of cells is counted using a cell counter. The results obtained from the isolated B11141 and B18013 mesothelial primary cells are shown in Figure 1 middle.

[0087] like Figure 1 As shown, compared with the basal medium, the above-mentioned media No. 1 to No. 14 all promoted the proliferation of primary gastric cancer mesothelial cells to varying degrees when used. Furthermore, the N2 supplement, insulin, niacin, non-essential amino acids, neuregulin 1, and fibroblast growth factor 7 were not essential for the proliferation of primary gastric cancer mesothelial cells. The highest proliferation effect was achieved when culturing primary gastric cancer mesothelial cells using media containing fetal bovine serum, Y-27632, epidermal growth factor, hydrocortisone, glutamine supplement, amphiregulin, and pancreatic transferrin.

[0088] Example 3

[0089] According to one embodiment of the present application, the effects of different concentrations of factors added to the culture medium on the proliferation of primary gastric cancer mesothelial cells are described.

[0090] Primary mesothelial cells were obtained from gastric cancer peritoneal lavage fluid samples (numbered B18013 and B11141) according to the method of step (2) of step 3 of Example 1. The cells were cultured using the culture medium of Example 2. The obtained primary mesothelial cells were cultured at a viable cell density of 2.6×10 4 pieces / cm 2 Inoculate into T75 culture flasks (2 million cells per flask) and mix well. After surface disinfection, place in a 37°C, 5% CO2 incubator for culture and expansion until the cells grow to more than 85%. Add 500 μL of 0.05% trypsin (rinse for 1 minute, aspirate and then add 500 μL of 0.05% trypsin to each well. Place in a 37°C, 5% CO2 incubator for 2 to 10 minutes until the cells are completely digested. After centrifugation at 1500 rpm for 4 minutes, discard the supernatant. Resuspend the cell pellet in DMEM / F12. Take 20 μL and add to a cell counting plate (specification: 50 plates / box). Count the total number of cells using a cell counter. The resulting cells are used for the following culture experiments.

[0091] Next, the following seven culture medium formulations were prepared for the experiment:

[0092] Recipe 1: The culture medium does not contain fetal bovine serum;

[0093] Formulation 2: The culture medium does not contain Y-27632;

[0094] Formulation 3: The culture medium does not contain epidermal growth factor;

[0095] Formulation 4: The culture medium does not contain hydrocortisone;

[0096] Formulation 5: The culture medium does not contain glutamine additives;

[0097] Formulation 6: The culture medium does not contain amphiregulin;

[0098] Formulation 7: The culture medium does not contain pancreatic transferrin.

[0099] Add 20 μL of 1x10 4 For each cell resuspension, 1 mL of the culture medium of formulas 1 to 7 was used to dilute the cell suspension.

[0100] When using the culture medium of Formula 1, 1 mL of prepared fetal bovine serum was added to each well of a 48-well plate seeded with primary cells, with final concentrations of fetal bovine serum of 2.5%, 5%, 10%, 20%, and 40%, respectively; and control wells (BC) were set up using the culture medium of Formula 1.

[0101] When using the culture medium of Formula 2, 1 mL of prepared Y-27632 was added to each well of a 48-well plate seeded with primary cells. The final concentrations of Y-27632 were 2.5 μM / mL, 5 μM / mL, 10 μM / mL, 20 μM / mL, and 40 μM / mL, respectively. Control wells (BC) were set up using the culture medium of Formula 2.

[0102] When using the culture medium of Formula 3, 1 mL of prepared epidermal growth factor was added to each well of a 48-well plate seeded with primary cells, with final concentrations of epidermal growth factor of 2.5 ng / mL, 5 ng / mL, 10 ng / mL, 20 ng / mL, and 40 ng / mL, respectively; and control wells (BC) were set up using the culture medium of Formula 3.

[0103] When using the culture medium of Formula 4, 1 mL of prepared hydrocortisone was added to each well of a 48-well plate seeded with primary cells. The final concentrations of hydrocortisone were 2.5 ng / mL, 5 ng / mL, 10 ng / mL, 20 ng / mL, and 40 ng / mL, respectively. Control wells (BC) were set up using the culture medium of Formula 4.

[0104] When using the culture medium of Formula 5, 1 mL of the prepared glutamine additive was added to each well of a 48-well plate seeded with primary cells. The volume ratio of the glutamine additive to the culture medium was 1:400, 1:200, 1:100, 1:50, and 1:25, respectively. Control wells (BC) were set up using the culture medium of Formula 5.

[0105] When using the medium of Formula 6, 1 mL of prepared amphiregulin was added to each well of a 48-well plate seeded with primary cells, with final concentrations of amphiregulin of 5 ng / mL, 10 ng / mL, 20 ng / mL, 40 ng / mL, and 80 ng / mL, respectively. Control wells (BC) were set up using the medium of Formula 6.

[0106] When using the culture medium of Formula 7, 1 mL of prepared pancreatic transferrin was added to each well of a 48-well plate seeded with primary cells. The volume ratio of pancreatic transferrin to culture medium was 1:400, 1:200, 1:100, 1:50, and 1:25, respectively. Control wells (BC) were set up using the culture medium of Formula 7.

[0107] When the cells were expanded to about 85% of the 48 wells, the cells were digested and counted, and the proliferation times were calculated by referring to the number of cells in the control wells (BC). The results were shown in Figures 2 to 8 .

[0108] like Figure 2 、 Figure 3 、 Figure 4 、 Figure 5 、 Figure 6 、 Figure 7 and Figure 8 As shown, the ratio is the ratio of the number of cells cultured in each culture medium for one generation to the number of cells cultured in the corresponding control well for one generation. A ratio greater than 1 indicates that the culture medium containing different concentrations of factors or small molecules is more effective in promoting cell proliferation than the culture medium in the control well; a ratio less than 1 indicates that the culture medium containing different concentrations of factors or small molecules is less effective in promoting cell proliferation than the culture medium in the control well.

[0109] according to Figures 2 to 8The results show that the volume ratio of fetal bovine serum to culture medium is greater than or equal to 2.5% and less than or equal to 40%, preferably greater than or equal to 10% and less than or equal to 40%; the concentration range of Rho protease inhibitor is greater than or equal to 2.5 μM / mL and less than or equal to 40 μM / mL, preferably greater than or equal to 2.5 μM / mL and less than or equal to 10 μM / mL, and the best preferred concentration is 2.5 μM; the concentration range of epidermal growth factor is greater than or equal to 2.5 ng / mL and less than or equal to 40 ng / mL, preferably greater than or equal to 2.5 ng / mL and less than or equal to 20 ng / mL, and the best preferred concentration is 2.5 ng / mL; the concentration range of hydrocortisone is greater than or equal to 2.5 ng / mL and less than or equal to 40 ng / mL, preferably greater than or equal to 2.5 ng / mL and less than or equal to 20 ng / mL, and the best preferred concentration is 2.5 ng / mL. Equal to 2.5 ng / mL and less than or equal to 10 ng / mL, the best preferred concentration is 5 ng / mL; the volume ratio of glutamine additive to the culture medium is greater than or equal to 1:400 and less than or equal to 1:25, preferably greater than or equal to 1:400 and less than or equal to 1:50, and the best preferred concentration is 1:50; the concentration range of amphiregulin is greater than or equal to 5 ng / mL and less than or equal to 80 ng / mL, preferably greater than or equal to 20 ng / mL and less than or equal to 80 ng / mL, and the best preferred concentration is 40 ng / mL; the volume ratio of pancreatic transferrin to the culture medium is greater than or equal to 1:400 and less than or equal to 1:25, preferably greater than or equal to 1:400 and less than or equal to 1:100, and the best preferred concentration is 1:200.

[0110] Example 4

[0111] According to one embodiment of the present application, a method for culturing and identifying primary mesothelial cells is described.

[0112] (1) A method for culturing primary mesothelial cells, such as Figure 18 As shown, specifically:

[0113] S102: Obtaining primary mesothelial cells;

[0114] S104: Primary mesothelial cells were cultured at a cell density of 1×10 4 pieces / cm 2 The cells were seeded into a culture dish and cultured using a culture medium for primary mesothelial cells.

[0115] According to the method of step (2) of step 3 of Example 1, primary mesothelial cells were obtained from gastric cancer peritoneal lavage fluid samples (numbered B11141, B18004, B18012, and B18013), and cultured using the culture medium of Example 2. The obtained primary mesothelial cells were cultured at a viable cell density of 1×10 4 pieces / cm 2The cells were seeded into 6-well plates (100,000 cells per well), mixed, and placed in a 37°C, 5% CO2 incubator after surface disinfection.

[0116] Observe the cultured primary mesothelial cells using a microscope. Figure 9 、 Figure 10 、 Figure 11 and Figure 12 As shown, the cells are closely arranged under the microscope with slightly irregular morphology.

[0117] (2) Immunohistochemical identification of primary mesothelial cells

[0118] 5 million cells were obtained from 2 cases of mesothelial primary cells (sample numbers B11041 and B11049) and fixed in 1 mL of 4% paraformaldehyde. The tissues fixed with 4% paraformaldehyde were embedded in paraffin and cut into 4 μm thick paraffin sections using a microtome. Conventional immunohistochemistry was then performed. The primary antibodies used were Calretinin (calcium-binding protein) and WT-1 (Wilms tumor protein, gene encoding). Figure 13 、 Figure 14 、 Figure 15 and Figure 16 Shown are the immunohistochemical detection results of two different samples (sample numbers B11041 and B11049) under different primary antibody conditions.

[0119] Example 5

[0120] According to one embodiment of the present application, Figure 19 As shown, the method for culturing primary mesothelial cells further comprises:

[0121] S202, obtaining peritoneal lavage fluid containing primary mesothelial cells;

[0122] S204, placing the peritoneal lavage fluid into a centrifuge tube, centrifuging the centrifuge tube, and removing the supernatant from the treated peritoneal lavage fluid to obtain a first peritoneal lavage fluid;

[0123] S206, adding a washing medium to the first peritoneal lavage fluid to resuspend the cell pellet, centrifuging the centrifuge tube again, and removing the supernatant of the treated first peritoneal lavage fluid again;

[0124] S208, determining whether there are blood cells in the first peritoneal lavage fluid after the supernatant is removed; if so, executing S210, otherwise executing S212;

[0125] S210, adding a lysis solution to lyse the blood cells, centrifuging the centrifuge tube again after lysis, removing the supernatant after treatment to obtain a second peritoneal lavage fluid, and executing S214;

[0126] S212, directly obtain the second peritoneal lavage fluid;

[0127] S214, basal culture medium was added to the second peritoneal lavage fluid to resuspend the cells to obtain primary mesothelial cells;

[0128] At S216, primary mesothelial cells were cultured at a cell density of 1×10 4 pieces / cm 2 The cells were seeded into a culture dish and cultured using a culture medium for primary mesothelial cells.

[0129] In this embodiment, the present invention obtains primary mesothelial cells from easily reproducible peritoneal lavage fluid, which can be obtained from the patient's body by professional medical personnel in professional medical institutions. This acquisition method can greatly ensure the activity and original characteristics of the cells, and the cells have strong proliferation ability, and the mesothelial primary cells can be quickly and effectively isolated. In addition, in the process of obtaining primary mesothelial cells, considering that there may be blood cell impurities in the peritoneal lavage fluid, which may affect the extraction effect of primary mesothelial cells, the use of blood cell lysis solution for lysis does not harm the primary mesothelial cells and can effectively remove blood cells.

[0130] Example 6

[0131] According to one embodiment of the present application, the culture cycle of primary mesothelial cells, cell number statistics, and Population Doubling (PD) value calculation are described.

[0132] According to the method of step 3 of step (2) of Example 1, primary mesothelial cells were obtained from three primary mesothelial cells (numbered B18004, B18012, and B18013). The primary mesothelial cells were cultured at a viable cell density of 2.4×10 4 pieces / cm 2 Inoculate in T12.5 culture flasks (300,000 cells per flask). Use a pipette to gently add 4 mL of the mesothelial primary cell culture medium of the present invention at room temperature to each T12.5 culture flask. After surface disinfection, place in a 37°C, 5% CO2 incubator (purchased from Thermo Fisher Scientific) for culture. After 5 to 9 days of cell culture, count the passages and count the days of culture until passage. The number of days of culture until passage is taken as a culture cycle. Continue to culture under the experimental conditions, amplify the amplified cells for different generations, count and record the corresponding culture cycle after each generation, and calculate the PD value according to the formula Population Doubling (PD) = 3.32 × log10 (total number of cells after digestion / initial number of cells seeded). (For the specific formula, see Chapman et al., Stem Cell Research & Therapy 2014, 5: 60).

[0133] like Figure 17 As shown in the figure, the growth curves of three primary cells cultured using the mesothelial primary cell culture medium of the present invention were drawn using Graphpad Prism software. The horizontal axis represents the number of days of cell culture, and the vertical axis is the cumulative cell proliferation multiple, which represents the multiple of cell expansion during the culture cycle. The larger the value, the more times the cells expand within a certain cycle, that is, the more cells are expanded. The slope represents the rate of cell expansion. Figure 17 It can be confirmed that after the primary mesothelial cells cultured in the culture medium of the present invention are continuously cultured and expanded for at least 50 days, the cell expansion rate remains basically unchanged and still has the ability to continue to expand.

[0134] In the present invention, the term "plurality" refers to two or more, unless otherwise specified. Terms such as "mounted," "connected," "connected," and "fixed" should be interpreted broadly. For example, "connected" can refer to fixed, removable, or integral connections; and "connected" can refer to direct or indirect connections through an intermediary. Those skilled in the art will understand the specific meanings of these terms in the present invention based on specific circumstances.

[0135] Throughout this specification, terms such as "one embodiment," "some embodiments," and "specific embodiments" mean that the specific features, structures, materials, or characteristics described in conjunction with that embodiment or example are included in at least one embodiment or example of the present invention. In this specification, schematic representations of these terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.

[0136] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. A culture medium for primary mesothelial cells, characterized in that include: basal culture medium; Additives, wherein the additives are Rho protease inhibitor, epidermal growth factor, hydrocortisone, glutamine additive, amphiregulin, pancreatic transferrin and fetal bovine serum; the concentration of the Rho protease inhibitor is 5 μM / mL, the concentration of the epidermal growth factor is 10 ng / mL, the concentration of the hydrocortisone is 10 ng / mL, the volume ratio of the glutamine additive to the culture medium is 1:100, the concentration of the amphiregulin is 20 ng / mL, the volume ratio of the pancreatic transferrin to the culture medium is 1:100, the volume ratio of the fetal bovine serum to the culture medium is equal to 10%, and the Rho protease inhibitor is Y-27632.

2. The culture medium for primary mesothelial cells according to claim 1, characterized in that The basal culture medium includes DMEM / F12 and antibiotics; The antibiotics include one or more of streptomycin, penicillin, amphotericin B and primocin.

3. The culture medium for primary mesothelial cells according to claim 2, wherein The concentration of streptomycin is greater than or equal to 25 μg / mL and less than or equal to 400 μg / mL; and / or The concentration of penicillin is greater than or equal to 25 U / mL and less than or equal to 400 U / mL; and / or The concentration of amphotericin B is greater than or equal to 0.25 μg / mL and less than or equal to 4 μg / mL; and / or The concentration of the Primocin is greater than or equal to 25 μg / mL and less than or equal to 400 μg / mL.

4. The culture medium for primary mesothelial cells according to claim 3, wherein The concentration of streptomycin is greater than or equal to 50 μg / mL and less than or equal to 200 μg / mL; and / or The concentration of penicillin is greater than or equal to 50 U / mL and less than or equal to 200 U / mL; and / or The concentration of amphotericin B is greater than or equal to 0.5 μg / mL and less than or equal to 2 μg / mL; and / or The concentration of the Primocin is greater than or equal to 50 μg / mL and less than or equal to 200 μg / mL.

5. The culture medium for primary mesothelial cells according to claim 4, characterized in that The concentration of streptomycin is 200 μg / mL; and / or The concentration of penicillin is 200 U / mL.

6. A method for culturing primary mesothelial cells, characterized in that: include: Obtaining primary mesothelial cells; The primary mesothelial cells were cultured at a cell density of 1×10 4 The primary mesothelial cells are seeded into a culture dish, and the primary mesothelial cells are cultured using the culture medium for primary mesothelial cells according to any one of claims 1 to 5.

7. The method for culturing primary mesothelial cells according to claim 6, characterized in that: The step of obtaining primary mesothelial cells specifically includes: Obtain peritoneal lavage fluid containing primary mesothelial cells; placing the peritoneal lavage fluid into a centrifuge tube, centrifuging the centrifuge tube, and removing the supernatant from the treated peritoneal lavage fluid to obtain a first peritoneal lavage fluid; adding a washing medium to the first peritoneal lavage fluid to resuspend the cell pellet, centrifuging the centrifuge tube again, and removing the supernatant from the treated first peritoneal lavage fluid to obtain a second peritoneal lavage fluid; Add basal culture medium to the second peritoneal lavage fluid to resuspend the cells to obtain primary mesothelial cells.

8. The method for culturing primary mesothelial cells according to claim 7, characterized in that: After adding a washing medium to the first peritoneal lavage fluid to resuspend the cell pellet, the centrifuge tube is centrifuged again, and the supernatant of the treated first peritoneal lavage fluid is removed again to obtain the second peritoneal lavage fluid. The steps specifically include: After adding a washing medium to the first peritoneal lavage fluid to resuspend the cell pellet, the centrifuge tube is centrifuged again, and the supernatant of the treated first peritoneal lavage fluid is removed again; determining whether blood cells are present in the first peritoneal lavage fluid after the supernatant is removed; if blood cells are present, adding a lysis solution to lyse the blood cells; centrifuging the centrifuge tube again after lysis; and removing the supernatant to obtain a second peritoneal lavage fluid; If blood cells were not present, a second peritoneal lavage was obtained directly.

9. The method for culturing primary mesothelial cells according to claim 7, characterized in that: The washing medium is a mixture of DMEM / F12 medium and 100 μg / mL Primocin.

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