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CN-121991888-A - Culture medium for rapid synchronous myogenic adipogenesis serum-free co-induction of fish stem cells and induction method

CN121991888ACN 121991888 ACN121991888 ACN 121991888ACN-121991888-A

Abstract

The invention relates to the field of in-vitro culture and induced differentiation of animal cells, in particular to a culture medium for rapid synchronous myogenic lipid-free serum co-induction of fish stem cells and an induction method. The method uses a serum-free induction culture medium with specific components, simultaneously meets nutrition, metabolism and fatty acid delivery conditions required by muscle cell fusion and fat drop formation in the same induction system, reduces process complexity and batch-to-batch fluctuation caused by step induction, realizes stable construction of a muscle-fat composite tissue, supports controllable supply and spectrum type regulation of key fatty acid components such as DHA, EPA and the like, and finally obtains the fish-source muscle-fat composite tissue with mature muscle fibers and fat drops simultaneously by starting synchronous differentiation when the cell fusion degree reaches a specified standard.

Inventors

  • ZHENG HONGWEI
  • XUE CHANGHU
  • ZHOU XUAN
  • LI ZHAOJIE
  • Ji Piyu
  • FENG TINGYU
  • ZHANG YUE

Assignees

  • 中国海洋大学
  • 青岛海洋食品营养与健康创新研究院

Dates

Publication Date
20260508
Application Date
20260409

Claims (9)

  1. 1. A culture medium for rapid synchronous myogenic adipogenic serum-free co-induction of fish stem cells, characterized by comprising a basal system, a co-differentiation core driver and a functional enhancement candidate: wherein the basic system comprises L-15 and DMEM/F12, and the volume ratio of the L-15 to the DMEM/F12 is (20-80): 80-20; The co-differentiation core driving factors comprise insulin, oleic acid, linoleic acid, DHA, EPA, ascorbic acid, taurine and transferrin, wherein the final concentration of the insulin is 1-50 mu g/mL, the final concentration of the oleic acid is 10-300 mu mol/L, the final concentration of the linoleic acid is 10-300 mu mol/L, the final concentration of the DHA is 5-200 mu mol/L, the final concentration of the EPA is 5-200 mu mol/L, the final concentration of the ascorbic acid is 10-500 mu mol/L, the final concentration of the taurine is 0.1-20 mmol/L, and the final concentration of the transferrin is 1-50 mu g/mL; The functional enhancement candidates comprise bovine serum albumin BSA and/or human serum albumin HSA, selenium and IGF-1, wherein the final concentration of the BSA and/or the HSA is 0.01-5% by mass volume, the final concentration of the selenium is 1-200 nmol/L, and the final concentration of the IGF-1 is 1-500 ng/mL.
  2. 2. The culture medium of claim 1, wherein the volume ratio of L-15 to DMEM/F12 is 1:1.
  3. 3. The medium of claim 1, wherein the co-differentiation core driver has a final concentration of insulin of 5-20. Mu.g/mL, oleic acid of 25-200. Mu.mol/L, linoleic acid of 25-200. Mu.mol/L, DHA of 10-150. Mu.mol/L, EPA of 10-150. Mu.mol/L, ascorbic acid of 25-200. Mu.mol/L, taurine of 0.5-5 mmol/L, and transferrin of 5-20. Mu.g/mL.
  4. 4. The medium of claim 1, wherein the final concentration of BSA and/or HSA in the functional enhancement candidate is 0.1-2% (w/v), selenium is added as Na 2 SeO 3 at a final concentration of 5-50 nmol/L, and IGF-1 is at a final concentration of 10-200 ng/mL.
  5. 5. A method of synchronously inducing differentiation of fish stem cells into muscle and adipose tissue in a serum-free condition of the culture medium according to any one of claims 1 to 4, comprising the steps of: (1) Respectively obtaining fish muscle satellite cells and fish fat-derived stem cells, respectively carrying out in vitro amplification culture in respective amplification culture mediums, carrying out passage when the cells reach 90-100% confluence in the amplification process according to the conventional method, collecting and counting the cells for subsequent co-culture; (2) Mixing and inoculating two types of cells into a culture container according to a preset cell number proportion, and establishing a co-culture system by using a co-culture amplification culture medium, wherein the cell number proportion is 7:3, 5:5 and 3:7, continuously culturing in a co-culture stage, dynamically monitoring the integral confluence degree, and considering that the co-culture system forms a continuous cell layer and has synchronous co-differentiation starting conditions when the integral confluence degree of the co-culture reaches or exceeds 90%; (3) Discarding the co-culture amplification medium, then adding the medium for rapid synchronous myogenic lipid-free serum-free co-induction of the fish stem cells to start co-differentiation culture, and defining the moment of first replacement of the medium for serum-free co-induction as co-differentiation 0 h; (4) And after the preset time point, evaluating the myogenic phenotype by MYH detection, evaluating the adipogenic phenotype by lipid drop staining, and judging that the fish-source myogenic-lipid composite tissue synchronous myogenic adipogenic is obtained when the MYH positive structure and the lipid drop accumulation phenotype appear in the same sample at the same time.
  6. 6. The method of claim 5, wherein the amplification medium in the step (1) is a basic culture solution prepared by mixing L-15 and DMEM/F12 according to a volume ratio of 1:1, and 10% serum and 1% neomycin diabody are added, and the co-culture amplification medium in the step (2) is a culture medium prepared by mixing L-15 and DMEM/F12 according to a volume ratio of 1:1, and 10% FBS is supplemented.
  7. 7. The method according to claim 5, wherein the co-culture system is considered to have a continuous cell layer and has a synchronous co-differentiation initiation condition when the surface of the culture in step (2) forms a continuous cell layer with little cell gap and no significant overlap drop-off occurs.
  8. 8. The method according to claim 5, wherein the step (3) is performed with 1 to 2 gentle washing with isotonic buffer after discarding the co-culture amplification medium.
  9. 9. The method of claim 5, wherein the liquid change in step (4) is performed once every 24 h times or a half-amount of liquid change.

Description

Culture medium for rapid synchronous myogenic adipogenesis serum-free co-induction of fish stem cells and induction method Technical Field The invention relates to the field of in-vitro culture and induced differentiation of animal cells, in particular to a culture medium for rapid synchronous myogenic lipid-free serum co-induction of fish stem cells and an induction method. Background In the construction of cell agricultural products such as cultured fish meat, muscle tissue is a key for determining the structural strength, fiber trend and protein content of a final product, and directly influences the elasticity, chewing feeling and tissue integrity of the final product, while fat tissue mainly contributes to energy density and flavor precursor generation, and influences juiciness and texture fineness through distribution and volume fraction of lipid droplets. Compared with terrestrial animals, the lipids of various marine fishes are naturally rich in n-3 long-chain polyunsaturated fatty acids such as DHA, EPA and the like, and the fatty acids not only have important nutritive value, but also are closely related to characteristic flavor and oxidation stability. Therefore, the key point of fish culture is not only to realize large-scale expansion of myogenic cells, but also to synchronously construct myo-lipid composite tissues with myofiber structures and lipid drop deposition in vitro, and further to realize the controllability of fatty acid composition and delivery mode, thereby improving the bionic degree and nutrition repeatability of the product. At present, a step induction strategy of asynchronism is usually adopted for muscle cells and fat cells, for example, a method for co-culturing and directional differentiation induction of muscle satellite cells and fat stem cells is disclosed in Chinese patent CN117586947A, and comprises the steps of co-culturing and co-differentiation, so that the muscle satellite cells and the fat-derived stem cells of the large yellow croaker can be effectively co-cultured, and the co-directional differentiation induction of the muscle satellite cells and the fat-derived stem cells can be realized, thereby providing a feasible scheme for mass production of high-quality cultured meat. The invention realizes the co-culture of the large yellow croaker muscle satellite cells and the fat stem cells, but the stepwise induction strategy of 'forming fat firstly and forming muscle later' leads to the differentiation period reaching 11 to 27 days, the operation flow becomes complicated due to the need of thoroughly replacing the culture medium twice, and the micro-environment fluctuation of the cells is easy to cause, the production efficiency and the process stability are limited, and the staged unidirectional exogenous signal induction mode also artificially cuts off the bidirectional paracrine cooperative interaction which is originally existed between the muscle cells and the fat cells, so that the complex tissue structure required by the meat culture is difficult to be truly simulated. Chinese patent CN121182738a discloses a method for producing cell culture meat by co-culturing large yellow croaker stem cell spheres in 3D, wherein myoblasts of large yellow croaker and precursor fat cells of large yellow croaker are mixed and inoculated into cell culture plates subjected to anti-adhesion treatment for proliferation culture, and the cells are spontaneously aggregated to form cell spheres and then subjected to differentiation culture, so that the cell culture meat is obtained. Can make the myoblast and precursor fat cell of large yellow croaker implement high-effective myoblast differentiation and adipogenic differentiation by proliferation culture and differentiation culture, and can effectively simulate the in-vivo microenvironment so as to produce the cultured meat with similar texture and taste to the traditional meat product. Although the invention provides an innovative thought for simulating in vivo microenvironment by constructing a 3D bracket-free cell sphere, the cell sphere formed by spontaneous aggregation has obvious in-batch variability and uncontrollability in size, shape and cell composition, the heterogeneity directly leads to the formation of nutrient, oxygen and metabolic waste gradients which are difficult to accurately regulate and control in the sphere, when the size of the sphere is increased, the cell in the central area is extremely easy to cause necrosis due to insufficient oxygen supply, and meanwhile, the core of the sphere and the outer cell face distinct microenvironment, so that the synchronicity and uniformity of myogenic and adipogenic differentiation are severely restricted, in addition, the technology depends on a miniaturized culture system such as a 96-well plate, the cell yield in unit volume is lower, and the evaporation problem caused by the edge effect of the porous plate further influences the stability and repeatability of experiments,