CN-121975727-A - Method for regulating and controlling fish stem cell co-culture synchronous induction myogenic adipogenic differentiation based on microcarrier matrix rigidity

Abstract

The invention relates to the technical field of fish cell culture, cell differentiation regulation and control and tissue construction of cell culture aquatic products, in particular to a method for synchronously inducing myogenic adipogenic differentiation based on microcarrier matrix rigidity regulation and control fish stem cell co-culture. The fish muscle satellite cells, the fish fat source stem cells and the two microcarriers are added into the same culture system together for suspension dynamic culture, wherein the culture system at least comprises two microcarriers, namely a high-rigidity microcarrier and a low-rigidity microcarrier. The invention realizes that the same culture system is adopted in a single reactor to penetrate the whole process, provides mechanical microenvironment difference by simultaneously configuring microcarriers with different rigidities, synchronously generates myogenic and adipogenic differentiation of myosatellite cells and adipose-derived stem cells in the same system under the condition of not additionally setting myogenic/adipogenic induction stages and not adding induction factors, and realizes controllable and batch-to-batch repeatable myoadipose tissue of products by microcarrier proportion and cell proportion.

Inventors

• ZHENG HONGWEI
• XUE CHANGHU
• ZHOU XUAN
• Ji Piyu
• FENG TINGYU
• LI ZHAOJIE
• WANG MINGHAO

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

20260505

Application Date

20260409

Claims (6)

1. 1. A method for synchronously inducing myogenic adipogenic differentiation based on microcarrier matrix rigidity regulation of fish stem cells is characterized in that fish muscle satellite cells, fish fat source stem cells and two types of microcarriers are added into the same culture system together for suspension dynamic culture, wherein the culture system at least comprises two types of microcarriers, namely a first type of high-rigidity microcarrier with the matrix rigidity of 20-200 kPa and a second type of low-rigidity microcarrier with the matrix rigidity of 1-20 kPa.
2. 2. The method of claim 1, wherein the microcarrier is one of a gelatin-based microcarrier, a chitosan-based microcarrier, a sodium alginate-based microcarrier, a cross-linked dextran matrix, a soy protein-based microcarrier, a pumpkin seed protein-based microcarrier, a pea protein-based microcarrier, a hyaluronic acid-based microcarrier, a chondroitin sulfate-based microcarrier, and a matrix hybrid-based microcarrier thereof.
3. 3. The method of claim 1, wherein the medium is a basal culture system for routine maintenance of cells, and wherein the simultaneous myogenic and adipogenic differentiation occurs independently of the addition of additional myogenic or adipogenic inducing factors during the simultaneous induction.
4. 4. The method of claim 1, wherein the high-stiffness microcarrier has a matrix stiffness of 30-70 kPa and the low-stiffness microcarrier has a matrix stiffness of 3-15 kPa.
5. 5. The method of claim 1, wherein the ratio of fish muscle satellite cells to fish adipose-derived stem cells is 9:1-1:9.
6. 6. The method of claim 1, wherein the initial seed number of fish muscle satellite cells and the high-rigidity microcarriers is 3.0X10 4 ~8.5×10 4 fish muscle satellite cells per 1mg high-rigidity microcarriers, and the initial seed number of fish adipose-derived stem cells and the low-rigidity microcarriers is 3.0X10 4 ~8.5×10 4 fish adipose-derived stem cells per 1mg low-rigidity microcarriers.

Description

Method for regulating and controlling fish stem cell co-culture synchronous induction myogenic adipogenic differentiation based on microcarrier matrix rigidity Technical Field The invention relates to the technical field of fish cell culture, cell differentiation regulation and control and tissue construction of cell culture aquatic products, in particular to a method for synchronously inducing myogenic adipogenic differentiation based on microcarrier matrix rigidity regulation and control fish stem cell co-culture. Background The degree of quality simulation of cell-cultured fish meat depends to a large extent on the co-construction of muscle tissue structure and fat deposition. The myofibrillar structure formed by muscle cell differentiation is the basis of texture, while intracellular lipids formed by adipocyte differentiation affect flavor release, mouthfeel moisturization and nutritional characteristics. If only a single muscle-source and lipid-source structure is obtained, it is often difficult to approximate natural fish meat in structure and flavor. Therefore, the synchronous differentiation and composite construction of myogenic and lipogenic cells are realized in the same production system, and the method is an important technical requirement for large-scale production of cell culture fish. In the prior art, the response difference of fish muscle satellite cells and adipose-derived stem cells to the culture environment is remarkable, and different optimal processes exist in the aspects of induction signals, serum levels, metabolic substrates, cell densities, micro-environmental factors and the like. Common strategies include stepwise induction or pot production, i.e. the expansion and induced differentiation of myogenic and lipogenic cells, respectively, followed by mixing or assembly at the post-treatment stage. However, the step strategy introduces additional operation links and time costs, increases pollution risk and reduces consistency of amplified production, and meanwhile, the post-mixing is difficult to realize fine control of spatial distribution, and the stability of tissue structure and consistency among batches are difficult to guarantee. Another class of strategies is to achieve simultaneous differentiation by exogenously chemically induced signals within the same system, such as the addition of adipogenic induction combinations, hormonal or lipid metabolism agonists, or the use of specific myogenic induction systems to drive myogenic differentiation. However, this method often requires switching of induction medium, setting of differentiation stage, and dependence on multicomponent induction factors, which results in complicated components of the culture system, increased cost, and increased batch-to-batch variation. More importantly, under the same chemical induction condition, the optimal induction intensity and time sequence of the myosatellite cells and the adipose-derived stem cells are difficult to meet simultaneously, and one type of cells are easy to differentiate sufficiently while the other type of cells differentiate limited, so that the myolipid proportion is uncontrollable. In addition to chemical signals, physical signals in the cellular microenvironment are also important factors in determining cell fate. The matrix rigidity can regulate and control cell proliferation and differentiation through cell adhesion, cytoskeletal tension and mechanical signal transduction. For myogenic and lipogenic lineages, the appropriate matrix stiffness is different, so that a differential microenvironment is constructed by taking the matrix stiffness as a core parameter, and physical regulation and control of differentiation directions are expected to be realized. Meanwhile, microcarrier suspension culture is a common route for mass production of adherence-dependent cells, and can meet the requirements of high specific surface area and bioreactor amplification. However, the traditional microcarrier system is mainly used for promoting proliferation, lacks a programmable physical microenvironment design facing the 'myoliposynchronous differentiation' target, and particularly lacks an implementation scheme for realizing dual-lineage synchronous differentiation in a single bioreactor without depending on an induction factor and only depending on microcarrier rigidity difference, so that a synchronous differentiation process with simplified flow and definite components is needed. Disclosure of Invention The technical problems to be solved by the invention are that in the prior art, myogenic cells and lipid-derived cells are difficult to synchronously differentiate in the same system, the components of the system are complex and the proportion of myoadipose tissues is difficult to control due to the dependence on complex induction factors, the traditional microcarrier system mainly promotes proliferation, the programmable physical microenvironment design facing the 'myoliposynchronous differe