WO2016195391A1

WO2016195391A1 - Method for isolating and culturing living cells from ancient extinct biological corpses or fossils

Info

Publication number: WO2016195391A1
Application number: PCT/KR2016/005850
Authority: WO
Inventors: 김은영; 홍기성; 정형민; 박세필
Original assignee: 주식회사 미래셀바이오; 건국대학교 글로컬산학협력단; 제주대학교 산학협력단
Priority date: 2015-06-02
Filing date: 2016-06-02
Publication date: 2016-12-08

Abstract

The present invention relates to a method for culturing cells from tissues of biological corpses, the method comprising the steps of: washing and disinfecting tissues of biological corpses; performing enzymatic treatment on the tissues; diluting the tissues with a first cell culture liquid and inoculating the diluted cell culture liquid on a plate; culturing cells from the tissues; and sub-culturing the cultured cells.

Description

How to isolate and culture living cells from ancient extinct carcasses or fossils

Disclosed are methods for isolating and culturing living cells from ancient extinct carcasses or fossils.

De-extinction refers to bringing back extinct creatures. The goal of extinction restoration is to conserve biodiversity and to identify relationships among different species, which has attracted much attention as the technology of biological cloning evolves. The Tasmanian Tiger, one of the cloning projects for extinct animals, has a well-preserved specimen stored in the museum as a relatively recent extinction, making it a priority for extinct animal restoration. Other species of American pigeons, Dodo birds, Cuban red macaws, and giant mosses from New Zealand are among the extinct species that are believed to be replicable.

Mammoths, which were said to have become extinct more than 3700 years ago, are also considered to be extinct. Mammoths have long noses and four-meter-long molars, which resemble elephants, but are extinct, characterized by their fur coverings. With the discovery of mammoth carcasses in Siberia, many researchers have predicted and analyzed that mammoth restoration is possible if DNA can be extracted from the mammoth carcasses. The process of restoring mammoths is a four-step process: first, extracting live somatic cells from frozen mammoth carcasses, extracting an egg from an elephant biologically similar to a mammoth, removing the nucleus from the egg, and removing the nucleus. The mammoth's somatic cell nucleus is implanted into the elephant uterus to give birth to the mammoth. In practice, however, there have been no successful cases of even separating and culturing intact cells from frozen mammoths.

Provided are methods for isolating and cultivating living cells from ancient extinct carcasses or fossils.

The problem to be solved is not limited to the above-mentioned problem, another task that is not mentioned will be clearly understood by those skilled in the art from the following description.

According to one embodiment, the steps of washing and disinfecting tissues of a biological carcass, enzymatically treating the tissues, diluting the tissues with primary cell culture and inoculating them on a plate, culturing cells from the tissues, And it provides a method for culturing cells from the tissue of the living body, comprising the step of subcultured the cultured cells.

According to one side, the step of washing and disinfecting the tissue of the biological carcass, the step of hydrating and washing the tissue with physiological saline, the step of disinfecting the washed tissue with alcohol and physiologically sterilized the tissue It may be to include the step of washing with saline.

According to one side, the step of enzymatic treatment of the tissue is carried out for 2 to 24 hours, the enzyme is collagenase type I, collagenase type II, collagenase type IV, Trypsin, EDTA, Dispase, And Accutase may be at least one selected from the group consisting of.

According to one side, after the step of enzyme treatment of the tissue, may further comprise the step of classifying the tissue by size.

According to one side, the plate may be coated with a coating material comprising at least one selected from the group consisting of Matrigel, collagen, gelatin, fibronectin, and laminin.

According to one side, the primary cell culture medium, cytokine-containing EGM2-MV, cytokine-free EBM-2, Ham F-10, F12, RPMI, DPBS, DMEM and MEM

It may be to include at least one selected from the group consisting of.

According to one side, the primary cell culture, may be one containing a serum of 15 to 30% concentration.

According to one side, the primary cell culture comprises an additive, the additive, EAA (essential amino acids), NEAA (non-essential amino acids), ITS mixture (insulin, transferrin, sodium selenite), PS (penicillin / streptomycin), and

It may be at least one selected from the group consisting of mercaptoethanol.

According to one side, the step of culturing the cells, adding a growth factor of 5 to 20ng / ml on the plate, and performing a half-media replacement every 2 to 3 days; may include.

According to one side, the growth factor, bFGF (Basic Fibroblast Growth Factor), EGF (Epidermal Growth Factor), IGF (Insulin like Growth Factor), VEGF (vascular endothelial growth factor), angiogenic growth factors (angiogenic growth factors), It may be at least one selected from the group consisting of ascorbic acid and HGF (Hepatocyte growth factor).

According to one side, after the passage of the cultured cells, it may further comprise the step of cryopreserving the passaged cells in a cryopreservation solution.

According to one side, the cryopreservation solution, at least one selected from the group consisting of serum, plasma, SR (Serum replacement), DMSO, glycerin (Glycerin), propanediol, and ethylene glycol (Ethylene glycol) It may be to include.

According to one embodiment, the method comprises culturing cells by culturing cells from the tissues of the living body, performing somatic cell nuclear transfer using the cultured cells, and producing cloned embryos from the somatic cells. Provides a method of producing cloned embryos.

According to one embodiment, it provides a method for producing a stem cell comprising culturing the cell by a method of culturing cells from the tissue of the living body, and producing stem cells using the cultured cells.

According to one embodiment, there is provided a method of producing a cloned animal comprising culturing the cell by a method of culturing a cell from the tissue of the living body, and producing a cloned animal using the cultured cell.

According to one embodiment, the cultured cells are provided by the method of culturing cells from the tissues of the organism.

The cell culture method of the present invention can be used to culture cells from dead bodies or fossils of extinct organisms. It is possible to produce cloned embryos and stem cells from cells cultured by the method of the present invention and ultimately restore extinct animals.

1 is a flow chart of a method for isolating and culturing live cells from ancient extinct organisms or fossils, according to one embodiment.

2 is a flow chart of a method for isolating and culturing live cells from ancient extinct organisms or fossils, according to one embodiment.

3 is a photograph of intracellular chromosomes according to one embodiment.

4 is an enlarged photograph showing that there are viable cells at specific sites in tissues by staining tissues according to an embodiment.

5 is a graph showing carbon dating results of tissues according to an embodiment.

6 is a nucleotide sequence of a mammoth mitochondrial nucleotide sequence and a primer registered in NCBI.

7 shows the pGEM-T vector.

8 shows the results of sequence analysis after cloning into a pGEM-T vector.

9 shows a part of the base sequence of pGEM-Cox1-1-T7.

10 and 11 show the results of BLAST analysis provided by NCBI.

Hereinafter, exemplary embodiments will be described in detail with reference to the accompanying drawings. Like reference numerals in the drawings denote like elements.

Various modifications may be made to the embodiments described below. The examples described below are not intended to be limited to the embodiments and should be understood to include all modifications, equivalents, and substitutes for them.

The terminology used herein is for the purpose of describing particular example embodiments only and is not intended to be limiting of examples. Singular expressions include plural expressions unless the context clearly indicates otherwise. In this specification, terms such as "comprise" or "have" are intended to indicate that there is a feature, number, step, action, component, part, or combination thereof described on the specification, and one or more other features. It is to be understood that the present invention does not exclude the possibility of the presence or the addition of numbers, steps, operations, components, components, or a combination thereof.

Unless defined otherwise, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by one of ordinary skill in the art. Terms such as those defined in the commonly used dictionaries should be construed as having meanings consistent with the meanings in the context of the related art and shall not be construed in ideal or excessively formal meanings unless expressly defined in this application. Do not.

In addition, in the description with reference to the accompanying drawings, the same components regardless of reference numerals will be given the same reference numerals and redundant description thereof will be omitted. In the following description of the embodiment, when it is determined that the detailed description of the related known technology may unnecessarily obscure the gist of the embodiment, the detailed description thereof will be omitted.

According to one side, the step of washing and disinfecting the tissue of the biological carcass, the step of hydrating and washing the tissue with physiological saline, the step of disinfecting the washed tissue with alcohol and physiologically sterilized the tissue Washing with saline may be performed by repeating 3 to 7 times each.

Since the tissue of the living organism may be freeze-dried, it is necessary to hydrate the tissue using physiological saline. Therefore, in order to hydrate the tissue and at the same time to clean the contaminants, the tissue is placed in a conical tube containing saline, and the tube is turned over and washed. Until the color of the saline becomes clear and transparent, it is preferable to perform the washing process three to seven times while replacing the saline. The washed tissue is immersed in a conical tube containing alcohol for 2 to 5 minutes, preferably about 3 minutes, and sterilized to prevent cell contamination. After alcohol disinfection, a washing process for washing the alcohol may be further included. Unlike the conventional method, a series of washing and disinfection processes may be repeatedly performed several times to remove the impurities from the lyophilized tissue and to hydrate the tissue. According to the above method, the hydrated tissue may be separated into a cell layer, and cells may be cultured from the separated cell layer.

According to one side, the washed and sterilized tissue is enzyme treated. According to one side, the enzyme may be at least one selected from the group consisting of collagenase type I, collagenase type II, collagenase type IV, Trypsin, EDTA, Dispase, and Accutase. The enzyme serves to break the junction between the cells constituting the tissue, to reduce the size of the tissue and to separate the cell layer from the tissue. Cells can be cultured from small tissues or cell layers isolated from tissues. Preferred enzymes which can be used are collagenase type I.

The enzyme may be prepared at a concentration of 0.1 to 2 w / v%. For example, the enzyme is dissolved in a buffer solution (Hank's Balanced Salt Solution (HBSS) with Calcium and magnesium at a rate of 1 g per 1 ml), and then diluted with a stock solution (stock solution 100 U / ul) (1000X stock), and 0.1 to 2 w / v% (or 50 to 500 U / ml).

The sterilized tissue is put in a solution containing the enzyme and chopped to a size of 1 mm or less, and then reacted for 2 to 24 hours in a 37 ° C shaking incubator. If the enzyme reaction time is less than 2 hours, the cell yield obtained from the tissue may be significantly reduced, and if the enzyme reaction time is more than 24 hours, the state of the obtained cells may be unstable and proliferation of the proliferating cells may not be possible.

According to one side, after the step of enzyme treatment of the tissue, may further comprise the step of classifying the tissue by size. The steps for classifying the tissues by size are as follows: 1) Enzymatically treated tissues are divided into conical tubes containing culture medium. 2) The tissues are obtained in order not to reach the bottom of the conical tube, and the tissues are sorted by size. The composition of the culture medium in the tube is, for example, high glucose DMEM containing 20 v / v% FBS, 1% v / v NEAA, 0.1% v / v b-merchaptanol, 1% v / v penicillin-streptomycine 15 ml conical tubes may be used.

Methods for classifying tissues by size may be used in various ways, for example, centrifugation, pipetting, and the like may be used. According to one embodiment, in order to give less physical stimulation to the tissue or cells, a method (political method) to obtain in order to sink to the bottom of the tube in consideration of the sedimentation rate according to the weight of the tissue may be preferable. Tissues sorted by size can be cultured on different plates, respectively.

It may be to include at least one selected from the group consisting of.

In addition, according to one side, the primary cell culture, may be to contain a serum of 15 to 30 v / v% concentration. Serum (eg, FBS) is albumin, globulin, fibronectin, fetuin, and anti-trypsin alpha2-macroglobulin, which promotes cell adhesion. It includes transferrin, and also contains various polypeptides, growth factors, hormones, amino acids, sugars, various nutrients such as keto acids, metabolites, inorganic substances, inhibitors, and the like. Therefore, when the concentration of the serum is less than 15%, the cells may not be cultured due to lack of nutrients in the culture, and when the concentration is greater than 30%, rather it may inhibit the proliferation of cells and cause excessive differentiation. Most preferably serum at a concentration of 20%. The serum that can be used can be, for example, Fetal Bovine Serum (FBS), Bovine Calf Serum (BCS), or horse serum (HS).

It may be at least one selected from the group consisting of mercaptoethanol.

According to one side, the step of culturing the cells, adding a growth factor of 5 to 20 ng / ml on the coating plate, and performing a half-media replacement every 2 to 3 days; may include.

According to one side, the growth factor, bFGF (Basic Fibroblast Growth Factor), EGF (Epidermal Growth Factor), IGF (Insulin like Growth Factor), VEGF (vascular endothelial growth factor), angiogenic growth factors (angiogenic growth factors), It may be at least one selected from the group consisting of ascorbic acid and HGF (Hepatocyte growth factor). The growth factor, as a substance capable of enhancing somatic cell culture, may be added to the culture plate so that the obtained somatic cells can stably proliferate early, and the most preferable growth factor may be bFGF.

The cell culture method includes the step of subcultured cultured cells, if it is confirmed that the cells are cultured. The cell passage method may be passaged from 1: 3 to 1:10 depending on the cell characteristics and the growth rate. If the passage rate is continuously different, the cell characteristics may change due to the difference in the number of proliferating cell populations. Therefore, it is generally desirable to keep the passage rate the same once it is determined. In addition, when it is confirmed that the cells proliferate stably, it is preferable to reduce the concentration of serum to about 10%. For example, at the second passage point when the passage culture is stabilized, a culture solution containing serum with reduced concentration may be used.

1 and 2 are flowcharts of a method for isolating and culturing live cells from ancient extinct organisms or fossils, according to one embodiment.

According to one embodiment, the step of removing impurities from a biological carcass or fossil tissue (110), washing and disinfecting the tissue (120), enzymatically treating the tissue (130), classifying the tissue by size And separating the cell layer (140). Inoculating the tissue on a plate to cultivate the cells (150), passaging the cultured cells (160) and cryopreserving the cultured cells (170), the tissue of the biological carcass To provide a method for culturing cells.

According to one embodiment, the step of washing and disinfecting the tissue 120, hydrating and washing the tissue using physiological saline (121), sterilizing the washed tissue using alcohol (122) ), And washing the sterilized tissue with physiological saline (123).

Further, according to one side, in the flow chart of Figures 1 and 2, the step of removing impurities from the biological carcass or fossil tissue (110), classifying the tissue by size, separating the cell layer (140), and the Cryopreservation of the cultured cells 170 may be modified or omitted as appropriate.

Hereinafter, the present invention will be described in more detail with reference to the following examples. The following examples are described for the purpose of illustrating the present invention, but the scope of the present invention is not limited thereto.

Example 1 Removal of Impurities

Impurities of ancient extinct carcasses and fossil tissues obtained were removed using forceps and surgical scissors.

Example 2: Cleaning and Disinfection

Tissues from which impurities were removed were washed three times in a petri dish containing physiological saline, cut into small chunks using forceps and scissors, and then placed in a 50 ml conical tube and continued until the saline solution appeared clear. The tube was then inverted and thoroughly washed (run five or more times). To prevent cell contamination during incubation, the previously washed tissues were transferred to a new 50 ml conical tube containing absolute alcohol and treated for approximately 3 minutes and washed again with physiological saline for at least 5 times to remove alcohol.

Example 3: Enzyme Treatment

After washing and disinfecting, the tissue was chopped into 0.1 w / v% collagenase type I solution and chopped using surgical scissors until it was smaller than 1 mm. The tissue and solution were transferred to a 50 ml conical tube. The reaction was carried out for 2 hours at 37 ℃ shaking incubator.

Example 4 Tissue and Cell Layer Separation

Enzymatically treated tissues and cells were stopped by enzymatic reaction by doubling the culture solution containing 10 v / v% FBS (or BCS (Bovine Calf Serum), HS (horse serum)) and stopping the enzymatic reaction. 50 ml conical tubes were placed in a rack and allowed to stand for at least 5 minutes to induce sedimented tissues and cells to settle below the tubes, and the supernatant solution was removed using a 50 ml pipette. After adding enough culture solution, the tube was repeatedly flipped up and down, mixed again and placed in a rack again, and washed twice to settle the tissue and cell layers. Tissue and cell layers need to be separated by size for culturing. To do this, use a 10 ml pipette to transfer 10 ml culture from a 50 ml conical tube to a 15 ml conical tube, and then add additional culture to settle and settle to approximately 4 large, medium and small sized tissues and cell layers. Divided into tubes.

Example 5 Inoculation of Tissues and Cells

Separated tissue and cell layer 4 groups contained primary culture medium, ie 20 v / v% FBS, 1 v / v% NEAA, 0.1 v / v% b-merchaptanol, 1 v / v% penicillin-streptomycine After diluting to an appropriate amount with a high glucose DMEM culture medium was inoculated in a 0.1% gelatin-coated culture dish and put in a 37 ℃ incubator to perform a primary culture.

After 2 days of primary cell culture, cells grown on the culture dish were identified under a microscope. It appeared to be located in the single cell group separated from the cells grown from the tissues first, 10ng / ml bFGF was added to help the proliferation of these cells, and the culture medium was replaced at intervals of 2 to 3 days while watching the proliferation state. On the 2nd day of culture, it was confirmed that the cells adhered to the culture dish.

Example 7: Passage culture and cryopreservation of cells

When cell proliferation was stabilized, a culture medium containing 10% serum, a high glucose DMEM medium containing 10% FBS, 1% NEAA, 0.1% b-merchaptanol, and 1% penicillin-streptomycine, was added. The culture was continued by changing. Proliferated cells were passaged and separated and cultured in a ratio of 1: 4 using TrypLE (Trypsin like enzyme) used for cell separation. In addition, cryopreservation of a large number of secured cells was used as a freezing solution containing 10% DMSO kinase and 70% serum. As a result of subculture, healthy growth of cultured cells was confirmed, and the cultured cells were cryopreserved.

Example 8: Identification of Chromosome

Chromosomal analysis was performed to determine the chromosomal characteristics of cells from ancient extinct organisms and fossils. The confirmation method is as follows. 1) 20 μl of colchicine solution at 100 μg / μl concentration was added to each culture vessel containing 10 ml of culture solution, and the culture vessel was gently shaken well and left at 37 ° C. for 2 hours. 2) Centrifuged for 5 minutes at 500 rpm. 3) The supernatant was removed and only a slight culture left. 4) The cells were suspended in 5 ml stock solution (0.075 M KCl) and left in a 37 ° C. constant temperature water bath for 25 minutes. 5) 5 drops of freshly prepared fixatives (methanol: glacial acetic acid = 3: 1, v / v) were added to each tube, and the tubes were mixed gently once or twice, followed by centrifugation at 1,200 rpm for 8 minutes. 6) After removing the supernatant, the cells were suspended by gently tapping the cells in 5 ml of fixed solution and allowed to stand at room temperature for 10 minutes. 7) After repeating the process 5-6 twice was suspended in 0.5 ml fixed solution. 8) A drop of cell suspension was added to the slide glass soaked in cold 70 v / v% ethanol. 9) The ethanol of the slides was dried on an alcohol lamp and dried in air. 10) Immerse in 5 w / v% Giemsa solution for 12 minutes. 11) After flipping the slide glass, the dye solution was washed with distilled water. 12) After drying in air, the coverslip was covered and observed under a microscope.

3 is a photograph of intracellular chromosomes according to one embodiment.

Example 9: Histochemical Dyeing

H & E staining and DAPI staining were performed to confirm the presence of cells in the tissues.

The radiocarbon dating was used to determine tissue dating.

Example 11: Genetic Analysis

In order to confirm that the cells cultured in Example 7 were cells of mammoths, gene analysis was performed. In the journal Nature, an internationally reliable paper, I refer to mammoth gene analysis published by Max Planck Institute in Germany in 2006. The mammoth DNA sequence is also registered in the National Center for Bioinformatics and Research (NCBI). Of the mammoth specific sequences published in Nature, Cox 1 (cytochrome c oxidase subunit 1) [Mammuthus primigenius (woolly mammoth)] was selected as a gene that can be used for phylogenetic analysis.

Based on the nucleotide sequence registered in the NCBI, a primer set for amplifying the Cox1 gene was designed. After extracting genomic DNA from the cultured cells of Example 7, Cox 1 gene was amplified by PCR using the primers. The amplified gene was cloned into pGEN-T-vector and sequenced. More specifically, nucleotide sequences of five clones were analyzed in the forward and reverse directions to finally confirm whether Cox1 genes were correctly expressed in the vector.

FIG. 7 shows a pGEM-T vector, and FIG. 8 shows the result of sequencing after cloning into a pGEM-T vector.

Referring to the analysis results of FIG. 7, in the case of pGEM-Cox1-1-T7, pGEM-Cox1-1-T9, and pGEM-Cox1-1-T10, Cox1 gene amplified by PCR was inserted into the pGEM-T vector. You can check it. Among them, whether the analyzed nucleotide sequence of pGEM-Cox1-1-T7 is consistent with the Mammoth-derived Cox1 gene, it was confirmed using BLAST of NCBI.

9 shows a part of the base sequence of pGEM-Cox1-1-T7.

10 and 11 show the results of BLAST analysis provided by NCBI.

Using BLAST provided by NCBI, it was confirmed that the analyzed sequence was 100% identical to the mitochondrial Cox1 gene registered in NCBI.

As a result of analyzing the DNA extracted from the cells cultured by the method of Examples 1 to 7, it was confirmed that the DNA is a mammoth specific gene Cox 1, it was confirmed that the cell is a mammoth cell.

The cultured somatic cells were used to investigate the possibility of production of exogenous somatic cell nuclear transfer cloned embryos and in vitro culture. i) Nucleated nuclei for somatic cell nuclear transfer using heterologous bovine oocytes matured in vitro for 22 hours, after removal of the cumulus cells in TL-HEPES culture supplemented with 0.1 w / v% hyaluronidase, were released. Only eggs were selected and used. For denuclearization, the oocytes were stained for 10 minutes in a TCM-HEPES solution containing 0.5 µg / ml Hoechst 33342 (manufactured by Sigma) and 7.5 µg / ml cytochalasin b, and the first polar body and nuclei were removed under a fluorescence microscope.ii) Donor cells to be used for nuclear transplantation were cultured ancient carcasses. Donor somatic cells during nuclear transfer were prepared by treating with 0.25 w / v% trypsin-EDTA solution, separating them into single cells, washing with 10% FBS-added somatic cell culture medium, and then again with D-PBS. iii) For somatic cell nuclear transfer, donor somatic cells were injected into the space between the zona pellucida of the nucleated nucleus and the cytoplasm of the nucleus removed with a somatic cell injection pipette. iv) Cell fusion was performed at 20 volts at 1 volt by direct current (DC) using LF101 Electro Cell Fusion Generator (NEPA GENE, Japan) in 0.3M mannitol solution. Was carried out. Cell fusion was observed after 30 minutes of electrical stimulation. v) Induced activation for 5 minutes in CR1aa culture medium containing 10 μM calcium ionomycin for fusion-proven nuclear transfer eggs in CR1aa culture medium containing 2 μM 6-dimethylaminopurine Incubated for 3 hours. Vi) After nuclear transfer, the activated eggs were transferred to a CR1aa solution containing 3 mg / ml fatty acid free BSA and cultured. At 24 hours, the embryos showing the divided segment were further cultured in CR1aa medium containing 10% bovine serum.

In addition, the results of the study of the production of cloned somatic cell nuclear transfer cloned embryos using the ancient mycelial cells obtained in this experiment as donor cells are shown in Table 1. Table 1: Somatic cloned embryo production and embryonic development by internuclear nuclear transfer (r = 4)

In four replicates, somatic cell nuclear transfer was performed using 178 mature oocytes. The cell fusion between ancient cells (M cell) and bovine egg showed 51.7%, and the percentage of egg split at day 2 of culture was 47.8. %, 33.7% of cells divided more than 8 cell stages at 4 days of culture.

Example 13: Method of Producing Cloned Animals

The process for producing a cloned animal using the cultured somatic cells is as follows. Production of cloned animals is divided into a process of producing cloned embryos and inducing implantation and pregnancy by transplanting the cloned embryos into surrogate mothers. In the production of cloned embryos, the nucleus of oocytes is best used for the production of cloned embryos, but if it is difficult to obtain homologous nuclei, the heterologous nuclei should be selected as shown in Example 12 of the present study. The method for producing cloned embryos through somatic cell nuclear transfer using donor cells is shown in Example 12. In addition, cloned blastocyst embryos produced by the above method for the production of cloned animals are implanted into the uterus of a synchronized-induced surrogate mother or the uterus of a natural-cycle surrogate mother to induce implantation, confirm estrus resumption, and ultrasound when pregnancy is maintained safely. Pregnancy is confirmed through a diagnosis.

Example 14 Step of Producing Stem Cells

The method for producing stem cells using the cultured somatic cells is as follows. Stem cells can be obtained from a somatic cell-derived pluripotent stem cell production process or from a somatic cell nuclear transfer cloned embryo-derived stem cell production method. i) Somatic cell-derived pluripotent stem cell production is to produce the respective virus introduced into the stem cell inducer Oct4 gene, Sox2 gene, Nanog gene, Lin gene, C-myc gene and Klf4 gene, each of the viruses Concentrating and mixing to prepare a suitable virus concentrate mixture, introducing these virus solutions into the prepared somatic cells, and culturing the somatic cells into which the genes are introduced. ii) Somatic cell nucleus cloned embryo-derived stem cell production is a method using the blastocyst embryo produced through Example 12. This method removes the zona pellucida of cloned embryos by using stem cell-derived stem cells, removes trophic germ cell layers through immunohistochemistry, recovers only internal cell masses and cultures them on supporting cells, and proliferates similar embryonic stem cell masses. The process of making a step is included.

Although the embodiments have been described by the limited embodiments and the drawings as described above, various modifications and variations are possible to those skilled in the art from the above description. For example, the techniques described may be performed in a different order than the described method, and / or the components described may be combined or combined in a different form than the described method, or replaced or substituted by other components or equivalents. Appropriate results can be achieved.

Therefore, other implementations, other embodiments, and equivalents to the claims are within the scope of the claims that follow.

Claims

Washing and disinfecting tissue of the biological carcass;

Enzymatically treating said tissue;

Diluting the tissue with primary cell culture to inoculate the plate;

Culturing the cells from the tissue; And

Subcultured with the cultured cells.
The method of claim 1,

The step of washing and disinfecting the tissue of the biological carcass,

Hydrating and washing the tissue with physiological saline;

Disinfecting the washed tissue with alcohol; And

Washing the sterilized tissue with saline; Method comprising culturing cells from the tissue of the living body.
The method of claim 1,

Enzymatically treating the tissue is performed for 2 to 24 hours,

The enzyme is used for culturing cells from tissues of a living body, which is at least one selected from the group consisting of collagenase type I, collagenase type II, collagenase type IV, Trypsin, EDTA, Dispase, and Accutase. Way.
The method of claim 1,

After enzymatically treating the tissue,

The method of culturing cells from the tissue of the living body, further comprising the step of classifying the tissue by size
The method of claim 1,

The plate is coated with a coating material comprising at least any one selected from the group consisting of Matrigel, collagen, gelatin, fibronectin, and laminin.
The method of claim 1,

The primary cell culture solution,

EGM2-MV with cytokines, EBM-2 without cytokines, Ham F-10, F12, RPMI, DPBS, DMEM and MEM
Comprising at least any one selected from the group consisting of, How to culture the cells from the tissues of the living body.
The method of claim 1,

The primary cell culture solution,

A method for culturing cells from tissues of a living body, comprising serum at a concentration of 15 to 30%.
The method of claim 1,

The primary cell culture solution contains an additive,

The additives include, but are not limited to, essential amino acids (EAA), non-essential amino acids (NEAA), ITS mixtures (insulin, transferrin, sodium selenite), penicillin / streptomycin (PS), and
Comprising at least any one selected from the group consisting of mercaptoethanol, a method for culturing cells from the tissue of a living body.
The method of claim 1,

The step of culturing the cells,

Adding 5 to 20 ng / ml growth factor on the plate and performing half media replacement at intervals of 2 to 3 days.
The method of claim 9,

The growth factor,

FGF2, basic Fibroblast Growth Factor (bFGF), Epidermal Growth Factor (EGF), Insulin like Growth Factor (IGF), vascular endothelial growth factor (VEGF), angiogenic growth factors, ascorbic acid and Hepatocyte growth and at least one selected from the group consisting of: a method for culturing cells from tissues of living organisms.
The method of claim 1,

After passaging the cultured cells,

Cryopreserving the passaged cells in a cryopreservation medium, further comprising the step of culturing cells from the tissue of the biological carcass.
The method of claim 11,

The cryopreservation medium,

Tissue of biological organisms, including at least one selected from the group consisting of serum, plasma, serum replacement (SR), DMSO, glycerin, propanediol, and ethylene glycol A method of culturing cells from.
Culturing the cells by the method of claim 1;

Somatic cell nuclear transfer using the cultured cells; And

Producing cloned embryos from the somatic cells; Cloned embryo production method comprising a.
Culturing the cells by the method of claim 1; And

Stem cell production method comprising the step of producing a stem cell using the cultured cells.
Culturing the cells by the method of claim 1; And

Cloning animal production method comprising the step of producing a clone animal using the cultured cells.
Cells cultured by the method of claim 1.