CN112481196B - Method for separating and primary culturing Haliotis discus hannai hepatopancreas cells - Google Patents
Method for separating and primary culturing Haliotis discus hannai hepatopancreas cells Download PDFInfo
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Abstract
The invention relates to a method for separating and primarily culturing Haliotis discus hannai Hance hepatopancreat cells, which comprises obtaining Haliotis discus hannai Hance hepatopancreat; treating the hepatopancreas by adopting a collagenase digestion method and sieving to obtain a sieving solution; carrying out multiple sedimentation and heavy suspension treatment on the sieved solution to obtain a cell suspension; inoculating the cell suspension into a six-hole cell culture plate, sealing by adopting a membrane, placing in an incubator, and replacing the complete culture medium of the abalone cells every 2 d. According to the invention, the Haliotis discus hannai hepatopancreas cells are treated by adopting a collagenase digestion method, so that purer and more sufficient Haliotis discus hannai primary hepatopancreas cells can be obtained in a shorter time, and more material choices are provided for in vitro experiments for researching the nutrient metabolism, the immunity and the like of the Haliotis discus hannai. And the adherence capacity of the hepatopancreas cells is further improved by optimizing experimental conditions and methods, the problem that cell aggregates are easy to agglomerate in the culture process is solved, and pure hepatopancreas cells with larger quantity are obtained.
Description
Technical Field
The invention belongs to the technical field of cell culture, and particularly relates to a method for separating and primarily culturing Haliotis discus hannai Hance hepatopancreat cells.
Background
Abalone is also called abalone, sea ear, etc., and belongs to the phylum Mollusca (Mollusca), Gastropoda (Gastropoda) and Haliotidae (Haliotidae), which is the first of eight major seafood in China. Because of rich nutrient substances, delicious taste and unique mouthfeel, the lotus seed cake is deeply favored by people (Yanlin, etc., 2019). Haliotis discus hanai is a breeding variety of abalones in the main north of China, and at present, the research on the aspects of nutrition metabolism and immune stress of Haliotis discus hanai is increasingly and widely carried out. However, the abalone culture cycle is long, and is easily affected by many factors such as environment, and the progress and efficiency of scientific experiment development are limited. The research of the in vitro cell level has the advantages of short research period, stable and controllable conditions, high repeatability and the like, establishes a stable and reliable abalone cell culture model, provides convenience for in vitro experiments and more material selections, and has important significance for further researching molecular mechanisms of nutrition metabolism, immunity and the like of the abalone.
The hepatopancreas is taken as the largest gland of the mollusk, plays an important role in various physiological processes such as digestion, absorption, metabolism, immunity and the like (Torone et al, 2011), and the hepatopancreas cells cultured in vitro can basically keep the characteristics of the hepatopancreas and can also eliminate the interference of other factors in vivo, thereby being used as an important tool for researching the specific nutritional metabolism process and the hepatopancreas function, and the cultured hepatopancreas cells of the Haliotis discus hannai can lay a foundation for researching the nutritional metabolism and the immunity of the abalone in the future. At present, in vitro culture methods of tissues such as blood, gill, mantle, kidney, foot muscle and the like (Lixia et al, 1997; Toraya et al, 2000) exist in Haliotis discus hannai, and a liver pancreatic cell culture method is not reported yet. The hepatopancreas tissue structure and the content are very complex, certain difficulty is caused to the separation process of hepatopancreas cells, partial bacteria are carried by the hepatopancreas cells, pollution is easily caused, and the problem of cell mass aggregation is easily caused in the culture process.
Disclosure of Invention
In view of the above, the present invention aims to overcome the defects of the prior art, and provide a method for separating and primary culturing ephantopus marmoreus cells, so as to solve the problems that in the prior art, certain difficulties are caused in the separation process of the ephantopus marmoreus cells, partial bacteria are carried in the ephantopus marmoreus cells, pollution is easily caused, and cell aggregates are easily aggregated in the culturing process.
In order to achieve the purpose, the invention adopts the following technical scheme: a method for separating and primary culturing Haliotis discus hannai hepatopancreas cells comprises the following steps:
obtaining the hepatopancreas of the haliotis discus hannai;
treating the hepatopancreas by adopting a collagenase digestion method and sieving to obtain a sieving solution;
carrying out multiple precipitation and resuspension treatment on the sieved liquid to obtain a cell suspension;
inoculating the cell suspension into a six-hole cell culture plate, sealing by adopting a membrane, placing in an incubator, and replacing the complete culture medium of the abalone cells every 2 days.
Further, the method for obtaining the hepatopancreas cells of Haliotis discus hannai comprises the following steps:
selecting healthy abalone bodies, and disinfecting the abalone bodies by using 75% alcohol;
peeling off the shell by using a sterilized dissecting tool, disinfecting the abalone soft body part by using 75% alcohol again, removing gonads and connective tissues wrapped outside the hepatopancreas by using forceps and scissors, and then cutting off the hepatopancreas;
and cleaning and disinfecting the hepatopancreas by using 75% alcohol, putting into sterile seawater, and placing in a rotary table for cleaning and disinfecting for 1 h.
Further, the processing of the hepatopancreas by collagenase digestion to obtain hepatopancreas cells comprises:
taking the hepatopancreas out of the seawater, and washing and disinfecting twice with D-PBS buffer solution pre-cooled by adding antibiotics;
cutting the hepatopancreas into 0.5-2mm 2 The block of (4) is put into a culture dish containing 0.25 percent of type IV collagenase to be digested for 10min, and the block is blown and beaten twice;
after digestion was complete, complete medium was added and the cells were ground through a 70 μm cell sieve to obtain a sieved solution.
Further, the multiple sedimentation and resuspension treatment of the sieved solution is performed to obtain a cell suspension, which includes:
collecting the sieved solution, adopting the rotation speed of 1000rpm and the duration of 3min, discarding the supernatant, then using the complete culture medium to resuspend the sediment and repeatedly centrifuging the sediment twice, and taking the sediment;
resuspending the precipitate, centrifuging at 600rpm for 2min, removing the supernatant, and collecting the precipitate;
resuspending the obtained precipitate with complete culture medium, centrifuging at 100rpm for 2min, collecting supernatant, and sieving with 70 μm cell sieve to obtain cell filtrate;
standing the cell filtrate for 5min, centrifuging at 100rpm for 30s, collecting supernatant, adding complete culture medium, diluting, and mixing to obtain cell suspension.
Further, each well of the six-well cell culture plate was 1.5ml, and the temperature of the incubator was 22 ℃.
Further, also comprises
Preparing a complete culture medium of haliotis discus hannai hepatopancreas cells, wherein the components of the culture medium comprise: l-15 basal medium, fetal calf serum, streptomycin mixed liquor 100x, gentamicin sulfate, amphotericin B, NaCl, KCl and CaCl 2 Magnesium sulfate, magnesium chloride.
Further, the method also comprises the following steps:
identifying cells and cell activities;
the identification of cells and cell activities comprises:
identifying the cells by a DAPI staining method;
cell activity was identified by trypan blue staining.
Further, the identification of the cells by the DAPI staining method comprises:
adding 200ul DAPI reagent into the complete culture medium, mixing, and dyeing at room temperature for 10 min;
the DAPI staining solution was aspirated and washed 3 times with PBS for 5 minutes each;
adding a proper amount of culture medium, and observing under a fluorescence microscope, wherein the excitation wavelength is 360-400 nm;
wherein the stained cells are cells.
Further, the identification of the cell activity by trypan blue staining method comprises:
trypan blue was diluted with PBS to a mass fraction of 0.4%;
adding 150ul trypan blue into the complete culture medium and 0.4% trypan blue solution according to the ratio of 9: 1;
observing under a microscope; in this case, the dead cells were stained blue, while the dead cells were stained colorless and transparent.
By adopting the technical scheme, the invention can achieve the following beneficial effects:
the invention provides a method for separating and primary culturing Haliotis discus hannai Hemsl cells, which comprises the steps of obtaining Haliotis discus hannai Hemsl liver pancreas; treating the hepatopancreas by adopting a collagenase digestion method and sieving to obtain a sieving solution; carrying out multiple precipitation and resuspension treatment on the sieved liquid to obtain a cell suspension; inoculating the cell suspension into a six-hole cell culture plate, sealing by adopting a membrane, placing in an incubator, and replacing the complete culture medium of the abalone cells every 2 days. According to the method, the haliotis discus hannai hepatopancreatic cells are treated by adopting a collagenase digestion method, so that purer and more sufficient primary haliotis discus hannai hepatopancreatic cells can be obtained in a shorter time, and more material choices are provided for in vitro experiments for researching nutrition metabolism, immunity and the like of the haliotis discus hannai. And the adherence capacity of the hepatopancreas cells is further improved by optimizing experimental conditions and methods, the problem that cell aggregates are easy to agglomerate in the culture process is solved, and pure hepatopancreas cells with larger quantity are obtained.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.
FIG. 1a is a cell morphology of the supernatant of the present invention at a centrifugation speed of 1000 rpm;
FIG. 1b is a cell morphology of the supernatant of the present invention at a centrifugation speed of 600 rpm;
FIG. 1c is a cell morphology of the supernatant of the present invention at a centrifugation speed of 400 rpm;
FIG. 1d is a cell morphology of the supernatant of the present invention at a centrifugation speed of 600rpm +400 rpm;
FIG. 1e is a cell morphology of the supernatant of the present invention at a centrifugation speed of 600rpm +200 rpm;
FIG. 1f is a cell morphology of the supernatant of the present invention at a centrifugation speed of 600rpm +100 rpm;
FIG. 2a is a diagram showing the morphology of the clumps of cells agglutinated during the cell culture process before the optimization of the method provided by the present invention;
FIG. 2b is a diagram showing the morphology of the clumps of cells agglutinated during the cell culture process after the optimization of the method provided by the present invention;
FIG. 3a is an electron micrograph of cells of the invention after 2h of initial culture;
FIG. 3b is an electron micrograph of the cells of the present invention after they have been cultured for 24 h;
FIG. 3c is an electron micrograph of cells of the present invention after initiation of culture for 120 h;
FIG. 4 is a graph showing the result of DAPI staining of cells cultured according to the present invention;
FIG. 5 is a graph showing the result of trypan blue staining of cells cultured according to the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be described in detail below. It should be apparent that the described embodiments are only some embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the examples given herein without any inventive step, are within the scope of the present invention.
The following describes a specific method for isolating and primary culturing ephora carnea cells provided in the embodiments of the present application with reference to the accompanying drawings.
As shown in fig. 1, the method for isolating and primary culturing ephatoncus hannai cells provided in the present application example comprises:
s101, obtaining the hepatopancreas of the haliotis discus hannai;
s102, treating the hepatopancreas by adopting a collagenase digestion method and sieving to obtain a sieving solution;
s103, carrying out multiple times of precipitation and heavy suspension treatment on the sieved solution to obtain a cell suspension;
s104, inoculating the cell suspension into a six-hole cell culture plate, sealing by adopting a membrane, placing in an incubator, and replacing the complete culture medium of the abalone cells every 2 d.
Complete media in this application include: l15 Medium (Gibco), fetal bovine serum (Biological Industries), streptomycin mixed liquor 100 × (Solibao), gentamicin sulfate (Solibao), amphotericin B (Solibao), NaCl (Sigma), KCl (Sigma), CaCl 2 (Sigma), magnesium sulfate (Sigma), magnesium chloride (Sigma).
Preparation of experimental reagents: 1. filtering natural seawater with 0.22 μm filter, autoclaving at 121 deg.C for 30min, cooling, adding penicillin (100IU/ml), streptomycin (100 μ g/ml), gentamicin (250 μ g/ml), and amphotericin B (2 μ g/ml), and standing. And adding the same content of antibiotics into the D-PBS solution for later use.
2. Weighing 500ml of L-15 dry powder, 10.1g of NaCl, 0.27g of KCL and 0.3g of CaCL 2 、0.5gMgSO 4 、O.9gMgCL 2 After fully dissolving to 500ml of ultrapure water, 5ml of streptomycin qing mixed solution (100x), 1ml of gentamycin sulfate (100mg/ml) and 1ml of amphotericin B (500mg/ml) are added, the culture medium after fully mixing is filtered and sterilized by a 0.22 mu m filter membrane, 15% of fetal bovine serum is added to prepare a complete culture medium, and the complete culture medium is placed for standby.
Experimental materials: haliotis discus hannai, 0.22 μm filter, syringe, T25 culture flask, 6-well cell culture plate, elbow forceps, surgical scissors, scalpel, weighing scoop, 100 μm cell sieve, 40 μm cell sieve, Pasteur pipette.
Sterilization of the experimental materials: sterilizing tools such as forceps and scissors and experimental consumables in an autoclave at 121 ℃ for 30min, and drying in a 55-degree oven for later use;
selection of experimental abalone for temporary culture: healthy abalones with good vitality and moderate weight are selected and put into a circulating seawater system containing an ultraviolet disinfection lamp for temporary culture.
The method for obtaining the hepatopancreatic cells of the haliotis discus hannai comprises the following steps:
selecting healthy abalone bodies, and disinfecting the abalone bodies by using 75% alcohol;
peeling off the shell by using a sterilized dissecting tool, sterilizing the abalone soft body part by using 75% alcohol again, removing gonads and connective tissues wrapped outside the hepatopancreas by using forceps and scissors, and then cutting off the hepatopancreas;
and cleaning and disinfecting the hepatopancreas by using 75% alcohol, putting the hepatopancreas into sterile seawater, and placing the hepatopancreas into a rotary table for disinfection and cleaning for 1 hour.
This application goes on in aseptic work platform, carries out comprehensive disinfection to the abalone body surface with 75% alcohol, peels off the shell back with the dissection instrument of sterilization, again with 75% alcohol disinfection abalone software part, get rid of the parcel behind gonad and the connective tissue outside the liver pancreas carefully with tweezers and scissors, cut down the liver pancreas and wash the disinfection back fast in 75% alcohol, put into the aseptic sea water that adds the antibiotic, arrange 60 commentaries on classics shaking table disinfection in and wash 1 h.
In some embodiments, the treating the hepatopancreas with collagenase digestion to obtain hepatopancreas cells comprises:
taking the hepatopancreas out of the seawater, and washing and disinfecting twice with D-PBS buffer solution pre-cooled by adding antibiotics;
cutting the hepatopancreas into pieces of 0.5-2mm2, sterilizing in a culture dish containing O.25% collagenase IV for 10min, and beating twice;
after digestion was complete, complete medium was added and the cells were ground through a 70 μm cell sieve to obtain a sieved solution.
Preferably, the multiple precipitation and resuspension treatment of the sieved solution is performed to obtain a cell suspension, and the method includes:
collecting the sieved solution, adopting the rotation speed of 1000rpm, keeping the duration for 3min, discarding the supernatant, then using the full culture medium to resuspend the precipitate and repeatedly centrifuging the precipitate twice, and taking the precipitate;
resuspending the precipitate, centrifuging at 600rpm for 2min, removing supernatant, and collecting the precipitate;
resuspending the obtained precipitate with complete culture medium, centrifuging at 100rpm for 2min, collecting supernatant, and sieving with 70 μm cell sieve to obtain cell filtrate;
standing the cell filtrate for 5min, centrifuging at 100rpm for 30s, collecting supernatant, adding complete culture medium, diluting, and mixing to obtain cell suspension.
Preferably, the six well cell culture plate is 1.5ml per well and the incubator is at a temperature of 22 ℃.
Specifically, the method for culturing the hepatopancreatic cells comprises the following steps:
(1) after the hepatopancreas were taken out of the seawater, they were washed and sterilized twice more with D-PBS buffer pre-cooled with antibiotics.
(2) The hepatopancreas are cut into pieces of 2mm to 0.5-2mm, and the pieces are put into a culture dish containing 0.25% collagenase IV to be dissolved for 10min, and are lightly beaten twice.
(3) After digestion was complete, complete medium containing serum was added to stop the digestion and the cells were ground through a 70 μm cell sieve.
(4) Collecting the sieved solution, centrifuging at 1000rpm for 3min, sucking and removing the supernatant to collect the precipitate, re-suspending the precipitate with a culture medium, repeating the centrifugation twice, and fully cleaning to remove mucus impurities and the like.
(5) The final pellet was resuspended, centrifuged at 600rpm for 2min, and the pellet collected by pipetting the supernatant.
(6) The pellet was resuspended in complete medium, centrifuged at 100rpm for 2min, the supernatant collected and sieved through a 70 μm cell sieve.
(7) And standing the cell filtrate for 5 minutes to generate a small amount of agglutinated masses, centrifuging at 100rpm for 30s, collecting supernatant, adding a proper amount of complete culture medium to dilute to a proper concentration, and lightly blowing and uniformly mixing.
(8) The diluted cell suspension is put into a six-hole cell culture plate, each hole is 1.5ml, the cell suspension is placed in a biochemical incubator at 22 ℃ after being sealed by a sealing film for culture, the cell state is observed, and the liquid is changed every two days.
In some embodiments, further comprising:
identifying cells and cell activities;
the identification of cells and cell activities comprises:
identifying the cells by a DAPI staining method;
cell activity was identified by trypan blue staining.
Wherein, DAPI staining: DAPI (4', 6-diamidino-2-phenylindole dihydrochloride) is a fluorescent dye that binds to most of the A, T bases in DNA and is commonly used for fluorescence microscopy. Because DAPI can penetrate intact cell membranes, it can be used for staining of both living and fixed cells. The cell nucleus was transfected with DAPI transfection reagent, as follows:
200ul of DAPI reagent was added to the medium, mixed well and stained at room temperature for 10 min.
The DAPI staining solution was aspirated and washed 3 times with PBS for 5 minutes each.
Adding a proper amount of culture medium, and observing under a fluorescence microscope with the excitation wavelength of 360-400 nm.
Wherein, the stained tissue is the cell, and the non-stained tissue is other tissues.
Trypan blue staining: the normal living cell has complete cell membrane structure, can exclude trypan blue and prevent the trypan blue from entering cells; and the cell with lost activity or incomplete cell membrane has increased permeability of the cell membrane and can be dyed into blue by trypan blue, so that the living cell and the dead cell can be distinguished very simply, conveniently and quickly by virtue of the trypan blue dyeing. And (3) staining the cultured cells by trypan blue 12 hours after the culture is started, and detecting the activity of the cells, wherein the specific operation is as follows:
trypan blue was diluted to 0.4% with PBS.
150ul of trypan blue (final concentration 0.04%) was added at a ratio of 9: 1 medium to 0.4% trypan blue solution.
Observing under a microscope: dead cells were stained in a distinct blue color, while viable cells were resistant to staining in a colorless, transparent state.
The experimental results using the above method are as follows:
the cell profile at different centrifugation speeds is shown in FIG. 1: FIG. 1a shows that the supernatant at 1000rpm is free of significant cell morphology; FIG. 1b shows that the supernatant at 600rpm is mostly irregular cell debris, impurities, etc.; FIG. 1c shows a small number of cells appearing in the supernatant at 400 rpm; FIG. 1d shows a small number of cells appearing in the 600rpm pellet +400rpm supernatant; FIG. 1e shows that 600rpm pellet +200rpm supernatant yielded a fraction of cells, but in a smaller number than 100 rpm; FIG. 1f shows that 600rpm pellet +100rpm supernatant yielded sufficient number of hepatopancreas cells with good morphological viability.
The cell processing culture method of this experiment can significantly reduce the appearance of cell agglutination clumps during the culture process, and fig. 2a is the cell agglutination clumps before optimization, specifically: cell agglutination lumps are easy to appear before optimization; fig. 2b shows the optimized cell clumps, which are specifically: after optimization, cell agglutination masses are obviously reduced.
FIG. 3a shows that the cells are uniformly dispersed after starting the culture for 2 hours; FIG. 3b shows that after 24h, the cells are round, smooth and transparent, have strong refractivity and rich stereoscopic impression, and most of the cells are attached to the wall. FIG. 3c shows that after 120h, a small amount of cells died, and other cells continued to grow and were better in activity.
Cell adherence condition: when 3ml of cell suspension is inoculated in each hole, most cells are in a suspension state, and individual cells are slightly attached to the wall and are easy to fall off; when 2ml of cell suspension is inoculated in each hole, part of cells are slightly attached to the wall and easily fall off when slightly shaking or changing liquid; when 1.5ml of cell suspension is inoculated in each hole, most cells are attached to the wall and are not easy to fall off, and the cells cannot fall off due to liquid change; the filtered cell suspension is diluted to a proper concentration, which is favorable for cell adherence, and the bottom of the cell culture bottle is coated with polylysine or gelatin, etc., so that the adherence efficiency of the cells can be improved.
As shown in FIG. 4, blue fluorescence was observed at 400nm excitation wavelength after DAPI staining using an Echo Revolve fluorescence microscope. The culture contents were confirmed to be cells, but other non-cellular substances.
As shown in fig. 5, after trypan blue staining, most of the cells rejected the staining and the cell viability was better.
In summary, the present invention provides a method for isolating and primary culturing haliotis discus hannai cells, comprising obtaining the liver pancreas of haliotis discus hannai; treating the hepatopancreas by adopting a collagenase digestion method and sieving to obtain a sieving solution; carrying out multiple precipitation and resuspension treatment on the sieved liquid to obtain a cell suspension; inoculating the cell suspension into a six-hole cell culture plate, sealing by adopting a membrane, placing in an incubator, and replacing the complete culture medium of the abalone cells every 2 days. According to the invention, the Haliotis discus hannai hepatopancreas cells are treated by adopting a collagenase digestion method, so that purer and more sufficient Haliotis discus hannai primary hepatopancreas cells can be obtained in a shorter time, and more material choices are provided for in vitro experiments for researching the nutrient metabolism, the immunity and the like of the Haliotis discus hannai. And the adherence capability of the hepatopancreas cells is further improved by optimizing experimental conditions and methods, the problem that cell aggregates are easy to agglomerate in the culture process is solved, and pure hepatopancreas cells with larger quantity are obtained.
The above description is only for the specific embodiments of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art can easily conceive of the changes or substitutions within the technical scope of the present invention, and all the changes or substitutions should be covered within the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the appended claims.
Claims (6)
1. A method for separating and primary culturing Haliotis discus hannai Hemsl cells is characterized by comprising the following steps:
obtaining the hepatopancreas of the haliotis discus hannai;
treating the hepatopancreas by adopting a collagenase digestion method and sieving to obtain a sieving solution;
carrying out multiple sedimentation and heavy suspension treatment on the sieved solution to obtain a cell suspension;
inoculating the cell suspension into a six-hole cell culture plate, sealing by adopting a membrane, placing in an incubator, and replacing the complete culture medium of the abalone cells every 2 d;
carrying out multiple precipitation and resuspension treatment on the sieved liquid to obtain a cell suspension, wherein the method comprises the following steps:
collecting the sieved solution, adopting the rotation speed of 1000rpm, keeping the duration for 3min, discarding the supernatant, then using the full culture medium to resuspend the precipitate and repeatedly centrifuging the precipitate twice, and taking the precipitate;
resuspending the precipitate, centrifuging at 600rpm for 2min, removing the supernatant, and collecting the precipitate;
resuspending the obtained precipitate with complete culture medium, centrifuging at 100rpm for 2min, collecting supernatant, and sieving with 70 μm cell sieve to obtain cell filtrate;
standing the cell filtrate for 5min, centrifuging for 30s at 100rpm, collecting supernatant, adding a complete culture medium, diluting and mixing uniformly to obtain a cell suspension; 1.5ml of each well of the six-well cell culture plate, and the temperature of the incubator is 22 ℃;
the method for treating the hepatopancreas by adopting a collagenase digestion method to obtain hepatopancreas cells comprises the following steps:
taking the hepatopancreas out of sterile seawater, and cleaning and disinfecting twice with D-PBS buffer solution pre-cooled by adding antibiotics;
cutting the hepatopancreas into 0.5-2mm 2 The block is put into a culture dish containing 0.25 percent of IV type collagenase to be digested for 10min, and the block is blown and beaten twice in the middle;
after digestion was complete, complete medium was added and the cells were ground through a 70 μm cell sieve to obtain a sieved solution.
2. The method of claim 1, wherein said obtaining hepatopancreas cells of Haliotis discus hannai, comprises:
selecting healthy abalone bodies, and disinfecting the abalone bodies by using 75% alcohol;
peeling off the shell by using a sterilized dissecting tool, sterilizing the abalone soft body part by using 75% alcohol again, removing gonads and connective tissues wrapped outside the hepatopancreas by using forceps and scissors, and then cutting off the hepatopancreas;
and cleaning and disinfecting the hepatopancreas by using 75% alcohol, putting into sterile seawater, and placing in a rotary table for cleaning and disinfecting for 1 h.
3. The method of claim 1 or 2, further comprising
Preparing a complete culture medium for Haliotis discus hannai hepatopancreas cells, wherein the culture medium comprises the following components: l-15 basal medium, fetal calf serum, streptomycin mixed liquor 100x, gentamicin sulfate, amphotericin B, NaCl, KCl, CaCl2, magnesium sulfate and magnesium chloride.
4. The method of claim 3, further comprising:
identifying cells and cell activities;
the identification of cells and cell activities comprises:
identifying the cells by a DAPI staining method;
cell activity was identified by trypan blue staining.
5. The method of claim 4, wherein identifying the cells using DAPI staining comprises:
adding 200ul of DAPI reagent into the complete culture medium, mixing, and dyeing at room temperature for 10 min;
the DAPI staining solution was aspirated and washed 3 times with PBS for 5 minutes each;
adding a proper amount of culture medium, and observing under a fluorescence microscope, wherein the excitation wavelength is 360-400 nm;
wherein the stained cells are cells.
6. The method of claim 4, wherein the identifying of cell activity using trypan blue staining comprises:
trypan blue was diluted with PBS to a mass fraction of 0.4%;
adding 150ul trypan blue into the complete culture medium and 0.4% trypan blue solution at a ratio of 9: 1;
observing under a microscope; in this case, the dead cells were stained blue, while the dead cells were stained colorless and transparent.
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| Application Number | Priority Date | Filing Date | Title |
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| CN202011531779.1A CN112481196B (en) | 2020-12-17 | 2020-12-17 | Method for separating and primary culturing Haliotis discus hannai hepatopancreas cells |
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| CN202011531779.1A CN112481196B (en) | 2020-12-17 | 2020-12-17 | Method for separating and primary culturing Haliotis discus hannai hepatopancreas cells |
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| CN112481196A CN112481196A (en) | 2021-03-12 |
| CN112481196B true CN112481196B (en) | 2022-09-13 |
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| KR20180024184A (en) * | 2016-08-29 | 2018-03-08 | 전남대학교산학협력단 | GnRH gene from Haliotis discus hannai and uses thereof |
| CN110926890A (en) * | 2019-10-21 | 2020-03-27 | 中国水产科学研究院南海水产研究所 | Method for extracting abalone hepatopancreas metabolite |
| CN111876369A (en) * | 2020-07-24 | 2020-11-03 | 中国海洋大学 | Rapid primary culture method and application of haliotis discus gill cells |
| CN111876370A (en) * | 2020-07-24 | 2020-11-03 | 中国海洋大学 | Abalone cell culture medium and abalone cell culture method |
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| WO2014062607A1 (en) * | 2012-10-15 | 2014-04-24 | Commercial Marine Biology Institute, Llc | Marine extract compositions and methods of use |
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Patent Citations (4)
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| KR20180024184A (en) * | 2016-08-29 | 2018-03-08 | 전남대학교산학협력단 | GnRH gene from Haliotis discus hannai and uses thereof |
| CN110926890A (en) * | 2019-10-21 | 2020-03-27 | 中国水产科学研究院南海水产研究所 | Method for extracting abalone hepatopancreas metabolite |
| CN111876369A (en) * | 2020-07-24 | 2020-11-03 | 中国海洋大学 | Rapid primary culture method and application of haliotis discus gill cells |
| CN111876370A (en) * | 2020-07-24 | 2020-11-03 | 中国海洋大学 | Abalone cell culture medium and abalone cell culture method |
Non-Patent Citations (2)
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| The effects of feeding Lactobacillus pentosus on growth, immunity, and disease resistance in Haliotis discus hannai Ino;Xiaolong Gao et al;《Fish Shellfish Immunol》;20180404;第78卷;全文 * |
| 鲍鱼褐藻胶裂解酶基因的原核表达及酶学性质;张齐等;《食品与生物技术学报》;20180615;第37卷(第6期);全文 * |
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