CN111154716B - In-vitro culture, identification and induced differentiation method of sebastes schlegeli hilgendorf myoblasts - Google Patents
In-vitro culture, identification and induced differentiation method of sebastes schlegeli hilgendorf myoblasts Download PDFInfo
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Abstract
The invention relates to an in vitro culture, identification and induced differentiation method of sebastes schlegeli hilgendorf myoblasts, belonging to the field of molecular biology. The sebastes schlegeli adult cells cultured by the method can be subcultured and used for basic research on the muscle growth of seawater sclerosteus fishes; can effectively induce and differentiate myoblasts in vitro and provide a basis for the research of the molecular mechanism of fusing monocytes into multinucleate cells in vivo.
Description
Technical Field
The invention belongs to the technical field of cell culture, and particularly relates to an in vitro culture, identification and induced differentiation method of sebastes schlegeli hilgendorf myoblasts.
Background
Sebastes schlegelii (Sebastes schlegelii), commonly known as Sebastes schlegelii, black heads and the like, is an important marine economic fish and excellent breed increasing and breeding variety, and is mainly distributed in Bohai sea, yellow sea and east sea in China. Sebastes schlegeli has a wide range of survival temperature, can live through the winter in deep water sea cages, is one of a few varieties suitable for being cultured in deep water cages in the north for a long time, is widely cultured in deep water cages in the coastal region in the north, but has a slow growth speed, and generally needs 2-3 years to reach the commodity specification. Growth is one of the most valuable economic traits for genetic improvement of sebastes schlegeli hilgendorf. The muscle of the fish accounts for 40% -60% of the total weight, is a main edible part and is also the core of the growth traits. The research on the related gene for regulating and controlling the muscle growth and development provides a foundation for adopting corresponding technical means to improve the muscle growth rate. The muscle cells cultured in vitro can provide basic experimental materials for researching the functional verification of the muscle growth related genes. Myoblasts (myoblasts) are stem cells derived from embryonic mesoderm, have the ability to self-renew and differentiate directionally, and are capable of differentiating to form muscle cells. Myoblasts cultured in vitro can achieve a complete process of myogenesis: proliferation, differentiation, fusion and formation of multinucleated myotubes. In teleost fish, studies on in vitro culture of muscle cells have been mainly conducted in salmon, carp and sea bream, but most of the cultured cells are fibroblasts. Compared with other vertebrates, no relevant report exists on the culture and induced differentiation of marine fish myoblasts. Through in vitro culture and induced differentiation of sebastes schlegeli hilgendorf myoblasts, basic experimental materials are provided for function verification of seawater fish muscle growth related genes and research of growth and development regulation and control mechanisms.
Disclosure of Invention
The invention aims to provide a method for in vitro culture, identification and induced differentiation of sebastes schlegeli hilgendorf myoblasts, provides experimental materials for in vitro verification of sebastes schlegeli hildorf muscle growth related genes, and provides a foundation for research on molecular mechanism of seawater sclerostes hilus muscle growth regulation.
The technical scheme adopted by the invention is carried out according to the following steps:
in-vitro culture method of sebastes schlegeli hilgendorf myoblasts
1) Sampling and disinfecting muscle tissue blocks of sebastes schlegeli hilgendorf: taking a small mass of sebastes schlegeli dorsalis, and transferring into a 50ml centrifuge tube containing 4% of three-antibody PBS (phosphate buffer solution);
2) primary culture of cells: firstly, moving the tissue block from a centrifuge tube to a 25ml container containing 4% double-resistant PBS, soaking for 5min, then sequentially passing through 25ml containers containing 3%, 2% and 1% triple-resistant PBS, soaking for 5min, and stirring at intervals so that the tissue block is in full contact with the PBS and sterilized; moving the tissue blocks to a container filled with 1% of three-antibody DMEM/F12; cut the tissue block to 3mm3Size; standing the tissue block solution cut in the step II, discarding the upper liquid and leaving the tissue blocks; adding a heavy suspension tissue block containing 1% of three-resistance DMEM/F12, standing, discarding the upper liquid again, and repeating for three times; transferring the tissue blocks into a culture bottle, uniformly placing the tissue blocks, inverting the culture bottle to enable the tissue blocks to be tightly combined on the culture bottle, and then adding the complete culture medium into the culture bottle in the forward direction;
3) subculturing of cells: the primary cells can be subcultured when growing to 50-60% abundance, all the subcultured cells are still cultured in an original culture bottle, and the 2 nd generation cells can be subcultured when growing to 75% abundance, and the cells are cultured in the original bottle; after the third generation, when the cell coverage rate reaches 90%, passage is carried out, and one bottle is transferred to two to three bottles; ② reagents used in passage, including culture medium, pancreatin and serum, are preheated for more than 0.5h in a 24 ℃ incubator; ③ the concrete steps of passage: discarding the original culture medium, washing off residual serum with 1ml serum-free culture medium, and discarding; adding 1ml of 0.25% pancreatin, treating for 2-3min, observing cell state, shaking culture flask to make cells float in culture medium when 90% of cells shrink into round shape, and adding serum to stop pancreatin digestion; finally adding a complete culture medium, uniformly mixing, and subculturing the cell absorbing suspension into another culture bottle;
4) freezing and recovering cells: firstly, sucking the culture medium in a culture bottle before digesting the cells by pancreatin in the step 3), and freezing for later use; centrifuging the obtained cell suspension at the temperature of 4 ℃ for 5min at 200g, removing supernatant, and suspending the cells by using 1ml of complete culture medium with precooling at the temperature of 4 ℃; adding 150ul of precooled DMSO into a precooled cryopreservation tube, adding a precooled complete culture medium, uniformly mixing, adding the cell suspension prepared in the previous step, and uniformly mixing the cryopreserved cells at 4 ℃ for 1 h; -20 ℃ for 3 h; -80 ℃ overnight; preserving in liquid nitrogen for a long time; fourthly, after the frozen cells are taken out of the liquid nitrogen, the freezing tube is subjected to water bath at 37 ℃ for 1-2min, and the water bath is stopped when only sporadic small ice blocks remain in the freezing tube; fifthly, centrifuging at 200g for 5min at 4 ℃, discarding the supernatant to remove DMSO, adding a fresh complete culture medium for resuspension, centrifuging, discarding the supernatant to further clean residual DMSO, adding 1ml of fresh complete culture medium for resuspension; sixthly, inoculating the cell suspension into a culture bottle, adding 3ml of unfreezing culture medium for standby cryopreservation, and after the cells are recovered, carrying out corresponding experiments after passage for more than two times, wherein the cell-related experiments are carried out in 2-3 d.
Furthermore, all instruments used in the operation process are placed in a closed container and are sterilized at high temperature, and the whole operation is carried out in an aseptic state.
Further, the complete culture medium comprises a mixed culture medium of the following components: the mass ratio of the MEM nonessential amino acids to the DMEM is 1%, the mass ratio of the DMEM to the DMEM is 1%, the DMEM to the DMEM is 10mg/mL penicillin, the DMEM to the DMEM is 10mg/mL streptomycin, the DMEM to the DMEM is 20% fetal calf serum, the DMEM to the DMEM: 1, mixing; when the mass ratio of the fetal calf serum in the complete culture medium used after the cells passed to the 10 th passage was adjusted to 15%, the rest components were unchanged.
Further, the serum-free medium is a DMEM/F12 medium containing 1% of double antibody by mass.
Further, the concentration of the pancreatin is 0.25%.
Further, the differentiation medium contains a mixed medium of the following components: 1% MEM nonessential amino acids by mass, 1% triantion by volume and 2% horse serum by mass; the mixed culture medium is prepared from L-15 and DMEM/F12 according to the mass ratio of 1: 1, mixing;
further, the triple antibody (BI, No. PB180424) is a mixed antibiotic consisting of 10000units/mL penicillin, 10mg/mL streptomycin and 1250units/mL nystatin; 1% of three antibody, namely adding 1mL of three antibody into 99mL of culture medium; the double antibody is a mixed antibiotic consisting of 10000units/mL penicillin and 10mg/mL streptomycin, and 1 percent of double antibody is added into 99mL of culture medium;
the invention also provides an identification method for identifying the cell species, which comprises the following steps: identification of cell species: determining a cell line source by comparing homology of an amplified muscle cell line 18S rRNA gene with a sebastes schlegeli muscle tissue 18S rRNA gene, designing a specific primer of the 18S rRNA gene, extracting myoblast total DNA and sebastes schlegeli muscle tissue total DNA, cloning a PCR product to a PMD-19T vector, converting the vector to DH5 alpha cell, and detecting to obtain a positive monoclonal strain to perform sequencing and comparative analysis; identifying cell types: determining the cell type by amplifying the marker gene Pax7 of the myosatellite cells and the marker gene MyoD of the myoblasts; rpl17 serves as an internal reference gene. Extracting total RNA of cells and total RNA of sebastes schlegeli muscle tissue by using a trizol method; inverting the cell RNA into cDNA by a gDNAeraser method, and amplifying gene sequences of Pax7, MyoD and Rpl17 by taking the cDNA as a template; performing sequence comparison analysis after PCR reaction;
wherein, the specific primers of the 18S rRNA gene are as follows:
18S-fw 5′-primer：5′-GTAAACCAAGAACTGAGGAGGAG-3′，
18S-rv 3′-primer：5′-GAGAAAAGCAGAAGTGGAATCA-3′；
the Pax7 primer was designed as follows:
Pax7-fw 5′-primer：5′-CCAAACGGCCAGATGAT-3′，
Pax7-rv 3′-primer：5′-ACAAAGCAGGGTTTGGA-3′；
the MyoD primers were designed as follows:
MyoD-fw 5′-primer：5′-ATGGAGCTGTCGGATATCT-3′，
MyoD-rv 3′-primer：5′-tgtaggacttgatagatcaggttg-3′。
the Rpl17 primer was designed as follows:
Rpl17-fw 5′-primer：5′-AGGCGACGCACCTACCG-3′，
Rpl17-rv 3′-primer：5′-CCTCTGGTTTGGGGACGA-3′
the invention also provides an induced differentiation method of subcultured cells, which comprises the following steps: 0-24 h: setting the time of inoculating the cells to be 0h, culturing the subculture cells by using a growth culture medium for 0-24h, wherein the growth culture medium is an L-15 culture medium containing 15% FBS, and the cells normally grow at 24 h; 24-48 h: changing a growth culture medium into a differentiation culture medium at 24h, wherein the differentiation culture medium is an L-15 culture medium containing 2% DHS, and cells begin to fuse at 48 h; 48-72 h: continuously culturing by using a differentiation culture medium, and fusing a large amount of cells in 72 hours; 72-96 h: continuously culturing in a differentiation culture medium, and almost all the cells are multi-core myotubes after cell fusion is finished at 96 h; secondly, photographing and observing the cells cultured by the growth medium and the differentiation medium every 24 hours, and recording the process from the mononuclear myocytes to the fusion of the mononuclear myocytes into the polynuclear myofibers; thirdly, staining the cell nucleus and the cell cytoplasm of 96h by using Hoechst and phalloidin respectively, and counting the proportion of the mononuclear cell and the polynuclear cell.
Compared with the prior art, the invention has the beneficial effects that:
the sebastes schlegeli adult cells cultured by the method can be subcultured and used for basic research on the muscle growth of seawater sclerosteus fishes; can effectively induce and differentiate myoblasts in vitro and provide a basis for the research of the molecular mechanism of fusing monocytes into multinucleate cells in vivo.
Drawings
FIG. 1 shows the growth state of cells in primary culture on different days: a. 5d, b, 7d, c, 10d, 15 d;
FIG. 2 shows the growth state of primary cultured cells of different generations.
FIG. 3 is a cell growth curve after two passages of cell cryopreservation recovery.
FIG. 4 shows the cell types (from top to bottom, Pax7, Myod and Rpl17) M, marker, generation 1, generation 5, generation 2, generation 10, generation 3, generation 25, generation 4, muscle tissue
FIG. 5 shows the statistics of the number of nuclei of cells cultured in growth medium and differentiation medium, where a is the growth medium, b is the differentiation medium, and c is the ratio of each fiber cell.
Detailed Description
The present invention will be described with reference to specific embodiments. The experimental articles referred to in this example were all commercially available products unless otherwise specified.
Example 1, primary culture of muscle cells of sebastes schlegeli, the specific method is as follows:
the tertiary antibiotic (BI, No. PB180424) is a mixed antibiotic consisting of 10000units/mL penicillin, 10mg/mL streptomycin and 1250units/mL nystatin; 1% of the three antibody, i.e., 1mL of the three antibody, was added to 99mL of the medium. FGFb (Solarbio, No. p 00032). Fetal bovine serum (BI, No. 04-001-1A). DMEM/F12(BI, No.01-172-1 ACS). L-15(Gibco, No. 11415-. 0.25% pancreatin (Gibco, No. 12605-028). Horse serum (BI, No. 04-004-1B).
1) Sampling and disinfecting muscle tissue blocks of sebastes schlegeli hilgendorf: selecting juvenile fish of sebastes schlegeli hilgendorf, which died at the age of 3-6 months, and sampling muscle tissue blocks. Secondly, before sampling, disinfecting the surface of the fish body with 75% alcohol, wiping the surface of the fish body with toilet paper, and repeating the steps for three times to remove bacteria on the surface of the fish body as far as possible. ③ when sampling, the skin of the fish is removed by using the scissors which are sterilized at high temperature (the scissors, the tweezers, the centrifuge tube, the small beaker and the penicillin bottle which are used in the experiment should be sterilized at high temperature in advance), and then the scalper is removed by using the scalpel, the small dorsal muscle is taken, and then the small dorsal muscle is moved into a 50ml centrifuge tube which is filled with PBS containing 4 percent of three-resistance antibody.
2) Primary culture of cells: firstly, a 50ml centrifuge tube containing 4% tris-resistant PBS is moved into a super clean bench for sterilization for more than 1h, and the centrifuge tube is sprayed with 75% alcohol for sterilization before being taken into the super clean bench (anyItems taken into the super clean bench should be disinfected). ② the tissue block is transferred from the centrifuge tube into a 25ml small beaker with 4 percent double-antibody (10000units/ml penicillin, 10mg/ml streptomycin) PBS for soaking for 5min by using forceps, and then is soaked for 5min in 25ml small beakers with 3 percent, 2 percent and 1 percent triple-antibody PBS. During this period, the PBS was stirred with forceps every 1min so that the tissue mass was completely contacted with PBS and sterilized. The primary pollution rate can be reduced to below 10% through the series of processes. ③ 1.5cm3The size tissue pieces were transferred from the small beaker to a penicillin bottle containing 1% tri-resistant DMEM/F12(2 mL). The tissue block in the penicillin bottle is cut into 3mm by scissors3The size can selectively cut larger tissue blocks in the cutting process, so as to avoid generating excessive smaller tissue blocks and muscle fibers. Fourthly, the penicillin bottle is kept still for half a minute, the upper liquid containing the small tissue blocks and the broken muscle fibers is discarded, and the partial tissue blocks are left. Then adding the heavy suspension tissue block containing 1% of three-resistance DMEM/F12, standing for half a minute, discarding the upper liquid, and repeating for three times to remove impurities except the tissue block to be cultured as far as possible. Fifthly, transferring the tissue blocks in the penicillin bottle to 25cm by using forceps2And (4) placing the tissue blocks in the culture bottle uniformly by using tweezers. The flask was inverted for 5h to hold the tissue pieces tightly together on the flask, and then the flask was turned upside down to add complete medium.
3) Subculturing of cells: the primary cells can be subcultured when growing to 50-60% abundance, all the subcultured cells are still cultured in an original culture bottle, and the 2 nd generation cells can be subcultured when growing to 75% abundance, and the cells are cultured in the original bottle; after the third generation, when the cell coverage rate reaches 90%, carrying out passage, and transferring two to three bottles in one bottle; ② reagents used in passage, including culture medium, pancreatin and serum, are preheated for more than 0.5h in a 24 ℃ incubator; ③ the concrete steps of passage: discarding the original culture medium, washing off residual serum with 1ml serum-free culture medium, and discarding; adding 1ml of 0.25% pancreatin, treating for 2-3min, observing cell state, shaking culture flask to make cells float in culture medium when 90% of cells shrink into round shape, and adding serum to stop pancreatin digestion; finally adding a complete culture medium, uniformly mixing, and subculturing the cell absorbing suspension into another culture bottle; 4) freezing and recovering cells: firstly, sucking the culture medium in a culture bottle before digesting the cells by pancreatin in the step 3), and freezing for later use; centrifuging the obtained cell suspension at the temperature of 4 ℃ for 5min at 200g, removing supernatant, and suspending the cells by using 1ml of complete culture medium with precooling at the temperature of 4 ℃; adding 150ul of precooled DMSO into a precooled cryopreservation tube, adding a precooled complete culture medium, uniformly mixing, adding the cell suspension prepared in the previous step, and uniformly mixing the cryopreserved cells at 4 ℃ for 1 h; -20 ℃ for 3 h; -80 ℃ overnight; preserving in liquid nitrogen for a long time; fourthly, after the frozen cells are taken out of the liquid nitrogen, the freezing tube is subjected to water bath at 37 ℃ for 1-2min, and the water bath is stopped when only sporadic small ice blocks remain in the freezing tube; fifthly, centrifuging at 200g for 5min at 4 ℃, discarding the supernatant to remove DMSO, adding a fresh complete culture medium for resuspension, centrifuging, discarding the supernatant to further clean residual DMSO, adding 1ml of fresh complete culture medium for resuspension; sixthly, inoculating the cell suspension into a culture bottle, adding 3ml of unfrozen culture medium for freezing and storing for later use, after the cells are recovered, carrying out corresponding experiments by more than two passages, and finding out that the cells are in a latent period at 0-1d according to a growth curve of 20 generations of muscle cells; 1-3d, entering logarithmic growth phase; 3-5d, stabilizing growth period; after 5 days, the decline period was entered. Cell-related experiments should be performed in 2-3 days, where cell proliferation is most vigorous and cell number is high. The cell has passed to 40 generations and is in good condition. The growth state of the cells in primary culture for different days is shown in figure 1, and the growth state of the cells in subculture for different generations is shown in figure 2.
FIG. 3 is a graph showing the growth curve of cells after two passages of cryopreservation and recovery. Growth curves were counted on a hemocytometer and plotted using GraphPad Prism 5 software.
Example 2
The method for identifying the cell species of subculture described in example 1, in this example, the 30 th passage cell was taken, and the specific method was as follows: identification of cell species: determining the source of a cell line by comparing homology of an amplified muscle cell line 18S rRNA gene with a sebastes schlegeli hilgendorf muscle tissue 18S rRNA gene, designing a specific primer of the 18S rRNA gene, extracting SSCM cell total DNA and sebastes schlegeli hilgendorf muscle tissue total DNA by using a phenol-chloroform method, wherein a PCR system comprises 2.5uL Taq enzyme buffer (10X), 2uL dNTPs (10mol/L), 0.5uL (10umol/L) of each primer, 1uLDNA template, 0.2uL Taq enzyme and 18.3uL water, and 25uL of the total system, mixing and carrying out PCR amplification, wherein the amplification conditions comprise pre-denaturation at 95 ℃ for 5min, denaturation at 95 ℃ for 30S, annealing at 59 ℃ for 30S, extension at 72 ℃ for 1min, circulation for 30 times, extension at 72 ℃ for 5min and heat preservation at 12 ℃; and (3) after electrophoresis detection of the PCR product, recovering, cloning to a PMD-19T vector, transforming to DH5 alpha cell, and detecting to obtain a positive monoclonal strain for sequencing and comparative analysis. Sequencing alignment analysis shows that the sequence similarity of the muscle cell line and sebastes schlegeli hilgendorf 18S rRNA is about 99.7%, which indicates that the cell line is derived from sebastes schlegeli hilgendorf. Identifying cell types: determining the types of passage cells by amplifying a marker gene Pax7 of a muscle satellite cell and a marker gene MyoD of a myoblast, and designing specific primers of a Pax7 gene and a MyoD gene; rpl17 serves as an internal reference gene. Extracting total RNA of cells and total RNA of sebastes schlegeli muscle tissue by using a trizol method. Inverting the cell RNA into cDNA by a gDNA Eraser method, and amplifying gene sequences of Pax7, MyoD and Rpl17 by taking the cDNA as a template; the PCR system and amplification conditions were as described above, with 59 deg.C (Pax7)/57 deg.C (MyoD)/60 deg.C (Rpl17) annealed for 30S, and 72 deg.C extended for 2min (Pax7)/1min (MyoD)/30S (Rpl 17); pax7 is a marker gene for muscle satellite cells, and is expressed in the cells of the first 10 generations and is not expressed in the cells of the 25 generations, which indicates that muscle satellite cells exist in the cells of the first ten generations; MyoD is a protein which is characterized by expression of myoblasts and fibroblasts, is expressed in 5, 10 and 25 generations, and is expressed more in 25 representatives; indicating myoblasts with stronger proliferative capacity in the cell line. The results are shown in FIG. 4. Myod is a marker gene of myoblasts, and the strong expression of Myod indicates that myoblasts gradually occupy the dominant position in cell lines.
The 18S primers were designed as follows:
18S-fw 5′-primer：5′-GTAAACCAAGAACTGAGGAGGAG-3′，
18S-rv 3′-primer：5′-GAGAAAAGCAGAAGTGGAATCA-3′；
the Pax7 primer was designed as follows:
Pax7-fw 5′-primer：5′-CCAAACGGCCAGATGAT-3′，
Pax7-rv 3′-primer：5′-ACAAAGCAGGGTTTGGA-3′；
the MyoD primers were designed as follows:
MyoD-fw 5′-primer：5′-ATGGAGCTGTCGGATATCT-3′，
MyoD-rv 3′-primer：5′-TGTAGGACTTGATAGATCAGGTTG-3′；
the Rpl17 primer was designed as follows:
Rpl17-fw 5′-primer：5′-AGGCGACGCACCTACCG-3′，
Rpl17-rv 3′-primer：5′-CCTCTGGTTTGGGGACGA-3′
example 3
Method for inducing differentiation of subcultured cells according to example 1: 0-24 h: setting the time for inoculating the cells to be 0h, setting the time to be 0-24h as a growth medium, wherein the growth medium is an L-15 medium containing 15% FBS, and the cells normally grow at 24 h; 24-48 h: changing a growth culture medium into a differentiation culture medium within 24 hours, wherein the differentiation culture medium is an L-15 culture medium containing 2% DHS, and cells begin to fuse within 48 hours; 48-72 h: continuously culturing in a differentiation culture medium for 72 hours to ensure that a large number of cells are fused; 72-96 h: continuously culturing in a differentiation culture medium, and almost all the cells are multi-core myotubes after cell fusion is finished at 96 h; secondly, photographing and observing the cells cultured by the growth medium and the differentiation medium every 24 hours, and recording the process from the mononuclear myocytes to the fusion of the mononuclear myocytes into the polynuclear myofibers; thirdly, 96h cells are stained with Hoechst and phalloidin respectively for nucleus and cytoplasm, and the proportion of mononuclear cells and multinuclear cells is counted, which is shown in figure 5.
The photographing instrument is a Nikon fluorescence inverted biological microscope. The method for staining Hoechst (Solarbio, No. C0021-1mL), phalloidin (Yeasen, No.40734ES75) is described in reference to the description. The ratio of monocytes to multinucleated cells was counted by Nikon fluorescence inverted biomicroscopy NIS-Elements BR software and the ratio map was plotted by GraphPad Prism 5 software.
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Claims (2)
1. An in vitro culture method of sebastes schlegeli hilgendorf myoblasts is characterized in that the method is as follows
1) Sampling and disinfecting muscle tissue blocks of sebastes schlegeli hilgendorf: taking a small mass of sebastes schlegeli dorsalis, and transferring into a 50ml centrifuge tube containing 4% of three-antibody PBS (phosphate buffer solution);
2) primary culture of cells: firstly, moving the tissue block from a centrifuge tube to a 25ml container containing 4% double-resistant PBS, soaking for 5min, then sequentially passing through 25ml containers containing 3%, 2% and 1% triple-resistant PBS, soaking for 5min, and stirring at intervals so that the tissue block is in full contact with the PBS and sterilized; moving the tissue blocks to a container filled with 1% of three-antibody DMEM/F12; cut the tissue block to 3mm3Size; standing the tissue block solution cut in the step II, discarding the upper liquid and leaving the tissue blocks; adding a heavy suspension tissue block containing 1% of three-resistance DMEM/F12, standing, discarding the upper liquid again, and repeating for three times; transferring the tissue blocks into a culture bottle, uniformly placing the tissue blocks, inverting the culture bottle to enable the tissue blocks to be tightly combined on the culture bottle, and then adding the complete culture medium into the culture bottle in the forward direction;
3) subculturing of cells: the primary cells can be subcultured when growing to 50-60% abundance, all the subcultured cells are still cultured in an original culture bottle, and the 2 nd generation cells can be subcultured when growing to 75% abundance, and the cells are cultured in the original bottle; after the third generation, when the cell coverage rate reaches 90%, passage is carried out, and one bottle is transferred to two to three bottles; ② reagents used in passage, including culture medium, pancreatin and serum, are preheated for more than 0.5h in a 24 ℃ incubator; ③ the concrete steps of passage: discarding the original culture medium, washing off residual serum with 1ml serum-free culture medium, and discarding; adding 1ml of 0.25% pancreatin, treating for 2-3min, observing cell state, shaking culture flask to make cells float in culture medium when 90% of cells shrink into round shape, and adding serum to stop pancreatin digestion; finally adding a complete culture medium, uniformly mixing, and subculturing the cell absorbing suspension into another culture bottle;
4) freezing and recovering cells: firstly, sucking the culture medium in a culture bottle before digesting the cells by pancreatin in the step 3), and freezing for later use; centrifuging the obtained cell suspension at the temperature of 4 ℃ for 5min at 200g, removing supernatant, and suspending the cells by using 1ml of complete culture medium with precooling at the temperature of 4 ℃; adding 150ul of precooled DMSO into a precooled cryopreservation tube, adding a precooled complete culture medium, uniformly mixing, adding the cell suspension prepared in the previous step, and uniformly mixing the cryopreserved cells at 4 ℃ for 1 h; -20 ℃ for 3 h; -80 ℃ overnight; preserving in liquid nitrogen for a long time; fourthly, after the frozen cells are taken out of the liquid nitrogen, the freezing tube is subjected to water bath at 37 ℃ for 1-2min, and the water bath is stopped when only sporadic small ice blocks remain in the freezing tube; fifthly, centrifuging at 200g for 5min at 4 ℃, discarding the supernatant to remove DMSO, adding a fresh complete culture medium for resuspension, centrifuging, discarding the supernatant to further clean residual DMSO, adding 1ml of fresh complete culture medium for resuspension; sixthly, inoculating the cell suspension into a culture bottle, adding 3ml of unfreezing culture medium for standby cryopreservation, after the cells are recovered, carrying out corresponding experiments after passage for more than two times, wherein the cell-related experiments are carried out in 2-3 d;
the complete culture medium comprises a mixed culture medium of the following components: the mass ratio of the MEM nonessential amino acids to the DMEM/F12 is 1%, the mass ratio of the MEM nonessential amino acids to the DMEM/F12, the mass ratio of the DMEM/F12 to the DMEM/FGF 10ng/mL, and the weight ratio of the DMEM/FGF to the fetal calf serum is 1: 1, mixing; when the mass ratio of the fetal calf serum in the complete culture medium used after the cells passed to the 10 th passage was adjusted to 15%, the rest components were unchanged.
2. The in vitro culture method of sebastes schlegeli hilgendorf myoblasts according to claim 1, wherein said serum-free medium is DMEM/F12 medium containing 1% double antibody by mass.
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| CN105200005A (en) * | 2014-08-13 | 2015-12-30 | 中国科学院海洋研究所 | Paralichthys olivaceus muscle satellite cell line establishing method, specific primer for identifying paralichthys olivaceus muscle satellite cell marker gene and application of specific primer |
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| CN101591638A (en) * | 2009-06-23 | 2009-12-02 | 中国水产科学研究院黄海水产研究所 | The construction process of kidney cell line of scophthalmus maximus |
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