KR102608766B1 - Medium composition for animal cell culture and method for culturing animal cells using the same - Google Patents

Abstract

Pertaining to a medium composition for culturing animal cells, according to a medium composition for culturing animal cells containing a basic medium and a yeast extract according to one aspect, cells can be effectively proliferated even in a basic medium that does not contain vitamins and organic acids, and the culture medium It has the effect of reducing the cost of cell culture media by minimizing the ingredients.

Description

Medium composition for animal cell culture and method for cultivating animal cells using the same {Medium composition for animal cell culture and method for culturing animal cells using the same}

The present invention relates to a medium composition for culturing animal cells.

With the continuous increase in population, problems with environmental pollution are emerging, and in particular, problems with marine environmental pollution are intensifying to a serious level. Since the 1960s, environmental pollution and indiscriminate fishing have caused some species to reach unsustainable levels, and marine environmental pollution caused by substances such as microplastics is gradually accumulating in the bodies of marine life, causing harm to humans who eat them. It affects even

Despite marine environmental pollution, the demand for marine products is increasing exponentially with population growth. In particular, the supply of crustaceans such as shrimp, crabs, and lobsters cannot meet demand, leading to an increase in aquaculture. However, with the increase in aquaculture, carbon gas emissions, a major cause of global warming, have worsened, and the supply method to meet demand is ultimately causing new environmental pollution.

Cultured meat is being proposed as a solution to curb environmental pollution and meet demand. Cultured meat can produce multiple individuals by extracting and proliferating cells from one individual, thus reducing carbon gas emissions and showing high production relative to area. In addition, cultured meat has the advantage of providing safe food that is not contaminated with heavy metals and microplastics, unlike marine products caught in the sea, because cells are proliferated in a laboratory incubator.

The most important thing to commercialize cultured meat is reducing the cost of producing cultured meat, and the thing that has the greatest impact on reducing the price of producing cultured meat is the cell culture medium. Various ingredients are added to the basic culture medium used when cultivating cultured meat. If cultured meat can be produced using a culture medium without the addition of these ingredients, it can greatly help reduce costs.

Republic of Korea Patent Publication No. 10-2021-0116551 Republic of Korea Patent Publication No. 10-2022-0040417

One aspect is to provide a medium composition for culturing animal cells including a basic medium and yeast extract.

Another aspect is to provide a method for cultivating animal cells, comprising culturing the cells in a medium composition for culturing animal cells containing a basic medium and a yeast extract.

One aspect provides a medium composition for culturing animal cells including a basic medium and yeast extract.

In one embodiment, the basic medium contains biotin, D-Ca pantothenate, a-ketoglutaric acid, choline chloride, and folic acid ( Folic acid, i-inositol, nicotinic acid, pyridoxine-HCl, riboflavin, thiamine-HCl, and penta-amino benzoic acid. It may not contain one or more vitamins selected from the group consisting of benzoic acid.

In one embodiment, the basic medium may not contain vitamins.

In one embodiment, the basic medium may not contain one or more organic acids selected from the group consisting of fumaric acid, malic acid, and succinic acid.

In one embodiment, the basic medium may not contain organic acids.

In the case of vitamins and organic acids, if excessive amounts are applied to cells, they may actually inhibit cell proliferation.

In one embodiment, the medium composition may contain 0.01 to 20, 0.01 to 10, 0.6 to 10, or 0.6 to 2% (w/v) of the yeast extract.

In this specification, the term "yeast extract" is a natural product that does not cause the problem of producing harmful substances such as acid-decomposed HVP (Hydrolyzed vegetable protein) because it is produced through autodigestion using NaCl, ethanol, or enzyme treatment after culturing yeast. It refers to a biological material and is used in microbial fermentation media or natural seasoning.

The yeast extract can be prepared by adding edible enzymes to edible yeast and hydrolyzing polypeptides of the edible yeast, and can be used for food.

In one embodiment, the yeast extract may be usable in food.

Yeast extract is known to contain minerals, vitamins, nucleic acids, peptides, and amino acids.

In one embodiment, the yeast is from the genus Saccharomyces ( Saccharomyces sp.), Wicherhamomyces sp., Pichia sp., and Hanseniaspora sp. It may be one or more types selected from the group consisting of.

The yeast extract may be a plant- or microbial-derived ingredient that replaces animal-based ingredients. The plant- or microbial-derived ingredients are highly suitable for use in biopharmaceutical and food applications.

In one embodiment, the animal cell may be one or more types selected from the group consisting of muscle stem cells, muscle cells, and progenitor cells thereof.

The muscle stem cells may be satellite cells or myoblasts, for example, but are not limited thereto.

As used herein, the term “muscle stem cell” refers to a cell having the characteristics of a muscle stem cell, including proliferation without transformation, unlimited proliferation, self-reproduction ability, and the ability to differentiate into muscle. Any cell that shows self-reproduction ability, unlimited proliferation ability, or muscle differentiation ability can be included without limitation. The self-reproduction ability and muscle differentiation ability can be confirmed using markers. Additionally, the muscle stem cells may be, for example, cells that exhibit self-renewal ability and expression of CD29, CD56, Oct4, Nanog, or Pax7. The type and origin of the muscle stem cells are not limited as long as they have differentiation ability and self-renewal ability. The muscle stem cells may be derived from, for example, mammals, humans, monkeys, pigs, horses, cows, chickens, ducks, sheep, dogs, cats, mice, or rabbits. In one embodiment, the muscle stem cells may be bovine or chicken muscle stem cells.

The muscle stem cells may be cells that express Pax7 or MyoD in the process of being induced into myoblasts.

As used herein, the term "myoblast" refers to a cell that is a precursor cell in the process of differentiating into a muscle cell and proliferates like a myogenic cell and can differentiate into a muscle cell (myogenic cell, myocyte) by fusion between cells. The “muscle cell” refers to a cell capable of forming muscle tissue generated from myoblasts through the differentiation process of myoblasts.

The muscle cells may be myocytes or myotubes, for example, myocytes, but are not limited thereto.

The progenitor cells refer to cells capable of generating cells differentiated into multiple lineages, such as myoblasts, fibroblasts, adipocytes, stromal cells, pericytes, smooth muscle cells, and endothelial cells. Progenitor cells differ from stem cells in that they typically do not have extensive self-replication capabilities.

The animal cells may further include fat cells (fat cells or adipocytes) and/or their progenitor cells, stromal cells (connective tissue) and/or their progenitor cells, or endothelial cells (blood vessels) and/or their progenitor cells. , but is not limited to this.

In one embodiment, the animal may be one or more species selected from the group consisting of cattle, sheep, pigs, poultry, crustaceans, and fish, but is not limited thereto.

In one embodiment, the animal cells may refer to animal-derived cells made by isolating cells from animal tissues and culturing them in vitro.

In one embodiment, the animal cells may be crustacean cells.

In one embodiment, the animal cells may be cells derived from shrimp, crab, or lobster.

As used herein, the term “culture media” refers to a material that supports the growth and survival of cells, including stem cells, in vitro.

As used herein, the term “basic medium” refers to a medium containing basic components for culturing cells. In general, when cells are cultured using only basic media, the cells often do not proliferate smoothly, so various ingredients are added for optimal cell proliferation.

In one embodiment, the basic medium is DMEM (Dulbecco's Modified Eagle's Medium), MEM (Minimal Essential Medium), BME (Basal Medium Eagle), RPMI 1640, DMEM/F-10 (Dulbecco's Modified Eagle's Medium: Nutrient Mixture F- 10), DMEM/F-12 (Dulbecco's Modified Eagle's Medium: Nutrient Mixture F-12), α-MEM (α-Minimal essential Medium), G-MEM (Glasgow's Minimal Essential Medium), IMDM (Isocove's Modified Dulbecco's Medium), It may be one or more selected from the group consisting of Knockout DMEM, E8 (Essential 8 Medium), SF-DMEM (serum free-Dulbecco Modified Eagle Medium), L15 Medium, and Grace's Insect Medium, but is not limited thereto.

In addition to the above medium, any medium used in the industry is sufficient, and the medium may contain amino acids, vitamins, inorganic salts, and other ingredients. For example, non-essential amino acids and L-glutamine, vitamins include vitamin B12, and inorganic salts include trace components (CuSO45H2O, Fe(NO3)39H2O, ZnSO4), phosphoenol pyruvate, and monoethanol. Amines (Momoethanolamine), Sodium selenite and Sodium bicarbonate, other ingredients include D-glucose, Linoleic acid, Lipoic acid and Sodium pyruvate ), hypoxantine sodium, putrescine hydrochloride (Putrescine HCl), and polyamine solution.

In one embodiment, the medium composition for culturing animal cells may not contain serum.

In one embodiment, the medium composition for culturing animal cells may be a medium containing serum.

The serum may be, but is not limited to, animal blood, and is preferably serum derived from the blood of mammals (e.g., pigs, horses, cows, goats, sheep, and dogs), and more preferably fetal bovine serum. serum, FBS).

In one embodiment, the serum is fetal bovine serum (FBS), human platelet lysate (hPL), human serum albumin (HSA), and human serum (HS). ), Platelet-Rich Plasma (PRP), platelet poor plasma (PPP), calf serum, horse serum, porcine serum and sheep serum. ), but is not limited thereto.

In one embodiment, the culture may be a suspension culture.

As used herein, the term “cell culture” refers to the process of artificially growing living cells in vitro under controlled conditions. In addition, a portion of an individual's tissue is aseptically removed, the intercellular connecting material is decomposed with enzymes, and the resulting suspension is spread on the flat bottom of a culture dish such as a bottle or Petri dish to grow and proliferate cells.

As used herein, the term “suspension culture” refers to culturing cells to be cultured in a floating state in a culture medium without being fixed to a substrate.

In the above culture process, SR (serum replacement) may be added to the medium to create a suspended state of the cultured cells, or the culture may be performed using a low-adhesion culture dish or bioreactor, but is not limited to this, and various suspended cultures used in the industry method can be used.

The culture may be growth and proliferation.

As used herein, the terms “growth and proliferation” mean an increase in the number of cells. The culture may be undifferentiated proliferation. The undifferentiated proliferation means that stem cells proliferate into cells with the same properties as the original cells, that is, with potency and self-renewal ability, without being differentiated into specific cells. . In this specification, the term "differentiation" refers to the phenomenon in which cells become specialized in structure or function while they divide and grow, that is, the shape or function of biological cells, tissues, etc. change to perform a given task. means that Measuring or determining the degree of differentiation into a specific cell type can be performed by methods well known in the art. Additionally, the differentiation can be characterized by cell surface markers (e.g., staining cells with tissue-specific or cell-label specific antibodies) and changes in cell morphology (e.g., using techniques such as flow cytometry or immunocytochemistry). For example, by examining cell morphology using light or confocal microscopy, measuring nuclear/cytoplasmic ratios, or by using techniques in the art such as polymerase chain reaction (PCR) and gene-expression profiling. It can be confirmed by measuring changes in gene expression using known techniques.

Another aspect provides a method of cultivating animal cells, comprising culturing the cells in a medium composition for culturing animal cells comprising a basic medium and a yeast extract.

In one embodiment, the basic medium may not contain vitamins.

In one embodiment, the basic medium may not contain organic acids.

In one embodiment, the animal cells may be crustacean cells.

In one embodiment, the culture may be a suspension culture.

According to a medium composition for animal cell culture containing a basic medium and yeast extract according to one aspect, cells can be effectively proliferated even in a basic medium that does not contain vitamins and organic acids, and the cost of cell culture medium is reduced by minimizing the culture medium components. There is an effect that can be done.

Figure 1 shows the results of confirming the difference in cell proliferation according to different culture media when culturing animal cells.
Figures 2a to 2c show the results of culturing animal cells in a medium containing yeast extract and confirming the difference in proliferation of animal cells compared to a medium with or without the addition of fetal bovine serum.
Figure 3 shows the results of confirming the difference in cell proliferation depending on the presence or absence of vitamins in the basic medium.
Figure 4 shows the results of confirming the difference in cell proliferation depending on the presence or absence of organic acids in the basic medium.
Figure 5 shows the results of confirming the difference in cell proliferation depending on the presence or absence of vitamins and organic acids in the basic medium.

Hereinafter, the present invention will be described in more detail through examples. However, these examples are for illustrative purposes only and the scope of the present invention is not limited to these examples.

The terms or words used in the specification and claims of the present invention are not to be construed as limited to their ordinary or dictionary meanings, and the inventor may appropriately define the concept of terms to explain his or her invention in the best way. It must be interpreted as meaning and concept consistent with the technical idea of the present invention based on principles.

Throughout the specification of the present invention, when a part is said to "include" a certain component, this means that it does not exclude other components but may further include other components, unless specifically stated to the contrary. .

Throughout the specification of the present invention, “A and/or B” means A or B, or A and B.

Example 1. Confirmation of cell proliferation by medium

To determine whether there was a difference in cell proliferation when the culture medium was changed during cell culture, crustacean-derived cells isolated from shrimp were cultured in commonly used basic media (DMEM-F12, L15 or Grace's insect media) at 0, 5 or 10. Culture was performed using medium supplemented with % (v/v) Fetal Bovine Serum (FBS), and the degree of cell proliferation was confirmed.

Specifically, crustacean cells were initially inoculated at ^1.5 To confirm the approximate tendency of cell proliferation, absorbance was measured at OD600, and the exact number of cultured cells was confirmed using a hemocytometer. The results are shown in Figure 1.

As shown in Figure 1, as a result of absorbance measurement and cell count confirmation using a hemocytometer, the highest cell proliferation was confirmed in Grace's insect media. Additionally, higher cell proliferation was observed in the culture medium containing fetal bovine serum than in the culture medium without the addition of fetal bovine serum. From the above results, it was confirmed that even if fetal bovine serum was included, the proliferation rate of crustacean cells was significantly different depending on the type of basic medium. Although Grace's insect media was suitable for the proliferation of crustacean cells, fetal bovine serum could be added to other basic culture media. It was found that even if added, cell proliferation occurred.

Example 2. Cell proliferation in medium containing yeast extract

In order to determine whether yeast extract can be used as an ingredient that can replace fetal bovine serum, which has a cell proliferation effect in the culture of animal cells, any changes in cell proliferation were examined when yeast extract was added. I checked to see if it was visible. Specifically, a culture medium was prepared by adding yeast extract to three types of basic media (DMEM-F12, L15 or Grace's insect media) at a concentration of 0.01, 0.6, 2 or 10% (w/v), and fetal bovine serum was added to the mixture. The proliferation of crustacean-derived cells isolated from shrimp, crab, or lobster was confirmed in the same manner as in Example 1, compared to the culture medium with or without the addition. The results are shown in Figure 2.

As shown in Figure 2, the highest cell proliferation rate was observed when Grace's insect media was used as a basic medium and yeast extract was added. From the above results, it was confirmed that adding yeast extract to the medium was more effective in cell proliferation than medium adding fetal bovine serum.

Example 3. Cell proliferation depending on the presence or absence of vitamins in the basic medium

Yeast extract is an extract obtained by extracting the water-soluble components of yeast and removing moisture. It contains amino acids, peptides, carbohydrates, organic acids, vitamins, and minerals. Similar to the components of yeast extract, the basic medium used during cell culture is composed of amino acids, sugars, organic acids, vitamins, organic acids, and minerals. Although the components are similar to those of yeast extract, there is a difference in content.

Yeast extract was added to a basic medium containing or not containing vitamins and the cells were cultured to see if there was a difference in cell proliferation depending on the presence or absence of vitamins contained in the basic medium, and whether the vitamin components of the basic medium were added to the yeast extract. I looked into whether it could be replaced with vitamins.

Specifically, a medium composition in which yeast extract was added at a concentration of 0.6% (w/v) to a base medium (Grace's insect media, serum free) excluding vitamin components, and a yeast extract was added to the base medium at a concentration of 0.6% (w/v). Cell proliferation was compared by preparing medium compositions added at a w/v) concentration. Crustacean cells were initially inoculated at ^1.5 Confirmed. The results are shown in Figure 3.

As shown in Figure 3, all crustacean cells showed no difference in cell proliferation depending on the presence or absence of vitamins. Through the above results, it was confirmed that only the vitamin components contained in the yeast extract are effective in proliferation of crustacean cells. Therefore, it was experimentally confirmed that the vitamin components of the yeast extract can replace the vitamin components of the basic culture medium (Grace's insect media). .

Example 4. Cell proliferation depending on the presence or absence of organic acids in the basic medium

Yeast extract was added to a basic medium containing or not containing organic acids and the cells were cultured to determine whether there was a difference in cell proliferation depending on the presence or absence of organic acids contained in the basic medium, and whether the organic acid component of the basic medium was added to the yeast extract. I looked into whether it could be replaced with an organic acid.

Specifically, a medium composition containing yeast extract added at a concentration of 0.6% (w/v) to a base medium (Grace's insect media, serum free) and removal of Fumaric acid, Malic acid and Succinic acid from the base medium. And we observed how crustacean cell proliferation changes in a medium composition containing yeast extract at a concentration of 0.6% (w/v). Cell proliferation was compared in the same manner as in Example 3, and the results are shown in Figure 4.

As shown in Figure 4, it was confirmed that the medium composition without the addition of fumaric acid, malic acid, and succinic acid was more effective for cell proliferation than the medium composition with the addition of fumaric acid, malic acid, and succinic acid. Fumaric acid, malic acid, and succinic acid rather promoted crustacean cell proliferation. It was confirmed that it inhibits . Through the above results, it was concluded that the organic acid component contained in the yeast extract alone was effective in cell proliferation, and that the organic acid added as a component of the basic medium actually inhibited cell proliferation.

Example 5. Cell proliferation depending on the presence or absence of vitamins and organic acids in the basic medium

Yeast extract was added to a basic medium containing or not containing vitamins and organic acids and the cells were cultured to determine whether there was a difference in cell proliferation depending on the presence or absence of vitamins and organic acids contained in the basic medium. We investigated whether it could be replaced with the vitamins and organic acids contained in yeast extract.

Specifically, a medium composition in which yeast extract was added at a concentration of 0.6% (w/v) to a base medium (Grace's insect media, serum free), vitamins and organic acids were removed from the base medium, and yeast extract was added to the base medium at a concentration of 0.6% (w/v). A medium composition added at a % (w/v) concentration was prepared and crustacean cells were cultured to confirm cell proliferation. Cell proliferation was compared in the same manner as in Example 3, and the results are shown in Figure 5.

As shown in Figure 5, cells cultured in a medium composition without adding vitamins and organic acids showed faster and higher proliferation than cells cultured in a medium composition with vitamins and organic acids added, and as in the results of Example 4, organic acids It was confirmed that cell proliferation was inhibited.

Taking the above results together, the vitamins contained in the yeast extract can replace the vitamins contained in the basic medium, and if the organic acid components contained in the basic medium are added in addition to the organic acids in the yeast extract, cell proliferation is inhibited. It was confirmed that excluding the vitamins and organic acids contained in the basic medium was effective for crustacean cell proliferation.

Claims (15)

As a basic medium, it is a basic medium that does not contain vitamins and does not contain one or more organic acids selected from the group consisting of fumaric acid, malic acid, and succinic acid; and
A medium composition for crustacean cell culture containing yeast extract.
delete delete delete According to paragraph 1,
A medium composition wherein the basic medium does not contain vitamins and organic acids.
According to paragraph 1,
A medium composition containing 0.01 to 10% (w/v) of the yeast extract.
According to paragraph 1,
The yeast is selected from the group consisting of Saccharomyces sp., Wicherhamomyces sp., Pichia sp., and Hanseniaspora sp. A medium composition of one or more types.
delete According to paragraph 1,
A medium composition wherein the crustacean cells are shrimp, crab or lobster derived cells.
According to paragraph 1,
A medium composition that does not contain serum.
According to paragraph 1,
The basic medium is DMEM (Dulbecco's Modified Eagle's Medium), MEM (Minimal Essential Medium), BME (Basal Medium Eagle), RPMI 1640, DMEM/F-10 (Dulbecco's Modified Eagle's Medium: Nutrient Mixture F-10), DMEM/F -12 (Dulbecco's Modified Eagle's Medium: Nutrient Mixture F-12), α-MEM (α-Minimal essential Medium), G-MEM (Glasgow's Minimal Essential Medium), IMDM (Isocove's Modified Dulbecco's Medium), Knockout DMEM, E8 (Essential 8 Medium), SF-DMEM (serum free-Dulbecco Modified Eagle Medium), L15 Medium, and Grace's Insect Medium.
According to paragraph 1,
A medium composition wherein the culture is a suspension culture.
As a basic medium, it is a basic medium that does not contain vitamins and does not contain one or more organic acids selected from the group consisting of fumaric acid, malic acid, and succinic acid; and
A method of cultivating crustacean cells, comprising culturing the cells in a medium composition for culturing crustacean cells containing yeast extract.
delete According to clause 13,
A culture method wherein the culture is a suspension culture.