CN110846271B

CN110846271B - Method for preparing comb shell muscle single cell suspension

Info

Publication number: CN110846271B
Application number: CN201911181731.XA
Authority: CN
Inventors: 孙秀俊; 刘志鸿; 周丽青; 杨爱国; 吴彪; 田吉腾
Original assignee: Yellow Sea Fisheries Research Institute Chinese Academy of Fishery Sciences
Current assignee: Yellow Sea Fisheries Research Institute Chinese Academy of Fishery Sciences
Priority date: 2019-11-27
Filing date: 2019-11-27
Publication date: 2021-06-25
Anticipated expiration: 2039-11-27
Also published as: CN110846271A

Abstract

The invention relates to a comb shell muscle preparation methodA method of single cell suspension belongs to the technical field of marine invertebrate cell biology, and comprises the following specific steps: cutting the adductor muscle tissue to 1-2mm³Small fragments of (a); adding enzyme mixed liquor for complete digestion, transferring the digested mixture into a centrifuge tube, centrifuging the mixture twice to collect cell precipitates, adding a precooled CMFSS solution into the cell precipitates, blowing and beating heavy suspension cells, filtering the cell precipitates, adding buffer solution CMFSS again, and blowing and heavy suspension to obtain scallop muscle single cell suspension; the method can realize low-cost, quick and efficient obtaining of the comb shell muscle single-cell suspension, ensures that the dissociated cells keep good activity, can be applied to dissociation, separation and purification of other marine shellfish cells and establishment of cell lines, and provides important reference data for the research of the comb shell single cells, gene functions and genetic breeding.

Description

Method for preparing comb shell muscle single cell suspension

Technical Field

The invention belongs to the technical field of marine invertebrate cell biology, and relates to a method for preparing comb shell muscle single cell suspension.

Background

Patinopecten yessoensis belongs to cold water shellfish, is originally distributed in the sea areas of Japan, Russian far east, Korean and the like, is one of the most excellent breeding varieties in the world scallop family, and is the scallop variety with the highest economic value in seawater culture shellfish in northern China at present. The comb shell muscle of the Japanese scallop is fat, fresh and tender, and rich in nutrition, is deeply favored by consumers at home and abroad, and is a high-grade marketable aquatic product in the aquatic product market. Although China is a big country for scallop cultivation, China is not a strong country for scallop cultivation, and the problems of relatively lagged genetic improvement research, relatively weak genetic basic research, deficient improved varieties and the like exist, so that the sustainable development of the scallop cultivation industry in China is seriously influenced and restricted. In recent years, more and more cultured Japanese scallops have the bottleneck problems of restricting the development of the culture industry, such as the reduction of the specification, the low output rate and the like. At present, the method for exploring the breeding way of improving the weight of the adductor muscles of the patinopecten yessoensis is a necessary way for the sustainable development of the patinopecten yessoensis industry, improving the economic benefit of culture production, accelerating the genetic improvement process of the weight characters of the adductor muscles and improving the quality characters of the patinopecten yessoensis.

The scallop adult tissues and organs are various and comprise mantle, adductor muscle, liver pancreas, kidney, haemolymph, gill, ventricle, auricle, intestinal tract, gonad and the like, which all provide abundant cell resources for the cell culture of the scallop. However, different tissues have unique physiological characteristics, which causes the culture of shellfish cells to be a worldwide problem. The adductor muscle tissue is the main edible part of the scallop, the existing experimental technology can not obtain the scallop muscle single cell suspension with better cell activity, and the research work of the cell culture, the establishment of a cell line, the functional gene verification and the like of the adult muscle tissue of the scallop is restricted. Therefore, the efficient preparation of the scallop muscle single-cell suspension is beneficial to establishing and optimizing the in vitro culture technology of the scallop adult tissue cells, provides important reference data for establishing the shellfish immortalized cell line, and has important theoretical and practical significance in the research fields of scallop functional genes, genetic breeding and the like.

Disclosure of Invention

The invention aims to provide a method suitable for preparing a comb shell muscle single-cell suspension, which is beneficial to quickly and efficiently dissociating comb shell muscle single cells to prepare a high-quality single-cell suspension and has important application values for dissociating, separating and purifying marine shellfish cells and establishing a cell line.

A method for preparing a comb shell muscle single cell suspension is characterized by comprising the following specific steps:

taking fresh adductor muscle tissue, adopting cleaning solution DPBS to wash the adductor muscle tissue, and shearing the tissue into 1-2mm³Small fragments of (a);

step two, adding an enzyme mixed solution into the tissue blocks cut in the step one, placing the tissue blocks in a water bath at the temperature of 28 ℃ for enzymolysis and digestion for 35-45min, and digesting until the tissue blocks disappear;

step three, completely transferring the tissue digestive juice into a new centrifugal tube;

step four, centrifuging the solution obtained in the step three at the temperature of 4 ℃ by 300 Xg for 1min, then centrifuging the cell supernatant by 600 Xg for 5min, and collecting cell precipitates;

adding pre-cooled special buffer CMFSS for scallop muscle cell dissociation into the collected cell sediment, and slightly sucking and blowing the cell sediment for 3-5 times by using a wide-mouth sterile gun head to resuspend the cells;

step six, repeating the step five for 1 time, filtering the resuspended cell mixed liquor by using a 40-micron cell sieve, centrifuging for 5min at the temperature of 4 ℃ at 600 Xg, collecting cell precipitates, adding pre-cooled scallop muscle cell dissociation special buffer solution CMFSS, and lightly washing and blowing 3-5 times of the resuspended cells to obtain scallop muscle single cell suspension;

and seventhly, detecting by using a cell counting plate to obtain the muscle single cells, and detecting the concentration and the activity of the single cells by using a 0.4% trypan blue staining method.

Step one, the cleaning solution DPBS is PBS buffer solution which does not contain calcium and magnesium ions, contains 0.04% of fetal bovine serum BSA by mass ratio and contains 400U/ml of penicillin and streptomycin;

the enzyme mixed solution of the second step comprises collagenase with the final concentration of 2mg/ml and dispase with the final concentration of 0.2mg/ml

The special buffer solution CMFSS for scallop muscle cell dissociation, which is prepared by ultrapure water, comprises the following components in final concentration: 25.5g/L NaCl,0.8g/L Na₂SO₄And 2.86g/L HEPES.

Compared with the prior art, the invention has the beneficial effects that:

the method for preparing the comb shell muscle single-cell suspension can realize low-cost, quick and efficient obtaining of the comb shell muscle single-cell suspension, ensures that dissociated cells keep good activity, can be applied to dissociation, separation and purification of other marine shellfish cells and establishment of cell lines, and provides important reference data for research on the single cells, gene functions and genetic breeding of the comb shells.

Drawings

FIG. 1 Trypan blue staining results (10 times objective) of a single cell suspension of scallop adductor muscle;

FIG. 2 Trypan blue staining results (20-fold objective lens) of the single cell suspension of scallop adductor muscle.

Note: the staining is made by Taifen blue, the blue color is dead cells or cell debris, and the colorless color represents living cells

Detailed Description

In order to explain the technical scheme of the invention more clearly, the invention is further described in detail with reference to the attached drawings. The specific embodiments described herein are merely illustrative of the invention and are not intended to be limiting.

Example 1

A method for preparing a comb shell muscle single cell suspension comprises the following specific steps:

firstly, dissecting scallop, cutting small pieces of adductor muscle tissue, placing the adductor muscle tissue in a sterile culture dish, adopting DPBS (tissue cleaning solution) added with special scallop tissue to wash the adductor muscle tissue for 3-5 times, and then cutting the tissue into 1-2mm³And placing the small fragments into a 15ml sterile centrifuge tube; the tissue cleaning solution DPBS special for scallop adductor muscle does not contain calcium and magnesium ions, contains 0.04 percent of fetal bovine serum BSA, and contains 400U/ml of penicillin and streptomycin.

Step two, adding 2ml of enzyme mixed liquor into a centrifugal tube, slightly and uniformly mixing the mixture by turning the centrifugal tube upside down to avoid mutual adhesion of tissue blocks, placing the centrifugal tube into a water bath at 28 ℃ for enzymolysis and digestion for 35-45min, and uniformly mixing the mixture by turning the centrifugal tube upside down for 3-5 times during digestion until the tissue blocks basically disappear; the enzyme mixture contains collagenase (2mg/ml) and dispase (0.2 mg/ml).

Step three, transferring the tissue digestive juice into a new sterile 15ml centrifuge tube by using a liquid transfer device with a sterile wide-mouth gun head, and washing the digestion tube for 1-2 times by using 3ml of precooled scallop muscle cell dissociation special buffer solution CMFSS;

step four, centrifuging for 1min at 4 ℃ by adopting a horizontal centrifuge at 300 Xg, removing residual tissue blocks, transferring cell supernatant into another 15ml sterile centrifuge tube, centrifuging for 5min at 600 Xg, and centrifuging and collecting cell precipitates;

step five, adding 5ml of pre-cooled scallop muscle cell dissociation special buffer CMFSS into the collected cell sediment, and lightly sucking and blowing the cell sediment for 3-5 times by using a wide-mouth sterile gun head to resuspend the cells; the special buffer solution CMFSS for scallop muscle cell dissociation is prepared from 25.5g/L NaCl and 0.8g/L Na₂SO₄And 2.86g/L HEPES.

And step six, repeating the step five for 1 time, filtering the resuspended cell mixed liquor by using a 40-micron cell sieve, slightly washing a centrifuge tube and the cell sieve by using 5ml of precooled CMFSS, centrifuging the cell mixed liquor by using a horizontal centrifuge 600 Xg for 5min at the temperature of 4 ℃, collecting cell precipitates, adding 200 mu l of precooled scallop muscle cell dissociation special buffer CMFSS, slightly washing and blowing the cell separated solution for 3-5 times by using a sterile wide-mouth gun head, and re-suspending the cells to obtain muscle single cell suspension.

Seventhly, detecting by using a cell counting plate to obtain the concentration of the muscle single cells more than 1000/mu l, and detecting the concentration and the activity of the single cells by using a 0.4% trypan blue staining method, wherein the proportion of living cells reaches more than 80%, and the method is shown in attached figures 1 and 2.

Claims

1. A method for preparing a comb shell muscle single cell suspension is characterized by comprising the following specific steps:

taking fresh adductor muscle tissue, washing the adductor muscle tissue by using cleaning solution DPBS, and shearing the tissue into small fragments for carrying out 1-2mm high-speed harvest; the cleaning solution DPBS is PBS buffer solution which does not contain calcium and magnesium ions and contains 0.04% of fetal bovine serum BSA, 400U/ml of penicillin and streptomycin by mass ratio;

step two, adding an enzyme mixed solution into the tissue blocks cut in the step one, placing the tissue blocks in a water bath at the temperature of 28 ℃ for enzymolysis and digestion for 35-45min, and digesting until the tissue blocks disappear; the enzyme mixed solution contains collagenase with the final concentration of 2mg/ml and dispase with the final concentration of 0.2 mg/ml;

step six, repeating the step five for 1 time, filtering the resuspended cell mixed liquor by using a 40-micron cell sieve, centrifuging for 5min at the temperature of 4 ℃ at 600 Xg, collecting cell precipitates, adding pre-cooled scallop muscle cell dissociation special buffer solution CMFSS, and lightly washing and blowing 3-5 times of the resuspended cells to obtain scallop muscle single cell suspension; the special buffer solution CMFSS for scallop muscle cell dissociation is prepared by ultrapure water, and comprises the following components in final concentration: 25.5g/L NaCl,0.8g/L Na₂SO₄And 2.86g/L HEPES;