CN113293128A

CN113293128A - Hexagrammos otakii spermatogonia culture medium and culture method

Info

Publication number: CN113293128A
Application number: CN202110189449.7A
Authority: CN
Inventors: 齐洁; 贺艳; 赵茜
Original assignee: Ocean University of China
Current assignee: Ocean University of China
Priority date: 2021-02-19
Filing date: 2021-02-19
Publication date: 2021-08-24

Abstract

The invention relates to a spermatogonia culture medium and a culture method of greenling, belonging to the field of cell biology, wherein the spermatogonia culture medium comprises the following components: mixed culture medium, 1% MEM nonessential amino acid by volume ratio, 1% double antibody by volume ratio, 1% fish serum by volume ratio, growth factor, nutrient substance, 5% fetal calf serum. The mixed culture medium is prepared from L-15 and DMEM/F12 according to the mass ratio of 1: 1 and mixing. The culture medium and the culture method are utilized to culture hexagrammos otakii spermatogonium separated and purified in vitro, spermatogonium with high purity and dry and proliferative activity can be obtained, and basic experimental materials are provided for the research of seawater fish spermatogonium transplantation and the regulation and control mechanism of spermatogonium proliferation and differentiation.

Description

Hexagrammos otakii spermatogonia culture medium and culture method

Technical Field

The invention belongs to the field of cell biology, and particularly relates to a spermatogonium culture medium for in-vitro short-term culture of hexagrammos otakii spermatogonia and a culture method.

Background

Spermatogonial Stem Cells (SSCs) are a class of adult stem cells with both proliferative and developmental potential that can undergo mitosis to form spermatogonial cells that are also drying, and can undergo meiosis to develop into female germ cells in females and male germ cells in males. However, the number of spermatogonial stem cells in the testis is rare, and in the case of adult mice, the number of spermatogonial stem cells only accounts for 0.02% -0.03% of the total germ cells in the testis, so that the establishment of an efficient method for purifying and culturing the spermatogonial cells is very important.

Hexagrammos otakii (Hexagrammos otakii) belongs to Sebastes Semicidae, Hexagrammos, is a cold-temperature offshore bottom layer fish, and is a common economic species in the Bohai sea and the east sea. The meat quality is delicious, and the meat is called northern rock spot. In recent years, the yield of hexagrammos otakii is reduced due to over-fishing, and large-scale artificial breeding becomes urgent. However, the laying amount of the hexagrammos otakii is small, and the eggs begin to be sticky about 20 minutes after being discharged, and the eggs are adhered to form blocks and attached to the reef at the bottom layer, so that the artificial breeding of the hexagrammos otakii is difficult due to the characteristic. The research on the proliferation and differentiation process and the in vitro culture of the Hexagrammos otakii spermatogonium can open the new direction of artificial propagation, avoid the limitation of the sexual maturity period and the spawning period, and provide new possibility for large-scale industrial culture.

Disclosure of Invention

The technical problem to be solved by the invention is to provide a culture medium and a culture method for in vitro culture of Hexagrammos otakii spermatogonium, and the culture medium and the culture method are used for culturing the Hexagrammos otakii spermatogonium which is separated and purified in vitro, so that the spermatogonium which has higher purity and dryness and proliferation activity can be obtained, and a basic experimental material is provided for the research of the regulation and control mechanism of seawater fish spermatogonium transplantation and spermatogonium proliferation and differentiation.

The technical scheme adopted by the invention is carried out according to the following steps:

hexagrammos otakii's spermatogonial cell culture medium, the spermatogonial cell culture medium include following composition: mixing culture medium, 1% MEM nonessential amino acid by volume ratio, 1% double antibody by volume ratio, 1% fish serum by volume ratio, growth factor, nutrient substance, 5% fetal calf serum; the mixed culture medium is prepared by mixing L-15 and DMEM/F12 according to the mass ratio of 1: 1;

the growth factor consists of the following components in concentration: 10ng/mL bFGF and 10mg/mL GDNF; the nutrient substance comprises the following components in concentration: 10ng/mL bovine insulin, 2mmol/L L-glutamine and 1mmol/L sodium pyruvate.

The double-resistant (BI) is a mixed antibiotic consisting of 10000units/mL penicillin and 10mg/mL streptomycin.

The invention also provides a method for separating, purifying and culturing greenling spermatogonium in vitro, which comprises the following steps:

1) sampling and disinfecting Hexagrammos otakii spermary tissue: taking a 13-15 month old male hexagrammos otakii spermary tissue, and transferring the tissue to a culture dish containing 10% of three-resistance 1 XPBS;

2) dissociation of the spermary cells: cleaning the testis tissue with 1 × PBS containing 5% of three-antibody for 2-3 times, and removing redundant tissue; secondly, continuously washing the treated tissue in 1 XPBS containing 5% of three-antibody for 2-3 times, transferring the tissue to a small container, and adding 1mL of serum-free L-15 culture medium; thirdly, shearing the tissue blocks into 1mm by using small sterile scissors³Sucking supernatant and collecting cells while leaving tissue blocks, subpackaging the tissue blocks in culture bottles, and adding dissociation solution for digestion; fourthly, digesting the tissues for 2 hours in a shaking table at the temperature of 28 ℃ at the rotating speed of 100 rpm/min; fifthly, after digestion, filtering the mixed solution to remove tissue blocks, and adding the same amount of FBS to neutralize enzyme reaction; sixthly, centrifuging for 5min twice at 400g, removing enzyme, and adding a serum-free L-15 culture medium for heavy suspension to obtain cell suspension;

3) purification of spermatogonia: percoll is centrifuged in discontinuous density gradient, the density gradient is 12 percent and 20 percent and 28 percent, liquid is slowly added into a centrifugal tube along the tube wall, firstly 28 percent solution is added, then 20 percent solution is added, and finally 12 percent solution is added, and the cell suspension obtained by separation is slowly added to the topmost layer of the centrifugal tube; ② centrifuging for 30min at 100g, enriching spermatogonial cells at the bottom of the centrifuge tube; ③ adding 1 XPBS to resuspend the sediment, washing for 2 times, and centrifuging for 5min at 400 g; fourthly, resuspending the precipitate by using the spermatogonial culture medium, transferring the precipitate to a culture plate, and putting the culture plate into an incubator for culture at the culture temperature of 24.8 ℃; after culturing for 24h, observing partial adherent cells, and transferring the non-adherent cells to a new culture plate for continuous culture; sixthly, after the cells are continuously cultured for 24 hours, the cells which are not attached to the wall are dyed by alkaline phosphatase, and whether the cells are dry or not is judged by observing the color development condition, so that purified spermatogonia are obtained;

4) short-term in vitro culture of spermatogonial cells: collecting spermatogonia purified by the above method, culturing in the spermatogonia culture medium, observing proliferation by periodic microscopic examination, and detecting dryness by alkaline phosphatase staining.

Furthermore, the dissociation solution is prepared by adding 1mg/mL collagenase I, 1mg/mL collagenase IV or 0.25% trypsin on the basis of a L-15 culture medium containing 0.05% DNase I.

Further, the triple antibody (BI) is a mixed antibiotic consisting of 10000units/mL penicillin, 10mg/mL streptomycin and 1250units/mL nystatin; 1% of three antibody, namely adding 1mL of three antibody into 99mL of culture medium; the double antibody (BI) is a mixed antibiotic consisting of 10000units/mL penicillin and 10mg/mL streptomycin; 1% of double antibody, namely adding 1mL of double antibody into 99mL of culture medium; the fish serum is self-made, blood is extracted from the spinal column of the Hexagrammos otakii through an injector, the Hexagrammos otakii is stood for half an hour at room temperature until the blood is layered, and the Hexagrammos otakii is centrifuged at 3000rpm/min for 5min to extract supernatant and stored at-20 ℃.

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

in the purification process of the Percoll method, concentration gradients of 10 percent, 12 percent and 28 percent are selected, and spermatogonia are positioned in the sediment under the concentration gradients, so that the spermatogonia are convenient to collect. Meanwhile, the purity of the greenling spermatogonium obtained by combining a plurality of differential wall-sticking methods is higher, and the greenling spermatogonium can be used for culturing and transplanting the spermatogonium; the screened mixed culture medium of DMEM/F12 and L-15 is used for culturing the Hexagrammos otakii spermatogonium, 5% serum and two cytokines of GDNF and bFGF are added into a culture system to maintain the dryness of the Hexagrammos otakii spermatogonium, and bovine insulin, sodium pyruvate, fish serum and the like are added to promote the proliferation of the Hexagrammos otakii spermatogonium, so that the culture system can realize the proliferation and the dryness maintenance of the Hexagrammos otakii spermatogonium in vitro, and theoretical and experimental bases are provided for the long-term culture and preservation of the spermatogonium.

Drawings

FIG. 1 shows the isolation and purification of spermatogonial stem cells: (A) mechanical methods, (B) 0.25% EDTA-pancreatin, (C) collagenase I, (D) collagenase IV, (E) cell stratification after Percoll centrifugation, (F) cells in the upper layer, (G) cells in the pellet.

Fig. 2 is cells obtained after differential adherence: (A) purified cells, (B) alkaline phosphatase detection of purified cells, (C) adherent support cells.

FIG. 3 shows the growth of SSCs and the results of alkaline phosphatase staining under different conditions in mixed media after 7 days of culture: (A) growth factor and 1% FBS added cell status, (B) growth factor and 5% FBS added cell status, (C) 5% FBS added cell status, and (D) alkaline phosphatase staining of B-picture cells.

FIG. 4 shows the growth of SSCs and the results of alkaline phosphatase staining under different conditions in L-15 medium after 7 days of culture: (A) growth factor and 1% FBS added cell status, (B) growth factor and 5% FBS added cell status, (C) 5% FBS added cell status, and (D) alkaline phosphatase staining of B-picture cells.

Detailed Description

The technical scheme of the present invention is further explained by the following examples, and the experimental materials and reagents involved in the examples are all commercially available finished products unless otherwise specified.

Example 1, separation and purification of Hexagrammos otakii spermatocyte, the specific method is as follows:

experimental reagent: triple antibody (BI), double antibody (BI), DMEM/F12(BI), L-15(Gibco), GDNF (Gibco), collagenase I (Sigma), collagenase IV (Sigma), Percoll (Sigma), bFGF (Solarbio), fetal bovine serum (BI), L-glutamine (Thermo Fisher), sodium pyruvate (Sigma), bovine insulin (Solarbio), trypsin (Gibco), BCIP/NBT alkaline phosphatase color development kit (Beyotime).

1) Preparation of the experiment: preparing a disposable culture dish, a 15mL centrifuge tube, scissors, tweezers, a penicillin vial, a gun head and the like, wherein the disposable culture dish, the 15mL centrifuge tube, the scissors, the tweezers, the penicillin vial, the gun head and the like are all required to be sterile;

2) greenling spermary tissue sampling and disinfection: firstly, selecting 13-month-old hexagrammos otakii, and sampling spermary tissues. Secondly, sterilizing the fish body, scissors, tweezers and the like by using 75% alcohol before sampling, removing surface bacteria as far as possible, completely taking out the spermary tissue, and transferring the spermary tissue to a culture dish containing 10% of three-resistance PBS (1 x);

3) dissociation of the spermary cells: cleaning the testis tissue with 1 × PBS (containing 5% of three antibodies) for 2-3 times, removing blood cells stained on the surface, and shearing off redundant connective tissues including macroscopic blood vessels and the like; secondly, continuously washing the treated tissue in 1 XPBS (containing 5% of three antibodies) for 2-3 times, transferring the tissue into a penicillin bottle, and adding 1mL of serum-free L-15 culture medium; thirdly, the tissue blocks are cut into 1mm pieces by using small sterile scissors³The supernatant was aspirated and collected (the collected cells were obtained by mechanical methods, see fig. 1A), while the tissue mass was left, and the tissue mass was dispensed into culture flasks and digested by adding enzymes (digestion system 10mL, 0.05% DNase I and 0.25% trypsin 1mg/mL (fig. 1B) or collagenase I (fig. 1C) or collagenase IV 1mg/mL (fig. 1D) in L-15 medium, it is evident that collagenase IV has the highest dissociation efficiency under the same conditions); fourthly, digesting the tissue for 2 hours in a shaking table at the temperature of 28 ℃ at the rotating speed of 100rpm/min, and blowing the dissociation liquid by using a gun head every half an hour; fifthly, after digestion, the mixed solution passes through a 70-micron screen and a 40-micron screen in sequence, tissue blocks are removed, and the same amount of FBS is added for neutralization enzyme reaction; sixthly, centrifuging for 5min twice at 400g, removing enzyme, and adding 2mL of L-15 culture medium for heavy suspension;

4) purification of spermatogonia: firstly, Percoll is centrifuged in a discontinuous density gradient way, the density gradient is 12%, 20% and 28%, a 15mL centrifuge tube is wetted by 1mL FBS, liquid is slowly added into the centrifuge tube along the tube wall after the FBS is removed by centrifugation, 28% of the liquid is added, 20% of the liquid is added, and 12% of the liquid is added finally, and the cell suspension obtained by separation is slowly added to the topmost layer; ② centrifuging for 30min at 100G, wherein the upper layer is mostly sperm and smaller somatic cells (figure 1F), spermatogonium is enriched at the bottom (figure 1G); ③ 1 XPBS will precipitate heavy suspension, washing 2 times, 400g centrifugation for 5 min; fourthly, resuspending the cells, and putting the cells into an incubator for culture, wherein the culture temperature is 24.8 ℃; after 24h of culture, partial cells adhere to the wall, most of the cells are supported by microscopic examination (figure 2C), and the cells which do not adhere to the wall are transferred to a new culture plate for continuous culture; sixthly, after culturing for 24 hours, collecting the cells which are not attached (figure 2A), and carrying out dryness detection on the cells which are not attached (figure 2B) by alkaline phosphatase staining, wherein microscopic examination shows that the cells which are not attached are almost all purple and are spermatogonium with dryness.

Example 2 short-term in vitro culture of Hexagrammos otakii spermatogonium

1) Cells obtained by Percoll and two-time differential adherence method are collected and placed on supporting cells to be cultured by using culture media with different components as follows respectively:

DMEM/F12+ 1% FBS + 1% diabody + 1% fish serum + 1% non-essential amino acids;

DMEM/F12+ 5% FBS + 1% diabody + 1% fish serum + 1% non-essential amino acids;

③ DMEM/F12+ 1% FBS + 1% double antibody + 1% fish serum + 1% non-essential amino acid +10ng/mL GDNF +10ng/mL bFGF +10ng/mL bovine insulin +2mmol/L L-glutamine +1mmol/L sodium pyruvate;

DMEM/F12+ 5% FBS + 1% double antibody + 1% fish serum + 1% nonessential amino acid +10ng/mL GDNF +10ng/mL bFGF +10ng/mL bovine insulin +2mmol/L L-glutamine +1mmol/L sodium pyruvate;

l-15+ 1% FBS + 1% double antibody + 1% fish serum + 1% non-essential amino acid;

sixthly, L-15%, 5% FBS, 1% double antibody, 1% fish serum and 1% nonessential amino acid (figure 4C);

seventhly, L-15+ 1% FBS + 1% double antibody + 1% fish serum + 1% nonessential amino acids +10ng/mL GDNF +10ng/mL bFGF +10ng/mL bovine insulin +2mmol/L L-glutamine +1mmol/L sodium pyruvate (FIG. 4A);

eighty percent (L-15 + 5% FBS + 1% diabody + 1% fish serum + 1% nonessential amino acids +10ng/mL GDNF +10ng/mL bFGF +10ng/mL bovine insulin +2mmol/L L-glutamine +1mmol/L sodium pyruvate (FIG. 4B);

ninthly, 1: 1 mixed culture medium, 1% FBS, 1% double antibody, 1% fish serum and 1% nonessential amino acid;

norr (R) 1: 1 mixed medium + 5% FBS + 1% diabody + 1% fish serum + 1% nonessential amino acids (FIG. 3C);

1: 1 Mixed Medium + 1% FBS + 1% double antibody + 1% Fish serum + 1% non-essential amino acids +10ng/mL GDNF +10ng/mL bFGF +10ng/mLBovine insulin +2mmol/L L-glutamine +1mmol/L sodium pyruvate (FIG. 3A);

1: 1 Mixed Medium + 5% FBS + 1% diabody + 1% fish serum + 1% non-essential amino acids +10ng/mL GDNF +10ng/mL bFGF +10ng/mL bovine insulin +2mmol/L L-Glutamine +1mmol/L sodium pyruvate (FIG. 3B).

2) The culture process comprises the following steps: DMEM/F12 culture medium group somatic cells are slow in adherence, and are not beneficial to separation of somatic cells and spermatogonial cells; after 7d of culture, under the condition that a mixed culture medium is used as a basic culture medium, cells in an experimental group (figure 3A) added with growth factors, nutrients and 1% FBS are proliferated, a small amount of cell clones exist, while the number of spermatogonium in the experimental group (figure 3B) added with the growth factors, the nutrients and 5% FBS is large, cell clone groups are formed and attached to supporting cells, and therefore, the fact that a certain amount of serum concentration is increased has a certain proliferation effect on the spermatogonium is shown; the cells did not proliferate significantly without the growth factor group (containing 5% FBS) (fig. 3C), and the cells attached to the supporting cells sporadically, indicating that cytokines and nutrients play a major role in the proliferation of spermatogonia; the cells of the group added with growth factors and 5% FBS are subjected to alkaline phosphatase staining (figure 3D), and the cells are found to be negative, while the cells attached to the cells are strong positive cells, which indicates that the group of culture media not only can promote the proliferation of spermatogonium, but also plays a positive role in maintaining the dryness of spermatogonium; similar to the above results, the effect was weaker than that of the mixed medium in the case of the L-15 minimal medium (FIG. 4). Thereby it is first

The components of the group culture medium are more suitable for short-term in-vitro culture of the Hexagrammos otakii spermatogonium.

The photographing instrument is a Nikon fluorescence inverted biological microscope. The alkaline phosphatase staining method is referred to the specification thereof.

Claims

1. A Hexagrammos otakii spermatocyte culture medium is characterized by comprising the following components: mixing culture medium, 1% MEM nonessential amino acid by volume ratio, 1% double antibody by volume ratio, 1% fish serum by volume ratio, growth factor, nutrient substance and 5% fetal calf serum; the mixed culture medium is prepared from L-15 and DMEM/F12 according to the mass ratio of 1: 1, mixing;

the growth factor consists of the following components with the concentration of 10ng/mLbFGF and 10mg/mL GDNF; the nutrient substance comprises the following components with the concentration of 10ng/mL bovine insulin, 2 mmol/LL-glutamine and 1mmol/L sodium pyruvate.

2. The Hexagrammos otakii spermatocyte medium of claim 1, wherein said diabesity is a mixed antibiotic consisting of 10000units/mL penicillin and 10mg/mL streptomycin.

3. The Hexagrammos otakii spermatocyte medium of claim 1, wherein said fish serum is Hexagrammos otakii serum.

4. A method for separating, purifying and culturing Hexagrammos otakii spermatogonium in vitro is characterized by comprising the following steps:

2) dissociation of the spermary cells: cleaning the testis tissue with 1 × PBS containing 5% of three-antibody for 2-3 times, and removing redundant tissue; secondly, continuously washing the treated tissue in 1 XPBS containing 5% of three-antibody for 2-3 times, transferring the tissue to a small container, and adding 1mL of serum-free L-15 culture medium; thirdly, shearing the tissue blocks into 1mm by using small sterile scissors³Sucking supernatant and collecting cells while leaving tissue blocks, subpackaging the tissue blocks in culture bottles, and adding dissociation solution for digestion; fourthly, digesting the tissues for 2 hours in a shaking table at the temperature of 28 ℃ at the rotating speed of 100 rpm/min; fifthly, after digestion, filtering the mixed solution to remove tissue blocks, and adding the same amount of FBS to neutralize enzyme reaction; sixthly, centrifuging for 5min twice at 400g, removing enzyme, andadding a serum-free L-15 culture medium for heavy suspension to obtain a cell suspension;

3) purification of spermatogonia: percoll is centrifuged in discontinuous density gradient, the density gradient is 12%, 20% and 28%, liquid is slowly added into a centrifugal tube along the tube wall, 28% solution is added firstly, 20% solution is added, and 12% solution is added finally, and the separated cell suspension is slowly added to the topmost layer of the centrifugal tube; ② centrifuging for 30min at 100g, enriching spermatogonial cells at the bottom of the centrifuge tube; ③ adding 1 XPBS to resuspend the sediment, washing for 2 times, and centrifuging for 5min at 400 g; resuspending the precipitate by using the spermatogonium culture medium of claim 1, transferring the resuspension to a culture plate, and culturing the suspension in an incubator at the culture temperature of 24.8 ℃; after culturing for 24h, observing partial adherent cells, and transferring the non-adherent cells to a new culture plate for continuous culture; sixthly, after the cells are continuously cultured for 24 hours, the cells which are not attached to the wall are dyed by alkaline phosphatase, and whether the cells are dry or not is judged by observing the color development condition, so that purified spermatogonia are obtained;

4) short-term in vitro culture of spermatogonial cells: collecting spermatogonia purified by the above method, culturing in the spermatogonia culture medium according to claim 1, observing proliferation by periodic microscopic examination, and detecting dryness by alkaline phosphatase staining.

5. The method of claim 3, wherein the dissociation solution is 1mg/mL collagenase IV added to 0.05% DNase IL-15.

6. The method of claim 3, wherein said tertiary antibody is a mixed antibiotic consisting of 10000units/mL penicillin, 10mg/mL streptomycin, and 1250units/mL nystatin.