AU2020102488A4 - Gobiocypris rarus swim bladder cell line and culture method and induced transformation method thereof - Google Patents
Gobiocypris rarus swim bladder cell line and culture method and induced transformation method thereof Download PDFInfo
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Abstract
The present invention discloses a Gobiocypris rarus swim bladder
cell line having a collection number of CCTCC NO:C 2019243, and
further discloses a construction and culture method for the Gobiocypris
rarus swim bladder cell line. The construction and culture method
specifically includes: primary culture of Gobiocypris rarus swim bladder
cells, subculture of the Gobiocypris rarus swim bladder cells, and
cryopreservation and resuscitation of the cells. Moreover, the present
invention further discloses an induced transformation method for the
Gobiocypris rarus swim bladder cell line. The induced transformation
method specifically includes the processes: preparation of an
N-Methyl-N-nitroso-N'-nitroguanidine solution, induction of Gobiocypris
rarus swim bladder cells by N-Methyl-N-nitroso-N'-nitroguanidine, and
low-serum culture after induction. In the Gobiocypris rarus swim bladder
cell line and the culture method and the induced transformation method
thereof disclosed by the present invention, a basis is provided for
applications of Gobiocypris rarus in aquatic animal transformation study,
and the applications of the Gobiocypris rarus in biological experiments
can be widened.
1
Drawings ofDescription
FIG. 1
FIG. 2
1
Description
Drawings ofDescription
FIG. 1
FIG. 2
Description
Technical Field
The present invention relates to the technical field of cytobiology, and particularly relates to a Gobiocypris rarus swim bladder cell line and a culture method and an induced transformation method thereof.
Background
Gobiocypris rarus belongs to Gobiocypris of Cyprinidae, is a special species in China, and is mainly distributed in the upper reaches of Yangtze River in Sichuan province. The Gobiocypris rarus has characteristics of small individual size (30-90 mm of an adult), is capable of growing adaptive to a wider temperature range (0-30°C), and is short in sexual maturity and life cycle, long in breeding season, high in spawning frequency, large in egg laying amount, transparent and observable in eggs and sensitive to environmental pollutants. Because of these characteristics, the Gobiocypris rarus becomes a tested organism with excellent aquatic ecotoxicology, and is recommended as a native test organism in China to be used in aquatic ecotoxicity testing.
However, the Gobiocypris rarus is only limited to be used in the aquatic ecotoxicity testing at present, and the application of Gobiocypris rarus in aquatic animal cell transformation researches has not been
Description
reported.
Therefore, to expand an application field of the Gobiocypris rarus and enable the Gobiocypris rarus to be widely applied to biological experiments, providing a novel Gobiocypris rarus swim bladder cell line and a culture method thereof is a problem that urgently needs to be solved by those skilled in the art. Moreover, to apply the Gobiocypris rarus to the aquatic animal cell transformation researches, providing an induced transformation method of the Gobiocypris rarus swim bladder cell line is also a problem that urgently needs to be solved by those skilled in the art.
Summary
In view of this, the present invention provides a Gobiocypris rarus swim bladder cell line and a culture method and an induced transformation method thereof. Thus, a basis is provided for applications of Gobiocypris rarus in aquatic animal transformation study, and the applications of the Gobiocypris rarus in biological experiments can be widened.
To achieve the above purposes, technical solutions of the present invention are as follows:
A Gobiocypris rarus swim bladder cell line has a collection number of CCTCC NO:C 2019243.
Specifically, the Gobiocypris rarus swim bladder cell line is classified and named as a Gobiocypris rarus swim bladder cell line GrSB, preserved in China Center for Type Culture Collection on September 26, 2019 and proved to survive, and has a collection registration number of
Description
CCTCC NO:C 2019243. A collection address is China Center for Type Culture Collection, Wuhan University, in Wuhan, China.
The preferred technical solutions have beneficial effects as follows: the Gobiocypris rarus swim bladder cell line disclosed in the present invention may perform long-term subculture and can be widely applied to biological experiments.
The present invention further provides a culture method of the above Gobiocypris rarus swim bladder cell line. The culture method specifically includes the following steps:
(1) Primary culture of Gobiocypris rarus swim bladder cells
taking healthy Gobiocypris rarus swim bladder tissues; cutting up the tissues for digestive treatment; and performing primary culture on the tissues by a first complete culture solution so as to obtain primary culture cells;
(2) Subculture of the Gobiocypris rarus swim bladder cells
performing digestive treatment on the primary culture cells; and collecting suspension cells to be inoculated to a second complete culture solution for subculture, thereby obtaining subculture cells, i.e., the Gobiocypris rarus swim bladder cell line; and
(3) Cryopreservation and resuscitation of the cells
performing digestive treatment on the subculture cells; mixing a precipitate obtained by centrifugal separation with cryopreservation fluid; and preliminarily refrigerating the mixture, and cryopreserving the mixture in liquid nitrogen for a long time;
Description
melting the mixture after cryopreservation; performing centrifugal separation; adding the obtained precipitate into the second complete culture solution for cleaning; then performing centrifugal separation again; and collecting cells to be inoculated to the second complete culture solution for culture.
The preferred technical solutions have beneficial effects as follows: according to the culture method for the Gobiocypris rarus swim bladder cell line disclosed in the present invention, the Gobiocypris rarus swim bladder cells are extracted from the healthy Gobiocypris rarus swim bladder tissues; and the Gobiocypris rarus swim bladder cell line (having the collection number of CCTCC NO:C 2019243) is obtained by virtue of the primary culture and the subculture, may perform the long-term subculture and can be widely applied to experiments. The cell line can be preserved for a long time after cryopreserved, can obtain biological activity again through resuscitation after cryopreserved, and may continuously perform the subculture.
Preferably, the first complete culture solution includes the following components in percentage by volume: 20% of fetal bovine serum, 1% of a triple antibiotic solution and 79% of an M199 culture solution.
The second complete culture solution includes the following components in percentage by volume: 10% of fetal bovine serum and 90% of an M199 culture solution.
Preferably, the triple antibiotic solution includes 100 U/mL of penicillin, 100 pg/nL of streptomycin and 0.25 pg/nL of amphotericin B; and the triple antibiotic solution adopts a product of a GIBCO, USA.
Description
The preferred technical solutions have beneficial effects as follows: the complete medium disclosed in the present invention can provide sufficient nutritive materials for the primary culture and subculture of the cells, and is suitable for Gobiocypris rarus swim bladder cell culture.
Preferably, the step (1) specifically includes the following steps:
(11) soaking fresh and healthy Gobiocypris rarus in 75% of alcohol for 1-2 min; taking swim bladder tissues in a sterile manner on a super clean bench; removing membrane components on the surfaces of the swim bladder tissues; rinsing the tissues with a PBS buffer solution for 2-3 times; and preserving the tissues in a preservation culture solution;
(12) taking out the swim bladder tissues; cutting the tissues into tissue blocks of 1 mm3 ; mixing the tissue blocks with the PBS buffer solution; performing centrifugal separation to remove the supernatant; collecting a precipitate; and repeatedly performing operations of mixing with the PBS buffer solution, performing centrifugal separation and removing the supernatant so as to obtain pretreated tissue blocks;
(13) adding a type II collagenase solution into the pretreated tissue blocks according to a volume ratio of 1:(5-10) for performing first digestive treatment, wherein a temperature of the first digestive treatment is 37°C, and time is 20-30 min; then performing centrifugal separation to remove the supernatant; and adding a first complete culture solution to perform centrifugal separation again, removing the supernatant, and collecting swim bladder cells;
(14) suspending the swim bladder cells with the first complete culture solution until cell density is higher than 2x1O' cells/mL; inoculating the cells into a cell culture flasks; culturing the cells at 27°C
Description
until the bottom of the cell culture flasks is full of the cells; sucking the cultured solution by a sucker; and cleaning the cells with the PBS buffer solution twice, thereby obtaining the primary culture cells.
The preferred technical solutions have beneficial effects as follows: according to the primary culture process disclosed in the present invention, the swim bladder cells are extracted from the fresh and healthy Gobiocypris rarus, and the cells grow stably by virtue of the primary culture, so that the primary culture cells can be obtained.
Preferably, the preservation culture solution in the step (11) includes the following components in percentage by volume: 5% of fetal bovine serum and 95% of an M199 culture solution.
Preferably, in the step (12), 5 mL of the PBS buffer solution is added into the tissue blocks of 1 mm2 ; a rotation speed of the centrifugal separation is 1000 rpm; and time is 3 min.
The preferred technical solutions have beneficial effects as follows: blood cells in the sampling process can be removed by virtue of low-speed centrifugal separation, thereby facilitating separation to obtain the swim bladder cells.
Preferably, a concentration of the type II collagenase solution in the step (13) is 0.1 mg/mL; and a rotation speed of the centrifugal separation is 1000 rpm, and time is 5 min.
Preferably, the step (2) specifically includes the following steps:
(21) adding a 0.25% of trypsin-EDTA solution into the primary culture cells for performing second digestive treatment; sucking the second digestive treatment solution by a sucker after the cells are digested
Description
to become round, wherein a temperature of the second digestive treatment is 20-26°C, and time is 30s-1 min; and adding a second complete culture medium to terminate digestion so as to obtain second digestive treatment cells;
(22) taking the second digestive treatment cells; slightly blowing the cells by the sucker; collecting suspension cells; inoculating the suspension cells into a cell culture flasks according to a ratio of 1:2; adding a second complete culture solution; culturing the cells at 27°C until the Gobiocypris rarus swim bladder cells grow into a single layer, thereby obtaining subculture cells;
(23) taking the subculture cells; and repeating the steps (21) to (22).
The preferred technical solutions have beneficial effects as follows: subculture is performed according to the subculture method disclosed in the present invention; and the cells are rapid in growth and stable in morphology.
Preferably, the 0.25% of trypsin-EDTA solution includes the following components in percentage by volume: 0.25% of trypsin, 0.1% of EDTA and the balance of a PBS solution.
Preferably, the ratio of 1:2 in the step (22) means that the subculture is performed according to a ratio of 1:2; the bottom wall of the cell culture flasks is 25 cm 2 ; and a volume of the second complete culture solution is 5 mL.
Preferably, the step (3) specifically includes the following steps:
(31) adding a 0.25% of trypsin-EDTA solution into subculture cells that are located in a logarithmic phase and have cell density of more than
Description
% in the step (2) for performing second digestive treatment; sucking the second digestive treatment solution by a sucker after the cells are digested to become round; and adding a fresh medium to terminate digestion so as to obtain second digestive treatment cells;
(32) performing centrifugal separation on the second digestive treatment cells in the step (31); removing the supernatant; adding cryopreservation fluid into the obtained precipitate; resuspending the cells according to a ratio of 5x106cells/mL; injecting the cells into cryovials; placing the cryovials in a programmed cooling box; preliminarily refrigerating the cells at -80°C for 16-24 h; and finally adding the cells into liquid nitrogen to be preserved for a long time;
(33) thawing the cells cryopreserved in the liquid nitrogen under a water bath condition of 37-40°C; centrifuging to remove the supernatant; adding the obtained precipitate into the complete culture solution for resuspending; performing centrifugal separation to remove the supernatant again; collecting Gobiocypris rarus swim bladder cells to be inoculated into the culture solution; culturing the cells at 27°C for 16-24 h; and replacing the culture solution for continuously culturing the cells, thereby obtaining the resuscitated Gobiocypris rarus swim bladder cells.
The preferred technical solutions have beneficial effects as follows: according to the cryopreservation and resuscitation method of the cells disclosed in the present invention, the cells can be cryopreserved for a long time, and may be adherent to walls after resuscitation; the bottle bottom is full of the cells within 16-24 h; and a proliferation rate is high.
Preferably, the 0.25% of trypsin-EDTA solution includes the following components in percentage by volume: 0.25% of trypsin, 0.1%
Description
of EDTA and the balance of a PBS solution; a temperature of the second digestive treatment is 20-26°C; and a rotation speed of the centrifugal treatment is 1000 rpm, and time is 5 min.
Preferably, the cryopreservation fluid includes the following components in percentage by volume: 20% of fetal bovine serum, 10% of dimethyl sulfoxide and 70% of an M199 culture solution.
The present invention further provides an induced transformation method for the Gobiocypris rarus swim bladder cell line. The induced transformation method specifically includes the following steps:
(A) Preparation of an N-Methyl-N-nitroso-N'-nitroguanidine solution
dissolving N-Methyl-N-nitroso-N'-nitroguanidine into deionized water to prepare a mother solution; and preparing an N-Methyl-N-nitroso-N'-nitroguanidine cell culture solution from the mother solution;
(B) Induction of Gobiocypris rarus swim bladder cells by N-Methyl-N-nitroso-N'-nitroguanidine
adding the 2-10-generation Gobiocypris rarus swim bladder cells according to claim 1 into a first complete culture solution; culturing the cells at 270 C for 16-24 h; and adding the N-Methyl-N-nitroso-N'-nitroguanidine cell culture solution to perform induced culture so as to obtain induced cells;
(C) Low-serum culture
Description
adding the induced cells into a culture solution A for culture; culturing the cells with a culture solution B; and observing a condition that a transforming focus of the cells occurs, thereby obtaining the Gobiocypris rarus swim bladder cell line subjected to induced transformation.
The preferred technical solutions have beneficial effects as follows: in the present invention, the Gobiocypris rarus swim bladder cell line is transformed with the N-Methyl-N-nitroso-N'-nitroguanidine (MNNG); the operation is simple and effective; and a reference base can be provided for aquatic animal cell transformation.
Preferably, the mother solution in the step (A) has a concentration of mg/mL; a concentration of the N-Methyl-N-nitroso-N'-nitroguanidine cell culture solution is 1-11 Ig/mL; the first complete culture solution in the step (B) includes the following components in percentage by volume: % of fetal bovine serum, 1% of a triple antibiotic solution and 79% of an M199 culture solution; and a temperature of the induced culture is 27 0C, and time is 24-48 h.
Preferably, the concentration of the N-Methyl-N-nitroso-N'-nitroguanidine cell culture solution may be 1 pg/mL, 3 pg/mL, 5 pg/mL, 7 pg/mL, 9 g/mL or 11 Ig/mL.
Preferably, the cleaning in the step (C) is that cleaning is performed with an HBSS solution for 1-3 times.
The culture solution A includes the following components in percentage by volume: 20% of fetal bovine serum and 80% of an M199
Description
culture solution; and a culture temperature of the culture solution A is 27 0C, and time is 2-3 weeks.
The culture solution B includes the following components in percentage by volume: 5% of fetal bovine serum and 95% of the M199 culture solution; and a culture temperature of the culture solution B is 27 0C, and time is 30-50 d.
The N-Methyl-N-nitroso-N'-nitroguanidine (MNNG) is a genotoxicity mutagenic agent, and produces induced mutation and carcinogenesis by virtue of diet, respiration or skin contact. However, mechanisms of nitrosamine chemical carcinogens to cytotoxicity and carcinogenicity are still unclear at present. In the present invention, transformation of the Gobiocypris rarus swim bladder cell line induced by the nitrosamine substance MNNG is researched in aspects such as morphology and chromosome, and a basis is provided for nitrosamine chemical substance induced aquatic animal cell mutagenesis.
Through the above technical solutions, compared with the prior art, the present invention discloses the Gobiocypris rarus swim bladder cell line and the culture method and the induced transformation method thereof, and has beneficial effects as follows:
(1) The Gobiocypris rarus swim bladder cell line disclosed in the present invention can perform long-term subculture, can be widely applied to the experiments, and has special practical values.
(2) According to the culture method for the Gobiocypris rarus swim bladder cell line disclosed in the present invention, specific operations are simple; the Gobiocypris rarus swim bladder cells are extracted from the
Description
fresh and healthy Gobiocypris rarus; and the stable Gobiocypris rarus swim bladder cell line is obtained by virtue of the primary culture and subculture. Moreover, the cryopreservation and resuscitation method for the Gobiocypris rarus swim bladder cell line is disclosed, so that the cell line is conveniently applied to the experiments.
(3) According to the induced transformation method for the Gobiocypris rarus swim bladder cell line disclosed in the present invention, specific operations are simple; and by virtue of induced culture of the N-Methyl-N-nitroso-N'-nitroguanidine, the Gobiocypris rarus swim bladder cell line can be transformed, and the basis is provided for nitrosamine chemical substance induced aquatic animal cell mutagenesis.
Description of Drawings
To more clearly describe the technical solution in the embodiments of the present invention or in the prior art, the drawings required to be used in the description of the embodiments or the prior art will be simply presented below. Apparently, the drawings in the following description are merely the embodiments of the present invention, and for those ordinary skilled in the art, other drawings can also be obtained according to the provided drawings without contributing creative labor.
Fig. 1 shows cell morphology observation of primary culture of Gobiocypris rarus swim bladder cells provided in Embodiment 1 of the present invention.
Description
Fig. 2 shows 5 0 th generation cell morphology observation of subculture of Gobiocypris rarus swim bladder cells provided in Embodiment 1 of the present invention.
Fig. 3 is a growth curve chart of Gobiocypris rarus swim bladder cells cultured by different media provided in Embodiment 1 of the present invention.
Fig. 4 is a growth curve chart of Gobiocypris rarus swim bladder cells cultured by fetal bovine serum (FBS) of different concentrations provided in Embodiment 1 of the present invention.
Fig. 5 is a growth curve chart of Gobiocypris rarus swim bladder cells cultured at different temperatures provided in Embodiment 1 of the present invention.
Fig. 6 shows cell morphology of normal Gobiocypris rarus swim bladder cells.
Fig. 7 shows cell morphology of Gobiocypris rarus swim bladder cells obtained by induced culture of 1 g/nL of MNNG in Reference example 1.
Fig. 8 shows cell morphology of Gobiocypris rarus swim bladder cells subjected to induced culture of 3 pg/mL of MNNG in Embodiment 2.
Fig. 9 shows cell morphology of Gobiocypris rarus swim bladder cells subjected to induced culture of 5 pg/mL of MNNG in Embodiment 3.
Description
Fig. 10 shows cell morphology of Gobiocypris rarus swim bladder cells subjected to induced culture of 7 pg/mL of MNNG in Embodiment 4.
Fig. 11 shows cell morphology of Gobiocypris rarus swim bladder cells subjected to induced culture of 9 pg/mL of MNNG in Embodiment 5.
Fig. 12 is a chromosome analysis diagram of Gobiocypris rarus swim bladder cells before and after MNNG transformation provided in the present invention.
Detailed Description
The technical solution in embodiments of the present invention will be clearly and fully described below. Apparently, the described embodiments are merely part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by those ordinary skilled in the art without contributing creative labor will belong to the protection scope of the present invention.
Embodiment 1
Embodiment 1 of the present invention discloses a Gobiocypris rarus swim bladder cell line having a collection number of CCTCC NO:C 2019243.
Specifically, the Gobiocypris rarus swim bladder cell line is classified and named as a Gobiocypris rarus swim bladder cell line GrSB,
Description
preserved in China Center for Type Culture Collection on September 26, 2019 and proved to survive, and has a collection registration number of CCTCC NO:C 2019243. A collection address is China Center for Type Culture Collection, Wuhan University, in Wuhan, China.
Embodiment 1 of the present invention further provides a culture method of the above Gobiocypris rarus swim bladder cell line. The culture method specifically includes the following steps:
(1) Primary culture of Gobiocypris rarus swim bladder cells
(11) fresh and healthy Gobiocypris rarus was soaked in 75% of alcohol for 1-2 min; swim bladder tissues were taken in a sterile manner on a super clean bench; membrane components on the surfaces of the swim bladder tissues were removed; the tissues were rinsed with a PBS buffer solution for 2-3 times; and the tissues were preserved in a preservation culture solution, wherein the first culture solution included 5% of fetal bovine serum and an M199 culture solution; and the preservation culture solution included the following components in percentage by volume: 5% of fetal bovine serum and 95% of the M199 culture solution;
(12) the swim bladder tissues were taken out; the tissues were cut into tissue blocks of 1 mm 2 ; 5 mL of a PBS buffer solution was added into a 15 ml of centrifuge tube; centrifugal separation was performed to remove the supernatant; a precipitate was collected; and operations of mixing with the PBS buffer solution, performing centrifugal separation and removing the supernatant were repeatedly performed so as to obtain pretreated tissue blocks, wherein a rotation speed of the centrifugal separation was 1000 rpm, and time was 3 min;
Description
(13) a type II collagenase solution was added into the pretreated tissue blocks according to a volume ratio of 1:(5-10) for performing first digestive treatment, wherein a temperature of the first digestive treatment was 37°C, and time was 20-30 min; then centrifugal separation was performed to remove the supernatant; and a first complete culture solution was added to perform centrifugal separation again, the supernatant was removed, and swim bladder cells were collected; wherein a concentration of the type II collagenase solution was 0.1 mg/mL; and a rotation speed of the centrifugal separation was 1000 rpm, and time was 5 min;
(14) the swim bladder cells were suspended with the first complete culture solution until cell density was higher than 2x10 5 cells/mL; the cells were inoculated into a cell culture flasks; the cells were cultured at 27 0C until the bottom of the cell culture flasks was full of the cells; the cultured solution was sucked by a sucker; and the cells were cleaned with the PBS buffer solution twice so as to obtain the primary culture cells;
(2) Subculture of the Gobiocypris rarus swim bladder cells
(21) a 0.25% of trypsin-EDTA solution was added into the primary culture cells for performing second digestive treatment; the second digestive treatment solution was sucked by a sucker after the cells were digested to become round; and a second complete culture medium was added to terminate digestion so as to obtain second digestive treatment cells; wherein the 0.25% of trypsin-EDTA solution included the following components in percentage by volume: 0.25% of trypsin, 0.1% of EDTA and the balance of a PBS solution;
Description
(22) the second digestive treatment cells were taken; the cells were slightly blown by the sucker; suspension cells were collected; the suspension cells were inoculated into a cell culture flasks according to a ratio of 1:2; a second complete culture solution was added to reach 5 mL/bottle; the cells were cultured at 27°C until the Gobiocypris rarus swim bladder cells growed into a single layer, thereby obtaining subculture cells, wherein the ratio of 1:2 was that the subculture was performed according to a ratio of 1:2; the bottom wall of the cell culture flasks was 25 cm 2 ; and a volume of the second complete culture solution was 5 mL;
(23) the subculture cells were taken; and the steps (21) to (22) were repeated;
(3) Cryopreservation and resuscitation of the cells
(31) a 0.25% of trypsin-EDTA solution was added into subculture cells (having cell density of about 2x10 6/mL) that were located in a logarithmic phase and had cell density of more than 90% in the step (2) for performing second digestive treatment; the second digestive treatment solution was sucked by a sucker after the cells were digested to become round; and a fresh medium was added to terminate digestion so as to obtain second digestive treatment cells; wherein the 0.25% of trypsin-EDTA solution included the following components in percentage by volume: 0.25% of trypsin, 0.1% of EDTA and the balance of the PBS solution; and a temperature of the second digestive treatment was -26 0C, and time was 30 s-1 min;
Description
(32) centrifugal separation was performed on the second digestive treatment cells in the step (31); the supernatant was removed; 2 mL of cryopreservation fluid was added into the obtained precipitate (including about 1xI07 cells); the cells were resuspended according to a ratio of x106cells/mL; the cells were injected into a cryovials; the cryovials was placed in a programmed cooling box; the cells were preliminarily refrigerated at -80°C for overnight; and finally the cells were added into liquid nitrogen to be preserved for a long time; wherein a rotation speed of the centrifugal separation was 1000 rpm, and time was 5 min; and the cryopreservation fluid included 20% of FBS, 10% of DMSO and 70% of an M199 culture medium;
(33) the cells were thawed under a water bath condition of 37-40°C after cryopreservation; centrifugal separation was performed to remove the supernatant; the obtained precipitate was added into the second complete culture solution; centrifugal separation was performed to remove the supernatant again; Gobiocypris rarus swim bladder cells were collected to be inoculated into the second complete culture solution; the cells were cultured at 27°C for 16-24 h; and the culture solution was replaced for continuously culturing the cells, thereby obtaining the resuscitated Gobiocyprisrarus swim bladder cells.
The first complete culture solution includes the following components in percentage by volume: 20% of fetal bovine serum, 1% of a triple antibiotic solution and 79% of an M199 culture solution; the triple antibiotic solution includes 100 U/mL of penicillin, 100 pg/mL of streptomycin and 0.25 pg/mL of amphotericin B; and the triple antibiotic solution adopted a product of a GIBCO, USA brand.
Description
The second complete culture solution includes the following components in percentage by volume: 10% of fetal bovine serum and 90% of the M199 culture solution.
A pH value of the M199 culture solution is 7.2-7.4.
The Gobiocypris rarus swim bladder cell line is cultured by the method disclosed in Embodiment 1 of the present invention; the cells can be subjected to subculture to 60 generations; and a subculture ratio is 1:3 or 1:4.
Result Validation
I. Gobiocypris rarus swim bladder cells subjected to primary culture in the step (1) of Embodiment 1 are subjected to cell morphology observation, and results are as shown in Fig. 1 of the description.
II. Gobiocypris rarus swim bladder cells subjected to subculture in the step (2) of Embodiment 1 are subjected to cell morphology observation, and results are as shown in Fig. 1 of the description.
III. Culture condition validation
1. Validation of culture media for the Gobiocypris rarus swim bladder cells
(1) Experimental objects
Control groups 1-4: four cell culture solutions such as DMEM, M199, MEM and L-15 were respectively selected; and FBS having a volume concentration of 10% was added for preparing a cell culture solution.
Description
(2) Experimental method
Cell density was adjusted to 5x104 mL-'; the four culture media were respectively inoculated a 6-well plate according to an amount of 2.5 mL/well; the cells were cultured in an incubator at 27°C; cells of 3 wells were taken out of each experimental group per day; the cells were collected by a Trypsin-EDTA digestion process and counted; the cells were totally cultured for 7 d and continuously counted for 7 times; and a growth curve of the cells was drawn. The results were as shown in Fig. 3 of the description.
2. Validation of serum concentrations in a culture process of the Gobiocypris rarus swim bladder cells
(1) Experimental grouping
Experimental groups 1-4: FBS was added into an M199 cell culture solution; and cell culture solutions having FBS volume concentrations of %, 10%, 15% and 20% were respectively prepared.
(2) Experimental method
Cell density was adjusted to 5x104 mL-'; the culture media of four serum concentrations were respectively inoculated a 6-well plate according to an amount of 2.5 mL/well; the cells were cultured in an incubator at 27°C; cells of 3 wells were taken out of each experimental group per day; the cells were collected by a Trypsin-EDTA digestion process and counted; the cells were totally cultured for 7 d and continuously counted for 7 times; and a growth curve of the cells was drawn. The results were as shown in Fig. 4 of the description.
Description
3. Culture temperatures in the culture process of the Gobiocypris rarus swim bladder cells
(1) Experimental grouping
Four different culture temperatures such as 15°C, 22°C, 27°C and 32°C were selected.
(2) Experimental method
A DMEM culture solution added with 10% of FBS was used; cell density was adjusted to 5x104 mL 1 ; cell suspension was inoculated a 6-well plate according to an amount of 2.5 mL/well, and placed in an incubator at four different culture temperatures; cells of 3 wells were taken out of each experimental group per day; the cells were collected by a Trypsin-EDTA digestion process and counted; the cells were totally cultured for 7 d and continuously counted for 7 times; and a growth curve of the cells was drawn. The results were as shown in Fig. 5 of the description.
As shown in Fig. 3, the Gobiocypris rarus swim bladder cell line has the best growth and proliferation in the M199 culture medium containing % of FBS at 27°C.
Embodiments 2-5
Embodiments 2-5 of the present invention further provide an induced transformation method for the Gobiocypris rarus swim bladder cell line disclosed in Embodiment 1. The induced transformation method specifically includes the following steps:
Description
(A) Preparation of an N-Methyl-N-nitroso-N'-nitroguanidine (MNNG) solution
The N-Methyl-N-nitroso-N'-nitroguanidine was dissolved into deionized water to prepare a 10 mg/mL of mother solution; and an N-Methyl-N-nitroso-N'-nitroguanidine cell culture solution having a concentration of 1-9 pg/mL was prepared from the mother solution; wherein a specific concentration was as shown in Table 1;
(B) Induction of Gobiocypris rarus swim bladder cells by N-Methyl-N-nitroso-N'-nitroguanidine
Gobiocypris rarus swim bladder cells subjected to subculture to reach the fifth generation in Embodiment 1 were cultured to a logarithmic growth phase; and the cells were added into the N-Methyl-N-nitroso-N'-nitroguanidine cell culture solution to be cultured at 27°C for 24-48 h so as to obtain induced cells; and
(C) Low-serum culture
The induced cells were separated; the induced culture solution was removed; the cells were cleaned with an HBSS solution for 1-3 times; and the cells were added into a high-serum culture solution to be cultured at 27°C for 2-3 weeks after cleaning; wherein the high-serum culture solution included 20% of FBS and an M199 culture solution;
the cells were cultured with a low-serum culture solution so as to obtain the Gobiocypris rarus swim bladder cell line subjected to induced transformation, wherein the low-serum culture solution includes 5% of FBS.
Description
Reference example 1
Only the concentration of the N-Methyl-N-nitroso-N'-nitroguanidine cell culture solution prepared from the mother solution in the step (A) of Embodiments 2-5 is replaced with Ig/mL, while other technical parameters are the same as those in Embodiments 2-5.
Reference example 2
Only the concentration of the N-Methyl-N-nitroso-N'-nitroguanidine cell culture solution prepared from the mother solution in the step (A) of Embodiments 2-5 is replaced with 11 Ig/mL, while the other technical parameters are the same as those in Embodiments 2-5.
Result Validation
I. Transformation culture results in Embodiments 2-5 are observed; and the numbers of transforming focuses produced by the induced Gobiocypris rarus swim bladder cells are as shown in Table 1 as follows:
Table 1
MNNG Concentration Number of transforming
focuses
Embodiment 2 3 g/mL +++
Embodiment 3 5 g/mL ++++
Embodiment 4 7 g/mL +++++
Embodiment 5 9 g/mL +
Reference example 1 Ig/mL
Description
Reference example 2 11 [g/mL
Notes: + represents occurrence of the transforming focuses; the more the + is, the greater the number of the transforming focuses is; represents no transforming focus.
Through the above results, the cells treated with the MNNG may produce transforming focuses of unequal numbers among normal cells after 10-30 d. In Reference example 1, the cells induced by an MNNG medium having an induced concentration of 1 pg/nL do not form any transforming focus; and in Reference example 2, cells induced by an MNNG medium having an induced concentration of 11 Ig/mL totally die due to an extremely high concentration. When the Gobiocypris rarus swim bladder cells are induced by an MNNG medium having an induced concentration of 3-9 pg/mL, the transforming focuses may occur. The cells induced by the MNNG media of different concentrations produce different numbers of the transforming focuses; and the transforming focuses are composed of dense overlapping cells. The transforming focuses have unequal sizes; smaller transforming focuses are composed of dozens of or hundreds of cells; and larger transforming focuses are visible to the naked eyes.
II. Observation of cell morphology of Gobiocypris rarus swim bladder cells before and after MNNG transformation
The fifth-generation Gobiocypris rarus swim bladder cells cultured to an exponental phase in the step (B) of Embodiments 2-5 are observed by a high power lens, and results are as shown in Fig. 6 of the description.
Description
The Gobiocypris rarus swim bladder cells subjected to induced transformation in the step (C) of Reference example 1 and Embodiments 2-5 are observed by the high power lens, and results are as shown in Figs. 7-11 of the description.
The results in Figs. 6-11 are as follows:
Fig. 6 shows normal Gobiocypris rarus swim bladder cells;
Fig. 7 shows Gobiocypris rarus swim bladder cells subjected to induced culture of 1 g/mL of MNNG in Reference example 1;
Fig. 8 shows Gobiocypris rarus swim bladder cells subjected to induced culture of 3 pg/mL of MNNG in Embodiment 2;
Fig. 9 shows Gobiocypris rarus swim bladder cells subjected to induced culture of 5 pg/mL of MNNG in Embodiment 3;
Fig. 10 shows Gobiocypris rarus swim bladder cells subjected to induced culture of 7 pg/mL of MNNG in Embodiment 4;
Fig. 11 shows Gobiocypris rarus swim bladder cells subjected to induced culture of 9 pg/mL of MNNG in Embodiment 5.
III. Chromosome analysis of transformed cells
(1) Experimental grouping
Blank control group: the fifth-generation Gobiocypris rarus swim bladder cells cultured to the exponental phase in the step (B) of Embodiments 2-5;
Control group: the Gobiocypris rarus swim bladder cells subjected to induced transformation in the step (C) of Reference example 1;
Description
Experimental group: the Gobiocypris rarus swim bladder cells subjected to induced transformation in the step (C) of Embodiments 2-5;
(2) Experimental method
Colchicine having a final concentration of 8-20 pg/mL was respectively added into the Gobiocypris rarus swim bladder cells in the blank control group, the control group and the experimental group; the cells were treated at 27°C for 4-8 h and then collected; the collected cells were subjected to hypotonic treatment with 0.075 mol/L of KCl for 25 min; 1 mL of pre-cooled Carnoy's fluid was added; centrifugation was performed at 1000 r/min for 5 min so as to remove the supernatant; and the cells were fixed with the pre-cooled Carnoy's fluid for 3 times, wherein fixation time was 15 min each time.
The cells were dripped by a cool dripping method, dried and then stained with 5% of Giemsa for 25 min.
Microscopic inspection was performed; 100 split phases were respectively selected for karyotype analysis and statistics; and chromosome analysis was performed. The results were as shown in Fig. 12.
(3) Experimental results
Fig. 12A shows a chromosome mode number 2n=50 of the normal Gobiocypris rarus swim bladder cells;
Fig. 12B shows a chromosome mode number 2n=50 of Gobiocypris rarus swim bladder cells induced by 1 g/mL of MNNG;
Description
Fig. 12C shows a chromosome mode number 2n=54 of Gobiocypris rarus swim bladder cells induced by 3 pg/mL of MNNG;
Fig. 12D shows a chromosome mode number 2n=72 of Gobiocypris rarus swim bladder cells induced by 5 pg/mL of MNNG;
Fig. 12E shows a chromosome mode number 2n=66 of Gobiocypris rarus swim bladder cells induced by 7 pg/mL of MNNG;
Fig. 12F shows a chromosome mode number 2n=96 of Gobiocypris rarus swim bladder cells induced by 9 pg/mL of MNNG.
Through the above results, the Gobiocypris rarus swim bladder cells induced by the MNNG of 3-9 pg/mL are all transformed and mutated.
Each embodiment in the description is described in a progressive way. The difference of each embodiment from each other is the focus of explanation. The same and similar parts among all of the embodiments can be referred to each other. For the device disclosed by the embodiments, because the device corresponds to a method disclosed by the embodiments, the device is simply described. Refer to the description of the method part for the related part.
The above description of the disclosed embodiments enables those skilled in the art to realize or use the present invention. Many modifications to these embodiments will be apparent to those skilled in the art. The general principle defined herein can be realized in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to these
Description
embodiments shown herein, but will conform to the widest scope consistent with the principle and novel features disclosed herein.
Claims (10)
1. A Gobiocypris rarus swim bladder cell line, having a collection number of CCTCC NO:C 2019243.
2. A culture method of the Gobiocypris rarus swim bladder cell line of claim 1, specifically comprising the following steps:
(1) primary culture of Gobiocypris rarus swim bladder cells
taking healthy Gobiocypris rarus swim bladder tissues; cutting up the tissues for digestive treatment; and performing primary culture on the tissues by a first complete culture solution so as to obtain primary culture cells;
(2) subculture of the Gobiocypris rarus swim bladder cells
performing digestive treatment on the primary culture cells; and collecting suspension cells to be inoculated to a second complete culture solution for subculture, thereby obtaining subculture cells, i.e., the Gobiocypris rarus swim bladder cell line;
(3) cryopreservation and resuscitation of the cells
performing digestive treatment on the subculture cells; mixing a precipitate obtained by centrifugal separation with cryopreservation fluid; and preliminarily refrigerating the mixture, and cryopreserving the mixture in liquid nitrogen for a long time;
melting the mixture after cryopreservation; performing centrifugal separation; adding the obtained precipitate into the second complete culture solution for cleaning; then performing centrifugal separation again;
Claims
and collecting cells to be inoculated to the second complete culture solution for culture.
3. The culture method of the Gobiocypris rarus swim bladder cell line according to claim 2, wherein the first complete culture solution comprises the following components in percentage by volume: 20% of fetal bovine serum, 1% of a triple antibiotic solution and 79% of an M199 culture solution;
the second complete culture solution comprises the following components in percentage by volume: 10% of fetal bovine serum and 90% of an M199 culture solution.
4. The culture method of the Gobiocypris rarus swim bladder cell line according to claim 2 or 3, wherein the step (1) specifically comprises the following steps:
(11) soaking fresh and healthy Gobiocypris rarus in 75% of alcohol for 1-2 min; taking swim bladder tissues in a sterile manner on a super clean bench; removing membrane components on the surfaces of the swim bladder tissues; rinsing the tissues with a PBS buffer solution for 2-3 times; and preserving the tissues in a preservation culture solution;
(12) taking out the swim bladder tissues; cutting the tissues into tissue blocks of 1 mm3 ; mixing the tissue blocks with the PBS buffer solution; performing centrifugal separation to remove the supernatant; collecting a precipitate; and repeatedly performing operations of mixing with the PBS buffer solution, performing centrifugal separation and removing the supernatant so as to obtain pretreated tissue blocks;
Claims
(13) adding a type II collagenase solution into the pretreated tissue blocks according to a volume ratio of 1:(5-10) for performing first digestive treatment, wherein a temperature of the first digestive treatment is 37C, and time is 20-30 min; then performing centrifugal separation to remove the supernatant; and adding a first complete culture solution to perform centrifugal separation again, removing the supernatant, and collecting swim bladder cells;
(14) suspending the swim bladder cells with the first complete culture solution until cell density is higher than 2x1O' cells/mL; inoculating the cells into a cell culture flasks; culturing the cells at 27°C until the bottom of the cell culture flasks is full of the cells; sucking the cultured solution by a sucker; and cleaning the cells with the PBS buffer solution twice, thereby obtaining the primary culture cells.
5. The culture method of the Gobiocypris rarus swim bladder cell line according to claim 2 or 3, wherein the step (2) specifically comprises the following steps:
(21) adding a 0.25% of trypsin-EDTA solution into the primary culture cells for performing second digestive treatment; sucking the second digestive treatment solution by a sucker after the cells are digested to become round, wherein a temperature of the second digestive treatment is 20-26°C, and time is 30s-1 min; and adding a second complete culture medium to terminate digestion so as to obtain second digestive treatment cells;
(22) taking the second digestive treatment cells; slightly blowing the cells by the sucker; collecting suspension cells; inoculating the
Claims
suspension cells into a cell culture flasks according to a ratio of 1:2; adding a second complete culture solution; culturing the cells at 27°C until the Gobiocypris rarus swim bladder cells grow into a single layer, thereby obtaining subculture cells;
(23) taking the subculture cells; and repeating the steps (21) to (22).
6. The culture method of the Gobiocypris rarus swim bladder cell line according to claim 2 or 3, wherein the step (3) specifically comprises the following steps:
(31) adding a 0.25% of trypsin-EDTA solution into subculture cells that are located in a logarithmic phase and have cell density of more than % in the step (2) for performing second digestive treatment; sucking the second digestive treatment solution by a sucker after the cells are digested to become round; and adding a fresh medium to terminate digestion so as to obtain second digestive treatment cells;
(32) performing centrifugal separation on the second digestive treatment cells in the step (31); removing the supernatant; adding cryopreservation fluid into the obtained precipitate; resuspending the cells according to a ratio of 5x106 cells/mL; injecting the cells into cryovials; placing the cryovials in a programmed cooling box at -80C overnight and then transferring the cryovials to liquid nitrogen for long-term storage.
(33) for reconstitution, thawing cell vials under running water at 37-40°C and subsequently centrifuging; removing the freezing medium; adding the obtained precipitate into the complete culture solution for resuspending;
Claims
performing centrifugal separation to remove the supernatant again; collecting Gobiocypris rarus swim bladder cells to be inoculated into the culture solution; culturing the cells at 27°C for 16-24 h; and replacing the culture solution for continuously culturing the cells, thereby obtaining the resuscitated Gobiocyprisrarus swim bladder cells.
7. The culture method of the Gobiocypris rarus swim bladder cell line according to claim 6, wherein the cryopreservation fluid comprises the following components in percentage by volume: 20% of fetal bovine serum, 10% of dimethyl sulfoxide and 70% of an M199 culture solution.
8. An induced transformation method for the Gobiocypris rarus swim bladder cell line of claim 1, specifically comprises the following steps:
(A) preparation of an N-Methyl-N-nitroso-N'-nitroguanidine solution
dissolving N-Methyl-N-nitroso-N'-nitroguanidine into deionized water to prepare a mother solution; and preparing an N-Methyl-N-nitroso-N'-nitroguanidine cell culture solution from the mother solution;
(B) induction of Gobiocypris rarus swim bladder cells by N-Methyl-N-nitroso-N'-nitroguanidine
adding the 2-10-generation Gobiocypris rarus swim bladder cells of claim 1 into a first complete culture solution; culturing the cells at 27°C
Claims
for 16-24 h; and adding the N-Methyl-N-nitroso-N'-nitroguanidine cell culture solution to perform induced culture so as to obtain induced cells;
(C) low-serum culture
adding the induced cells into a culture solution A for culture; culturing the cells with a culture solution B; and observing a condition that a transforming focus of the cells occurs, thereby obtaining the Gobiocypris rarus swim bladder cell line subjected to induced transformation.
9. The induced transformation method for the Gobiocypris rarus swim bladder cell line according to claim 8, wherein the mother solution in the step (A) has a concentration of 10 mg/mL; a concentration of the N-Methyl-N-nitroso-N'-nitroguanidine cell culture solution is 1-11 Ig/mL; the first complete culture solution in the step (B) comprises the following components in percentage by volume: 20% of fetal bovine serum, 1% of a triple antibiotic solution and 79% of an M199 culture solution; and a temperature of the induced culture is 27C, and time is 24-48 h.
10. The induced transformation method for the Gobiocypris rarus swim bladder cell line according to claim 8, wherein the cleaning in the step (C) is that cleaning is performed with an HBSS solution for 1-3 times;
the culture solution A comprises the following components in percentage by volume: 20% of fetal bovine serum and 80% of an M199
Claims
culture solution; and a culture temperature of the culture solution A is 27 0C, and time is 2-3 weeks;
the culture solution B comprises the following components in percentage by volume: 5% of fetal bovine serum and 95% of the M199 culture solution; and a culture temperature of the culture solution B is 27 0C, and time is 30-50 d.
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