Development of novel stem cell line derived from paralichthys olivaceus fillet and use thereof

Abstract

The present invention relates to a novel stem cell line derived from a Paralichthys olivaceus fillet and use thereof and, more specifically, to: a stem cell line (Accession No. KCLRF-BP-00545) derived from a Paralichthys olivaceus fillet; cultured meat comprising the stem cell line; and a food composition comprising the cultured meat. The stem cell line according to the present invention has pluripotency, thus enabling differentiation into various types of cells, exhibits an excellent growth rate even at low FBS concentrations, and is easily stored, and thus can be effectively used as a main raw material for the mass production of economical aquatic cell-cultured foods.

Description

Development of a novel stem cell line derived from flounder fillet and its use

[0001] This application claims priority to Republic of Korea Patent Application No. 10-2024-0076502, filed on June 12, 2024, the entire disclosure of which is incorporated herein by reference.

[0002] The present invention relates to the development of a novel stem cell line derived from the fillet of flatfish (Paralichthys olivaceus) and its use, and more particularly, to a stem cell line derived from the fillet of flatfish (Accession No. KCLRF-BP-00545), cultured meat containing the stem cell line, a food composition containing the cultured meat, and a method for producing a stem cell line derived from the fillet of flatfish.

[0003]

[0004] Research is being conducted on alternative seafood as a sustainable alternative to fish supply. These alternative seafood products can be broadly categorized into plant-based seafood using plant-based ingredients and cultured seafood products, which replicate the tissue of seafood by isolating and culturing seafood cells. Research on cultured seafood has been ongoing since attempts to expand goldfish fillets in vitro as a protein source for long-term space travel, and the number of companies conducting research in this area is increasing worldwide.

[0005] The development of aquatic cell-cultured foods requires optimization of the cell line of interest, a growth medium that provides nutrients to the cells, a food-compatible scaffold that provides a three-dimensional structure for cell growth and proliferation, and a bioreactor that provides a closed environment. In particular, aquatic cell lines, which serve as the foundation, have physiological characteristics that are favorable for in vitro culture and mass production, such as safety from diseases, high tolerance to hypoxia, high buffering capacity, and relatively low-temperature growth conditions, making them excellent food materials. Cell lines being studied as raw materials for aquatic cell-cultured foods are largely divided into embryonic stem cell lines, adult stem cell lines, and induced pluripotent stem cell lines. Embryonic stem cell lines are derived from cells inside the blastocyst formed in the early stage of development and have the advantage of being pluripotent and having consistent quality. However, obtaining blastocysts themselves is difficult, their lifespan is short, and proliferation / differentiation into mature cell forms requires a lot of time and resources. Adult stem cell lines are undifferentiated progenitor cells present in specific organs and tissues. They possess the pluripotency to differentiate into specific cell types associated with the isolated organ or tissue. They minimize artificial manipulation, thus eliminating GMO concerns. They are easier to obtain than blastocysts and can be cultured at relatively low cost. However, they have limited proliferation potential and significant inter-individual variation. Induced pluripotent stem cell lines are embryonic stem cell-like cells generated by reprogramming somatic cells. While they can maintain consistent quality, they are expensive to produce and maintain, and are not free from GMO concerns. To utilize these cell lines as raw materials for aquatic cell-cultured foods, self-renewal and differentiation capabilities are particularly important. Research on various cell types, such as muscle cells, adipocytes, and fibroblasts related to meat quality, is essential. However, research and background knowledge on fish stem cell lines are limited, and established protocols for producing complex tissues in vitro are lacking.

[0006]

[0007] Accordingly, the inventors of the present invention established novel stem cells from the fillet of flatfish (Paralichthys olivaceus), which is widely distributed in the waters of our country and is a mainstay of the Asian aquaculture industry, and confirmed that the stem cells can be used as a raw material for excellent aquatic cell cultured food products through culture, thereby completing the present invention.

[0008]

[0009] Accordingly, the purpose of the present invention is to provide a fillet-derived stem cell line (accession number KCLRF-BP-00545) of flatfish (Paralichthys olivaceus).

[0010] Another object of the present invention is to provide cultured meat containing the stem cell line.

[0011] Another object of the present invention is to provide a food composition comprising the cultured meat.

[0012] Another object of the present invention is to provide a method for producing a stem cell line derived from flounder fillet.

[0013] Another object of the present invention is to provide a primer set composition for identifying a cell type capable of differentiation of a manufactured fish stem cell line.

[0014]

[0015] To achieve the above purpose, the present invention provides a stem cell line (accession number KCLRF-BP-00545) derived from the fillet of flatfish (Paralichthys olivaceus).

[0016] In order to achieve another object of the present invention, the present invention provides cultured meat containing the stem cell line.

[0017] In order to achieve another object of the present invention, the present invention provides a food composition comprising the cultured meat.

[0018] In order to achieve another object of the present invention, the present invention provides a method for producing a stem cell line derived from flounder fillet, comprising the following steps:

[0019] a) A step of crushing the fillet tissue of flounder into a size of 5 mm or less;

[0020] b) A step of treating the crushed tissue with a collagen-decomposing enzyme and then crushing it again;

[0021] c) a step of treating the above-mentioned pulverized tissue with a protein-decomposing enzyme;

[0022] d) A step of filtering the decomposed tissue using a porous membrane having a size of 70 ㎛ and 30 ㎛;

[0023] e) a step of removing debris from the filtered cells; and

[0024] f) A step of attaching cells from which debris has been removed according to step e) above to a culture vessel.

[0025] In order to achieve another object of the present invention, the present invention provides a primer set composition for identifying a cell type capable of differentiating a manufactured fish stem cell line, which is specific for PPARγ, LPL, FAS, FAP, PDGFR, Fibronectin, SOX9, and COMP.

[0026]

[0027] The present invention is described in detail below.

[0028]

[0029] The present invention provides a stem cell line (accession number KCLRF-BP-00545) derived from the fillet of flatfish (Paralichthys olivaceus).

[0030]

[0031] In the present invention, the flounder (Paralichthys olivaceus) is a marine fish belonging to the family Paridae of the order Plaice, and is also widely known as flatfish in Korea. It is characterized by its eyes slanted to the left, a large mouth, and sharp teeth. It is distributed in the western Pacific Ocean, including Korea, Japan, and China, and mainly inhabits sandy bottoms at depths of 10 to 200 m. Its body length is generally about 40 to 60 cm, its weight is about 1 to 3 kg, and its back is yellow-brown, while its belly is white. Flounder is recognized as a high-quality fish in Japan due to its excellent taste and high catch rate. In particular, Korea has the world's highest level of technology for flounder farming, so it is widely used for food.

[0032] Meanwhile, the stem cell line according to the present invention is characterized by being derived from the fillet of the aforementioned flounder. The term "fillet" refers to fish flesh separated from the bones by cutting it lengthwise parallel to the fish's spine, with most of the scales removed and consisting primarily of skeletal muscle and fat.

[0033] The inventors established an immortalized stem cell line by continuously subculturing the cells obtained by extracting fillets from healthy flounder fry, crushing and filtering the tissue, and depositing the international patent on this line with the Korea Cell Line Research Foundation (KCLRF), an international depository, in accordance with the Budapest Treaty on May 16, 2024 (Deposit No. KCLRF-BP-00545). That is, the stem cell line according to the present invention is derived from the fillet, which is the meat of flounder widely consumed as food in Korea, and in particular, as confirmed by the inventors of the present invention, the stem cell line according to the present invention is a multipotent stem cell line that expresses all multipotent markers (see Example 3), and specifically has the characteristic of being able to differentiate into any one of muscle cells, adipocytes, fibroblasts, neural progenitor cells, and chondrocytes (see Example 4), so that aquatic cell cultured food manufactured using this as a raw material can exhibit excellent meat quality and flavor, and also has an excellent growth rate even in a low concentration of FBS (Fetal Bovine Serum) (see Example 6), and when stored frozen, it exhibits a survival rate of 80%, making it easy to store, and thus, it is also economically feasible for mass production of aquatic cell cultured food.

[0034]

[0035] Accordingly, the present invention provides cultured meat comprising a stem cell line derived from the fillet of the above-described flounder.

[0036] The term 'cultured meat' above is also called synthetic meat, clean meat, slaughter-free meat, lab-grown meat, in-vitro meat, cell-based meat, or cell-cultured meat, and refers to lean meat produced using cell engineering technology without livestock farms by culturing animal stem cells. That is, the cultured meat according to the present invention may mean lean meat obtained in the form of a lump by culturing a stem cell line derived from the fillet of the above-described flounder. In this sense, the cultured meat 'including the stem cell line' derived from the fillet of the flounder may mean cultured meat 'including the stem cell line itself', and may also mean cultured meat 'including new cells formed by the growth, division, and / or differentiation of the stem cell line'.

[0037] When producing cultured meat by culturing stem cell lines, stem cell lines do not grow thicker than a certain level. Therefore, in order to produce cultured meat as thick as regular lean meat, the stem cell lines can be cultured using a cell culture support or scaffold. Accordingly, the cultured meat according to the present invention may be cultured meat that includes a cell culture support or scaffold for effective stem cell culture.

[0038] The above cell culture support or scaffold is widely known in the art and may be a cell culture support or scaffold composed mainly of gelatin, chitosan, polyvinyl alcohol, agar, agarose, collagen, alginate, glucomannan and / or cellulose, but is not limited thereto, and any cell culture support or scaffold known in the art may be used without limitation for the purpose of applying a stem cell line as an edible substance and culturing the same to produce cultured meat.

[0039] The cultured meat according to the present invention may further include fat and / or coloring agents for flavor and aesthetic appeal, in addition to the stem cell line. The fat may be included by co-culturing adipocytes injected during the production of the cultured meat, but is not limited thereto. The above coloring agent refers to a compound that gives color to food, and in order to reproduce the color required for cultured meat, artificial coloring agent, natural coloring agent, natural extract (e.g., beet extract, pomegranate fruit extract, cherry extract, carrot extract, red cabbage extract, red seaweed extract), modified natural extract, natural juice (e.g., beet juice, pomegranate juice, cherry juice, carrot juice, red cabbage juice, red seaweed juice, dragon fruit juice), modified natural juice, FD&C (Food Drug & Cosmetics) Red No. 3 (erythrosine), FD&C Green No. 3 (fast green FCF), FD&C Red No. 40 (allura red AC), FD&C Yellow No. 5 (tartazine), FD&C Yellow No. 6 (sunset yellow FCF), FD&C Blue No. 1 (brilliant blue FCF), FD&C Blue No. 2 (indigotine), titanium dioxide, annatto, anthocyanin, betanin, beta-APE 8 carotenal, beta-carotene, black currant, burnt sugar, canthaxanthin, caramel, carmine / carminic acid, cochineal extract, curcumin, lutein, carotenoids, monascin, paprika, riboflavin, saffron, turmeric, and combinations thereof can be used, but are not particularly limited thereto. Additionally, a coloring agent such as nitrite and ascorbic acid, erythorbic acid, or a salt thereof that promotes the coloring of the nitrite can be further added as a coloring aid.

[0040]

[0041] In addition, the present invention provides a food composition comprising the cultured meat.

[0042]

[0043] In the present invention, the food composition is suitable or edible for consumption by humans and / or non-human animals, and may be added by uniformly or unevenly crushing the cultured meat into a size ranging from 1 μm to 10 cm depending on the type or purpose of the food, or may mean added in the form of a dried powder, but is not limited thereto.

[0044] In the present invention, the content of the composition is not particularly limited depending on the purpose or aspect of use, and may be, for example, 0.01 to 99 wt%, preferably 0.5 to 50 wt%, and more preferably 1 to 30 wt%, based on the total weight of the composition. In addition, the food composition according to the present invention may further include additives such as acceptable carriers, excipients, or diluents in addition to the active ingredient. The composition of the present invention may include 0.1 to 99.9 wt% of the cultured meat produced by the method of the present invention, and 99.9% to 0.1 wt% of the carrier.

[0045] In the present invention, the food may be at least one selected from the group consisting of confectionery, bread, noodles, special nutritional foods, foods for special medical purposes, sauces, seasoned foods, pickles, stews, processed meat products, packaged meat, processed seafood products, and instant foods. However, the above-described foods and foods described below are merely examples, and the present invention is not limited to these examples.

[0046] The above 'confectionery' refers to a product made by roasting, puffing, frying, etc. using grain flour as the main ingredient, or a product made by adding food or food additives thereto, and may be a snack product including cultured meat according to the present invention in particular.

[0047] The above 'bread' refers to a product made by kneading wheat flour or other grains, sugar, oil, eggs, etc. as main ingredients, with or without fermentation, or a product made by kneading cream, sugar, eggs, etc. as main ingredients, freezing the dough, and cooking the dough, and may in particular be a pizza, pie, or hot dog containing the cultured meat according to the present invention.

[0048] The above 'noodles' are made by molding, heat-treating, drying, etc. using grain flour or starch as the main ingredient, and may be raw noodles, boiled noodles, dried noodles, or fried noodles, including cultured meat according to the present invention.

[0049] The above 'special nutritional food' refers to a food manufactured and / or processed by mixing food and nutritional ingredients for specific subjects requiring special nutritional management, such as infants / toddlers, pregnant / nursing women, etc., and means formula milk, infant formula, growth formula, weaning food for infants / toddlers, weight control formula, food for pregnant / nursing women, nutritional formula for the elderly, etc., and in particular, it may be a formula or weaning food containing the cultured meat according to the present invention as an ingredient.

[0050] The above 'special medical purpose food' refers to a food manufactured and / or processed to be supplied through oral or tube feeding for the purpose of replacing part or all of a meal for a person who has a limited ability to normally consume, digest, absorb or metabolize, or who has a physiologically different nutritional requirement from the general public due to a clinical condition such as disease or surgery, and who needs sufficient nutritional supply or who needs restriction or supplementation of some nutritional components.

[0051] The above 'soy sauce' refers to a product manufactured and / or processed by culturing yeast, etc. on plant / animal raw materials or fermenting / aging / aging a mixture of salt, etc. with meju, etc. as the main raw material, and may be, in particular, a mixed soy sauce or mixed sauce including cultured meat according to the present invention.

[0052] The above-mentioned 'seasoned food' is used for the purpose of enhancing flavor in manufacturing, processing, and cooking food, and may be, in particular, a sauce or spice processed product containing cultured meat according to the present invention.

[0053] The above 'pickled or braised food' refers to a product pickled or heated by adding salt, vinegar, sugar or soy sauce to animal / vegetable raw materials, and may be kimchi, pickled food or braised food including cultured meat according to the present invention in particular.

[0054] The above 'processed meat products or packaged meat' refers to ham, sausage, bacon, dried and stored meat, seasoned meat, extracted meat products, easy-to-cook meat sets, processed meat-containing products, and packaged meat processed using meat or processed meat products as the main ingredient, and in particular, may be ham, sausage, or easy-to-cook meat sets containing cultured meat according to the present invention.

[0055] The above 'processed aquatic products' refers to products manufactured and / or processed by using aquatic products as the main ingredient and going through processes such as grinding and drying, or by adding food or food additives thereto. In particular, it may be processed fish products such as fish cakes and fish sausages containing cultured meat according to the present invention, salted seafood, and dried fish, but is not limited thereto.

[0056] The above 'instant food' refers to food that is consumed immediately or after a simple cooking process such as heating, and may be, in particular, raw food, dumplings, and instant / convenience food containing cultured meat according to the present invention.

[0057]

[0058] In addition, the present invention provides a method for producing a stem cell line derived from flounder fillet, comprising the following steps:

[0059] a) A step of crushing the fillet tissue of flounder into a size of 5 mm or less;

[0060] b) A step of treating the crushed tissue with collagen or protein-decomposing enzyme and then crushing it again;

[0061] c) A step of centrifuging the above-mentioned pulverized tissue, resuspending the pellet, and filtering it using a porous membrane of 70 μm and 30 μm in size;

[0062] d) a step of removing debris from the filtered cells; and

[0063] e) A step of attaching and culturing cells from which the above debris has been removed to a culture vessel.

[0064]

[0065] Below, the above manufacturing method is described in detail for each step.

[0066]

[0067] a) A step of crushing the fillet tissue of flounder into a size of 5 mm or less;

[0068]

[0069] Step a) of the method for producing a stem cell line according to the present invention is a step of preparing and crushing a fillet for producing a stem cell line derived from flounder fillets. The flounder is preferably a healthy flounder fry with a body length of 30 cm or less, preferably 7 to 25 cm, but is not limited thereto. The prepared flounder is euthanized, disinfected by soaking in 65% to 75% ethanol, and then the ethanol is removed. The scales and epidermis are removed using a scalpel or scalpel, and only the fillet is extracted. Thereafter, the fillet tissue is crushed into a size of 5 mm or less, preferably 4 mm or less, and more preferably 1 to 3 mm using a single cell separator or dissociator known in the art.

[0070]

[0071] b) A step of treating the crushed tissue with collagen or protein-decomposing enzymes and then crushing it again;

[0072]

[0073] Step b) of the method for producing a stem cell line according to the present invention is a step of treating the tissue pulverized in step a) with a collagenase (e.g., collagenase type I, II, III, IV, V, VI VII, VIII, or XI) and / or a proteinase (e.g., trypsin) to dissolve the tissue, and then further crushing the tissue using a single cell separator or dissociator. The treatment with the collagenase and / or the treatment with the proteinase may be performed simultaneously or sequentially as separate steps, and the process of crushing the tissue may be performed as a separate step after treating each enzyme or after treating all enzymes, and if necessary, this step may be repeated multiple times in the method for producing the present invention.

[0074]

[0075] c) A step of centrifuging the above-mentioned crushed tissue, resuspending the pellet, and filtering using a porous membrane;

[0076]

[0077] Step c) of the stem cell line production method according to the present invention is a step for isolating cells from the pulverized tissue. To this end, the pulverized tissue is first centrifuged at 200 g to 400 g, preferably 300 g, for 8 to 12 minutes, preferably 10 minutes, to obtain a pellet. The obtained pellet is then resuspended and filtered using a porous membrane to perform cell separation. The porous membrane may preferably be a cell strainer having a size of 70 μm or 30 μm, but is not limited thereto, and it is apparent that a person skilled in the art can perform cell separation appropriately using a strainer of a required size.

[0078]

[0079] d) a step of removing debris from the filtered cells;

[0080]

[0081] Step d) of the stem cell line production method according to the present invention is a step of removing debris such as cell debris, which is unnecessary foreign matter, in addition to the separated cells. This step can be performed using a debris removal solution known in the art.

[0082]

[0083] e) A step of attaching and culturing cells from which the debris has been removed to a culture vessel;

[0084]

[0085] Step e) of the stem cell line production method according to the present invention is a step of inoculating and culturing the finally isolated cells in a culture vessel coated with an extracellular matrix. The inoculated cells are then cultured while changing the medium every 1-2 days. Once a monolayer is formed, the cells are removed and transferred to a new flask for continuous subculture. As subculture continues, the isolated cell line develops a fibroblast-like cell morphology and can achieve spontaneous immortalization, thereby exhibiting the characteristics of a stem cell line.

[0086]

[0087] Meanwhile, in order to confirm whether the cell line manufactured after step e) corresponds to a stem cell, the method for manufacturing a stem cell line according to the present invention

[0088] f) a step of confirming whether the cultured cells have pluripotency; or

[0089] g) a step of identifying the cell type into which the cultured cells can differentiate;

[0090] One or more of the following steps may be performed.

[0091]

[0092] Typically, whether cultured cells are pluripotent is determined by checking whether the cells express pluripotency markers known as POU5F1 (POU domain, class 5, transcription factor 1), SOX-2 (Sex determining region Y-box 2), NANOG, and KLF4 (Kruppel-like factor 4). Furthermore, determining which cell types the cultured cells can differentiate into is determined by individually checking whether the cells express specific markers associated with muscle cells, adipocytes, fibroblasts, neural progenitor cells, and chondrocytes.For example, in one embodiment of the present invention, in the case of muscle cells, the expression of PAX7 (paired box 7), MyoD (myoblast determination protein 1), MyoHC (myosin heavy chain), and MyoG (myogenin), in the case of adipocytes, the expression of PPARγ (Peroxisome proliferator-activated receptor gamma), LPL (lipoprotein lipase), and FAS (fatty acid synthase), in the case of fibroblasts, the expression of Vimentin, FAP (fibroblast activation protein alpha), PDGFR alpha (platelet derived growth factor receptor alpha), and Fibronectin, in the case of neural progenitor cells, the expression of Nestin, Aldh1l1 (aldehyde dehydrogenase 1 family member L1), MAG (myelin-associated glycoprotein) of oligodendrocytes, and Slc6a4 (solute carrier family 6 member 4 marker) of serotonergic neurons, and in the case of chondrocytes, the expression of SOX9 (sex-determining region Ybox 9) and Expression of COMP (cartilage oligomeric matrix protein) was confirmed.

[0093] In one embodiment of the present invention, RNA is obtained from cultured cells, cDNA is prepared therefrom, and then an amplification product is generated by PCR using a primer specific to the gene as a template to determine whether the cell has pluripotency and / or the cell type into which the cell can differentiate is determined. However, the present invention is not limited thereto, and a person skilled in the art can easily determine whether the cell has pluripotency and / or the cell type into which the cell can differentiate by determining whether the gene / protein is expressed using a method for determining whether the gene or protein is expressed that is well known in the art.

[0094] Meanwhile, in order to confirm the cell type into which cells can differentiate, when the following primer sets are used to confirm the expression of genes / proteins, as used in one embodiment of the present invention, the expression of the corresponding genes / proteins can be easily identified: i) a set of forward primers having a base sequence according to SEQ ID NO: 19 and reverse primers having a base sequence according to SEQ ID NO: 20, for confirming the expression of PPARγ (Peroxisome proliferator-activated receptor gamma); ii) a set of forward primers having a base sequence according to SEQ ID NO: 21 and reverse primers having a base sequence according to SEQ ID NO: 22, for confirming the expression of LPL (lipoprotein lipase); iii) a set of forward primers having a base sequence according to SEQ ID NO: 23 and reverse primers having a base sequence according to SEQ ID NO: 24, for confirming the expression of FAS (fatty acid synthase); iv) A set of forward primers having a base sequence according to SEQ ID NO: 27 and a set of reverse primers having a base sequence according to SEQ ID NO: 28 for confirming FAP (fibroblast activation protein alpha) expression; v) A set of forward primers having a base sequence according to SEQ ID NO: 29 and a set of reverse primers having a base sequence according to SEQ ID NO: 30 for confirming PDGFR alpha (platelet derived growth factor receptor alpha) expression; vi) A set of forward primers having a base sequence according to SEQ ID NO: 31 and a set of reverse primers having a base sequence according to SEQ ID NO: 32 for confirming Fibronectin expression;vii) a set of forward primers having a base sequence according to SEQ ID NO: 41 and a set of reverse primers having a base sequence according to SEQ ID NO: 42 for confirming the expression of SOX9 (sex-determining region Ybox 9); and viii) a set of forward primers having a base sequence according to SEQ ID NO: 43 and a set of reverse primers having a base sequence according to SEQ ID NO: 44 for confirming the expression of COMP (cartilage oligomeric matrix protein).

[0095]

[0096] The stem cell line according to the present invention is derived from the fillet of flounder, has multipotent differentiation ability, can differentiate into various types of cells, exhibits excellent growth rate even in low concentrations of FBS, and is easy to store, so it can be usefully used as a main raw material for mass production of economical aquatic cell cultured foods.

[0097]

[0098] Figure 1 shows microscopic photographs of cells isolated from flounder fillets. (A) is a microscopic photograph of a primary cell (passage 0 generation), (B) is a microscopic photograph of a cell from passage 1, (C) is a microscopic photograph of a cell from passage 2, and (D) is a microscopic photograph of a cell from passage 50.

[0099] Figure 2 shows a phylogenetic tree of the stem cell line (PCS-PO) according to the present invention.

[0100] Figure 3 shows the results of confirming the expression of markers related to the pluripotency of a stem cell line (PCS-PO) according to the present invention.

[0101] Figures 4a and 4b show the results of confirming the expression of markers according to each cell type in order to confirm the cell type into which the stem cell line (PCS-PO) according to the present invention can differentiate.

[0102] Figure 5 shows the results of confirming the cell proliferation activity of the stem cell line (PCS-PO) according to the present invention.

[0103] Figure 6 shows the proliferation activity of the stem cell line (PCS-PO) according to the present invention according to the FBS concentration (2.5%, 5%, 10%, 20%).

[0104] Figure 7 shows the results of manufacturing a stem cell line (PCS-PO) according to the present invention in the form of freeze-dried powder.

[0105] Figures 8a and 8b show the results of encapsulating stem cell lines (PCS-PO) using microcarriers (alginate) according to the present invention (Figure 8a) and the results of confirming the survival of PCS-PO cells through fluorescent staining (Figure 8b).

[0106]

[0107] The present invention is described in detail below. However, the following examples are merely illustrative of the present invention, and the scope of the present invention is not limited to the following examples.

[0108]

[0109] Example 1. Establishment of a flounder fillet cell line

[0110] Healthy flounder fry (7–25 cm) were purchased from a fishery seed company in South Chungcheong Province. The purchased flounder were euthanized with Tricaine methanesulfonate (Sigma, USA) and disinfected in 70% ethanol after they became motionless. The ethanol was removed, scales and cuticles were removed using a scalpel, and fillets were excised. The tissues were disrupted using sterile scissors and a gentleMACS Dissociator (Miltenyi, Germany). The fish were washed in wash media (DMEM (welgene, Korea) supplemented with 9 mM NaHCO3 and 20 mM HEPES), 1% Antibiotic-Antimycotic (Gibco, USA), and 0.15% Gentamycin (Sigma, USA)). To dissolve the tissue, 0.2% collagenase solution (Worthington, USA) with 1% Antibiotic-Antimycotic was added and reacted at 23℃ for 60 minutes. The tissue was further disrupted using a gentleMACS Dissociator, and 0.4% Trypsin (Sigma, USA) was added and secondary dissolution was performed at 23℃ for 20 minutes. After neutralization using DMEM (adjusted to 9 mM NaHCO3 and added 20 mM HEPES) supplemented with 15% Horse serum (Sigma, USA), 1% Antibiotic-Antimycotic, and 0.15% Gentamycin, centrifugation was performed, and the pellet was resuspended and filtered using a 70 μm and 30 μm strainer.The filtered pellet was removed from debris using Debris Removal Solution (Miltenyi, Germany), and finally suspended in DMEM medium (adjusted with 9 mM NaHCO3 and 20 mM HEPES) supplemented with 20% FBS and 1% Antibiotic-Antimycotic. The cells were inoculated onto TC-Treated Multiple 6-well plates (Corning, USA) coated with γ-irradiated poly-L-lysine (Sigma, USA) and Laminin (Sigma, USA). The inoculated flounder cells were cultured at 23°C, and the medium was changed once every 1–2 days. Primary cultured flounder fillet cells were mainly composed of muscle-like cells, and it was observed that they grew together with fibroblast-like cells. After forming a monolayer, the cells were detached using 0.2% trypsin and a scraper, and continuously transferred to new flasks for subculture. Currently, the flounder fillet cells morphologically show the form of fibroblast-like cells and are believed to have been naturally immortalized through subculture for more than 50 generations over one year (see Fig. 1). The isolated flounder fillet cells were named Pulmuone's cultured seafood cell - Paralichthys olivaceus (PCS-PO) cells and were internationally patented and deposited with the Korea Cell Line Research Foundation (KCLRF), an international depository institution, in accordance with the Budapest Treaty on May 16, 2024 (Deposit No. KCLRF-BP-00545).

[0111]

[0112] Example 2. COI gene analysis of PCS-PO cell line

[0113] To identify the species of origin of PCS-PO cells, cytochrome oxidase subunit 1 (COI) gene sequence analysis was performed. DNA was extracted from PCS-PO cells using MagPurix 12s Automated Nucleic Acid Purification System (Zinexts Life Science Corp, Taiwan), and the VF2(I0) / FISH R2 primer set (Genetic Analysis Method for Food Raw Material Authenticity Determination, National Institute of Food and Drug Safety Evaluation, see Table 1) and TaKaRa Taq TM PCR was performed using Hot Start Version (TAKARA, USA). PCR products were confirmed using a QIAxcel (Qiagen, Germany) automated electrophoresis device, and the PCR products were sequenced. The determined base sequences were compared for homology using NCBI's BLAST analysis program. In addition, a phylogenetic tree was constructed using the Mega11 program's neighbor-joining method, and bootstrap analysis was performed with 1,000 iterations.

[0114] Name Base Sequence Sequence Number VF2(I) 5'- TGT AAA ACG ACG GCC AGT CAA CCA ACC ACA AAG ACA TTG GCA C -3'1 FISH R 2 5'- CAG GAA ACA GCT ATG ACA CTT CAG GGT GAC CGA ATC AGA A -3'2

[0115] As a result, the COI gene sequence of PCS-PO cells showed 100% genetic homology with the COI sequence of flounder, and it was confirmed that it was distinguished from closely related species in the phylogenetic tree (see Figure 2).

[0116]

[0117] Example 3. Verification of multipotency of PCS-PO cells

[0118] To verify the pluripotency of PCS-PO cells (passage 38), RNA was isolated using an RNA extraction kit (Qiagen, Germany) and then synthesized into cDNA using a RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher, USA). PCR was then performed using primers (see Table 2) for POU5F1 (POU domain, class 5, transcription factor 1), SOX-2 (Sex determining region Y-box 2), NANOG, and KLF4 (Kruppel-like factor 4), which are pluripotency markers involved in cell self-renewal, development, and differentiation regulation. PCR reaction conditions included 1 cycle (initial denaturation) at 95°C for 5 minutes, followed by 30 cycles of denaturation at 95°C for 30 seconds, binding at 55°C for 30 seconds (except for SOX2 and KLF4 at 60°C), and extension at 72°C for 30 seconds, followed by a final extension at 72°C for 7 minutes. PCR products were confirmed using QIAxcel.

[0119] Name Base sequence Sequence number POU5f1-F5'- CAG ACC CGA GCC TCC TCT AT -3'3POU5f1-R5'- GAC CCT TGA TCT CCG TTG AG -3'4SOX-2-F5'- GCA CAA CTC GGA GAT AAG C -3'5SOX-2-R5'- TGG TCT GCT GGG AAT AGG -3'6NANOG-F5'- AGT CAC CGA CCT GAG CAA CT -3'7NANOG-R5'- GTG GCT GAC ATA CCT GGT GA -3'8KLF4-F5'- GCA ACT GCA CCA TCT CAC AG -3'9KLF4-R5'- GGA ACT GCA TGG AGG ATG AC -3'10

[0120] As a result, POU5F1 was identified at 305 bp, SOX-2 at 503 bp, NANOG at 300 bp, and KLF4 at 300 bp (see Table 3 and Figure 3), indicating that PCS-PO cells have multipotency, that is, the ability to differentiate into various cells, and thus have high utility as a raw material for aquatic cell culture food.

[0121] TargetPOU5F1SOX-2NANOGKLF4PCS-PO (Passage 38)DetectionDetectionDetectionDetection

[0122] Example 4. Analysis of differentiation types of PCS-PO cells

[0123] Since PCS-PO are multipotent stem cells capable of differentiating into specific cell types, we examined the types of cells into which PCS-PO can differentiate. To verify cell types, we utilized specific markers related to muscle cells, adipocytes, fibroblasts, neural progenitor cells, and chondrocytes.

[0124] In the case of muscle cells, PAX7 (paired box 7), MyoD (myoblast determination protein 1), MyoHC (myosin heavy chain), and MyoG (myogenin), which are expressed during the muscle proliferation / differentiation stage, were confirmed. In the case of adipocytes, the expression of the adipogenic transcription factor PPARγ (Peroxisome proliferator-activated receptor gamma) and the adipogenic effectors LPL (lipoprotein lipase) and FAS (fatty acid synthase) was confirmed. In the case of fibroblasts, the expression of the intracellular protein Vimentin, the surface proteins FAP (fibroblast activation protein alpha) and PDGFR alpha (platelet derived growth factor receptor alpha), and the secreted protein Fibronectin was confirmed. For neural progenitor cells, Nestin of neural progenitor cells, Aldh1l1 (aldehyde dehydrogenase 1 family member L1) of astrocytes, MAG (myelin-associated glycoprotein) of oligodendrocytes, and Slc6a4 (solute carrier family 6 member 4 marker) of serotonergic neurons were selected according to the cell type into which they are differentiated. For chondrocytes, the expression of SOX9 (sex-determining region Ybox 9), a chondrogenic transcription factor important for differentiation and regeneration, and COMP (cartilage oligomeric matrix protein), an extracellular matrix synthesized by chondrocytes, was confirmed.

[0125] To this end, RNA was isolated from PCS-PO cells using an RNA extraction kit, then synthesized into cDNA using a RevertAid First Strand cDNA Synthesis Kit, and the PCR product was amplified using QIAxcel (see Table 4 primers).

[0126] 구분서열의 명칭염기서열Annealing Temp(℃)서열번호근육세포Pax7b-F5'- AGC TAG CGG CAT TCA ACC AT -3'60(℃)11Pax7b-R5'- GTG TTG TGG CTG TGA GGA GA -3'12MyoD-F5'- CTA ACC AGA GGC TGC CHA AG -3'60(℃)13MyoD-R5'- CAT GCC ATC WGA GCA GTT GG -3'14MyoHC-F5'- GGC TGG TGG AAA AGA ACA AGG ATC CAC TG -3'58(℃)15MyoHC-R5'- TGC AGA CAC CGT CTG GAA G -3'16MyoG-F5'- GAG TCT GTC TGG GGG TGT TG -3'60(℃)17MyoG-R5'- ACT GCA GAG ATG CTG TCC AC -3'18지방세포PPARγ-F5'- CTC TTC ACT GAG CAG AGG AAC C -3'59.5(℃)19PPARγ-R5'- GTC GTC CAT CTT TAC CTA CGG TC-3'20LPL-F5'- GCA CAA GCT GAA AAT GCA GG -3'58.5(℃)21LPL-R5'- CAT CAT GAA CAG GAG AGC TTG TG -3'22FAS-F5'- CTG CAC TTT GCC GCT ACT ACC -3'59.5(℃)23FAS-R5'- GCA GAC CTT CTT TCA TCC ACA AC -3'24섬유아세포Vimentin-F5'- CCT GCC GAA CTT CTC ATC TC -3'60(℃)25Vimentin-R5'- TCC TGG AGA GTG CAT GCT AA -3'26FAP-F5'- GGT TCG CTT TCT CCA TTT AGG TG -3'59(℃)27FAP-R5'- GTC GCT CAT CAC TTT GTA GAA ACT G -3'28PDGFR-F5'- CCG ACT TTG TGT TTT CAG GC -3'56.5(℃)29PDGFR-R5'- GGA CTT TGG GTA ATG AGT GC -3'30Fibronectin-F5'- GTT TTC CAT GTG TGT GTC CTC C -3'59(℃)31Fibronectin-R5'- GCA TCA GCA GCT AAA CAA ATG C -3'32신경전구세포Nestin-F5'- GCA CCC AAC TTT ACA CCA CAC C -3'60(℃)33Nestin-R5'- TCA TCC TGT TTT ATG CCA TCG TC -3'34Aldh1l1-F5'- GGC AGG GCA TTC TGA GCA A -3'60(℃)35Aldh1l1-R5'- GCC TCT TTA GCA GCA GTC ACG -3'36MAG-F5'- GCG ACG AGA ATC TTG AGT TGA CAT -3'60(℃)37MAG-R5'- ACG GGG TTA CTC TCT ACC TCG C -3'38Slc6a4-F5'- CTG TTG ATG GCG GTG TTC G -3'60(℃)39Slc6a4-R5'- TGT AGT AGA AGG CTA TGT AGA GGG CT -3'40연골세포SOX9-F5'- CCG TTT GTG GAG GAA GCA GAG -3'59.5(℃)41SOX9-R5'- TCA TCG ACG TCA AAG CTC CC -3'42COMP-F5'- CAG GTC AGC GTC TCT TGT TTA TG -3'57.5(℃)43COMP-R5'- CAT CAG AGT CCT CCT GCA GAG -3'44.

[0127] As a result, as can be confirmed in Figures 4a and 4b, PAX7 expressed in muscle cells was confirmed at 365 bp, MyoD at 178 bp, MyoHC at 177 bp, and MyoG at 570 bp, and PPARγ, a marker related to fat cells, was confirmed at 542 bp, LPL at 225 bp, and FAS at 453 bp. Vimentin, expressed in fibroblasts, was identified at 470 bp, Fibronectin at 311 bp, PDGFR at 119 bp, and FAP at 283 bp. In the case of markers related to neural progenitor cells, Nestin was identified at 443 bp, Aldh1l1 at 421 bp, MAG at 352 bp, and Slc6a4 at 166 bp. SOX9, related to chondrocytes, was identified at 419 bp, and COMP at 572 bp.

[0128] That is, PCS-PO cells were confirmed to be capable of differentiating into muscle cells, adipocytes, fibroblasts, neural progenitor cells, and chondrocytes. Muscle cells are the main component of fish fillets, while other cells play an important role in flavor and texture.

[0129]

[0130] Example 5. Verification of proliferation activity of PCS-PO cells

[0131] To verify the proliferation activity of PCS-PO cells, 5 x 10 PCS-PO (Passage 39) cells were cultured 4Cells were seeded into TC-Treated Multiple 6-Well Plates at a concentration of 100 cells / ml. Cells were cultured at 23°C for 7 days, and then detached using trypsin and a scraper every other day, followed by centrifugation at 300g at 4°C for 10 minutes. The supernatant was removed, and 1 ml of DMEM (adjusted to 9 mM NaHCO3 and supplemented with 20 mM HEPES) supplemented with 20% FBS and 1% Antibiotic-Antimycotic was inoculated, resuspended, and the cell number was confirmed using a LunaII automated cell counter (Logos Biosystems, Korea). The medium was replaced every two days, and the doubling time was calculated by the following formula.

[0132] Doubling Time = Duration(h) x Log(2) χ [Log(final conc.) - Log(initial conc.)]

[0133] As a result, the number of cells increased over time, increasing approximately 7-fold compared to the initial cell concentration after 7 days of culture, and the doubling time was confirmed to be 60.57 hours (see Figure 5). This shows a higher proliferation rate compared to the flounder embryonic stem cell line disclosed in a previous paper, which increased approximately 6-fold compared to the initial cell concentration after 7 days of culture.

[0134]

[0135] Example 6. Proliferation activity in low concentration FBS

[0136] To verify proliferation activity in low concentration FBS, PCS-PO (Passage 39) cells were seeded at 5 x 10 4Cells were seeded into TC-Treated Multiple 6-Well Plates at a concentration of cells / ml, and then cultured at 23°C for 7 days with adjusted FBS concentrations of 2.5, 5, 10, and 20%. The cells were removed using trypsin and a scraper at daily intervals, centrifuged at 300g at 4°C for 10 minutes, and the supernatant was removed. 1 ml of DMEM (adjusted to 9 mM NaHCO3 and added 20 mM HEPES) supplemented with 2.5, 5, 10, and 20% FBS and 1% Antibiotic-Antimycotic was inoculated and resuspended, and the cell number was confirmed using a LunaII automated cell counter.

[0137] As a result, the growth rate was higher as the concentration of FBS increased, and in particular, even at FBS concentrations of 5 and 10%, cell proliferation activity increased by about 5 times or more compared to the existing cell concentration, confirming that PCS-PO cells are capable of stable growth even at low concentrations of FBS (see Fig. 6). This means that aquatic culture products can be manufactured more economically when PCS-PO cells are used.

[0138]

[0139] Example 7. Storage characteristics of PCS-PO cells

[0140] PCS-PO cells were cultured to form a monolayer in a tissue culture-treated T-25 flask (Cornin, USA) for long-term storage. The cells were detached using trypsin and a scraper, and then trypsin was removed. 5 x 10 cells were then cultured in DMEM (adjusted to 9 mM NaHCO3 and 20 mM HEPES) supplemented with 1% antibiotic-antimycotic. 5 ~ 1 x 10 6After adjusting the concentration to cells / ml, the medium was removed and 1 ml of Cellbanker 1 (Zenoaq, Japan) was inoculated and dispensed into Nunc Cryo Tube Vials (SPL, Korea). The cells were left to stand at -70°C for 1 day and then stored in liquid nitrogen. To confirm the revival of PCS-PO cells, PCS-PO cells stored in liquid nitrogen for more than 100 days were thawed as quickly as possible at 23°C and suspended in DMEM (adjusted with 9 mM NaHCO3 and 20 mM HEPES) supplemented with 10% FBS and 1% Antibiotic-Antimycotic. The cells were centrifuged at 300g at 4°C for 10 minutes to remove the preservative. Afterwards, 1 ml of 10% FBS and 1% Antibiotic-Antimycotic-added DMEM (adjusted to 9 mM NaHCO3 and added 20 mM HEPES) was added to suspend the cells, stained using Trypan Blue Solution (Gibco, USA), and then cell viability and cell count were checked using a LunaII automated cell counter, and then re-cultured in a tissue culture-treated T-25 flask.

[0141] As a result, PCS-PO cells stored in liquid nitrogen formed a monolayer within 7 days in DMEM (adjusted with 9 mM NaHCO3 and 20 mM HEPES) medium supplemented with 10% FBS and 1% Antibiotic-Antimycotic, and showed an average survival rate of 80%.

[0142]

[0143] Example 8. Safety verification of PCS-PO cells

[0144] To verify the safety of PCS-PO cells, aerobic bacteria, Escherichia coli / coliforms, Mycoplasma, and heavy metals were tested. Aerobic bacteria and E. coli / coliforms were identified after 48 hours of incubation at 37°C using 3M Petrifilm aerobic count (3M, USA) and 3M Petrifilm E. coli / coliform (3M, USA). For Mycoplasma, all mycoplasma species, a major source of contamination in cell culture, were identified using the BioMycoX Mycoplasma qPCR Detection Kit (CellSafe, Korea). For heavy metals, lead (Pb), arsenic (As), and cadmium (Cd) were measured by inductively coupled plasma mass spectrometry (ICP-MS) using iCA RQ ICP-MS (Thermo Fisher, USA), and mercury (Hg) was measured by quantitative method (gold amalgam method) by atomic absorption spectrometry using DMA-80 (Milestone, Italy) (Food Code, Ministry of Food and Drug Safety).

[0145] Safety verification results confirmed that aerobic bacteria, Escherichia coli / coliform bacteria, and Mycoplasma were not detected. Heavy metal levels were confirmed to be lower in PCS-PO cells compared to the original fish, and compliance with legal heavy metal standards for aquatic products was confirmed (see Table 5).

[0146] SampleAerobic bacteriaEscherichia coli / coliform groupMycoplasmaLead(mg / Kg)Cadmium(mg / Kg)Total mercury(mg / Kg)Arsenic(mg / Kg)PCS-PONot detectedNot detectedNot detected0.002NDND0.001Flound flounder---0.002ND0.045±0.0010.327±0.009

[0147] * Legal standards for microbiological data for aquatic products: - Bacterial count: Animal-derived frozen aquatic products that have been sanitized and placed in containers / packages for distribution and consumption by the final consumer: n=5, c=2, m=100,000, M=500,000

[0148] - Escherichia coli: Animal-derived frozen seafood products sanitized and placed in containers / packages for distribution and consumption by end consumers: n=5, c=2, m=0, M=10

[0149] * Legal standards for heavy metals in fish (fish meat):

[0150] - Lead: 0.5 mg / kg or less, Cadmium: 0.2 mg / kg or less, Total Mercury: 0.5 mg / kg or less

[0151]

[0152] Example 9. Utilization of PCS-PO cells

[0153] Methods for utilizing PCS-PO cells as raw materials for aquatic cell culture foods include powder production through freeze-drying and capsule formation using microcarriers (Alginate).

[0154] First, PCS-PO cells were cultured for production in powder form, then the medium was removed, washed with phosphate-buffered saline (PBS), and the cells were detached using trypsin and a scraper, after which the trypsin was removed. After washing with PBS, the cells were centrifuged to remove the supernatant, and the cells were recovered with distilled water, pre-frozen at -70°C for more than 24 hours, and then lyophilized for 48 hours (see Figure 7).

[0155] Next, for capsule formation using microcarriers, PCS-PO cells were seeded and mixed in a 1% alginate solution, encapsulated into beads through a reaction with calcium ions, and cultured for 1 week. Afterwards, fluorescent staining was performed using a Live / Dead Cell Double Staining Kit (Sigma, USA) and Hoechst 33342 (Thermo Fisher, USA), and it was confirmed that PCS-PO cells were viable (see Figs. 8a and 8b).

[0156] In addition, it is not particularly limited to the above-described utilization methods and can be utilized in various forms considering the desired texture, etc., so it is easy to apply to aquatic cell culture foods.

[0157]

[0158] The stem cell line according to the present invention is derived from the fillet of flounder, has multipotent differentiation ability, can differentiate into various types of cells, shows excellent growth rate even in low concentrations of FBS, and is easy to store, so it can be usefully used as a main raw material for mass production of economical aquatic cell cultured foods, and thus has high potential for industrial use.

[0159]

[0160] [Accession number]

[0161] Name of depositor: Korea Cell Line Research Foundation (KCLRF)

[0162] Accession number: KCLRFBP00545

[0163] Date of acceptance: 20240516

[0164] [Correction pursuant to Rule 91, November 14, 2024]

Claims

1. Stem cell line derived from fillet of flounder (Paralichthys olivaceus) (Accession number KCLRF-BP-00545).

2. A stem cell line according to claim 1, characterized in that the stem cell line has multipotentiality.

3. In the first paragraph, the stem cell line is characterized in that it is capable of differentiating into at least one selected from the group consisting of muscle cells, fat cells, fibroblasts, neural progenitor cells, and chondrocytes.

4. Cultured meat containing the stem cell line of clauses 1 to 3.

5. In the fourth paragraph, the cultured meat is characterized in that it is produced using a stem cell line cultured using a cell culture support or scaffold.

6. Cultured meat according to claim 5, characterized in that the cell culture support or scaffold is composed of gelatin, chitosan, polyvinyl alcohol, agar, agarose, collagen, alginate, glucomannan and / or cellulose.

7. A food composition comprising the cultured meat of paragraph 4.

8. A food composition according to paragraph 5, characterized in that the food is at least one selected from the group consisting of confectionery, bread, noodles, special nutritional foods, foods for special medical purposes, soy sauce, seasoned foods, pickles, stews, processed meat products, packaged meat, processed aquatic products, and instant foods.

9. Method for producing a stem cell line derived from flounder fillet, comprising the following steps: a) A step of crushing the fillet tissue of flounder into a size of 5 mm or less; b) A step of treating the crushed tissue with collagen or protein-decomposing enzyme and then crushing it again; c) A step of centrifuging the above-mentioned crushed tissue, resuspending the pellet, and filtering using a porous membrane; d) a step of removing debris from the filtered cells; and e) A step of attaching and culturing cells from which the above debris has been removed to a culture vessel.

10. A method for producing a stem cell line derived from flounder fillet, characterized in that the flounder of step a) in paragraph 9 has a body length of 7 to 25 cm.

11. A method for producing a stem cell line derived from flounder fillet, characterized in that in the 9th paragraph, the centrifugation in step c) is performed at 200 g to 400 g.

12. A method for producing a stem cell line derived from flounder fillet, characterized in that in the 9th paragraph, the centrifugation in step c) is performed for 8 to 12 minutes.

13. In paragraph 7, after step e), f) a step of confirming whether the cultured cells have pluripotency; or g) a step of identifying the cell type into which the cultured cells can differentiate; A method for producing a stem cell line derived from flounder fillet, characterized in that it further comprises one or more of the steps.

14. A primer set composition for identifying a cell type capable of differentiating a manufactured fish stem cell line, characterized in that it comprises at least one primer set selected from a group consisting of primer sets listed below: i) A set of forward primers having a base sequence according to SEQ ID NO: 19 and reverse primers having a base sequence according to SEQ ID NO: 20 for confirming expression of PPARγ (Peroxisome proliferator-activated receptor gamma); ii) A set of forward primers having a base sequence according to SEQ ID NO: 21 and reverse primers having a base sequence according to SEQ ID NO: 22 for confirming LPL (lipoprotein lipase) expression; iii) A set of forward primers having a base sequence according to SEQ ID NO: 23 and reverse primers having a base sequence according to SEQ ID NO: 24 for confirming FAS (fatty acid synthase) expression; iv) A set of forward primers having a base sequence according to SEQ ID NO: 27 and reverse primers having a base sequence according to SEQ ID NO: 28 for confirming the expression of FAP (fibroblast activation protein alpha); v) A set of forward primers having a base sequence according to SEQ ID NO: 29 and reverse primers having a base sequence according to SEQ ID NO: 30 for confirming expression of PDGFR alpha (platelet derived growth factor receptor alpha); vi) A set of forward primers having a base sequence according to SEQ ID NO: 31 and reverse primers having a base sequence according to SEQ ID NO: 32 for confirming fibronectin expression; vii) a set of forward primers having a base sequence according to SEQ ID NO: 41 and reverse primers having a base sequence according to SEQ ID NO: 42 for confirming the expression of SOX9 (sex-determining region Ybox 9); and viii) A set of forward primers having a base sequence according to SEQ ID NO: 43 and reverse primers having a base sequence according to SEQ ID NO: 44 for confirming the expression of COMP (cartilage oligomeric matrix protein).

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