CN111849862B

CN111849862B - Separation and culture method for fish high-fat storage hepatocytes

Info

Publication number: CN111849862B
Application number: CN202010590409.9A
Authority: CN
Inventors: 苗宗余; 蔡磊; 黄韧; 李建军; 魏远征; 叶惠欣; 曾进
Original assignee: Guangdong Laboratory Animals Monitoring Institute
Current assignee: Guangdong Laboratory Animals Monitoring Institute
Priority date: 2020-06-24
Filing date: 2020-06-24
Publication date: 2022-06-07
Anticipated expiration: 2040-06-24
Also published as: CN111849862A

Abstract

The invention discloses a separation and culture method of fish high-fat storage hepatocytes. a. Selecting juvenile goby of 45-55 days, obtaining liver tissue of the juvenile goby under aseptic condition, and dispersing the liver tissue into blocks to obtain tissue blocks; b. placing a cell sieve with the aperture of 20 mu m in the trypsin stop solution, and arranging the cell sieve with the aperture of 50 mu m above the cell sieve with the aperture of 20 mu m; c. placing the tissue block in a cell sieve with the aperture of 50 microns, continuously adding a trypsin liquid into the cell sieve with the aperture of 50 microns to digest the tissue block, continuously digesting in a flowing manner, collecting cells on the cell sieve with the aperture of 20 microns, and cleaning with 10% FBS to obtain high-fat storage hepatic cells; d. inoculating the high-fat storage hepatic cells into a culture medium for culturing. The high-fat storage hepatic cells can be stable for one week after being attached to the wall, the adipocytes can survive for about 1 month, and the period can be used for cell level experiments, so that the problem that mature adipocytes of fishes are difficult to separate and culture is solved.

Description

Separation and culture method for fish high-fat storage hepatocytes

Technical Field

The invention relates to the field of cell biology, in particular to a separation and culture method of fish high-fat storage hepatocytes.

Background

In scientific research, in order to further verify the accuracy and authenticity of experimental results, verification experiments at a cell level are generally required, and the cell sources used at the cell level are commercial cells and primary cell culture. Primary cell culture is the first culture in vitro, after cells are taken from donor biopsies, to allow them to grow and reproduce continuously under artificial conditions. The cells retain the basic properties of the original cells, are the basis of various experimental researches, and are the first step for establishing various cell lines. The primary cell culture comprises the culture of differentiable primary stem cells and the culture of primary mature cells; the key of in vitro cell culture is how to separate and obtain cells with complete morphology and high in vitro metabolic activity. In recent years, many researchers have made extensive studies on how to obtain highly active and stable cells, i.e., cell separation techniques, including shear digestion (Anderson, n.g. (1953) Science), ex vivo collagenase perfusion (Berry, m.n., & Friend, d.s. (1969) The Journal of cell biology), in situ collagenase perfusion (Seglen, P.O. (1976) Academic Press). To date, the following methods have been mainly used for cell culture: tissue block adherent culture (wuning, wu chenolong. (2007)), mixed culture (Bhatia, s.n., Balis, u.j., (1999) The FASEBjournal), monolayer collagen culture, co-culture system, etc., The above-mentioned commonly used primary mature cell culture can only maintain The morphology and function of The cell within 1-2 weeks, and then gradually degenerates and dies, thereby limiting The application of primary mature cell culture. The separation and culture method of liver mature adipocytes is more rarely reported, and the general cell culture selection is basically to culture primary stem cells, then to perform differentiation induction to form liver adipocytes, and then to perform experimental verification.

Disclosure of Invention

The invention aims to provide a separation and culture method of high-fat storage hepatic cells of fishes, which can obtain the high-fat storage hepatic cells of gobies.

The invention discloses a separation and culture method of fish high-fat storage hepatocytes, which comprises the following steps:

a. selecting juvenile goby of 45-55 days, obtaining liver tissue of the juvenile goby under aseptic condition, and dispersing the liver tissue into blocks to obtain tissue blocks;

b. placing a cell sieve with the aperture of 20 mu m in the trypsin stop solution, and arranging the cell sieve with the aperture of 50 mu m above the cell sieve with the aperture of 20 mu m;

c. placing the tissue blocks in a cell sieve with the aperture of 50 microns, continuously adding a trypsin liquid into the cell sieve with the aperture of 50 microns to digest the tissue blocks, continuously digesting in a flowing manner, collecting cells on the cell sieve with the aperture of 20 microns, and washing the cells by using an M199 culture medium containing 10% FBS to obtain high-fat storage hepatocytes;

d. high-fat storage hepatocytes were seeded in serum-free M199 medium (Gibco. Lot:2044485) at 27. + -. 1 ℃ with 5% CO₂Culturing under the condition. The culture medium was changed every other day. After the cells are stably cultured for one week after being attached to the wall, the fat cells can survive for about 1 month, and the period can be used for cell level experiments.

Dispersing the liver tissue into blocks to obtain tissue blocks with the size of 1 +/-0.1 mm²And (5) small blocks.

The trypsin stop solution was M199 medium (FBS, Gibco, Lot:2036224C) containing 10% FBS by volume.

The trypsin liquid is the trypsin liquid containing 0.25 percent of mass fraction.

The flow-sustained digestion is flow-sustained digestion until the tissue mass disappears.

According to the invention, two cell sieves with different apertures are stacked, and mature adipocytes with uniform size of 20-50 μm can be collected by placing a 50 μm cell sieve on a 20 μm cell sieve through a double-cell sieve according to the diameter of the collected cells.

The culture container for culturing the high-fat storage hepatic cells can be selected according to needs, mature adipocytes attached to the wall can be obtained, whether the cells can sink or not is the key for attaching the wall, and whether the cells sink or not is the key for the growth time of the target fish and the separation method of the cells, so that the highest activity of the cells is ensured. The high-fat storage hepatocytes separated by the separation method can grow adherently.

The culture condition is determined according to the growth environment of a cell donor, the living environment temperature of the goby is 27 +/-1 ℃, and the optimal temperature is the same as the living temperature of the goby after different temperature tests in indoor culture; at the temperature, the adherent growth of the cells is facilitated, and the survival time of the cells is longer.

The method for separating and culturing the fish high-fat storage hepatic cells can efficiently separate the high-fat storage hepatic cells of the goby, and culture the goby according to the conditions of the invention, so that the goby can be stable for one week after adherence, the fat cells can survive for about 1 month, and the fat cells can be used for cell level experiments in the period, thereby solving the problem of difficult separation and culture of mature fat cells of the fish.

Description of the drawings:

FIG. 1 is a schematic view of a goby mature fat cell collection apparatus in which 1 cell sieve having a pore size of 50 μm; 2. a cell sieve with the aperture of 20 mu m; 3. a sterile culture dish; 4. digestion stop solution (M199 medium containing 10% FBS);

FIG. 2 is a culture diagram of mature adipocytes of goby, wherein as shown in the figure, the adipocytes occupy 3/4 of cell volume, the cell membrane, cytoplasm and lipid droplet interface are clear, and the adipocytes are mature and can grow adherent to a special culture medium;

FIG. 3 is a graph of the staining of mature adipocytes of goby, as shown by Oli Red staining, with arrows showing lipid droplets and membrane structure of parietal cells.

FIG. 4 shows the mature fat cell morphology of goby after 60 days, the fat drops in the cells are full, the boundary with the cell wall is not obvious, and the cells basically have no adherence phenomenon;

FIG. 5 shows that 50d cells (left side) can grow adherently and the cell morphology presents fusiform (arrow) compared with the morphology of 60d goby mature adipocytes, and 60d adipocytes (right side) cannot grow adherently because lipid droplets are full and the cell membrane cannot stretch (arrow).

The specific implementation mode is as follows:

the following examples are further illustrative of the present invention and are not intended to be limiting thereof.

Example 1:

(1) selecting 50d juvenile goby of goby, obtaining liver tissue under aseptic condition, slowly showering liver tissue block with aseptic PBS for three times, placing in the middle of cell sieve with aperture of 50 μm, dispersing tissue block into 1 + -0.1 mm with aseptic apparatus²Small blocks;

(2) placing a 50 μ M pore size cell sieve over a 20 μ M pore size cell sieve, the 20 μ M pore size cell sieve being seated in M199 medium (Gibco. Lot:2044485) containing 10% FBS by volume fraction;

(3) firstly, rinsing an upper layer and a lower layer of cell sieves by using a serum-free M199 culture medium, slowly and continuously dropwise adding a trypsin liquid with the mass fraction of 0.25% onto a tissue block, and continuously digesting the tissue block in a flowing manner until the tissue block disappears;

(4) collecting cells on a 20-micron cell sieve, wherein the size of the cells is 20-50 microns, slowly and gently washing the cells for 3 times by using FBS with the volume fraction of 10%, and counting;

(5) at 1 × 10⁶Per cm²The cell amount was plated on 24-well plates, and serum-free M199 medium supplemented with disantipenicilline (100. + -. 10IU/mL) and streptomycin (100. + -. 10. mu.g/mL) was added at 2 mL/well, 27. + -. 1 ℃ with 5% CO₂The culture was overnight and the culture medium was changed every other day. After the cells are stably cultured for one week after being attached to the wall, the adipocytes can survive for about 1 month, and can be used for cell level experiments in the period, and the mature adipocytes of the goby during the culture period are shown in FIG. 2.

(6) The results of the staining with oil red are shown in FIG. 3.

According to the method, goby livers before 45 days and after 55 days are respectively selected as raw materials, and are separated and cultured according to the method, and the results show that the livers before 45 days are immature, fat cells are incompletely differentiated, the liver volume is too small, and the requirement on the sample amount cannot be met; 60d (as shown in figure 4) of goby liver cells are full of fat, the fat cells cannot distinguish cell membranes and cytoplasm, and mature fat cells obtained by digestion grow in a suspension manner, so that later-stage experiment operation is not facilitated; in the 50d goby liver cells, fat droplets account for 3/4, the cell membrane, cytoplasm and fat droplet interface are clear, and the fat cells mature and can grow in a special culture medium in an adherent manner (as shown in figure 5).

According to the invention, a tissue block is digested by flowing trypsin, a cell sieve with the aperture of 20 microns at the lower layer of the double-cell sieve is designed to be located in a culture medium containing 10% FBS, and experiments show that sufficient fat cells cannot be separated due to too short pancreatin digestion time and the fat cells are broken due to too long separation time, so that complete cells cannot be obtained; therefore, the invention adopts a flowing trypsin digestion method, the trypsin is continuously contacted with the tissue blocks on the cell sieve with the aperture of 50 mu m, the liquid amount of the trypsin is slowly added, the liquid is increased to a certain volume and then automatically falls on the lower 20 mu m cell sieve, the 20 mu m cell sieve is located in the enzyme stopping liquid, the digestion reaction is stopped in time, then the cells with the size less than 20 mu m, such as collagen cells, fibroblasts and the like, are filtered and removed through the 20 mu m cell sieve, and finally the target cells in the 20 mu m cell sieve are collected.

Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A separation and culture method of fish high fat storage hepatocytes is characterized by comprising the following steps:

b. placing a cell sieve with the aperture of 20 mu m in the trypsin termination solution, and arranging a cell sieve with the aperture of 50 mu m above the cell sieve with the aperture of 20 mu m;

c. placing the tissue block in a cell sieve with the aperture of 50 microns, continuously adding a trypsin liquid into the cell sieve with the aperture of 50 microns to digest the tissue block, continuously digesting in a flowing manner, collecting cells on the cell sieve with the aperture of 20 microns, and cleaning the cells by using a culture medium containing 10% FBS to obtain high-fat storage hepatic cells;

d. inoculating the high-fat storage hepatic cells into a culture medium for culturing.

2. The method for separating and culturing the fish high-fat storage hepatocytes as claimed in claim 1, wherein the inoculation of the high-fat storage hepatocytes into the culture medium is an inoculation of the high-fat storage hepatocytes into a serum-free M199 culture mediumMedium, 5% CO at 27. + -. 1 ℃ C₂Culturing under the condition, and replacing culture solution every other day.

3. The method for separating and culturing fish high-fat storage hepatocytes of claim 1 or 2, wherein the liver tissues are dispersed into pieces to obtain tissue pieces, and the size of the tissue pieces is 1 ± 0.1mm²And (5) small blocks.

4. The method for separating and culturing fish high-fat storage hepatocytes as claimed in claim 1 or 2, wherein the trypsin stop solution is M199 medium containing 10% FBS.

5. The method for separating and culturing fish high-fat storage hepatocytes of claim 1 or 2, wherein the trypsin solution is a trypsin solution containing 0.25% by mass of trypsin.

6. The method for separating and culturing fish high-fat storage hepatocytes as claimed in claim 1 or 2, wherein the flow-sustaining digestion is flow-sustaining digestion until the tissue mass disappears.