CN106479960B - Construction method of spinibarbus grahami fin cell line - Google Patents

Abstract

The invention relates to a construction method of a spiny barbel cell line, which comprises the following steps: 1) obtaining a light barb barbel fin: sterilizing the fish body by combining methylene blue and alcohol; 2) primary culture: DMEM/F12 or L-15 culture solution containing cell growth factor, fetal calf serum, penicillin, streptomycin, gentamicin and amphotericin B is adopted for culture; 3) subculturing: when the cell culture solution is transferred to the 8 th generation, the cell culture solution is changed into a basic culture solution, and the establishment of the spiny barbel cell line is successful. The spiny cell line of the barbel grahami obtained by the construction method can be continuously passaged and directly applied to biological characteristic research, can meet the requirements of saving and theoretical research on germplasm resources of economic species of the spiny barbel grahami, can be repeatedly frozen and thawed, and can be applied to important researches such as cell biology, virology, toxicology, molecular biology, genetics, immunology and the like.

Description

Construction method of spinibarbus grahami fin cell line

Technical Field

The invention relates to a method for establishing a cell line by utilizing a spinibarbus grahami fin tissue, belonging to the technical field of freshwater aquatic organism cell culture and ultralow temperature cryopreservation.

Technical Field

Barcheilus spinulosus (Spinibarbus hollandi), commonly known as Cyprinus carpioi, Cyprinus nipponensis, Anseria nipponica, October, Judas, Roughage belonging to the family Cyprinidae (Cyprinidae) Barcheilus (Spinibarbus) of the family Spinibarbus subfamily. Historically, the spiny barbel is the main capture object for several fishermen in domestic water systems, and the maximum individual reaches more than 25 kilograms. Mainly distributed in Yangtze river, Qiantangjiang river, Minjiang river, Jiulongjiang river, Zhujiang river, Yuanjiang river, Taiwan island and Hainan island. According to the survey of the Chinese domestic red river system in recent years, the population decline of the spinibarbus grahami is serious, and the population of the spinibarbus grahami is sporadically seen in the red river basin. The reason for the drastic reduction of the population number of the spinibarbus grahami is as follows: the fishing strength is high, and the development and protection consciousness of the cascade power station is weak. Through years of research, the research institute of Kunming animals of Chinese academy of sciences breaks through the artificial propagation of the Sinocyclocheilus grahami, realizes the preservation and recovery of the individual level of the economic fish, and further industrialization realizes the protection of the population level, but the research of the cell level is not carried out, so the inventor throws the eye to the somatic cell culture of the Sinocyclocheilus grahami.

The fish cell culture is started in the 60's of the 20 th century, more than 280 cell lines are established until the development, only more than 50 cell lines are established after more than 30 years of Chinese fish cell culture, and the species for establishing the cell lines is less than 1.5 percent of the total number of Chinese fishes (more than 3500 Chinese fishes), so that the establishment speed of the cell lines is very slow, the attention is not enough, and the participation of workers is less likely to be one of the reasons; secondly, compared with mammalian cell culture, the fish cell culture is more easily polluted, so that the failure rate is increased, most of the existing patents or articles attribute the phenomenon to the technical problem, and the quality problem of the fish body is rarely emphasized, and in the practice of the cell culture of the barbel grahami, the living state of the fish body directly influences the success or failure of cell line construction; in addition, because of the limitation of the wild habitat of the spinibarbus grahami, the understanding of the life habit of the spinibarbus grahami is less, and the difficulty of cell culture of the spinibarbus grahami is increased to a certain extent. Therefore, the successful construction of the sinocyclocheilus grahami fin cell line is based on the long-term understanding of wild and healthy state habits of sinocyclocheilus grahami. The same report as the present invention is not found through the literature search.

Disclosure of Invention

Aiming at the technical problems, the invention aims to provide a construction method of a barbel grahami fin cell line, which is simple and easy to operate, so as to meet the requirements of quick karyotype analysis, germplasm resource preservation and cell level adaptability research of the barbel grahami.

Specifically, the invention provides a construction method of a sinocyclocheilus grahami fin cell line, which comprises the following steps:

1) obtaining a light barb barbel fin: soaking a bobbichilus grahami fish body with 20-30mg/L methylene blue to sterilize for 20-30 minutes, and then anaesthetizing the bobbichilus grahami fish body with 80-120ppm MS-222; disinfecting the bobbichilus grahami fish body by 75% alcohol, taking a fin strip of the bobbichilus grahami fish body, and adding HBSS disinfectant to soak the taken bobbichilus grahami fish body fin tissue; after soaking for 15-30 minutes, cleaning fin tissues for 3-5 times by using HBSS (hepatitis B SS) disinfectant;

2) primary culture: cutting the fin tissue obtained in the step 1) into small tissue pieces of about 100mm3, inoculating the small tissue pieces into a cell culture bottle, inverting the small tissue pieces in an incubator at 22-26 ℃ overnight, adding primary culture solution the next day, starting primary culture in the incubator at 22-26 ℃, replacing the primary culture solution by half every three days, beginning to migrate cells from the tissue pieces on days 7-10, and growing into cell monolayers in 20-30 days to obtain primary fin cells;

3) subculturing: after 80% of primary fin cells are paved at the bottom of a bottle, starting passage, sucking out primary culture solution in a culture bottle, suspending the cells by a trypsin digestion method containing 0.1-0.2% of trypsin, adding 1-2mL of passage culture solution to neutralize trypsin for reaction after the cells become round, carrying out passage for the first time according to a ratio of 1:1, then carrying out passage according to a ratio of 1:2, and continuously culturing in an incubator at a temperature of 22-26 ℃; and (4) passage is carried out once every 4-6 days, when the cell culture solution is transferred to the 8 th generation, the cell culture solution is changed into a basic culture solution, and the establishment of the spinulo grahami cell line is successful.

Further, the HBSS disinfectant is a mixed solution of Hanks balanced salt solution containing penicillin with the concentration of 200-400IU/mL, streptomycin with the concentration of 200-400 mu g/mL, gentamicin with the concentration of 30-60 mu g/mL and amphotericin B with the concentration of 30-60 mu g/mL.

Further, the basic culture solution is a mixed solution of DMEM/F12 or L-15 culture solution containing fetal bovine serum accounting for 10-15% of the total volume and cell growth factors with the concentration of 6-10 ng/L.

Further, the primary culture solution is a mixed solution of DMEM/F12 or L-15 culture solution containing 15-25% of fetal bovine serum, 8-12ng/L of cell growth factor, 200-400IU/mL of penicillin, 200-400. mu.g/mL of streptomycin, 30-60. mu.g/mL of gentamicin and 30-60. mu.g/mL of amphotericin B.

Preferably, the subculture solution is a mixed solution of DMEM/F12 or L-15 culture solution containing 10-20% of fetal bovine serum, 8-10ng/L of cell growth factor, 20-40 mug/mL of gentamicin and 20-40 mug/mL of amphotericin B in terms of total volume.

Further, the pH values of the primary culture solution, the subculture solution and the basal culture solution were all 7.0-7.2, and the osmotic pressure was 280-320 mOsm/L.

Further, the construction method also comprises the following steps of freezing and recovering the cells:

1) preparing a cell cryopreservation solution: the frozen stock solution comprises fetal bovine serum and DMSO, and the volume ratio of the frozen stock solution is 9: 1, mixing the components in a volume ratio, and preparing the components in situ;

2) freezing and storing cells: taking cells in logarithmic phase, digesting by the pancreatin, centrifuging the cell suspension at 800-; adding the prepared cell freezing solution into the cell sediment, resuspending to make the cell concentration about 1 × 106/mL, transferring 1mL of cell suspension into a 1.8mL freezing tube, placing the freezing tube in a programmed cooling box, placing the freezing tube for 24-48 hours at minus 80 ℃, and then placing the freezing tube in liquid nitrogen for long-term storage;

3) cell recovery: taking the freezing tube out of the liquid nitrogen, putting the tube into a 37 ℃ water bath kettle, and quickly shaking the tube until the tube is melted; then transferring the thawed cells into a 15mL centrifuge tube in a sterile operation platform, adding an equivalent amount of basic culture solution, centrifuging at 800-; resuspending the cells with the basic culture solution, transferring the cells into a cell culture flask, culturing the cells in an incubator at 22-26 ℃ for 5-7 days, and growing the cells into a monolayer.

The percentages used in the steps of the present invention are volume percentages.

The invention has the following remarkable effects:

1. the construction method provided by the invention is simple and easy to operate, the repeatability of the construction method is strong, and the constructed cell line has good stability.

2. Compared with the blood short-term culture for karyotyping, the cells which are just migrated from the fin tissue block obtained by the construction method have activity and large cell amount, can be used for chromosome analysis and repeated freeze thawing, and can be applied to important researches such as cell biology, virology, toxicology, molecular biology, genetics, immunology and the like.

3. The spiny cell line of the spiny barbel constructed by the invention is in a fiber-forming shape, can be continuously passaged and directly applied to biological characteristic research, and meets the requirements of storage, theoretical research and application of the germplasm resources of the spiny barbel.

4. The cell culture is not an independent event, and needs to be combined with the biological characteristics of the fish, and the spinibarbus grahami fin cell obtained by the construction method is the first cell line of the spinibarbus grahami, so that a foundation is laid for the application of the spinibarbus grahami in immunology and toxicology.

Drawings

Fig. 1 is a graph showing the process of migration of fin tissue out of cells in primary culture of barbel grahami.

Fig. 2 is a graph showing a monolayer of fin cells in primary culture of barbel grahami.

Fig. 3 is a graph showing the effect of different sterilization modalities on cell migration of a tissue mass of a sinocyclocheilus grahami's fin.

Fig. 4 is a graph showing the effect of different culture solutions on the growth of light-barbus-gracilis fin cells.

Detailed Description

The above aspects of the invention and other aspects of the invention will be apparent from the detailed description below.

Example 1

1. Preparation of HBSS disinfectant and cell culture solution

HBSS disinfectant: antibiotics were added to HBSS to a penicillin concentration of 300IU/mL, a streptomycin concentration of 300. mu.g/mL, a gentamicin concentration of 40. mu.g/mL, and an amphotericin B concentration of 40. mu.g/mL.

Basic culture solution: adding cell growth factor and fetal calf serum into DMEM/F12 culture solution to make the concentration of cell growth factor 8ng/L, the fetal calf serum accounts for 15% of the total volume.

Primary culture solution: adding cell growth factors, fetal calf serum and antibiotics into DMEM/F12 culture solution to ensure that the concentration of the cell growth factors is 12ng/L, the fetal calf serum accounts for 20 percent of the total volume, the concentration of penicillin is 300IU/mL, the concentration of streptomycin is 300 mug/mL, the concentration of gentamicin is 50 mug/mL, and the concentration of amphotericin B is 50 mug/mL.

Subculture solution: adding cell growth factor, fetal calf serum and antibiotics into DMEM/F12 culture solution to make the concentration of the cell growth factor be 8ng/L, the fetal calf serum account for 20% of the total volume, the concentration of gentamicin be 20 mug/mL, and the concentration of amphotericin B be 20 mug/mL.

Adjusting the pH value of the culture solution to 7.2 and the osmotic pressure to 300mOsm/L, and storing in a refrigerator at 4 ℃ for later use.

2. Primary culture

Soaking and disinfecting with 20mg/l methylene blue for 30 minutes, and then anesthetizing the barbus pallidum by using 100ppm MS-222; and sterilizing the fish body with 75% alcohol, placing in a superclean bench, taking the fin with a sterile dissecting instrument, placing in a 12-hole plate, and adding HBSS disinfectant to soak the fin tissue. After 30 minutes of soaking, the fin tissue was washed 3 times with HBSS disinfectant, cut into small pieces of tissue, and inoculated to 25cm²In a cell culture flask, the flask was inverted overnight at 26 ℃ and 4mL of primary culture medium was added the next day, primary culture was initiated in the 26 ℃ incubator, the medium was changed half a day every three days, and on day 8, cells began to migrate from the tissue mass (FIG. 1) and a monolayer of adult cells was grown for 25 days (FIG. 2).

3. Subculturing

Passage was initiated after 80% of the primary fin cells had spread to the bottom of the flask. The specific subculture method comprises the following steps: sucking out primary culture solution in a culture bottle, adding 1.5mL of 0.1% trypsin-EDTA solution, digesting for 2 minutes, after cells become round, adding 2mL of subculture solution to neutralize pancreatin reaction, slightly blowing the bottom of the bottle to make adherent cells fall off, carrying out first passage according to a ratio of 1:1, carrying out passage according to a ratio of 1:2, and continuously culturing in an incubator at 26 ℃; the cell line was successfully established when the culture medium was changed to basal medium every 5 days after passage to passage 8. At present, the cell passage has passed to 20 generations.

4. Cell cryopreservation and recovery

(a) Preparing a cell cryopreservation protective solution: the frozen stock solution comprises fetal bovine serum and DMSO according to the ratio of 9: 1, mixing the components in the volume ratio, and preparing the mixture just before use.

(b) Freezing and storing cells: cells in the logarithmic growth phase were taken, digested with the above-mentioned pancreatin, and the cell suspension was centrifuged at 1000rpm for 8 minutes, and the supernatant was discarded. Adding the prepared cell freezing medium into the cell sediment, and re-suspending to make the cell concentration about 1X 10⁶And (2) transferring 1mL of cell suspension into a 1.8mL cryopreservation tube, placing the cryopreservation tube into a programmed cooling box, placing the tube at-80 ℃ for 24 hours, and then placing the tube into liquid nitrogen for long-term storage.

(c) Cell recovery: and taking the freezing tube out of the liquid nitrogen, putting the tube into a 37 ℃ water bath kettle, and quickly shaking the tube until the tube is melted. The thawed cells were then transferred to a 15mL centrifuge tube in a sterile operating station and an equal amount of basal medium was added and centrifuged at 800rpm for 8 minutes to remove the supernatant. The cells were resuspended in basal medium and transferred to a cell culture flask and cultured in an incubator at 26 ℃ for 7 days to form a monolayer.

Example 2

1. Preparation of HBSS disinfectant and cell culture solution

HBSS disinfectant: antibiotics were added to HBSS to a penicillin concentration of 400IU/mL, a streptomycin concentration of 400. mu.g/mL, a gentamicin concentration of 30. mu.g/mL, and an amphotericin B concentration of 30. mu.g/mL.

Basic culture solution: adding cell growth factor and fetal calf serum into the L-15 culture solution to make the concentration of the cell growth factor be 10ng/L, and the fetal calf serum account for 15% of the total volume.

Primary culture solution: adding cell growth factor, fetal calf serum and antibiotics into the L-15 culture solution to ensure that the concentration of the cell growth factor is 10ng/L, the fetal calf serum accounts for 20 percent of the total volume, the concentration of penicillin is 300IU/mL, the concentration of streptomycin is 300 mug/mL, the concentration of gentamicin is 40 mug/mL, and the concentration of amphotericin B is 40 mug/mL.

Subculture solution: adding cell growth factor, fetal calf serum and antibiotics into the L-15 culture solution to make the concentration of the cell growth factor be 10ng/L, the fetal calf serum account for 20% of the total volume, the concentration of gentamicin be 30 mug/mL and the concentration of amphotericin B be 30 mug/mL.

Adjusting the pH value of the culture solution to 7.0 and the osmotic pressure to 320mOsm/L, and storing in a refrigerator at 4 ℃ for later use.

2. Primary culture

Soaking and disinfecting with 30mg/L methylene blue for 20 minutes, and then anaesthetizing the spiny barbel by using 80ppm MS-222; and sterilizing the fish body with 75% alcohol, placing in a superclean bench, taking the fin with a sterile dissecting instrument, placing in a 12-hole plate, and adding HBSS disinfectant to soak the fin tissue. Soaking for 15 min, cleaning fin tissue with HBSS disinfectant for 5 times, cutting into small pieces, and inoculating to 25cm²In a cell culture flask, the flask was inverted overnight in a 24 ℃ incubator, 5mL of primary culture solution was added the next day, primary culture was initiated in a 24 ℃ incubator, the culture solution was changed half a day every three days, and on day 7, cells began to migrate from the tissue mass, and a monolayer of cells grew for 30 days.

3. Subculturing

Passage was initiated after 80% of the primary fin cells had spread to the bottom of the flask. The specific subculture method comprises the following steps: sucking out primary culture solution in a culture bottle, adding 1mL of 0.2% trypsin-EDTA solution, digesting for 2 minutes, after cell rounding, adding 2mL of subculture solution to neutralize pancreatin reaction, slightly blowing the bottom of the bottle to make adherent cells fall off, performing first passage according to a ratio of 1:1, performing passage according to a ratio of 1:2, and continuously culturing in an incubator at 24 ℃; the cell line was successfully established when the culture medium was changed to basal medium every 5 days after passage to passage 8. At present, the cell passage has passed to 20 generations.

4. Cell cryopreservation and recovery

(a) Preparing a cell cryopreservation protective solution: the frozen stock solution comprises fetal bovine serum and DMSO, and the volume ratio of the frozen stock solution is 9: 1, mixing the components in the volume ratio, and preparing the mixture just before use.

(b) Freezing and storing cells: cells in the logarithmic growth phase were taken, digested with the above-mentioned pancreatin, and the cell suspension was centrifuged at 800rpm for 10 minutes, and the supernatant was discarded. Adding the prepared cell freezing medium into the cell sediment, and re-suspending to make the cell concentration about 1X 10⁶And (2) transferring 1mL of cell suspension into a 1.8mL cryopreservation tube, placing the cryopreservation tube into a programmed cooling box, placing the tube at-80 ℃ for 24 hours, and then placing the tube into liquid nitrogen for long-term storage.

(c) Cell recovery: and taking the freezing tube out of the liquid nitrogen, putting the tube into a 37 ℃ water bath kettle, and quickly shaking the tube until the tube is melted. The thawed cells were then transferred to a 15mL centrifuge tube in a sterile operating station and an equal amount of basal medium was added and centrifuged at 800rpm for 8 minutes to remove the supernatant. The cells were resuspended in basal medium and transferred to a cell culture flask and cultured in a 24 ℃ incubator for 9 days to form a monolayer.

Example 3

1. Preparation of HBSS disinfectant and cell culture solution

HBSS disinfectant: antibiotics were added to HBSS to a penicillin concentration of 200IU/mL, a streptomycin concentration of 200. mu.g/mL, a gentamicin concentration of 60. mu.g/mL, and an amphotericin B concentration of 60. mu.g/mL.

Basic culture solution: adding cell growth factor and fetal calf serum into DMEM/F12 culture solution to make the concentration of cell growth factor 10ng/L, the fetal calf serum accounting for 10% of the total volume.

Primary culture solution: adding cell growth factors, fetal calf serum and antibiotics into DMEM/F12 culture solution to ensure that the concentration of the cell growth factors is 12ng/L, the fetal calf serum accounts for 15 percent of the total volume, the concentration of penicillin is 200IU/mL, the concentration of streptomycin is 200 mug/mL, the concentration of gentamicin is 30 mug/mL, and the concentration of amphotericin B is 30 mug/mL.

Subculture solution: adding cell growth factors, fetal calf serum and antibiotics into DMEM/F12 culture solution to make the concentration of the cell growth factors be 10ng/L, the fetal calf serum account for 15% of the total volume, the concentration of gentamicin be 20 mug/mL and the concentration of amphotericin B be 20 mug/mL.

Adjusting the pH value of the culture solution to 7.2 and the osmotic pressure to 300mOsm/L, and storing in a refrigerator at 4 ℃ for later use.

2. Primary culture

Soaking and disinfecting with 25mg/l methylene blue for 25 minutes, and then anesthetizing the spinulobarbus by using 120ppm MS-222; and sterilizing the fish body with 75% alcohol, placing in a superclean bench, taking the fin with a sterile dissecting instrument, placing in a 12-hole plate, and adding HBSS disinfectant to soak the fin tissue. After 20 minutes of soaking, the fin tissue was washed 4 times with HBSS disinfectant, cut into small pieces of tissue, and inoculated to 25cm²In a cell culture flask, the flask was inverted overnight in a 22 ℃ incubator, 5mL of primary culture medium was added the next day, primary culture was initiated in a 22 ℃ incubator, the medium was changed half a day every three days, and on the 10 th day, cells began to migrate from the tissue mass, and a monolayer of cells was grown for 30 days.

3. Subculturing

Passage was initiated after 80% of the primary fin cells had spread to the bottom of the flask. The specific subculture method comprises the following steps: sucking out primary culture solution in a culture bottle, adding 1mL of 0.2% trypsin-EDTA solution, digesting for 2 minutes, after cell rounding, adding 2mL of subculture solution to neutralize pancreatin reaction, slightly blowing the bottom of the bottle to make adherent cells fall off, performing first passage according to a ratio of 1:1, performing passage according to a ratio of 1:2, and continuously culturing in an incubator at 22 ℃; the cell line was successfully established when the culture medium was changed to basal medium every 7 days after passage to passage 8. At present, the cell passage has passed to 20 generations.

4. Cell cryopreservation and recovery

(a) Preparing a cell cryopreservation protective solution: the frozen stock solution comprises DMEM/F12 culture solution, fetal bovine serum and DMSO, and the weight ratio of the frozen stock solution is 4.5: 4.5: 1, mixing the components in the volume ratio, and preparing the mixture just before use.

(b) Freezing and storing cells: cells in the logarithmic growth phase were taken, digested with the above-mentioned pancreatin, and the cell suspension was centrifuged at 1200rpm for 6 minutes, and the supernatant was discarded. Adding the prepared cell freezing medium into the cell sediment, and re-suspending to make the cell concentration about 1X 10⁶And (2) transferring 1mL of cell suspension into a 1.8mL cryopreservation tube, placing the cryopreservation tube into a programmed cooling box, placing the tube at-80 ℃ for 48 hours, and then placing the tube into liquid nitrogen for long-term storage.

(c) Cell recovery: and taking the freezing tube out of the liquid nitrogen, putting the tube into a 37 ℃ water bath kettle, and quickly shaking the tube until the tube is melted. The thawed cells were then transferred to a 15mL centrifuge tube in a sterile operating station and an equal amount of basal medium was added and centrifuged at 1000rpm for 6 minutes to remove the supernatant. The cells were resuspended in basal medium and transferred to a cell culture flask and cultured in an incubator at 22 ℃ for 9 days to form a monolayer.

Example 4

The fish bodies of the barbel grahami of example 1 were compared using different disinfection methods.

The spiny barbel in example 1 was subjected to the most common potassium permanganate soaking sterilization, methylene blue soaking sterilization, alcohol sterilization, potassium permanganate + alcohol combined sterilization, and methylene blue + alcohol combined sterilization, and other steps were the same as those in example 1. The results show (see fig. 2) that potassium permanganate disinfection and potassium permanganate + alcohol combined disinfection exhibit lower tissue block cell migration rates throughout the primary culture process, and as the number of culture days increases, some tissue blocks migrating out of the cells will slough off and will not migrate out of the cells anew attached; under the condition of no pollution, the methylene blue and alcohol combined disinfection can obtain higher cell migration rate of the tissue blocks, the fin tissue blocks can basically migrate out of the cells along with the increase of the culture days, and the number of the fallen tissue blocks is less.

Because of the light barb ba cheilus grahami lives in flowing water environment, the cell is changeful to be contaminated, and the effect of joint disinfection is better some, but to light barb ba cheilus grahami, the toxicity of potassium permanganate is great, has also influenced the emigration of tissue piece cell when reducing the pollution rate, consequently, methylene blue + alcohol joint disinfection is the more suitable disinfection mode.

Example 5

The base solutions of the cell culture solutions of example 1 were compared with each other using different culture solutions.

The culture solution of example 1 was replaced with several culture solutions commonly used for culturing fish-type cells, DMEM (high-sugar), DMEM/F12, L-15, M199, M1640, and the effect of these different culture solutions on the growth of the light-barbed barbel fin cells was compared (see fig. 3). The results show that the growth rate and the adherence rate of the cells are different under different culture solution conditions. After one day of cell culture, DMEM/F12 and L-15 were more adherent cells, followed by DMEM and DMEM (high glucose), while M199 and M1640 medium were the least adherent cells; the cell number increases to different degrees under various culture conditions along with the increase of the culture time, wherein the growth rate of the cells in the DMEM/F12 and L-15 culture solution is fastest, the growth rate of the cells in the DMEM and DMEM (high glucose) is second, the growth rate of the cells in the M199 and M1640 is slowest, the cell number is not obviously increased, and the cell number in the DMEM/F12 and L-15 culture solution is about twice of the cell number in the DMEM and DMEM (high glucose). Therefore, for the spinibarbus grahami fin cells, DMEM/F12 and L-15 were the best, and the other four culture solutions were not suitable for cell growth.

It will be appreciated by those skilled in the art that, although specific embodiments of the invention have been described herein for purposes of illustration, various modifications may be made without deviating from the spirit and scope of the invention. Therefore, the detailed description and examples of the invention should not be construed as limiting the scope of the invention. The invention is limited only by the appended claims. All documents cited in this application are incorporated herein by reference in their entirety.

Claims (5)

1. A method for constructing a spinulo grahami cell line is characterized by comprising the following steps:

1) obtaining a light barb barbel fin: soaking a bobbichilus grahami fish body with 20-30mg/L methylene blue to sterilize for 20-30 minutes, and then anaesthetizing the bobbichilus grahami fish body with 80-120ppm MS-222; disinfecting the bobbichilus grahami fish body by 75% alcohol, taking a fin strip of the bobbichilus grahami fish body, and adding HBSS disinfectant to soak the taken bobbichilus grahami fish body fin tissue; after soaking for 15-30 minutes, cleaning fin tissues for 3-5 times by using HBSS (hepatitis B SS) disinfectant; the HBSS disinfectant is a mixed solution of Hanks balanced salt solution containing penicillin with the concentration of 200-400IU/mL, streptomycin with the concentration of 200-400 mu g/mL, gentamicin with the concentration of 30-60 mu g/mL and amphotericin B with the concentration of 30-60 mu g/mL;

2) primary culture: cutting the fin tissue obtained by the step 1) into 100mm³Inoculating the small tissue blocks into a cell culture bottle, inverting in an incubator at 22-26 ℃ overnight, adding primary culture solution the next day, starting primary culture in the incubator at 22-26 ℃, replacing the primary culture solution half every three days, starting cell migration from the tissue blocks on the 7 th-10 th day, and growing into cell monolayers in 20-30 days to obtain primary fin cells;

3) subculturing: after 80% of primary fin cells are paved at the bottom of a bottle, starting passage, sucking out primary culture solution in a culture bottle, suspending the cells by a trypsin digestion method containing 0.1-0.2% of trypsin, adding 1-2mL of passage culture solution to neutralize trypsin for reaction after the cells become round, carrying out passage for the first time according to a ratio of 1:1, then carrying out passage according to a ratio of 1:2, and continuously culturing in an incubator at a temperature of 22-26 ℃; the method is characterized in that the method is carried out once every 4-6 days, when the generation is transferred to the 8 th generation, a cell culture solution is changed into a basic culture solution, the establishment of the barbel grahami fin cell line is successful, and the basic culture solution is a mixed solution of 10-15% fetal calf serum accounting for the total volume and 6-10ng/L cell growth factor-containing DMEM/F12 or L-15 culture solution.

2. The method of construction of claim 1, wherein: the primary culture solution is a mixed solution of DMEM/F12 or L-15 culture solution containing 15-25% of fetal bovine serum, 8-12ng/L of cell growth factor, 200-400IU/mL of penicillin, 200-400 mu g/mL of streptomycin, 30-60 mu g/mL of gentamicin and 30-60 mu g/mL of amphotericin B.

3. The method of constructing a sinocyclocheilus grahami cell line according to claim 1, wherein the method comprises the steps of: the subculture solution is a mixed solution of DMEM/F12 or L-15 culture solution containing 10-20% of fetal calf serum, 8-10ng/L of cell growth factors, 20-40 mug/mL of gentamicin and 20-40 mug/mL of amphotericin B.

4. The method of constructing a sinocyclocheilus grahami cell line according to claim 1, wherein the method comprises the steps of: the pH values of the primary culture solution, the subculture solution and the basic culture solution are all 7.0-7.2, and the osmotic pressure is 280-320 mOsm/L.

5. The method of constructing a grahami cell line according to any one of claims 1 to 4, wherein the method comprises the steps of: the construction method also comprises the following steps of freezing and recovering the cells:

1) preparing a cell cryopreservation solution: the frozen stock solution comprises fetal bovine serum and DMSO, and the volume ratio of the frozen stock solution is 9: 1, mixing the components in a volume ratio, and preparing the components in situ;

2) freezing and storing cells: taking cells in logarithmic phase, digesting by the pancreatin, centrifuging the cell suspension at 800-; adding the prepared cell freezing medium into the cell sediment, and resuspending to make the cell concentration 1X 10⁶Transferring 1mL of cell suspension into a 1.8mL cryopreservation tube, placing the cryopreservation tube into a programmed cooling box, placing the tube for 24-48 hours at-80 ℃, and then placing the tube into liquid nitrogen for long-term storage;

3) cell recovery: taking the freezing tube out of the liquid nitrogen, putting the tube into a 37 ℃ water bath kettle, and quickly shaking the tube until the tube is melted; then transferring the thawed cells into a 15mL centrifuge tube in a sterile operation platform, adding an equivalent amount of basic culture solution, centrifuging at 800-; resuspending the cells with the basic culture solution, transferring the cells into a cell culture flask, culturing the cells in an incubator at 22-26 ℃ for 5-7 days, and growing the cells into a monolayer.

Images