CN115141791A

CN115141791A - Pelteobagrus fulvidraco kidney cell separation and culture method

Info

Publication number: CN115141791A
Application number: CN202210604513.8A
Authority: CN
Inventors: 赵涛; 罗智; 谭肖英
Original assignee: Tongwei Dafeng Feed Co ltd
Current assignee: Tongwei Dafeng Feed Co ltd
Priority date: 2022-05-31
Filing date: 2022-05-31
Publication date: 2022-10-04

Abstract

The invention relates to the technical field of biotechnology and discloses a method for separating and culturing kidney cells of pelteobagrus fulvidraco, which comprises the following steps: (1) Selecting juvenile pelteobagrus fulvidraco, sterilizing the fish body, quickly taking the kidney of the fish body through aseptic dissection on a super clean bench, and immediately putting the fish body into PBS containing penicillin-streptomycin-gentamicin three-resistant solution for cleaning; (2) Soaking the cleaned kidney in a culture medium containing penicillin-streptomycin-gentamicin three-antibody solution, and cutting into pieces; (3) Digesting the cut kidney by using a mixed solution of collagenase I and pancreatin digestive juice, collecting suspension, filtering, centrifuging, removing blood cells, adding a complete culture medium to resuspend and precipitate to obtain cell suspension, and centrifuging to obtain cell precipitate; (4) Adding sufficient complete culture medium into the precipitate, fully suspending and mixing, subpackaging the suspension and mixing solution into 25cm2 cell culture bottles, and culturing at constant temperature of 28 ℃.

Description

Pelteobagrus fulvidraco kidney cell separation and culture method

Technical Field

The invention relates to the technical field of biotechnology, in particular to a method for separating and culturing kidney cells of pelteobagrus fulvidraco.

Background

Pelteobagrus fulvidraco (Pelteobagrus fulvidraco) is a common omnivorous freshwater fish in the genus of Pseudobagrus in the family of Pseudobagaceae. Mainly distributed in water systems of Chinese Zhujiang, minjiang, xiangjiang, yangtze, yellow, haihe, songhua and Heilongjiang. In recent years, the pelteobagrus fulvidraco breeding technology is mature day by day, the yield is increased year by year, and the pelteobagrus fulvidraco breeding technology is developed into a famous and excellent aquaculture variety of fresh water. However, when the aquaculture industry is rapidly developed, the culture scale and the culture density of the pelteobagrus fulvidraco are gradually enlarged, and meanwhile, the nutrition of the feed is unbalanced, so that various diseases of the pelteobagrus fulvidraco are frequently caused. Therefore, the basic research on the pelteobagrus fulvidraco is beneficial to the healthy and rapid development of the breeding industry of the pelteobagrus fulvidraco. However, in the aspect of basic research on pelteobagrus fulvidraco, there is still no breakthrough in the isolation and culture of primary kidney cells.

The research on the cell level of fishes is greatly developed at home and abroad. In 1962, a first fish cell line, namely rainbow trout gonad cell line (RGT-2), was established by Wolf and Quimby, and gradually, a fat head fish muscle cell line, tilapia mossambica kidney cell line, snakehead kidney cell line, turbot muscle cell line, SWT embryonic cell line SWT, atlantic trout visceral tissue cell line AS, carangid juvenile fish cell line and the like, including sea fresh water fish cell lines, were also established successively. The establishment of cell lines is premised on the isolation and culture of primary cells, and the culture of primary fish cells is an optimal model for simulating the living state of fishes, and is widely applied to aspects such as etiology, nutrition, pharmacology, physiology, cell biology, environmental toxicology and the like. At present, no published patent and paper about the isolation and culture of primary kidney cells of pelteobagrus fulvidraco is found. The literature reports that the separation and culture of primary cells of some freshwater fishes only refer to the separation and culture method of mammalian cells, but the separation and culture method cannot be completely applied, and meanwhile, the cell culture conditions for the separation of different fishes and different tissues are different, so that the separated cells are unstable, and the success rate is low. Therefore, the establishment of a reliable and high-feasibility method for separating and culturing the primary kidney cells of the pelteobagrus fulvidraco is beneficial to further research on the gene function of the pelteobagrus fulvidraco.

Disclosure of Invention

Technical problem to be solved

Aiming at the defects of the prior art, the invention provides a method for separating and culturing kidney cells of pelteobagrus fulvidraco, which has the advantages of good effect of separating the kidney cells, more obtained cells, good cell growth in the culture process and the like, and solves the problem of difficulty in separating and culturing the nerve cells of the pelteobagrus fulvidraco.

(II) technical scheme

In order to realize the purposes of good separation effect of the kidney cells, more obtained cells and good cell growth in the culture process, the invention provides the following technical scheme: a method for separating and culturing primary kidney cells of pelteobagrus fulvidraco comprises the following steps:

(1) Selecting juvenile pelteobagrus fulvidraco, sterilizing the fish body, quickly taking the kidney of the fish body through aseptic dissection on a super clean bench, and immediately putting the fish body into PBS containing penicillin-streptomycin-gentamicin three-resistant solution for cleaning;

(2) Soaking the cleaned kidney in a culture medium containing penicillin-streptomycin-gentamicin three-antibody solution, and cutting into pieces;

(3) Digesting the cut kidney with mixed solution of collagenase I and pancreatin digestive juice, collecting suspension, filtering, centrifuging, removing blood cells, adding complete culture medium to resuspend and precipitate to obtain cell suspension, and centrifuging to obtain cell precipitate

(4) Adding sufficient complete culture medium into the precipitate, fully suspending and mixing, subpackaging the suspension and mixing solution into 25cm2 cell culture bottles, and culturing at 28 ℃.

Preferably, the juvenile pelteobagrus fulvidraco in the step (1) has the weight of about 8g, is bred in an indoor aquarium before the kidney tissue is taken, and is wiped with 75% alcohol before the kidney tissue for disinfection.

Preferably, the penicillin content in the penicillin-streptomycin-gentamicin-containing triple-resistant PBS or the culture medium solution in the step (1) or (2) is 200U/ml, the streptomycin content is 0.2mg/ml, and the gentamicin content is 100 mu g/ml.

Preferably, in the step (3), the concentration of collagenase I in the collagenase I and pancreatin mixed digestive juice is 2mg/ml, the concentration of pancreatin is 0.25%, the adding amount is 5ml mixed digestive juice per gram kidney tissue, and the digestion is carried out for 1.5-2h at 28 ℃.

Preferably, the complete medium composition in the step (4) comprises: l-15 basal medium, 100U/ml penicillin, 20mmol/L HEPES, 0.1mg/ml streptomycin, 50. Mu.g/ml gentamycin, 2mmol/L L-glutamine and 20% superfine fetal bovine serum.

Preferably, all the liquid of the method is filtered through a 0.22 μm sieve.

Preferably, in the method, the concentrations of penicillin, streptomycin and gentamicin in PBS or a three-antibody mixed solution in a culture medium for washing kidney tissues are respectively twice of the concentrations in the culture medium, the antibiotics for washing the tissues are mainly used for killing bacteria on the tissues, the antibiotics in the culture medium are mainly used for inhibiting the growth of the bacteria in the process of culturing cells, and the selection of the antibiotics with proper concentrations in the process of culturing the cells is very important because the antibiotics with high concentrations have adverse effects on the growth and survival of the cells.

(III) advantageous effects

Compared with the prior art, the invention provides a method for separating and culturing the kidney cells of the yellow catfish, which has the following beneficial effects:

1. according to the method for separating and culturing the kidney cells of the pelteobagrus fulvidraco, repeated experiments are carried out by using the reported methods for separating and culturing the kidney cells of the fishes and the mammals, but the methods have poor separation effect on the kidney cells of the pelteobagrus fulvidraco, the number of the obtained cells is small, the survival capability is weak, and experimental research is difficult to carry out. Based on the technical scheme, the applicant finally provides the technical scheme of the invention through searching separation and culture conditions for several times, solves the problem of difficult separation of the kidney cells of the pelteobagrus fulvidraco, and explores a good cell culture method. The method lays a good foundation for research on the related theoretical basis of the pelteobagrus fulvidraco, and provides reference for establishment of cell separation culture methods of other species.

Compared with the related fish and mammal cell separation and culture technology reported in the existing literature and patents, the invention establishes the method for separating and culturing the primary kidney cells of the pelteobagrus fulvidraco, can solve the problem of difficult separation and culture of the neural cells of the pelteobagrus fulvidraco, has good kidney cell separation effect and more obtained cells, has good cell growth and stable physiological state in the culture process, and meets the requirements of primary culture.

Drawings

Fig. 1 is a cell viability table of pelteobagrus fulvidraco primary kidney cells isolated in example 1 and comparative example 1, which are cultured for 24 h;

fig. 2 is an isolated primary kidney cell of pelteobagrus fulvidraco of the present invention;

fig. 3 shows the cell morphology of the primary kidney cells of pelteobagrus fulvidraco isolated in example 1 of the present invention cultured for 72 h;

fig. 4 shows the cell morphology of the primary kidney cells of pelteobagrus fulvidraco isolated in example 1 and comparative example 1 of the present invention after 72h culture.

Detailed Description

Example 1

The specific operation steps of the method for separating and culturing the kidney cells of the pelteobagrus fulvidraco are as follows:

(1) Selecting juvenile pelteobagrus fulvidraco with the weight of about 8g, feeding the juvenile pelteobagrus fulvidraco in an indoor aquarium, normally feeding the juvenile pelteobagrus fulvidraco with commercial feed, before dissecting and taking the kidney, anaesthetizing the fish with MS-222 (100 mg/L water), taking out the juvenile pelteobagrus fulvidraco, wiping the fish body with 75% alcohol, cutting off the gill arch to bleed, immediately placing the fish on a sterile super clean bench for dissection, quickly taking out the kidney on ice, immediately soaking and cleaning the fish in PBS containing penicillin-streptomycin-gentamicin (the penicillin content is 200U/ml, the streptomycin content is 0.2mg/ml and the gentamicin content is 100 mu g/ml) twice, and fully removing impurities on the kidney to obtain kidney tissues, wherein the kidney tissues are shown in figure 1;

(2) The cleaned kidney is put into a prepared penicillin-streptomycin-gentamicin-containing triantion culture medium (the penicillin content is 200U/ml, the streptomycin content is 0.2mg/ml, and the gentamicin content is 100 mu g/ml), the tissue is cut into 1-2mm & lt 3 & gt by using a high-temperature sterilized ophthalmic scissors, then the tissue fragments are transferred into a 15ml centrifuge tube together with the culture medium, the centrifuge tube is centrifuged at 1000rpm for 10min, the supernatant is discarded, and then 5ml of collagenase I and pancreatin mixed digestive juice (the collagenase I concentration is 2mg/ml, and the trypsin concentration is 0.25%) is added into the tissue fragments, and the tissue fragments are digested at the constant temperature of 28 ℃ for 1.5-2h.

(3) Adding a neutralization culture medium (100U/ml penicillin, 0.1mg/ml streptomycin, 50 mu g/ml gentamicin, 2mmol/L L-glutamine, and L-15 culture medium of 10% fetal calf serum) with the same volume as the enzyme solution into the digested tissue suspension to terminate the digestion, collecting the cell suspension in a 50ml sterile centrifuge tube, passing through a 100-mesh screen, collecting filtrate in a new centrifuge tube, centrifuging at room temperature for 10min at 1000rpm, sucking and discarding the supernatant, retaining the precipitate, sucking and discarding the excess blood cells by using a sterile gun head, adding a complete culture medium into the kidney cells, re-suspending, centrifuging at 1000rpm for 5min, sucking and discarding the supernatant, retaining the precipitate, sucking and then discarding the blood cells, re-suspending the cells, centrifuging, repeating for 3 times, and fully purifying to obtain pure kidney cells.

(4) Depending on the size of the pellet, complete medium was added, the cells were resuspended thoroughly, the cells were aliquoted into 25mm2 cell culture flasks (5 ml per flask), the cell density was maintained at 105 cells/ml, and the morphology of the cells was observed by inverted microscopy, as shown in FIG. 2. As can be seen from the figure, the cells are uniformly dispersed and in good condition, and are suitable for subsequent culture, and after observation, the cells are cultured in a constant-temperature cell culture box at 28 ℃.

The complete medium comprises the following components: l-15 basic culture medium, 100U/ml penicillin, 20mmol/L HEPES, 0.1mg/ml streptomycin, 50 mu g/ml gentamycin, 2mmol/L L-glutamine and 20% superfine fetal calf serum, wherein the pH of the culture medium is about 7.6.

(5) After 24h of culture, the culture solution in the original culture flask was aspirated away, 5ml of fresh complete culture solution was added, and the mixture was further placed in a constant temperature incubator at 28 ℃.

(6) The primary kidney cells of the pelteobagrus fulvidraco cultured for 72h are shown in fig. 3, and it can be seen from the figure that most of the kidney cells begin to grow adherent to the skin, are fusiform and are stable in state.

2. Separated pelteobagrus fulvidraco primary kidney cell activity detection method

After 24h of culture, appropriate amount of primary kidney cells were aspirated, trypan blue was added for staining (cell suspension was mixed well with 0.4% trypan blue solution at 1.

Live cell rate (%) = total number of live cells/(number of live cells + number of dead cells) × 100%

Cell viability detection results show that the primary pelteobagrus fulvidraco kidney cells separated by the method have the viable cell rate of over 90 percent, as shown in fig. 4, the cells grow well and are complete in shape, the requirements of primary cell culture are met, and the technical scheme is feasible.

As a supplementary note to example 1, based on the same basic experimental protocol, the following experiment was performed by adjusting some experimental parameters, wherein the kidney tissue was digested in L-15 medium containing 20% extra fetal calf serum, wherein the collagenase concentration of type I was 2mg/ml, the trypsin concentration was 0.1%, the digestion conditions were 28 ℃ for 3 hours. The results show that the cells are well dispersed, and the cell viability reaches more than 85% by 0.4% trypan blue staining observation, which is basically the same as the results of example 1, and indicate that the selection condition of the scheme is feasible.

Comparative example 1 separation of primary kidney cells of pelteobagrus fulvidraco

Basically the same as example 1, except for the difference in fish body size, the embodiment is as follows:

selecting pelteobagrus fulvidraco with the weight of about 100 g.

After 24h of culture, the cells observed by 0.4% trypan blue staining showed that the cell viability was low, only about 60%, as shown in fig. 4. After 72h of culture, most cells were clustered, the number of adherent cells was small, the cells were not uniformly dispersed, and most cells were deformed as shown in FIG. 4B. The result shows that the kidney tissue of the juvenile fish can obviously enhance the cell separation effect and has stronger cell activity, and simultaneously, the technical scheme of the embodiment 1 of the invention has reliable selection conditions.

In summary, the separation and culture method of the kidney cells of the pelteobagrus fulvidraco is used for repeated experiments by using the reported separation and culture methods of the kidney cells of the fishes and the mammals, but the separation effect of the methods on the kidney cells of the pelteobagrus fulvidraco is poor, the obtained cells are few, the viability is weak, and experimental research is difficult to perform. Based on the technical scheme, the applicant finally provides the technical scheme of the invention through several times of groping on separation and culture conditions, solves the problem of difficult separation of the kidney cells of the pelteobagrus fulvidraco, and explores a good cell culture method. The method lays a good foundation for research on the related theoretical basis of the pelteobagrus fulvidraco, and provides reference for establishment of cell separation culture methods of other species.

It should be noted that the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. Without further limitation, an element defined by the phrases "comprising a," "8230," "8230," or "comprising" does not exclude the presence of additional like elements in a process, method, article, or apparatus that comprises the element.

Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.

Claims

1. A method for separating and culturing kidney cells of pelteobagrus fulvidraco is characterized by comprising the following steps:

(2) Soaking the cleaned kidney in a culture medium containing penicillin-streptomycin-gentamicin three-antibody solution, and shearing;

(3) Digesting the cut kidney by using a mixed solution of collagenase I and pancreatin digestive juice, collecting suspension, filtering, centrifuging, removing blood cells, adding a complete culture medium to resuspend and precipitate to obtain cell suspension, and centrifuging to obtain cell precipitate;

(4) Adding sufficient complete culture medium into the precipitate, fully suspending and mixing, subpackaging the suspension and mixing solution into 25cm2 cell culture bottles, and placing at 28 ℃ for constant-temperature culture.

2. The method for separating and culturing the kidney cells of the pelteobagrus fulvidraco according to claim 1, which is characterized in that: the juvenile pelteobagrus fulvidraco in the step (1) is about 8g in weight, and is fed into an indoor aquarium before being taken as kidney tissue.

3. The method for separating and culturing the kidney cells of the pelteobagrus fulvidraco according to claim 1, which is characterized in that: the fish body disinfection operation in the step (1) is to wipe the fish body with 75% alcohol.

4. The method for separating and culturing the kidney cells of the pelteobagrus fulvidraco according to claim 1, which is characterized in that: the step (1) or (2) is to put the penicillin-streptomycin-gentamicin three-antibody solution in PBS or culture medium, and the operation is to put the penicillin-streptomycin-gentamicin three-antibody solution in PBS or culture medium with the concentration of 200U/ml penicillin, 0.2mg/ml streptomycin and 100 mu g/ml gentamicin for soaking for 10min.

5. The method for separating and culturing the kidney cells of the pelteobagrus fulvidraco according to claim 1, which is characterized in that: in the step (3), the concentration of collagenase I in the collagenase I and pancreatin mixed digestive juice is 2mg/ml, the concentration of pancreatin is 0.25 percent, the adding amount is 5ml mixed digestive juice added into each gram of kidney tissue, and the digestion is carried out for 1.5 to 2 hours at the temperature of 28 ℃.

6. The method for separating and culturing the kidney cells of the pelteobagrus fulvidraco according to claim 1, which is characterized in that: the complete culture medium in the step (4) is an L-15 culture medium containing penicillin with the concentration of 100U/ml, streptomycin with the concentration of 0.1mg/ml, gentamycin three-antibody solution with the concentration of 50 mu g/ml, HEPES with the concentration of 20mmol/L, glutamine with the concentration of 2mmol/L and special fetal calf serum with the concentration of 20 percent.