CN115747154B - Culture medium for long-term culture of fish muscle stem cells - Google Patents

Abstract

The invention firstly provides a dry culture medium for long-term culture of fish muscle stem cells, which is prepared from a fish muscle cell growth culture medium and a grass goldfish muscle cell condition culture medium according to the following steps of 1: 1. The invention also provides a culture method for effectively preventing the loss of the stem cells of the fish muscle, which comprises the following steps: in primary culture, half a day of liquid is changed until a continuous cell monolayer grows; after the first passage is successful, the method of replacing the culture medium for half of the liquid change every day between passage intervals is still adopted until the 5 th passage culture; after the 5 th subculture, the daily exchange of the medium between passage intervals was changed to the method of total medium exchange. The half-replacement method of the culture medium comprises the following steps: before passage, the old culture medium is collected, centrifuged, filtered and sterilized, and then added into the dry culture medium of fresh fish muscle stem cells according to a proportion, and the mixture is uniformly mixed for liquid exchange and culture of the fish muscle stem cells.

Description

A culture medium for long-term culture of fish muscle stem cells

Technical Field

The invention belongs to the technical field of stem cell and animal cell culture, and in particular relates to a culture medium for long-term culture of fish muscle stem cells.

Background Art

With the continuous improvement of the level of human social and economic development, the global consumption of meat products, including fish, has also been increasing. In recent years, cell-cultured meat (also known as cultured meat) has attracted widespread attention due to its traceability, green safety, and taste closer to traditional meat. Among them, cell-cultured fish can provide a new technical approach for the sustainable development of fishery resources and to meet the growing demand for fish. In the future, with the continuous maturity of cell-cultured fish technology and the realization of large-scale production, "cell factories" are expected to gradually replace aquaculture farms and become a new food revolution.

Satellite cells in fish muscle tissue are a type of adult stem cell, also known as muscle stem cells, which have high proliferation potential and are an important seed cell for producing cell-cultured fish meat. Cultivating fish muscle stem cells in vitro, causing them to proliferate in large quantities, and then inducing them to differentiate into mature muscle fibers is a common technical approach to obtaining cell-cultured fish meat.

However, the study found that with the increase of in vitro culture time and generations, the stemness of fish muscle stem cells will gradually decrease, and eventually differentiate into myoblasts, leading to the loss of stem cell lines. He et al. found that the stemness of Xu's flatfish muscle stem cells can only be maintained until about the 10th generation (doi:10.1016/j.gene.2021.145869). Therefore, how to maintain the stemness of fish muscle stem cells for a long time is a technical bottleneck problem that needs to be solved urgently in cell cultured fish. The present invention intends to develop a culture medium that can be used to maintain the stemness of fish muscle stem cells for a long time, which is of great scientific significance for promoting the development of cell cultured fish.

Summary of the invention

The purpose of the present invention is to provide a culture medium for culturing fish muscle stem cells, that is, a culture medium for long-term cultivation of seed cells for producing cell cultured fish meat: muscle stem cells; thereby achieving the effect of long-term maintenance of the pluripotency of fish muscle stem cells cultured in vitro.

The dry culture medium for culturing fish muscle stem cells provided by the present invention is a fish muscle cell growth culture medium, and is added with the supernatant of the conditioned culture medium in which the fish muscle cells have been cultured;

The addition ratio of the supernatant is 1:1.

As a specific description of the embodiment, the supernatant of the conditioned medium cultured with fish muscle cells is obtained by inoculating fish muscle cells into fish muscle cell growth medium, culturing for 1 day, and then filtering and sterilizing the supernatant with a 0.45 μm filter membrane.

The fish muscle cells, as specifically described in the embodiment, are grass goldfish CAM cells;

As a specific record of the embodiment, the fish muscle cell growth medium is an L-15 medium containing 20% fetal bovine serum, 10% fish muscle extract, 120ng/mL basic fibroblast growth factor (bFGF) and 20ng/mL epidermal growth factor (EGF), with a pH value of 7.0.

Furthermore, antibiotics are added to the fish muscle cell growth medium;

As a specific description of the embodiment, the antibiotics are added with a final concentration of 100 IU/mL penicillin and 100 μg/mL streptomycin.

Another aspect of the present invention provides a culture method for effectively preventing the loss of dryness/pluripotency of fish muscle stem cells, which is to inoculate and culture fish muscle stem cells, and then in the primary culture, half of the medium is replaced every day until a continuous cell monolayer grows; after the first successful subculture, until the fifth subculture, the daily medium replacement between subculture intervals still adopts the method of half-replacing the culture medium; after the fifth subculture, the daily medium replacement between subculture intervals is changed to the method of full replacement of the culture medium; the method of half-replacing the culture medium is: before subculture, the old culture medium is collected and centrifuged and filtered for sterilization, and then added to the dry culture medium of fresh fish muscle stem cells at a ratio of 1:1, mixed, and used for the medium replacement and culture of fish muscle stem cells.

By using the fish muscle stem cell stemness culture medium and the culture method for maintaining the stemness of muscle stem cells for a long time provided by the present invention, the stemness of grass goldfish muscle stem cells can be maintained for more than 25 generations.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1: The results of stemness detection of grass goldfish muscle stem cells after five subcultures in the stemness medium SCM-D. A, pax7 (green) immunofluorescence staining results; B, myoD (red) immunofluorescence staining results; C, DAPI (blue) staining results; D, merged results; E, light microscopy results.

Figure 2: The results of stemness detection of grass goldfish muscle stem cells after 1, 5, 10 and 25 subcultures in the stemness medium SCM-E. Figures A, F, K and P are the results of pax7 (green) immunofluorescence staining, Figures B, G, L and Q are the results of myoD (red) immunofluorescence staining, Figures C, H, M and R are the results of DAPI (blue) staining, Figures D, I, N and S are the merged results, and Figures E, J, O and T are the results of light microscopy.

Figure 3: Primary culture and subculture results of grass goldfish muscle stem cells in the stem culture medium SCM-E. Figure A shows the primary culture cell monolayer of grass goldfish muscle stem cells, and Figures B-E show the grass goldfish muscle stem cell monolayers at the 5th, 10th, 15th and 25th generations, respectively.

DETAILED DESCRIPTION

The present invention provides a dry culture medium for culturing fish muscle stem cells, which is a fish muscle cell growth culture medium added with a conditioned culture medium; the conditioned culture medium is the supernatant of a culture medium for culturing fish muscle cells;

As a specific record of the embodiment, the fish muscle cell growth medium is an L-15 medium containing 20% fetal bovine serum, 10% fish muscle extract, 1% double antibody solution, 20ng/mL basic fibroblast growth factor (bFGF) and 20ng/mL epidermal growth factor (EGF), with a pH value of 7.0.

However, other growth media for culturing fish muscle cells can also be selected, such as growth medium for rainbow trout muscle cells (doi:10.1007/s00441-010-1071-8), growth medium for turbot muscle cells (doi:10.1016/j.jnutbio.2017.08.015), growth medium for Atlantic salmon muscle cells (doi:10.1186/1471-2199-10-80), growth medium for olivaceous flounder muscle cells (doi:10.7717/peerj.1519), growth medium for Xu's flatfish muscle cells (doi:10.1016/j.gene.2021.145869) and growth medium for humpback seabass muscle cells (doi:10.1007/s10695-020-00841-5), etc.

The conditioned medium is the culture medium supernatant obtained by culturing grass goldfish muscle cells (CAM) in the above-mentioned fish muscle cell growth medium;

It can also be muscle cells from other fish, such as rainbow trout muscle cells (doi: 10.1007/s00441-010-1071-8), turbot muscle cells (doi: 10.1016/j.jnutbio.2017.08.015), Atlantic salmon muscle cells (doi: 10.1186/1471-2199-10-80), Japanese flounder muscle cells (POMSCS) (doi: 10.7717/peerj.1519), Xu's flathead muscle cells (doi: 10.1016/j.gene.2021.145869), humpback seabass muscle cells (CAM) (doi: 10.1007/s10695-020-00841-5), etc.

As a specific record of the embodiment, the method for preparing the conditioned medium is to inoculate grass goldfish CAM cells into the fish muscle cell growth medium, culture at 28°C for 24 hours, collect the culture medium supernatant, centrifuge at 300×g for 5 minutes to remove dead cells and solid impurities, and then filter and sterilize with a 0.45μm filter membrane.

The muscle extract is prepared by homogenizing the muscle tissue of the fish, bathing the homogenate in a 60°C water bath, adjusting the pH value of the supernatant to 7.0 with 0.1 g/mL NaHCO ₃ , and then filtering and sterilizing with a 0.22 μm filter membrane to obtain the muscle extract.

A specific preparation method thereof is as follows:

S1: Select healthy adult fish, remove all muscle tissue above the lateral line, add L-15 basal medium at a ratio of 3 mL/g tissue, and homogenize at 4°C.

S2: Incubate at 60℃ for 1 hour, mix once every 10 minutes. Centrifuge at 8000×g for 2 hours at 4℃, and collect the supernatant.

S3: The pH value of the supernatant was adjusted to 7.0 with 0.1 g/mL NaHCO ₃ , and sterilized by suction filtration using a 0.22 μm filter membrane in a clean bench.

The present invention is described in detail below by taking the separation and culture of grass goldfish muscle stem cells as an example.

Example 1: Design and optimization of stem cell culture medium for fish muscle stem cells

The design and optimization of the stemness culture medium for fish muscle stem cells was carried out on the basis of L-15 culture medium. Five different stemness culture media were prepared by adding different types and concentrations of fetal bovine serum, growth factors, muscle extract and grass goldfish muscle cell (CAM) conditioned medium to L-15 culture medium. Then, their ability to maintain the stemness of grass goldfish muscle stem cells was compared, thereby screening out the optimal stemness culture medium for fish muscle stem cells, which can be used to maintain the stemness of fish muscle stem cells for a long time.

The five designed stem culture media for fish muscle stem cells were named SCM-A, B, C, D and E, respectively, and were used to culture grass carp muscle stem cells, and the best stem culture media were screened according to the stemness maintenance time of muscle stem cells. The formulas of these five stem culture media are: SCM-A, 79% L-15 medium + 20% FBS + 1% double antibody; SCM-B, 69% L-15 medium + 20% FBS + 10% muscle extract + 1% double antibody; SCM-C, 79% L-15 medium + 20% FBS + 1% double antibody + 20ng/mL bFGF + 20ng/mL EGF; SCM-D, 69% L-15 medium + 20% FBS + 10% muscle extract + 1% double antibody + 20ng/mL bFGF + 20ng/mL EGF; SCM-E, 50% SCM-D + 50% grass carp muscle cell (CAM) conditioned medium.

The results showed that grass carp muscle stem cells could not adhere to the wall and grow in the stem culture media SCM-A, B and C, but could successfully adhere to the wall and proliferate and grow and be passaged in the stem culture media SCM-D. However, the stemness of the cells was lost when they were passaged to the fifth generation (Figure 1). The stemness was best maintained in the stem culture media SCM-E and could be maintained to at least the 25th generation (Figure 2).

Among them, the preparation method of grass goldfish muscle cell (CAM) conditioned medium is: inoculate grass goldfish CAM cells into the above-mentioned fish muscle cell growth medium, culture at 28°C for 24 hours, collect the culture medium supernatant, centrifuge at 300×g for 5 minutes to remove dead cells and solid impurities, and then filter and sterilize with a 0.45μm filter membrane.

The preparation method of the fish muscle extract is as follows: select healthy adult grass goldfish, take out all the muscles above the lateral line, put them into a clean beaker, weigh them accurately, and cut them into pieces with ophthalmic scissors to a size of ⁵ mm2. Add L-15 basal medium at a ratio of 3 mL/g tissue and homogenize at 4°C. Water bath at 60°C for 1 hour, mix once every 10 minutes. Centrifuge at 8000×g for 2 hours at 4°C, and discard the precipitate. Adjust the pH of the supernatant to 7.0 with 0.1 g/mL _NaHCO3 , filter and sterilize with a 0.22μm filter membrane in a clean bench, and store at -20°C for later use after aliquoting.

The 1% double antibody solution is a mixed solution of penicillin sodium and streptomycin with a final concentration of 1.0×10 ⁵ IU/L.

Example 2: Dissociation and primary culture of grass carp muscle stem cells

Euthanize the grass goldfish and disinfect with alcohol. In a clean bench, take the muscle above the lateral line of the fish and put it into a 50-mL centrifuge tube pre-filled with 20 mL of 10% double antibody D-hank's solution (8 g NaCl, 0.4 g KCl, 0.06 g KH ₂ PO ₄ , 0.08 g _{Na 2} HPO ₄ ·12H ₂ O and 0.35 g NaHCO ₃ , dissolved in 1 L ultrapure water, adjusted to pH 7.0), and place it on ice. Take three culture dishes and add 5 mL of 10% double antibody D-hank's solution to each dish. Then, use tweezers to take out the muscle from the above 50-mL centrifuge tube, put the muscle into three culture dishes in turn, and wash it 3 times. After that, the cleaned muscle tissue was placed in a 3.5-cm dish pre-filled with 500 μL D-hank's solution (containing 10% double antibody), and the muscle was cut into pieces with ophthalmic scissors until it could be sucked up by the 1-mL gun tip. The preliminarily cut tissue was transferred to a 50-mL centrifuge tube, and 1-2 mL of D-hank's solution containing 10% double antibody was aspirated to wash and recover the muscle tissue remaining in the dish and gun tip. The volume of the muscle tissue suspension in the centrifuge tube was controlled within 3 mL. Dispase type Ⅱ with a final concentration of 2.5 mg/mL and Collagenase D with a final concentration of 10 mg/mL were added to the muscle tissue suspension, and then 50 μL of 100 mM CaCl ₂ was added, and the mixture was mixed by blowing. The mixture was digested for 1.5 h in a water bath shaker at 170 rpm and the temperature was 28°C. After 45 min, the mixture was taken to the clean bench and mixed by blowing, and then continued to shake for 45 min. The tissue and cell suspension after enzymatic hydrolysis were first filtered with a 70μm filter membrane to remove large tissue pieces. Before filtering, the filter membrane was moistened with 5mL of D-hank's solution. 12mL of fish muscle stem cell culture medium was added to rinse the filter membrane. The filtrate was collected and centrifuged at 300×g for 5min, and the supernatant was discarded. The cell pellet was resuspended with 2mL of fish muscle stem cell culture medium, transferred to a 3.5-cm small dish, and cultured in a 28℃ biochemical incubator. After 3 days, a monolayer of grass goldfish muscle stem cells growing on the wall could be observed.

Example 3: Establishment and culture of grass goldfish muscle stem cell line

When the cell plating rate at the bottom of the culture dish reaches ₉₀ %-95%, the old culture medium in the culture bottle is sucked away, and 2mL PE solution (8.0g NaCl, 0.2g KCl, 0.2g _KH2PO4 , 3g _Na2HPO4 · _12H2O and _0.2g EDTA-2Na, dissolved in 1L ultrapure water, adjusted to pH 7.0) is added to wash the cell monolayer once to remove dead cells and residual serum. 2mL PE solution is added again, and after the cells appear to be in a contracted state, the PE solution is discarded. Subsequently, 0.125% trypsin is added, digested for 40-50 seconds, and the side wall of the culture bottle is gently tapped to make all the cells fall off. Then, 2mL of fresh fish muscle stem cell dry culture medium is quickly added, and the cell suspension is gently blown with a pipette to form a cell suspension, and inoculated into a new cell culture dish at a "one-to-two" passage ratio, and cultured in a 28°C biochemical incubator. The results are shown in Figure 3. During the primary culture and subculture process, the cell morphology of grass goldfish muscle stem cells did not change significantly, and they were spindle-shaped and could be stably propagated to the 25th generation.

In the experiment, it was found that the following fluid replacement measures can effectively avoid the loss of the stemness of fish muscle stem cells. In the primary culture, half of the fluid is replaced every day until a continuous cell monolayer grows. After the first successful subculture, until the fifth subculture, the daily fluid replacement between subcultures still adopts the method of half-replacing the culture medium. After the fifth subculture, the daily fluid replacement between subcultures is changed to the method of replacing all the culture medium. The method of half-replacing the culture medium is: before subculture, collect the old culture medium, centrifuge and filter sterilize, add it to the fresh dry culture medium of fish muscle stem cells in a 1:1 ratio, mix well, and use it for fluid replacement and culture of fish muscle stem cells.

Example 4: Detection of stemness of grass goldfish muscle stem cells

Paired homeobox transcription factor 7 (PAX7) is highly expressed in muscle stem cells of animals, plays an important role in maintaining and stimulating the self-renewal and proliferation of muscle stem cells, and is a specific marker of muscle stem cells.

The stemness of grass goldfish muscle stem cells can be identified by immunofluorescence staining of the muscle stem cell marker pax7. The specific method is: first, fix the grass goldfish muscle stem cells in the immunostaining fixative for 10 minutes, and then add PBS to wash 3 times. Then add 0.2% TritonX-100, and after permeabilization at room temperature for 10 minutes, add PBS and wash 3 times. After that, add immunostaining blocking solution and block for 1 hour. After the blocking is completed, discard the blocking solution, add the primary antibody (pax7 and myoD), and incubate at 4°C overnight. The next day, equilibrate at room temperature for 30 minutes, add PBS to wash 3 times, add the secondary antibody, and incubate at room temperature for 1 hour. After that, add PBS to wash 3 times, and then add DAPI to stain the cell nucleus. After that, add PBS to wash 3 times, and observe and photograph under an inverted fluorescence microscope.

The results showed that during the subculture process, the stemness marker pax7 in the muscle stem cells of the grass goldfish remained expressed, at least until the 25th generation. This shows that the fish muscle stem cell stemness culture medium provided by the present invention can maintain the stemness of fish muscle stem cells for a long time, at least until the 25th generation.

Claims (8)

1. A dry culture medium for culturing fish muscle stem cells, characterized in that the dry culture medium is a fish muscle cell growth medium, and is supplemented with a supernatant of a conditioned medium in which fish muscle cells have been cultured; The supernatant is obtained by inoculating fish muscle cells into fish muscle cell growth medium and filtering and sterilizing after culturing for 1 day; The fish muscle cell growth medium is an L-15 medium containing 20% fetal bovine serum, 10% fish muscle extract, 120ng/mL basic fibroblast growth factor and 20ng/mL epidermal growth factor, with a pH value of 7.0. 2. The dry culture medium according to claim 1, characterized in that the supernatant is added in a ratio of 1:1. 3. The dry culture medium as claimed in claim 1, wherein the fish muscle cells are grass goldfish CAM cells. 4. The dry culture medium as claimed in claim 1, characterized in that antibiotics are also added to the fish muscle cell growth medium. 5. The dry culture medium according to claim 4, characterized in that the antibiotics are penicillin and streptomycin added at a final concentration of 100 IU/mL and 100 mg/mL respectively. 6. Use of the dry culture medium according to claim 1 in culturing fish muscle stem cells. 7. Use of the dry culture medium according to claim 1 in preparing a product for culturing fish muscle stem cells. 8. A culture method for effectively preventing the loss of stemness of fish muscle stem cells, characterized in that the method is to use the dry culture medium described in claim 1 for culture, to inoculate and culture the fish muscle stem cells, and then in the primary culture, half of the culture medium is replaced every day until they grow into a continuous cell monolayer; after the first successful subculture, until the fifth subculture, the daily culture change between subculture intervals still adopts the method of half-replacing the culture medium; after the fifth subculture, the daily culture change between subculture intervals is changed to the method of full replacement of the culture medium; the method of half-replacing the culture medium is: before subculture, the old culture medium is collected and centrifuged and filtered for sterilization, and then added to the fresh dry culture medium of the fish muscle stem cells in a 1:1 ratio, mixed, and used for the replacement of the culture medium and culture of the fish muscle stem cells.