Culture medium for suspension domestication culture of fibroblast stem cells and domestication method

Abstract

The invention discloses a culture medium for suspension domestication culture of fibroblast stem cells and a domestication method. The culture medium comprises a low-serum culture medium and a serum-free culture medium; the low-serum culture medium comprises amino acid, vitamins, trace elements, serum and proliferation promoting factors; the serum-free culture medium comprises amino acid, trace elements, growth factors, hydrolysates, lipids and proliferation promoting factors; the proliferation promoting factor comprises any one or a combination of at least two of insulin, ethanolamine or transferrin. By means of the autonomous domestication method, the suspension cell domestication efficiency is improved, and the suspension cells obtained through domestication have the advantages of being high in density, good in stability, high in dispersity, high in multiplication speed and the like.

Description

Technical Field

[0001] This invention belongs to the field of animal cell culture technology, and relates to a culture medium and a method for the suspension domestication culture of fibroblast stem cells. Background Technology

[0002] With the continued development of the global economy, the demand for meat products continues to rise. However, traditional meat production cannot meet this demand, leading to a series of problems such as environmental pressure and animal welfare. Cell-cultured meat technology is a new type of food synthesis biotechnology that has emerged in recent years. Cell-cultured meat is generally produced by culturing animal cells in vitro, using animal muscle and fat cells as raw materials. It is edible meat produced by cultivating animal cells in vitro and using biomanufacturing methods, based on the growth patterns of meat within the animal's body. "In other words, meat can be consumed without farming or slaughtering, with controllable taste and flavor, and the ability to artificially adjust protein content and quality, fat content, and reduce saturated fatty acid content. Compared with traditional farming methods, cell-cultured meat technology shows significant advantages in environmental resource protection, animal welfare, and public health."

[0003] High costs are a major problem in the large-scale production of cell-cultured meat. To further improve the production efficiency of cell-cultured meat, reduce costs, and promote the development and industrialization of cell-cultured meat technology, establishing a stable and efficient serum-free suspension cell culture technology system is a key technology for low-cost large-scale preparation of cell-cultured meat.

[0004] Commonly used seed cell sources for cultured meat include fibroblasts, muscle stem cells (MuSCs), and mesenchymal stem cells (MSCs). These seed cells are isolated, extracted, and induced to differentiate into stable, adherent cell lines. The culture medium used for adherent culture is typically basal medium supplemented with serum. The production process of cultured meat relies on edible scaffolds for cell expansion and digestion, making the process complex, resulting in low cell density, high production costs, and difficulties in scale-up. In contrast, serum-free suspension cell culture systems, compared to common adherent cultures, can grow more cells per unit volume, thus enabling the production of more cultured meat products.

[0005] A self-acclimation culture method for suspension cells was adopted, utilizing both culture medium (chemical regulation) and culture method (physical regulation) to gradually screen cells adapted to suspension growth. This enabled cells to grow in suspension in serum-free medium, exhibiting advantages such as high cell density, good stability, high dispersibility, and rapid multiplication rate. However, significant technical bottlenecks and optimization opportunities remain in improving the acclimation efficiency of suspension cell lines through rapid and efficient self-acclimation methods and developing low-cost serum-free culture medium systems to support high-density cell growth.

[0006] Therefore, there is an urgent need to provide a method for acclimating fibroblast stem cells into suspension, which can improve the efficiency of acclimating suspension cells and enable the acclimated suspension cells to have characteristics such as high density, good stability, high dispersibility, and fast multiplication rate. Summary of the Invention

[0007] To address the shortcomings of existing technologies and practical needs, this invention provides a culture medium and domestication method for the suspension domestication culture of fibroblast stem cells. This method domesticates avian fibroblasts from adherent growth to suspension growth, ultimately achieving suspension culture and high-density expansion of avian fibroblasts, providing sufficient cell clusters for subsequent large-scale production and the preparation of cell-cultured meat. The purpose of this invention is to rapidly and effectively improve the efficiency of domesticating suspension cells through a self-domestication method. The domesticated suspension cells exhibit high density, good stability, high dispersibility, and rapid multiplication rate.

[0008] To achieve this objective, the present invention adopts the following technical solution:

[0009] In a first aspect, the present invention provides a culture medium for suspension domestication culture of fibroblast stem cells, the culture medium comprising a low serum culture medium and a serum-free culture medium;

[0010] The low serum culture medium includes: amino acids, vitamins, trace elements, serum, and proliferative factors;

[0011] The serum-free culture medium includes: amino acids, trace elements, growth factors, hydrolysates, lipids, and proliferation-promoting factors;

[0012] The proliferative factors include any one or a combination of at least two of insulin, ethanolamine, or transferrin.

[0013] This invention improves the efficiency of domesticating suspended cells through an autonomous domestication method. The domesticated suspended cells have the characteristics of high density, good stability, high dispersibility, and fast multiplication rate.

[0014] In this invention, these components simulate the growth environment of cells in vivo, ensuring that cells can effectively divide and proliferate stably in vitro.

[0015] Insulin's main function is to promote cellular uptake of glucose and amino acids, accelerate protein synthesis, significantly improve cell growth rate and survival rate under serum-free / low-serum culture conditions, and provide energy and material basis. Transferrin's main function is to transport iron ions (to meet the needs of respiration and DNA synthesis) and scavenge free iron to prevent oxidative damage; in serum-free culture media, it can increase cellular iron uptake efficiency by 30%-50%, enhancing metabolism and proliferation. Ethanolamine, as a precursor to brain phosphate, participates in phospholipid synthesis, and its main functions are to maintain stable cell membrane flow, regulate cell metabolism and gene expression, and enhance cell adhesion and proliferation rate. Insulin regulates metabolism to create conditions for other components; transferrin maintains enzyme activity and protects cells, working with insulin to ensure physiological functions; ethanolamine improves cell membrane state and enhances nutrient utilization, and the three form a synergistic network mechanism. This invention achieves a breakthrough in culture medium performance, optimizes the cellular nutritional environment, enhances cell adaptability and stress resistance, promotes the development of cell culture technology, and has broad application prospects and commercial value.

[0016] Preferably, the amino acids in the low serum culture medium include any one or a combination of at least two of tyrosine, cystine, glutamic acid, glutamine, histidine, proline, or asparagine.

[0017] Preferably, the vitamins in the low serum culture medium include any one or a combination of at least two of the following: choline chloride, folic acid, inositol, nicotinamide, riboflavin, D-calcium pantothenate, pyridoxal hydrochloride, or vitamin B12.

[0018] Preferably, the trace elements in the low serum culture medium include any one or a combination of at least two of the following: iron, copper, zinc, selenium, manganese, or molybdenum.

[0019] Preferably, the concentration of tyrosine in the low serum culture medium is 50-100 mg / L (e.g., 50 mg / L, 80 mg / L, or 100 mg / L), the concentration of cysteine ​​is 30-80 mg / L (e.g., 30 mg / L, 50 mg / L, or 80 mg / L), the concentration of glutamate is 2-10 mg / L (e.g., 5 mg / L, 8 mg / L, or 10 mg / L), the concentration of glutamine is 250-600 mg / L (e.g., 250 mg / L, 500 mg / L, or 600 mg / L), the concentration of histidine is 30-60 mg / L (e.g., 30 mg / L, 50 mg / L, or 60 mg / L), the concentration of proline is 8-20 mg / L (e.g., 8 mg / L, 10 mg / L, or 20 mg / L), and the concentration of asparagine is 3-10 mg / L (e.g., 3 mg / L, 8 mg / L, or 10 mg / L).

[0020] Preferably, the concentration of iron in the low serum culture medium is 0.1~0.4 mg / L (e.g., 0.1 mg / L, 0.2 mg / L, or 0.4 mg / L), the concentration of copper is 0.0008~0.002 mg / L (e.g., 0.0008 mg / L, 0.001 mg / L, or 0.002 mg / L), the concentration of zinc is 0.05~0.45 mg / L (e.g., 0.1 mg / L, 0.2 mg / L, or 0.4 mg / L), the concentration of selenium is 0.0008~0.003 mg / L (e.g., 0.001 mg / L, 0.002 mg / L, or 0.003 mg / L), and the concentration of manganese is 0.0001~0.002 mg / L (e.g., 0.0001 mg / L, 0.001 mg / L, or 0.002 mg / L). The concentration of molybdenum is 0.0001 to 0.0004 mg / L (e.g., 0.0001 mg / L, 0.0002 mg / L, or 0.0003 mg / L).

[0021] Preferably, the serum mass percentage in the low serum culture medium is 1% to 3% (e.g., 1%, 2% or 3%).

[0022] Preferably, the concentration of insulin in the low serum culture medium is 1-10 mg / L (e.g., 2 mg / L, 5 mg / L or 8 mg / L), the concentration of ethanolamine is 0.12-1.5 mg / L (e.g., 0.5 mg / L, 1 mg / L or 1.5 mg / L), and the concentration of transferrin is 2-5 mg / L.

[0023] Preferably, the amino acids in the serum-free culture medium include any one or a combination of at least two of tyrosine, cystine, glutamic acid, glutamine, histidine, proline, or asparagine.

[0024] Preferably, the trace elements include any one or a combination of at least two of iron, copper, zinc, or selenium.

[0025] Preferably, the growth factors include recombinant epidermal growth factor and / or recombinant insulin-like growth factor.

[0026] Preferably, the hydrolysate includes: soybean hydrolysate and / or yeast hydrolysate.

[0027] Preferably, the proliferative factor includes any one or a combination of at least two of insulin, ethanolamine, or transferrin.

[0028] Preferably, the lipids include any one or a combination of at least two of linoleic acid, cholesterol, or phosphatidylcholine.

[0029] Preferably, the serum-free culture medium contains tyrosine at a concentration of 80–300 mg / L (e.g., 100 mg / L, 200 mg / L, or 300 mg / L), cysteine ​​at a concentration of 50–300 mg / L (e.g., 100 mg / L, 200 mg / L, or 300 mg / L), glutamate at a concentration of 5–20 mg / L (e.g., 10 mg / L, 15 mg / L, or 20 mg / L), glutamine at a concentration of 300–1000 mg / L (e.g., 500 mg / L, 800 mg / L, or 1000 mg / L), histidine at a concentration of 30–120 mg / L (e.g., 50 mg / L, 100 mg / L, or 120 mg / L), proline at a concentration of 15–50 mg / L (e.g., 15 mg / L, 20 mg / L, or 30 mg / L), and asparagine at a concentration of 5–15 mg / L (e.g., 5 mg / L, 10 mg / L, 20 mg / L, or 30 mg / L). (mg / L or 15 mg / L).

[0030] Preferably, the serum-free culture medium contains 30-120 mg / L of iron (e.g., 30 mg / L, 80 mg / L, or 120 mg / L), 0.001-0.005 mg / L of copper (e.g., 0.001 mg / L, 0.003 mg / L, or 0.005 mg / L), 0.5-0.9 mg / L of zinc (e.g., 0.5 mg / L, 0.7 mg / L, or 0.9 mg / L), and 0.001-0.005 mg / L of selenium (e.g., 0.001 mg / L, 0.003 mg / L, or 0.005 mg / L).

[0031] Preferably, the concentration of recombinant epidermal growth factor in the serum-free culture medium is 5-15 μg / L (e.g., 5 μg / L, 10 μg / L or 15 μg / L), and the concentration of recombinant insulin-like growth factor is 20-30 μg / L (e.g., 20 μg / L, 25 μg / L or 30 μg / L).

[0032] Preferably, the concentration of soybean hydrolysate in the serum-free culture medium is 500-2000 mg / L (e.g., 500 mg / L, 1000 mg / L or 2000 mg / L), and the concentration of yeast hydrolysate is 500-2000 mg / L (e.g., 500 mg / L, 1000 mg / L or 2000 mg / L).

[0033] Preferably, the concentration of insulin in the serum-free culture medium is 1-5 mg / L (e.g., 1 mg / L, 2 mg / L or 5 mg / L), the concentration of ethanolamine is 0.5-6 mg / L (e.g., 0.5 mg / L, 2 mg / L or 6 mg / L), and the concentration of transferrin is 5-20 mg / L (e.g., 5 mg / L, 10.0 mg / L or 20.0 mg / L).

[0034] Preferably, the concentration of linoleic acid in the serum-free culture medium is 0.05~5 mg / L, the concentration of cholesterol is 0.1~5 mg / L, and the concentration of phosphatidylcholine is 0.5~10 mg / L.

[0035] In this invention, these amino acids each play an indispensable role in the life activities of cells: tyrosine provides for the synthesis of proteins and active substances; cystine maintains the redox balance of cells and resists damage; glutamate serves as a nitrogen / carbon source for energy and participates in multiple metabolic pathways; glutamine supports rapid cell proliferation and regulates osmotic pressure and acid-base balance; histidine regulates cell signal transduction and enzyme activity; proline maintains the stability of protein structure and preserves cell morphology and function; and asparagine participates in nucleic acid and protein synthesis and helps in the transmission of genetic information.

[0036] Manganese participates in the activation of various intracellular enzymes and has an important impact on cellular energy metabolism and antioxidant defense systems; molybdenum, as a component of some enzymes, participates in cellular sulfur and nitrogen metabolism. The synergistic effect of these trace elements helps maintain intracellular physiological homeostasis and promotes healthy cell growth in low serum environments.

[0037] In a second aspect, the present invention provides the use of the culture medium for suspension acclimatization culture of fibroblast stem cells described in the first aspect in the preparation of products for suspension acclimatization culture of fibroblast stem cells.

[0038] Thirdly, the present invention provides a method for acclimating fibroblast stem cells into suspension, the method comprising the following steps:

[0039] (1) The adherent cells of fibroblast stem cells were digested, the cell suspension was collected, and the cell pellet was obtained by centrifugation;

[0040] (2) The cell pellet obtained in step (1) is resuspended using the low serum medium in the medium described in the first aspect, and the resuspended cells are inoculated for suspension culture.

[0041] (3) The cell pellet obtained in step (1) or the cells obtained after suspension culture in step (2) are inoculated with the serum-free culture medium in the first aspect and then cultured in suspension.

[0042] In step (2) of this invention, the serum concentration in the low-serum culture medium is decreased in a gradient of 3%, 2%, and 1%. Some adherent cells are highly dependent on serum during the initial acclimatization process because serum contains abundant nutrients, including adhesion factors or calcium ions that can cause cell clumping during suspension culture. Cell clumping limits the uptake of nutrients from the culture medium and affects dissolved oxygen transport to the cells, leading to cell death. As the cells gradually adapt through passage, their demand for serum decreases. Therefore, the serum content in the culture medium is gradually reduced to allow the cells to adapt to continued suspension culture in a serum-free medium.

[0043] Preferably, the digestion process in step (1) includes: when the cell adhesion rate is greater than or equal to 90%, digestion is performed using digestive enzymes.

[0044] Preferably, the digestive enzyme includes pancreatic enzymes.

[0045] Preferably, the mass percentage of the pancreatic enzyme is 0.15% to 0.35% (e.g., 0.15%, 0.2% or 0.35%).

[0046] Preferably, the digestion process takes 3 to 5 minutes (e.g., 3 minutes, 4 minutes or 5 minutes).

[0047] Preferably, the centrifugation speed is 230~300 g (e.g. 230 min, 250 g or 300 g) and the time is 4~6 min (e.g. 4 min, 5 min or 6 min).

[0048] Preferably, the suspension culture of the cells after inoculation and resuspension in step (2) includes: continuous passage culture for 5 to 10 times (e.g., 5, 8 or 10 times) after inoculation, with a passage interval of 2 to 4 days (e.g., 2 days, 3 days or 4 days).

[0049] Preferably, the inoculation density in step (2) is 0.5 × 10⁻⁶. 6 ~3.0×10 6 cells / mL (e.g., 0.5 × 10⁻⁶) 6 cells / mL, 2.0×10 6 cells / mL or 3.0 × 10 6 (cells / mL).

[0050] Preferably, the culture temperature for the continuous subculture is 35~37℃ (e.g., 35℃, 36℃ or 37℃), the carbon dioxide concentration is 5%~6% (e.g., 5% or 6%), and the rotation speed is 80~200 rpm / min (e.g., 80 rpm / min, 100 rpm / min or 200 rpm / min).

[0051] Preferably, the suspension culture in step (3) includes: continuous subculturing 20 to 50 times (e.g., 20, 40 or 50 times) after inoculation, with a subculturing interval of 3 to 4 days (e.g., 3 or 4 days) before tax.

[0052] Preferably, the inoculation density in step (3) is 1.0 × 10⁻⁶. 6 ~2.0×10 6 cells / mL (e.g., 1×10⁻⁶ cells / mL) 6 cells / mL, 1.5×10 6 cells / mL or 2.0 × 10 6 (cells / mL).

[0053] Preferably, the culture temperature for the continuous subculture is 35~37℃ (e.g., 35℃, 36℃ or 37℃), the carbon dioxide concentration is 5%-6% (e.g., 5% or 6%), and the rotation speed is 80~200 rpm / min (e.g., 80 rpm / min, 100 rpm / min or 200 rpm / min).

[0054] Fourthly, the present invention provides the use of the culture medium for suspension domestication culture of fibroblast stem cells as described in the first aspect or the method described in the third aspect in the preparation of products for cultured meat.

[0055] Compared with the prior art, the present invention has the following beneficial effects:

[0056] (1) The suspension cells obtained by domestication in this invention are fully adapted to suspension culture in serum-free medium, and the cell density can reach 6.0 × 10⁻⁶. 6 ~15.0×10 6 With a cell / mL ratio, the viability was maintained at over 98%, enabling the domestication and suspension culture of poultry fibroblast stem cells for cell-cultured meat. The selected suspension cells showed no clumping, good single-cell dispersion, high growth density, and fast doubling time, which improved efficiency and reduced costs for large-scale production of poultry fibroblast-cultured meat.

[0057] (2) The method of the present invention improves the efficiency of adapting to suspended growth cells and shortens the acclimatization period by 1 to 3 months;

[0058] (3) This invention expands the cell culture meat technology system and fills the gap in the autonomous domestication and suspension technology for cell culture poultry fibroblast stem cells. Attached Figure Description

[0059] Figure 1 A diagram showing the state of avian fibroblast stem cells after complete domestication using the culture medium described in Example 1 of this invention;

[0060] Figure 2 A diagram illustrating the domestication and growth of avian fibroblast stem cells cultured using the culture medium described in Example 1 of this invention;

[0061] Figure 3 A statistical chart showing the doubling time of avian fibroblast stem cells cultured using the culture medium described in Example 1 of this invention;

[0062] Figure 4 This is a growth curve of avian fibroblast suspension cells cultured using the culture medium described in Example 1 of the present invention. Detailed Implementation

[0063] To further illustrate the technical means and effects of this invention, the following description, in conjunction with embodiments and accompanying drawings, provides a further explanation of the invention. It is understood that the specific embodiments described herein are merely illustrative of the invention and not intended to limit it.

[0064] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0065] In this invention, amino acids were purchased from Sigma-Aldrich or Thermo Fisher Scientific, vitamins from Sigma-Aldrich and Thermo Fisher Scientific, trace elements from Sigma-Aldrich and AMRESCO, serum from Thermo Fisher Scientific (Gibco brand, catalog number: 10099141), insulin from Sigma-Aldrich (catalog number: I0516, derived from bovine pancreas), ethanolamine from Sigma-Aldrich (catalog number: E0135, ≥98%), transferrin from Thermo Fisher Scientific (Gibco brand, catalog number: 11107018, recombinant human transferrin), polyvinyl alcohol from Sigma-Aldrich (catalog number: P8136, degree of hydrolysis 86-89%), and recombinant insulin-like growth factor-1 from PeproTech (catalog number: 100-11).

[0066] Example 1

[0067] This embodiment provides a culture medium for the suspension domestication culture of fibroblast stem cells, the culture medium including a low serum culture medium and a serum-free culture medium.

[0068] In the low serum culture medium: the concentrations of tyrosine were 55 mg / L, cystine 35 mg / L, glutamate 8.1 mg / L, glutamine 520 mg / L, histidine 32 mg / L, proline 15.5 mg / L, asparagine 7.5 mg / L, iron 0.42 mg / L, copper 0.0012 mg / L, zinc 0.43 mg / L, selenium 0.009 mg / L, manganese 0.0001 mg / L, molybdenum 0.0001 mg / L, insulin 2 mg / L, ethanolamine 0.32 mg / L, and transferrin 3 mg / L.

[0069] In serum-free culture medium: tyrosine concentration was 150 mg / L, cysteine ​​concentration was 210 mg / L, glutamate concentration was 8.5 mg / L, glutamine concentration was 800 mg / L, histidine concentration was 52 mg / L, proline concentration was 24 mg / L, asparagine concentration was 10.5 mg / L, iron concentration was 60 mg / L, copper concentration was 0.0015 mg / L, zinc concentration was 0.5 mg / L, selenium concentration was 0.0015 mg / L, recombinant epidermal growth factor concentration was 10 μg / L, recombinant insulin-like growth factor concentration was 5 μg / L, soybean hydrolysate concentration was 1000 mg / L, yeast hydrolysate concentration was 500 mg / L, insulin concentration was 5 mg / L, ethanolamine concentration was 1.2 mg / L, transferrin concentration was 5 mg / L, and linoleic acid concentration was 0.1 mg / L. The concentration of cholesterol was 0.22 mg / L, and the concentration of phosphatidylcholine was 1.0 mg / L.

[0070] Example 2

[0071] This embodiment provides a culture medium for the suspension domestication culture of fibroblast stem cells, the culture medium including a low serum culture medium and a serum-free culture medium.

[0072] In the low serum culture medium: the concentrations of tyrosine were 50 mg / L, cystine 30 mg / L, glutamate 2 mg / L, glutamine 250 mg / L, histidine 30 mg / L, proline 8 mg / L, asparagine 3 mg / L, iron 0.1 mg / L, copper 0.0008 mg / L, zinc 0.05 mg / L, selenium 0.0008 mg / L, manganese 0.00012 mg / L, molybdenum 0.0001 mg / L, insulin 1 mg / L, ethanolamine 0.12 mg / L, and transferrin 3 mg / L.

[0073] In serum-free culture medium: tyrosine concentration was 80 mg / L, cysteine ​​concentration was 50 mg / L, glutamate concentration was 5 mg / L, glutamine concentration was 300 mg / L, histidine concentration was 30 mg / L, proline concentration was 15 mg / L, asparagine concentration was 5 mg / L, iron concentration was 74 mg / L, copper concentration was 0.001 mg / L, zinc concentration was 0.5 mg / L, selenium concentration was 0.001 mg / L, recombinant epidermal growth factor concentration was 5 μg / L, recombinant insulin-like growth factor concentration was 20 μg / L, soybean hydrolysate concentration was 500 mg / L, yeast hydrolysate concentration was 500 mg / L, insulin concentration was 4 mg / L, ethanolamine concentration was 0.5 mg / L, transferrin concentration was 10 mg / L, linoleic acid concentration was 0.18 mg / L, and cholesterol concentration was 0.20 mg / L. The concentration of phosphatidylcholine was 1.45 mg / L.

[0074] Example 3

[0075] This embodiment provides a culture medium for the suspension domestication culture of fibroblast stem cells, the culture medium including a low serum culture medium and a serum-free culture medium.

[0076] In the low serum culture medium: the concentrations of tyrosine were 100 mg / L, cystine 80 mg / L, glutamate 10 mg / L, glutamine 600 mg / L, histidine 60 mg / L, proline 20 mg / L, asparagine 10 mg / L, iron 0.4 mg / L, copper 0.002 mg / L, zinc 0.45 mg / L, selenium 0.003 mg / L, manganese 0.002 mg / L, molybdenum 0.0004 mg / L, insulin 10 mg / L, ethanolamine 1.5 mg / L, and transferrin 5 mg / L.

[0077] In serum-free culture medium: tyrosine 300 mg / L, cysteine ​​300 mg / L, glutamate 20 mg / L, glutamine 1000 mg / L, histidine 120 mg / L, proline 50 mg / L, asparagine 15 mg / L, iron 80 mg / L, copper 0.005 mg / L, zinc 0.9 mg / L, selenium 0.005 mg / L, recombinant epidermal growth factor 15 μg / L, recombinant insulin-like growth factor 30 μg / L, soybean hydrolysate 1000 mg / L, yeast hydrolysate 1000 mg / L, insulin 6 mg / L, ethanolamine 6 mg / L, transferrin 8 mg / L, and linoleic acid 0.15 mg / L. The concentration of cholesterol was 0.10 mg / L, and the concentration of phosphatidylcholine was 1.2 mg / L.

[0078] Example 4

[0079] This embodiment provides a culture medium for the suspension domestication culture of fibroblast stem cells. The only difference from Example 1 is that the proliferative factor in the low serum culture medium does not contain insulin, and its mass fraction is allocated to ethanolamine and transferrin in a proportional manner.

[0080] Example 5

[0081] This embodiment provides a culture medium for the suspension domestication culture of fibroblast stem cells. The only difference from Example 1 is that the proliferative factor in the low serum culture medium does not contain ethanolamine, and its mass fraction is allocated to insulin and transferrin in proportion.

[0082] Example 6

[0083] This embodiment provides a culture medium for the suspension domestication culture of fibroblast stem cells. The only difference from Example 1 is that the proliferative factor in the low serum culture medium does not contain transferrin, and its mass fraction is allocated to ethanolamine and insulin in a proportional manner.

[0084] Example 7

[0085] This embodiment provides a culture medium for the suspension domestication culture of fibroblast stem cells. The only difference from Example 1 is that the growth factor ethanolamine is replaced with polyvinyl alcohol in an equal amount in the low serum culture medium.

[0086] Example 8

[0087] This embodiment provides a culture medium for the suspension domestication culture of fibroblast stem cells. The only difference from Example 1 is that the proliferating factor insulin is replaced with an equal amount of recombinant insulin-like growth factor in the low serum culture medium.

[0088] Example 9

[0089] This embodiment provides a culture medium for the suspension domestication culture of fibroblast stem cells. The only difference from Example 1 is that the serum-free culture medium does not contain insulin as the proliferative factor, and its mass fraction is allocated to ethanolamine and transferrin in a proportional manner.

[0090] Example 10

[0091] This embodiment provides a culture medium for the suspension domestication culture of fibroblast stem cells. The only difference from Example 1 is that the serum-free culture medium does not contain ethanolamine as the proliferative factor, and its mass fraction is allocated to insulin and transferrin in proportion.

[0092] Example 11

[0093] This embodiment provides a culture medium for the suspension domestication culture of fibroblast stem cells. The only difference from Example 1 is that the serum-free culture medium does not contain transferrin as the proliferative factor, and its mass fraction is allocated to ethanolamine and insulin in a proportional manner.

[0094] Example 12

[0095] This embodiment provides a culture medium for the suspension domestication culture of fibroblast stem cells. The only difference from Example 1 is that the proliferation-promoting factor ethanolamine is replaced with polyvinyl alcohol in an equal amount in the serum-free culture medium.

[0096] Example 13

[0097] This embodiment provides a culture medium for the suspension domestication culture of fibroblast stem cells. The only difference from Example 1 is that the proliferating factor insulin is replaced with an equal amount of recombinant insulin-like growth factor in the serum-free culture medium.

[0098] Example 14

[0099] This embodiment provides a culture medium for the suspension domestication culture of fibroblast stem cells. The only difference from Example 1 is that the low serum culture medium does not contain manganese as a trace element, and its mass fractions are proportionally allocated to iron, copper, zinc, selenium and molybdenum.

[0100] Example 15

[0101] This embodiment provides a culture medium for the suspension domestication culture of fibroblast stem cells. The only difference from Example 1 is that the low serum culture medium does not contain molybdenum as a trace element, and its mass fractions are proportionally allocated to iron, copper, zinc, selenium and manganese.

[0102] Example 16

[0103] This embodiment provides a culture medium for the suspension domestication culture of fibroblast stem cells. The only difference from Example 1 is that the low serum culture medium does not contain copper as a trace element, and its mass fractions are proportionally allocated to iron, molybdenum, zinc, selenium and manganese.

[0104] Example 17

[0105] This embodiment provides a culture medium for the suspension domestication culture of fibroblast stem cells. The only difference from Example 1 is that the trace element molybdenum is replaced with chromium in an equal amount in the low serum culture medium.

[0106] Example 18

[0107] This embodiment provides a culture medium for the suspension domestication culture of fibroblast stem cells. The only difference from Example 1 is that the trace element manganese in the low serum culture medium is replaced with nickel in equal amounts.

[0108] Comparative Example 1

[0109] This comparative example provides a culture medium for the suspension acclimatization culture of fibroblast stem cells, which differs from Example 1 only in that the low serum culture medium does not contain insulin, ethanolamine, and transferrin.

[0110] Comparative Example 2

[0111] This comparative example provides a culture medium for the suspension domestication culture of fibroblast stem cells, which differs from Example 1 only in that the serum-free culture medium does not contain insulin, ethanolamine, and transferrin.

[0112] Test Example 1

[0113] This test case provides a suspension domestication method for fibroblast suspension cell lines derived from SPF chicken embryos, suitable for cell cultured meat. After domestication, the density and cell viability of fibroblast stem cells were detected.

[0114] (1) Adherent cell digestion treatment: When the confluence rate of adherent cells reaches 90%, trypsin is used for digestion, and the cell suspension is collected and centrifuged to obtain cell pellet; the trypsin concentration is 0.25%, the digestion time is 3 min, and the centrifugation parameters are 300 g centrifugation for 5 min.

[0115] (2) Low serum acclimatization suspension culture: The cell pellet from step (1) was resuspended in a suspension culture container shake flask using the low serum culture medium (containing 3% newborn calf serum) described in each example and comparative example. The cells were gently dispersed by blowing air to make them appear as single cells. The cell seeding density was 3.0 × 10⁶ cells / year. 6 The working volume is 1 / 5 of the culture container volume. The culture temperature is 37℃, the carbon dioxide concentration is 5%, the humidity is 80%, and the shaking speed is 200 rpm / min. The culture is passaged at 4-day intervals, and the culture is repeated 5 times. Samples need to be taken and counted every day during the acclimatization process.

[0116] (3) Serum-lowering adaptation and screening culture: Based on the periodic counting results, cells from the suspension culture in step (2) that cannot adapt to medium change or passage are discarded. Cells that can adapt are transferred to the low-serum culture medium (containing 2% newborn calf serum) described in each example and comparative example, with a cell seeding density of 3.0 × 10⁶ cells / year. 6 The cells / mL working volume was 1 / 5 of the culture container volume. The culture temperature was 37℃, the carbon dioxide concentration was 5%, the humidity was 80%, and the shaking speed was 200 rpm / min. The culture was passaged at 4-day intervals, and the culture was repeated 5 times.

[0117] The concentrations of newborn calf serum were then reduced to 2%, 1%, 0.5%, and 0%, and cultured sequentially for 5 consecutive passages. In this example, if the cell passage ratio was less than 1:4, all passages were performed by centrifugation at 300g for 5 min. Once significant proliferation was observed in the suspension culture, the cells were further cultured at 3.0 × 10⁻⁶ cells / mL. 7 Cells were cryopreserved at a density of cells / mL for future use. The cryopreservation solution consisted of 10% DMSO, 10% serum, and 80% low serum culture medium. The cryopreservation tubes were placed in a -80°C freezer for 24 hours using a programmed cooling box, and then transferred to a liquid nitrogen tank for long-term storage.

[0118] (4) Passage the suspension cells screened in step (3) into serum-free culture medium at a cell seeding density of 2.0 × 10⁶ cells / year. 6 The cell density was set at 1 / 5 of the culture vessel volume, with a working volume of 1 / 5 of the vessel volume. The culture temperature was 37℃, CO2 concentration was 5%, humidity was 80%, and the shaker speed was 200 rpm / min. Cells were passaged every 3 days. Cell clumping and dispersion were observed until the cell density reached 4.0 × 10⁶ cells / mL after three days of culture.6 Cells per mL with a viability of over 95%, good dispersibility, and no large clumps are suitable for stable continuous passage. After every 5 consecutive passages, cells fully adapted to suspension growth can be selected at a density of 3.0 × 10⁶ cells / mL. 7 Cells were cryopreserved at a density of cells / mL for future use in research and development. The cryopreservation solution consisted of 10% DMSO and 90% serum-free culture medium. The cryopreservation tubes were placed in a -80°C freezer for 24 hours using a programmed cooling box, and then transferred to a liquid nitrogen tank for long-term storage.

[0119] (5) After completing the serum-free acclimatization in step (4), the obtained fully suspended cells were cultured for 3 days, and the cell density, cell viability, cell diameter, doubling time and cell dispersion status were detected.

[0120] (6) Transfer the suspension cells obtained in step (5) to a shake flask for expansion culture. After culturing for 72 h, wait until the cell density reaches 5.0 × 10⁻⁶. 6 ~8.0×10 6 Cells / mL, viability 98%, cell diameter 15-21 μm, doubling time 25-40 h, at 2.0 × 10⁶ cells / mL. 7 ~4.0×10 7 Cells / mL were cryopreserved to serve as the original suspension cell library PCB for subsequent use in establishing MCB and WCB libraries. The cryopreservation solution consisted of 10% DMSO and 90% serum-free culture medium. The cryopreservation tubes were placed in a -80°C freezer for 24 hours using a programmed cooling box, and then transferred to a liquid nitrogen tank for long-term storage.

[0121] Table 1

[0122]

[0123]

[0124] As shown in Table 1, the cell density of fibroblast suspended stem cells cultured using the culture medium described in Examples 1-3 of this invention can reach 8.0 × 10⁻⁶. 6The cell density was [number of cells / mL], cell viability reached 98%, cell diameter reached 21 μm, doubling time was 25 h, and cells were in a single dispersed state, indicating that the serum-free, fully suspended fibroblasts obtained by the domestication and screening of this invention have excellent performance. Examples 4-8 and Comparative Example 1 show that the proliferative factors insulin, ethanolamine, and transferrin work synergistically in the low-serum culture medium to promote the proliferation of fibroblast stem cells. Examples 9-11 and Comparative Example 2 demonstrate that the proliferative factors insulin, ethanolamine, and transferrin work synergistically in the serum-free culture medium to promote the proliferation of fibroblast stem cells. Examples 12-13 show that compared with Example 1, cell growth density was significantly reduced and doubling time was prolonged. This indicates that although IGF-1 and insulin are structurally homologous and have overlapping signaling pathways, IGF-1 cannot completely replace the role of insulin in promoting fibroblast stem cell proliferation, disrupting the precise synergistic balance among insulin, ethanolamine, and transferrin. The results of Examples 14-16 demonstrate that the trace elements iron, copper, zinc, selenium, manganese, and molybdenum in the low-serum culture medium of the present invention require a specific combination to synergistically promote the proliferation of fibroblast stem cells. Example 17, compared to Example 1, showed a significant decrease in cell growth density and viability, indicating that chromium cannot replace molybdenum's specific role in promoting fibroblast proliferation and maintaining normal cell metabolism, thus disrupting the synergistic effect of the trace element combination. Example 18, compared to Example 1, showed a deterioration in cell growth status and a prolonged doubling time, indicating that the addition of nickel cannot achieve the biological functions of manganese and may even have a toxic inhibitory effect on cells, further confirming that manganese is indispensable in this specific trace element combination.

[0125] The state of avian fibroblast stem cells after complete domestication of the culture medium described in Example 1 is shown in the figure below. Figure 1 As shown, the suspension cells domesticated by this invention are morphologically regular and plump, and exist in a single, dispersed suspension state without significant cell clustering; the growth diagram of fibroblast stem cells domesticated is shown below. Figure 2 As shown, this demonstrates that the serum-free culture medium of the present invention can support cells from suspension adaptation to stable passage, with stable cell growth curves and strong adaptability to the suspension environment; the statistical graph of fibroblast doubling time is shown in the figure. Figure 3 As shown, the culture medium of the present invention can significantly shorten the cell doubling time and improve the proliferation efficiency of suspension cells; the growth curve of fibroblast suspension cells is shown in the figure. Figure 4 As shown, this indicates high cell growth density, a high plateau phase, and a delayed death phase.

[0126] Test Example 2

[0127] Avian fibroblasts, obtained by digestion of chicken embryo tissue with trypsin and which had been acclimated to suspension in a serum-free environment, were cultured in shake flasks using the serum-free culture medium described in the examples and comparative examples. Cell samples were collected for analysis of cell protein content.

[0128] Using a protein assay kit (Thermo #23227), the cultures obtained from the domestication culture of each group in Test Example 1 were subjected to cell precipitation, cell lysis, and standard preparation. The absorbance at 480 nm was measured using a multi-mode microplate reader. All sample / standard / blank readings were averaged, and a standard curve was generated by plotting absorbance versus protein concentration. The resulting protein content detection range was compared with the original adherent cell protein content, using the original adherent cell protein content as the baseline level.

[0129] Results: Fibroblast suspended stem cells cultured using the culture media described in Examples 1-3 of this invention showed that the protein content of the cells after serum-free suspension culture was comparable to that of the original adherent cells. This indicates that the culture media of this invention, while increasing cell growth rate, did not affect the normal metabolic activity and protein synthesis capacity of the cells, thus maintaining the functional integrity of the cells. Examples 4-8 and Comparative Example 1 demonstrate that the proliferative factors insulin, ethanolamine, and transferrin work synergistically in the low-serum culture medium, which is crucial for maintaining normal protein synthesis metabolism in cells. The absence of any one factor leads to a decrease in protein synthesis capacity, and complete absence results in extremely low protein content. Examples 9-11 and Comparative Example 2 demonstrate that the proliferative factors insulin, ethanolamine, and transferrin work synergistically in the serum-free culture medium, and their concentration ratio is essential for maintaining high levels of protein synthesis in cells. Examples 12-13 show a decrease in protein synthesis capacity compared to Example 1. This indicates that although IGF-1 and insulin are structurally homologous and have overlapping signaling pathways, IGF-1 cannot completely replace the role of insulin in promoting fibroblast proliferation, disrupting the precise synergistic balance among insulin, ethanolamine, and transferrin. The results of Examples 14-16 demonstrate that the trace elements iron, copper, zinc, selenium, manganese, and molybdenum in the low-serum culture medium of the present invention require a specific combination to synergistically ensure normal cellular metabolism and protein synthesis pathways. The absence of any one trace element severely weakens the cell's protein production capacity. Example 17, compared to Example 1, shows a significant decrease in cellular protein content, indicating that chromium cannot replace molybdenum's specific role in promoting fibroblast proliferation and maintaining normal cellular metabolism, thus disrupting the synergistic effect of the trace element combination. Example 18, compared to Example 1, shows a decrease in total cellular protein production, indicating that the addition of nickel cannot achieve the biological functions of manganese and may even have a toxic inhibitory effect on cells, further confirming that manganese is indispensable in this specific trace element combination.

[0130] In summary, this invention improves the efficiency of domesticating suspended cells through an autonomous domestication method, and the domesticated suspended cells have the characteristics of high density, good stability, high dispersibility, and fast multiplication rate.

[0131] The applicant declares that the detailed method of the present invention is illustrated by the above embodiments, but the present invention is not limited to the above detailed method, that is, it does not mean that the present invention must rely on the above detailed method to be implemented. Those skilled in the art should understand that any improvements to the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. A culture medium for the suspension acclimatization culture of fibroblast stem cells, characterized in that, The culture medium includes low-serum culture medium and serum-free culture medium; The low serum culture medium includes: amino acids, vitamins, trace elements, serum, and proliferative factors; The serum-free culture medium includes: amino acids, trace elements, growth factors, hydrolysates, lipids, and proliferation-promoting factors; The proliferative factors include any one or a combination of at least two of insulin, ethanolamine, or transferrin.

2. The culture medium according to claim 1, characterized in that, The amino acids in the low serum culture medium include any one or a combination of at least two of the following: tyrosine, cystine, glutamic acid, glutamine, histidine, proline, or asparagine. Preferably, the vitamins in the low serum culture medium include any one or a combination of at least two of the following: choline chloride, folic acid, inositol, nicotinamide, riboflavin, D-calcium pantothenate, pyridoxal hydrochloride, or vitamin B12. Preferably, the trace elements in the low serum culture medium include any one or a combination of at least two of the following: iron, copper, zinc, selenium, manganese, or molybdenum.

3. The culture medium according to claim 1 or 2, characterized in that, The low serum culture medium contains tyrosine at a concentration of 50–100 mg / L, cystine at a concentration of 30–80 mg / L, glutamate at a concentration of 2–10 mg / L, glutamine at a concentration of 250–600 mg / L, histidine at a concentration of 30–60 mg / L, proline at a concentration of 8–20 mg / L, and asparagine at a concentration of 3–10 mg / L. Preferably, the concentration of iron in the low serum culture medium is 0.1~0.4 mg / L, the concentration of copper is 0.0008~0.002 mg / L, the concentration of zinc is 0.05~0.45 mg / L, the concentration of selenium is 0.0008~0.003 mg / L, the concentration of manganese is 0.0001~0.002 mg / L, and the concentration of molybdenum is 0.0001~0.0004 mg / L. Preferably, the serum mass percentage in the low serum culture medium is 1% to 3%; Preferably, the concentration of insulin in the low serum culture medium is 1-10 mg / L, the concentration of ethanolamine is 0.12-1.5 mg / L, and the concentration of transferrin is 2-5 mg / L.

4. The culture medium according to any one of claims 1-3, characterized in that, The amino acids in the serum-free culture medium include any one or a combination of at least two of the following: tyrosine, cystine, glutamic acid, glutamine, histidine, proline, or asparagine. Preferably, the trace elements include any one or a combination of at least two of iron, copper, zinc, or selenium; Preferably, the growth factors include: recombinant epidermal growth factor and / or recombinant insulin-like growth factor; Preferably, the hydrolysate includes: soybean hydrolysate and / or yeast hydrolysate; Preferably, the proliferative factor includes any one or a combination of at least two of insulin, ethanolamine, or transferrin; Preferably, the lipids include any one or a combination of at least two of linoleic acid, cholesterol, or phosphatidylcholine.

5. The culture medium according to any one of claims 1-4, characterized in that, The serum-free culture medium contains tyrosine at a concentration of 80–300 mg / L, cystine at a concentration of 50–300 mg / L, glutamate at a concentration of 5–20 mg / L, glutamine at a concentration of 300–1000 mg / L, histidine at a concentration of 30–120 mg / L, proline at a concentration of 15–50 mg / L, and asparagine at a concentration of 5–15 mg / L. Preferably, the concentration of iron in the serum-free culture medium is 30-120 mg / L, the concentration of copper is 0.001-0.005 mg / L, the concentration of zinc is 0.5-0.9 mg / L, and the concentration of selenium is 0.001-0.005 mg / L. Preferably, the concentration of recombinant epidermal growth factor in the serum-free culture medium is 5-15 μg / L, and the concentration of recombinant insulin-like growth factor is 20-30 μg / L. Preferably, the concentration of soybean hydrolysate and yeast hydrolysate in the serum-free culture medium is 500-2000 mg / L. Preferably, the concentration of insulin in the serum-free culture medium is 1-5 mg / L, the concentration of ethanolamine is 0.5-6 mg / L, and the concentration of transferrin is 5-20 mg / L. Preferably, the concentration of linoleic acid in the serum-free culture medium is 0.05~5 mg / L, the concentration of cholesterol is 0.1~5 mg / L, and the concentration of phosphatidylcholine is 0.5~10 mg / L.

6. The use of the culture medium for suspension culture of fibroblast stem cells according to any one of claims 1-5 in the preparation of products for suspension culture of fibroblast stem cells.

7. A method for acclimatizing fibroblast stem cells into suspension, characterized in that, The fibroblast stem cell suspension domestication method includes the following steps: (1) The adherent cells of fibroblast stem cells were digested, the cell suspension was collected, and the cell pellet was obtained by centrifugation; (2) The cell pellet obtained in step (1) is resuspended using the low serum medium of any one of the media described in claims 1-5, and the resuspended cells are inoculated for suspension culture. (3) The cell pellet obtained in step (1) or the cells obtained after suspension culture in step (2) are inoculated with serum-free culture medium of any one of claims 1-5 and then cultured in suspension.

8. The method for acclimatizing fibroblast stem cells according to claim 7, characterized in that, The digestion process described in step (1) includes: when the cell adhesion rate is greater than or equal to 90%, digestion is performed using digestive enzymes; Preferably, the digestive enzyme includes pancreatic enzymes; Preferably, the pancreatic enzyme has a mass percentage content of 0.15% to 0.35%; Preferably, the digestion process takes 3 to 5 minutes; Preferably, the centrifugation speed is 230~300 g and the time is 4~6 min.

9. The method for acclimatizing fibroblasts according to claim 7 or 8, characterized in that, The suspension culture of cells after inoculation and resuspension in step (2) includes: continuous passage culture 5 to 10 times after inoculation, with a passage interval of 2 to 4 days before. Preferably, the inoculation density in step (2) is 0.5 × 10⁻⁶. 6 ~3×10 6 cells / mL; Preferably, the culture temperature for the continuous subculture is 35~37℃, the carbon dioxide concentration is 5%-6%, and the rotation speed is 80~200 rpm / min; Preferably, the suspension culture in step (3) includes: continuous subculturing 20 to 50 times after inoculation, with a subculturing interval of 3 to 4 days before tax; Preferably, the inoculation density in step (3) is 1.0 × 10⁻⁶. 6 ~2.0×10 6 cells / mL; Preferably, the culture temperature for continuous subculturing is 35~37℃, the carbon dioxide concentration is 5%~6%, and the rotation speed is 80~200 rpm / min.

10. The use of the culture medium for suspension domestication culture of fibroblast stem cells as described in any one of claims 1-5 or the method of any one of claims 7-9 in the preparation of products for cultured meat.

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