KR20140041249A - A composition for cryopreservation of stem cells or primary cells comprising plant-derived human serum albumin, plant protein hydrolysate and lipid - Google Patents

Abstract

The present invention relates to a composition and a usage for the cryopreservation of stem cells or primary cells including a plant-derived human serum albumin, a lipid, and a plant protein hydrolysate as active ingredients, and more specifically, to a composition for the cryopreservation of stem cells or primary cells including a plenty of plant-derived human serum albumin, lipid, and plant protein hydrolysate as active ingredients and a method for the cryopreservation of stem cells or primary cells using the same, wherein the plant-derived human serum albumin maintains animal-free and xeno-free properties and reduces the death of cells due to freezing.

Description

TECHNICAL FIELD The present invention relates to a composition for cryopreservation of stem cells or primary cultured cells comprising recombinant human serum albumin, lipid and plant protein hydrolyzate derived from plants as an active ingredient, and to a composition for cryopreservation of stem cells or primary cultured cells comprising plant-derived human serum albumin, plant protein hydrolysate and lipid}

The present invention relates to a composition for cryopreserving stem cells or primary cultured cells containing plant-derived recombinant human serum albumin, lipid and plant protein hydrolyzate as an active ingredient, and more particularly to a composition for cryopreservation of stem cells or primary cultured cells Derived recombinant human serum albumin, lipid, and plant protein hydrolysates sufficient to reduce cell death due to freezing while maintaining animal-free and xeno-free in long-term storage of the plant A composition for cryopreserving a stem cell or a primary cultured cell comprising as an active ingredient; And a method for cryopreserving stem cells or primary cultured cells using the same.

Stem cells are cells that can multiply indefinitely while maintaining undifferentiated state and can differentiate into specific functions and forms when given a certain environment and conditions. Human embryonic stem cells are capable of continuously self-renewing under appropriate in vitro culture conditions, and are capable of differentiating into all kinds of cells of the human body, resulting in pluripotency, In addition to understanding the basic knowledge of growth, it is important to develop a cell therapy system that is the fundamental treatment method of human injury or various diseases, and to study the various effects of various drug candidates, The range of results is expanding. Despite the surge in the demand for adult stem cells and human embryonic stem cells, there is still a shortage of techniques and methods for maintaining undifferentiated adult and human embryonic stem cells for long term storage, .

Primary cells are primary cells that are directly cultured directly from animal tissues or organs, and are normal cells that are obtained directly from living organisms. These cells grow to some extent with cell division when they are cultured, but have aging limitations if they continue to pass. Despite these limitations, primary cultured cells are used in the production of biologics because they have the advantage of being similar to the actual reactions of living organisms. However, due to the above limitations, it is necessary to preserve the cells in a frozen state.

Therefore, in order to use stem cells or primary cultured cells for clinical trials and cell therapy, it is very important to develop a preservation solution that does not use animal-derived factors and to develop suitable preservation methods for primary cultured cells and stem cells.

Stem cells can be preserved while preserving the characteristics and survival rate of stem cells themselves, such as pre-differentiation ability and versatility, and there are refrigeration preservation and cryopreservation. The cold storage method is a method of storing stem cells at low temperature and developed for short term transportation and storage. However, the cold storage method has a limitation in long-term storage depending on the demand of stem cells and primary cultured cells, since it is possible to store for a short period of time within one week, more precisely for 1 to 3 days at low temperature (4 ° C). On the other hand, the cryopreservation method is a method of storing cells, tissues and organs at a very low temperature of -70 ° C to -196 ° C to stop all the metabolic reactions in the cells to semi-persist the desired stem cells. This method is the ideal method for semi-permanent storage of stem cells and primary cultured cells for research and therapeutic purposes, and is used to store stem cells and primary cultured cells in all non-commercial or commercial cell banks.

However, in the process of freezing and thawing, the formation of ice crystals and cell damage due to imbalance of ion and osmotic pressure inevitably can not be avoided, resulting in increased damage due to freezing and thawing processes. In order to minimize these damages and to maximize survival rate and stem cell function after freezing and thawing, development of freezing preservative and development of freezing method to minimize damage accompanied by freezing and thawing according to biochemical and physical characteristics of the cells very important.

In general, intracellular ice crystallization, which occurs during cryopreservation of animal cells, is the major cause of cell damage. Therefore, freeze inhibitors are used to prevent this, and mainly DMSO (dimethyl sulfoxide) or glycerol is used. These are permeating-type cryoprotectants that effectively reduce the formation of ice crystals during freezing and thawing. Specifically, glycerol acts as a salt-buffer to bind to a metal ion and dehydrate the cell itself, thereby protecting the cell by reducing the volume of the entire ice crystal during freezing of the cell. In the case of DMSO, it partially blocks the formation of puncture by dissolving the cell membrane and interferes with the hydrogen bonding between water molecules, thereby inhibiting damage to cells by ice crystals. However, DMSO and glycerol are toxic to cells, resulting in ionic and osmotic imbalance during freezing and thawing, and the generation of free radicals, which can not be avoided Lt; / RTI >

Therefore, in order to solve this problem to a certain extent, a method using a combination of a freeze inhibitor and a vehicle capable of minimizing ion and osmotic imbalance and free radical formation is widely used I am using it. However, Fetal Bovine Serum (FBS) and Human Serum, which are components of commonly used refrigerant, are difficult to control due to lot to lot variation, Human-derived primary cultured cells and stem cells, which are intended to be stored in an infectious source transmitted by human serum, or the like, may be exposed and cause some immune reactions. In recent years, there have been reported techniques using recombinant human serum albumin produced from microorganisms or animal cells to eliminate the problems of quality control of the refrigerant component and problems caused by the infectious agent. However, the use of such recombinant human serum albumin also has some problems with the quality control of refrigerant components, and there is still a possibility of exposure of infectious agents that can be infected with animals. Therefore, a new refrigerant Is required.

In order to develop a composition capable of effecting cryopreservation of stem cells and primary cultured cells because the kinds and composition ratios of components contained in the composition for cryopreservation can be changed according to the type of cells to be cryopreserved, The components suitable for primary cultured cells and the proper compositional ratio of the components should be determined. Therefore, in order to develop a composition for cryopreservation of stem cells or primary cultured cells having both safety and stability, it is desirable to provide a composition capable of effectively causing cryopreservation of stem cells and primary cultured cells, Proper compositional ratios should be developed.

Under these circumstances, the present inventors have made intensive efforts to develop an animal-free and non-heterologous substance and a method which can be used for the cryopreservation of stem cells or primary cultured cells. As a result, it has been found that a plant- In the case of using a composition for cryopreservation comprising serum albumin, lipid and plant protein hydrolyzate, it is possible to safely freeze the stem cells and primary cultured cells without contamination of heterologous proteins, and even if thawed, And that the cell survival rate after thawing is improved rather than the case of using a cryoprotectant containing recombinant human serum albumin derived from a conventional bovine serum protein or microorganism, and completed the present invention.

It is an object of the present invention to provide a method of freezing a stem cell or a primary cell, which comprises recombinant human serum albumin derived from plants, lipid and plant protein hydrolyzate as an active ingredient, And to provide a composition for preservation.

Another object of the present invention is to provide a method for lyophilizing stem cells or primary cultured cells using the above composition.

In order to achieve the above object, the present invention provides a method for cryopreserving a stem cell or a primary cell comprising a plant-derived recombinant human serum albumin, a lipid and a plant protein hydrolyzate as an active ingredient, Lt; / RTI >

The composition for cryopreservation according to the present invention contains a recombinant human serum albumin derived from a plant, a lipid and a hydrolyzate of a plant protein, and does not use an animal factor and maintains a xeno-free state. Preservation of prion protein is low risk of infection and can be safely used. In addition, it is possible to effectively prevent the cell death due to freezing and to stably maintain the characteristics of the primary cultured cells or the stem cells, including components that have a great effect on the cryopreservation of stem cells or primary cultured cells, Very useful for long-term storage. The composition for cryopreservation, which contains all of the plant-derived recombinant human serum albumin, lipid and plant protein hydrolyzate, and the composition ratio thereof are not known, and were first identified by the present inventors. In addition, the composition for cryopreservation comprising the combination of the plant-derived recombinant human serum albumin, lipid and plant protein hydrolyzate not only maintains the function of the preserved cells but also allows the cell survival rate to be higher than that of the conventional cryopreservation composition It can be used for the cryopreservation of stem cells or primary cultured cells which need to be preserved for self-treatment and the like.

The term "cryopreservation " in the present invention means to keep the cells stable over a long period of time through freezing. Cells are generally mutated at a rate of about 1 in 10,000 in culture, and when cells continue to be transplanted over a long period of time, they become a different cell population than the original cell population, It may be lost by subculture. It may also be infected with mycoplasma or the like during subculture. Due to this problem, the cells are frozen and preserved before the inherent characteristics of the cells disappear, and cryopreservation of the cells is carried out so that they can be taken out when needed. In particular, in the case of stem cells, effective cryopreservation is considered to be more important in stem cells since healthy stem cells must be immediately available for use as a therapeutic agent. Cryopreservation may be carried out through conventional methods in the art for freezing and preserving cells, examples of which include, but are not limited to, vitrification methods, slow freezing methods, or bulk cryopreservation Do not. For the purpose of the present invention, the cryopreservation method includes, without limitation, any method capable of effectively preserving stem cells or primary cultured cells. In one embodiment of the present invention, the vitrification and slow freezing methods were used (Example 3).

Cryopreservation of cells is carried out by treating the cells to be frozen with a cryoprotectant, which may additionally contain several components to prevent cell damage by the cryoprotectant. In the case of the composition for cryopreservation according to the present invention, it is preferable that the composition contains a plant recombinant human serum albumin (hHSA), a lipid and a plant protein hydrolyzate, and the cryopreservation of stem cells or primary cultured cells Thus improving safety and stability.

The term "plant-derived recombinant human serum albumin" in the present invention means a protein obtained by transforming a plant cell using the amino acid sequence of a part of human serum albumin having a total or biological activity of human serum albumin. The human serum albumin can be used as an animal-free alternative which is equivalent to or superior to serum in cryopreservation, and is added to the composition for cryopreservation to stabilize cells. In particular, the recombinant human serum albumin of the present invention has an advantage of being able to minimize the risk of an animal or viral infectious agent produced from a plant transformant, and thus is useful for the utilization of stem cells or primary cultured cells, It has usefulness. In addition, the plant-derived recombinant human serum albumin contained in the composition of the present invention has an advantage that there is substantially no lot to lot variation between the human serum albumin and human serum albumin derived from animal cells or serum.

In the composition of the present invention, the plant-derived recombinant human serum albumin may be contained in an amount of 5 to 50 parts by weight, preferably 10 to 40 parts by weight, based on 100 parts by weight of the total composition, But is not limited thereto.

Information on human serum albumin that can be used in the present invention can be obtained by using a known database such as the National Institute of Health (NCBI) GenBank, for example, a protein having an Accession Number of NP_000468 (NM_000477) Do not. In addition, the human serum albumin may be a protein defined by the amino acid sequence of SEQ ID NO: 1, more preferably 70% or more, preferably 80% or more, more preferably 90% or more, , More preferably 95% or more, and most preferably 98% or more. The present invention includes all amino acid sequences that are substantially contained in the composition of the present invention and have biological activity to inhibit cell damage upon cell freezing. In addition, it is obvious that a protein variant having an amino acid sequence in which some sequences are deleted, modified, substituted or added is also included within the scope of the present invention, provided that the amino acid sequence has the same or corresponding biological activity as human serum albumin. The polynucleotide encoding the human serum albumin has a nucleotide sequence of 70% or more, preferably 80% or more, more preferably 90% or more, still more preferably 95% or more, Or more, and most preferably 98% or more as a nucleotide sequence having a biological activity that substantially inhibits cell damage upon cell freezing is included in the scope of the present invention.

The plant-derived recombinant human serum albumin may be obtained by transforming with an expression vector containing a polynucleotide encoding human serum albumin, or may be obtained by using recombinant human serum albumin derived from commercially available plant.

In the present invention, the term "vector" is an expression vector capable of expressing a target protein in a suitable host cell, wherein the expression vector comprising the polynucleotide encoding the human serum albumin comprises a polynucleotide encoding human serum albumin, Refers to a recombinant polynucleotide molecule comprising a regulatory sequence that is possibly linked to a vector capable of transforming into a plant and expressing a desired protein in a plant for the purposes of the present invention. The plant expression vector is not particularly limited as long as it is an expression vector comprising a polynucleotide encoding the human serum albumin.

The term "transformed" in the present invention means that DNA is introduced into a host cell and DNA can be cloned as a factor of chromosome or by integration of chromosome integration. Thus, foreign DNA is introduced into cells to artificially induce a genetic change This is the phenomenon that causes it. Any transformation method of the present invention can be used, and can be easily carried out according to a conventional method in the art.

The kind of the plant transformant producing the recombinant human serum albumin of the present invention is not particularly limited as long as it can express human serum albumin, for example, tobacco, rice or soybean transformant. In one embodiment of the present invention, rAlbumin ACF, plant-derived recombinant human serum albumin from Sheffield, recombinant human serum albumin produced in rice, and Cellastim, a plant-derived recombinant human serum albumin from InVitria, were used.

The term "lipid" in the present invention is an organic substance including fat, wax, sterol, fat soluble vitamin, monoglyceride, diglyceride, triglyceride, It has a good melting property in organic solvent. For the purpose of the present invention, the lipid includes any kind of lipid molecule that plays a role of inhibiting cell damage upon cryopreservation of stem cells or primary cultured cells, including, for example, soy lecithin, a plant phospholipid, Cholesterol, arachidonic acid, DL-a-tocopherol acetate, linoleic acid, linolenic acid, myristic acid, oleic acid, , Palmitoleic acid, palmitic acid or stearic acid. The lipid may include, but is not limited to, cholesterol, which is preferably a sterol. In one embodiment of the present invention, cholesterol, arachidonic acid, DL-a-Tocopherol acetate, linolenic acid, linoleic acid, myristic acid, , Lipid concentrate containing oleic acid, palmitoleic acid, palmitic acid, stearic acid, and Tween 80 was used.

The lipid plays a role of reducing the membrane damage when the cell membrane is weakened when it is frozen, for example, when the cell membrane is dissolved and weakened by the freezing inhibitor such as DMSO. Particularly, when cholesterol is contained, the ratio of cholesterol / phospholipid can be increased, and the damage of the cell membrane can be remarkably reduced. The lipid may be included in an amount of 0.1 part by weight to 5.0 parts by weight based on 100 parts by weight of the total composition of the present invention, preferably 0.1 part by weight to 1.0 part by weight based on 100 parts by weight of the total composition.

In one embodiment of the present invention, a lipid concentrate containing cholesterol was used as a representative lipid. When lipid is additionally contained in the composition for cryopreservation in addition to human serum albumin, it is confirmed that the survival rate of the cryopreserved cells is remarkably increased (Example 4).

In the present invention, the term "plant protein hydrolyzate" is a substance containing an amino acid or / and a peptide, and is a substance containing an amino acid and / or a peptide prepared by hydrolyzing a plant protein. The plant protein hydrolyzate can be produced by hydrolyzing a plant protein using a specific enzyme or the like, but is not limited thereto. For the purpose of the present invention, the plant protein hydrolyzate is not particularly limited as long as it contains an amino acid capable of reducing damage to cells in cryopreservation of stem cells or primary cultured cells. For example, tobacco, rice or Soy protein hydrolyzate. The plant protein hydrolyzate may be directly hydrolyzed using an enzyme or the like, or commercially available plant protein hydrolyzate may be used. In one embodiment of the present invention, hydrolyzed soybean protein was used as hydrolyzed plant protein, and ULTRAPEP SOY or ULTRAPEP YE from Sheffield was purchased and used.

In addition, the plant protein hydrolyzate may be directly hydrolyzed using an enzyme or the like, or may be further purified. The plant protein hydrolyzate is preferably selected from the group consisting of valine, leucine, isoleucine, methionine, threonine, lysine, phenylalanine or tryptophan, And non-essential amino acids such as essential amino acids, such as arginine, asparagine, glutamine, glycine, histidine, serine, tyrosine or proline, But are not limited thereto.

The plant protein hydrolyzate includes essential amino acids and / or non-essential amino acids that can be used as basic energy sources of cells, and supplies nutrients to the cells at the time of freezing and thawing, Contributes to height. In one embodiment of the present invention, the effect of the plant protein hydrolyzate was confirmed by using a soybean protein hydrolyzate, and as a result, the plant protein hydrolyzate was further added to the composition of the present invention comprising plant-derived recombinant human serum albumin and lipid , The survival rate of stem cells was remarkably increased. Thus, it was confirmed that the cryopreservation efficiency could be increased when the plant protein hydrolyzate was further contained (Example 5).

Particularly, the composition of the present invention contains 1 to 40 parts by weight, preferably 1 to 20 parts by weight, more preferably 1 to 10 parts by weight of the plant protein hydrolyzate per 100 parts by weight of the total composition When the weight part is included, the cryopreservation efficiency can be maximized.

More preferably, the composition of the present invention comprises 10 to 40 parts by weight of plant-derived recombinant human serum albumin, 0.1 to 1 part by weight of lipid and 1 to 10 parts by weight of plant protein hydrolyzate, based on 100 parts by weight of the total composition But are not limited thereto.

In addition, the composition may further comprise a freeze inhibitor.

In the present invention, the term "freeze inhibitor" refers to a substance capable of inhibiting the formation of ice crystals and ionic and osmotic pressure which are inevitably accompanied by freezing and thawing processes in keeping cells, tissues or organs at extremely low temperatures of -80 ° C to -200 ° C. Means a substance used to minimize damage to cells due to imbalance. For the purpose of the present invention, the above-mentioned freezing inhibitor includes, without limitation, any substance capable of reducing cell damage in freezing. Examples thereof include permeable freezing inhibitors such as dimethylsulfoxide (DMSO), glycerol, propylene glycol, ethylene glycol, , Carboxymethylcellulose salts, carboxymethylcellulose (CMC), polysaccharides, disaccharides, and the like, but are not limited thereto. In one embodiment of the present invention, dimethylsulfoxide (DMSO) is used as a representative example of the freezing inhibitor. The freeze inhibitor may be included in an amount of 1 part by weight to 15 parts by weight, preferably 5 parts by weight to 10 parts by weight, based on 100 parts by weight of the total composition of the present invention.

In addition, the compositions of the present invention further comprise a buffer selected from the group consisting of citrate, phosphate, succinate, tartrate, fumarate, gluconate, oxalate, lactate, acetate, histidine and Tris And may be selected from the group consisting of sodium chloride, potassium chloride, boric acid, sodium borate, mannitol, glycerin, propylene glycol, polyethylene, glycols, maltose, sucrose, erythritol, arabitol, xylitol, sorbitol trihalose and glucose And may further include one or more isotonic agents. It may also contain a cell death inhibitor, Rho associated kinase inhibitor, catalase, zVAD-fmk.

The composition of the present invention comprising the plant-derived recombinant human serum albumin, lipid and plant protein hydrolyzate can be very useful for cryopreservation of cells, particularly stem cells or primary cultured cells.

The term "stem cell" in the present invention means an undifferentiated cell having self-replicating ability and differentiation-promoting ability. Stem cells include subpopulations such as pluripotent stem cells, multipotent stem cells, and unipotent stem cells, depending on the differentiation ability. The pluripotent stem cell refers to a cell capable of differentiating into all the tissues or cells constituting the living body. The pluripotent stem cells are not all kinds of cells, but are capable of differentiating into pluripotent tissues or cells. ≪ / RTI > However, the term "pluripotent stem cell" refers to a cell having an ability to differentiate into a specific tissue or cell.

Examples of the pluripotent stem cells include ES cell, undifferentiated germline cell (EG cell), and degenerative stem cell (iPS cell). Examples of pluripotent stem cells include mesenchymal stem cells (derived from fat, Adult stem cells such as hematopoietic stem cells (derived from bone marrow or peripheral blood), nervous system stem cells, reproductive stem cells, and the like are examples of the pluripotent stem cells, And committed stem cells, which exist only in the low state and make only the hepatocytes with vigorous division after the activation. In one embodiment of the present invention, it has been confirmed that the composition of the present invention can be used safely and stably for cryopreservation of stem cells by using bone marrow-derived mesenchymal stem cells and cord-derived stem cells.

In the present invention, the term "primary cell" is a cell isolated from a tissue extracted from an individual without manipulation of a gene or the like, and expresses the function of a biological organ / tissue. Primary cultured cells are separated from skin, vascular endothelium, bone marrow, fat, and cartilage, and are used as a cell therapy agent for studying the functions of tissues and cells, or for recovering lost tissues. In one embodiment of the present invention, it has been confirmed that the composition of the present invention can be safely and stably used for cryopreservation of stem cells by using a foreskin-derived fibroblast as a representative primary cultured cell (FIG. 9).

The stem cells and primary cultured cells are not particularly limited as long as they can be stably cryopreserved by the composition of the present invention. Examples thereof include human, monkey, pig, horse, cow, sheep, , And rabbit-derived cells. The stem cells or primary cultured cells are preferably human stem cells or primary cultured cells, but are not limited thereto.

In one embodiment of the present invention, bone marrow-derived mesenchymal stem cells and umbilical cord-derived stem cells were used as stem cells, and human dermal fibroblasts were used as primary cultured cells. This was suspended and frozen in the cryopreservation composition of the present invention including all of plant-derived recombinant human serum albumin, lipid and plant protein hydrolyzate, and thawed to confirm cell viability and cell morphology. As a result, it was confirmed that the composition of the present invention exhibited a high cell survival rate and a healthy form as compared with other compositions containing recombinant human serum albumin derived from fetal bovine serum or microorganism (FIGS. 4 to 7). Particularly, when the cells are frozen using the slow freezing method, when the composition of the present invention further comprises recombinant human serum albumin and a plant protein hydrolyzate in lipid, the cell survival rate is higher than that of the recombinant human serum albumin and lipid alone Showed more improved results, indicating that it can be used very stably when freezing stem cells and primary cultured cells when all of the recombinant human serum albumin, lipid and plant protein hydrolysates are included (FIG. 3). In particular, the plant-derived human serum albumin, lipid and plant protein hydrolysates contained in the composition of the present invention are all derived from plants and have animal-free and non-diverse characteristics. Therefore, diseases that can be spread in animals such as mad cow disease And the like, so that the composition of the present invention has stability as well as safety.

In another aspect, the present invention provides a method for cryopreserving stem cells or primary cultured cells using the composition.

The composition, stem cells, primary cultured cells and cryopreservation are as described above.

The cryopreservation method includes, but is not limited to, freezing the stem cell or the primary cultured cell treated with the composition.

The cooling of stem cells or primary cultured cells treated with the composition for cryopreservation of the present invention can be carried out through conventional methods in the art for cooling stem cells or primary cultured cells. Examples thereof include Vitrification method, But is not limited to, a slow freezing method. The vitrification-freezing method can be performed by freezing the temperature by 1 DEG C per minute at a constant rate using a device such as a controlled-rate freezer (CRF), and when it reaches -80 DEG C, it is preferably stored in a nitrogen tank . The gentle freezing can be carried out by placing the vial containing the cell-containing cryopreservation suspension in a refrigeration container box containing isopropyl alcohol and cooling the cryoprecipitate at a constant temperature for 12 to 24 hours in an ultra- But is not limited thereto. In addition, the cooled cells can be stored in a liquid nitrogen tank and taken out and used again when necessary. In one embodiment of the present invention, the stem cells and the primary cultured cells suspended in the composition of the present invention are frozen using vitrification and slow freezing methods, and then stored in liquid nitrogen.

When the composition and method of the present invention is used, the cell survival rate of stem cells and primary cultured cells can be maintained at a high level without using fetal bovine serum (FBS) or animal-derived factors, The present invention is useful in the related pharmaceutical and medical industries where animal-free and xeno-free are required since the exposure to the liver is essentially blocked and thus safe in the living body application.

FIG. 1 shows the cryopreservation effect of a cryopreservative (HSA) containing a recombinant human serum albumin and a cryopreservative (HSA + Lipid) supplemented with a lipid concentrate containing cholesterol. The cryopreservation agent The resulting mesenchymal stem cells were frozen for 7 days and then thawed to confirm cell viability.
FIG. 2 shows the cryopreservation effect of a cryopreservative (HSA) containing recombinant human serum albumin and a cryopreservative (HSA + Lipid) supplemented with a lipid concentrate containing cholesterol. Derived mesenchymal stem cells were frozen for 7 days, thawed, and cultured for 3 days to confirm the cell growth rate.
FIG. 3 shows the effect of the composition of the present invention depending on the presence or absence of the plant protein hydrolyzate in order to confirm the effect of the plant protein hydrolyzate used in the composition of the present invention. The embryo-derived stem cells frozen using the above method were stored for 2 weeks and then thawed, and the survival rate of the cells was confirmed by means of Propidium Iodide (PI) staining, and the results are shown in a graph.
FIG. 4 is a graph showing the results of a comparison between the cryopreservative (FBS) containing fetal bovine serum (FBS), the cryopreservative (HSA) comprising microbial-derived recombinant human serum albumin and the plant-derived recombinant human serum albumin, After one day of freezing treatment using the plant protein / lipid of the present invention containing the hydrolyzate for 2 weeks and then thawing and inoculating the culture dish, bone marrow mesenchymal stem cells (BONE Marrow Mesenchymal Stem Cell ) Was observed through a microscope. The upper portion is photographed at a magnification of 50x, and the lower portion is photographed at a magnification of 100x. When the composition of the present invention (Plant protein / Lipid) is used, a healthy cell state is exhibited as compared with the case of using a cryoprotectant containing a serum protein or microorganism-derived recombinant human serum albumin.
FIG. 5 is a graph showing the results of a cryoprotectant (FSA) containing frozen storage agent (FBS) containing fetal calf serum, a cryopreservation agent (HSA) containing microorganism-derived recombinant human serum albumin and a plant- (Plant protein / Lipid) for 2 weeks and then thawed to confirm the cell viability of bone marrow-derived mesenchymal stem cells through propidium iodide staining and reanalyzed the same.
Fig. 6 is a graph showing the results of a cryoprotectant (HSA) containing frozen storage agent (FBS) containing fetal bovine serum, a frozen storage agent (HSA) containing microorganism-derived recombinant human serum albumin, and a plant- The cells were frozen for 2 weeks using a preservative (Plant protein / Lipid), and thawed and inoculated into a culture dish. After 1 day, the cells were observed with a microscope to observe the state of Umbilical Cord Matrix Stem Cell . The upper portion is photographed at a magnification of 50x, and the lower portion is photographed at a magnification of 100x.
Fig. 7 is a graph showing the results of a cryoprotectant (HSA) containing frozen storage agent (FBS) containing fetal calf serum, recombinant human serum albumin derived from microorganism, And the cell viability of the umbilical cord stem cells was confirmed by propidium iodide staining after being frozen for 2 weeks and then thawed as in the case of bone marrow-derived mesenchymal stem cells using a preservative (Plant protein / Lipid).
FIG. 8 is a graph showing the results of the freezing and thawing of frozen-thawed human serum albumin (HSA) containing frozen storage agent (FBS) containing fetal bovine serum, frozen storage agent (HSA) containing microbial-derived recombinant human serum albumin, and plant-derived recombinant human serum albumin, The stem cells from the umbilical cord were frozen in the vitrification method using CRF equipment using a preservative (Plant protein / Lipid), immediately transferred to the LN2 tank, stored for 2 weeks and thawed to measure the cell viability using propidium iodide ) ≪ / RTI > staining.
Fig. 9 is a graph showing the results of a cryoprotectant (HSA) containing frozen storage agent (FBS) containing fetal bovine serum, a frozen storage agent (HSA) containing microbial recombinant human serum albumin, and a frozen storage agent containing plant-derived recombinant human serum albumin, lipid and plant protein hydrolyzate Derived fibroblasts were frozen and stored for 2 weeks using a plant protein / lipid, followed by thawing, and cell viability was confirmed by propidium iodide staining. The results are shown in the graph.

Hereinafter, the present invention will be described in more detail by way of examples. However, the examples are illustrative of the present invention, and the contents of the present invention are not limited to the following examples.

Example  1: Preparation of stem cells and primary cultured cells

Bone marrow mesenchymal stem cells, Umbilical Cord Matrix Stem Cells and fibroblasts from bone marrow, Umbilical cord, and Foreskin donated from patients, respectively, Fibroblast) was directly extracted. CB-MS-GM (CEFO, Seoul, Korea) culture medium was used as the culture medium of the stem cells and CB-HDF-GM (CEFO. Seoul, Korea) culture medium was used as the fibroblast culture medium. The cells were cultured in an incubator at 37 ° C, 5% CO ₂ , and the cultures were exchanged every 3 days.

All the experiments of the present invention were conducted through procedures and methods according to the Act on Bioethics and Safety under the approval of Cell Bio Bioethics Committee of the Institution.

Example  2: recombinant human serum albumin derived from plants, lipid and plant protein The hydrolyzate  Preparation of Composition for Cryopreservation Containing

In order to prepare a composition for cryopreservation which can safely and stably preserve stem cells and primary cultured cells, the inventors used recombinant human serum albumin, lipid and plant protein hydrolyzate derived from plants as an effective ingredient. At this time, as recombinant human serum albumin derived from plants, rAlbumin ACF, a plant-derived recombinant human serum albumin of Sheffield, recombinant human serum albumin produced from rice, or recombinant human serum albumin of plant origin (InVitria) Cholesterol lipid concentrate (Invitrogen) was used as the lipid, UltraPep Soy (Sheffield Was used.

As the basal medium, MS-GM (CEFO., Korea) was used for stem cells and CB-HDF-GM (CEFO., Seoul, Korea) for primary cultured cells. Plant-derived recombinant human serum albumin protein was used mainly in 10 to 40% in this experiment, and especially when it contained 40%. When the composition of the present invention contains 20% of plant-derived recombinant human serum albumin protein, the plant protein hydrolyzate is mainly contained at 5%, and when the plant-derived recombinant human serum albumin protein contains 10%, the plant protein hydrolyzate Was included to 10%.

The composition of the cryopreservative of the present invention prepared using the active ingredient was as shown in Table 1 below.

The main composition of the cryopreservation composition of the present invention Base badge
(Basal Medium) Freezing inhibitor
(Cryoprotectant) Serum and nutrients Etc MS-GM (CEFO., Korea) or CB-HDF-GM (CEFO., Seoul, Korea) DMSO
5-15% FBS 10-50% - HAS 10-50% - Plant-derived recombinant human serum albumin and lipid
10-50% Plant protein hydrolyzate
1-20%

Example  3: Cold / thaw treatment of stem cells

The bone marrow mesenchymal stem cells, Umbilical cord matrix stem cells and fibroblasts cultured through the method of Example 1 were collected and washed with PBS (Phosphate-buffered saline) , Recovered and stained with propidium iodide (PI) to count the number of cells. Then, a cryopreservative containing a fetal bovine serum (FBS) as a main component so that the stem cells or the fibroblasts become 1 x 10 ⁶ cells / ml; A cryopreservative containing a microorganism-derived recombinant human serum albumin (HAS) as a main component; Was suspended in a plant-derived recombinant human serum albumin (Plant Protein, rhAlbumin) of Example 2 and a cryoprotectant containing lipid as a main component.

Slow freezing method and vitrification method using CRF (Controlled-Rate Freezer) equipment were used as the cell freezing method, respectively.

In the slow freezing method, vials containing cells suspended in the cryopreservation agent are placed in a refrigerator container containing isopropyl alcohol, and then the temperature is gradually lowered at a constant rate in an ultra-low temperature refrigerator below -70 ° C for at least 12 hours And then transferred to a liquid nitrogen tank. In the vitrification method using the CRF equipment, the vial suspending the cryopreservation agent was placed in the CRF equipment and the inside temperature of the vial was decreased by 1 ° C per minute to reach -180 ° C. The vial temperature Upon reaching -80 ° C, the vial was immediately taken out and transferred to the LN2 tank, a liquid nitrogen tank.

The thawing of the frozen cells was performed as follows.

After 2 weeks of freezing, cryopreserved stem cells and primary cultured cells were removed and rapidly thawed in a constant temperature water bath at 37 ° C. Then, vials containing cells were cleaned using 70% Cells were washed with Basal Medium, and cells were inoculated on a culture dish and cultured for 24 hours to confirm the effect of the composition for cryopreservation of the present invention.

Example  4: Confirmation of lipid freezing preservation effect

In order to confirm whether the lipid is contained as an active ingredient in the frozen storage liquid and can reduce the damage during the cooling / thawing of stem cells, human serum albumin protein and lipid concentrate, The cryoprotectant was prepared in the same manner as in Example 1.

Composition of the main active ingredient of frozen storage liquid to confirm the effect of lipid Base badge
(Basal medium) Freezing inhibitor
(Cryoprotectant) Serum and nutrients
(Serum & Nutrients) Etc
(Other) MS-GM (CEFO., Korea) DMSO (dimethyl sulfoxide)
5-15% Human Serum Albumin 5-50% - Human Serum Albumin 5-50% Cholesterol Lipid Concentrate
0.1% -1%

The composition of the lipid concentrate was as shown in Table 3 below.

Composition of lipid concentrate Component Arachidonic acid Cholesterol DL-α-Tocopherol acetate Linoleic acid Linolenic acid Myristic acid Oleic acid Palmitoleic acid Palmitic acid Pluronic® F-68 Steraric acid Tween® 80 Mg / L 2 220 70 10 10 10 10 10 10 100,000 10 2,200

The frozen storage solution was prepared according to the composition shown in Table 2, and umbrella-derived stem cells (UCMSC) of 1 x 10 ⁶ cells / mL were suspended therein and frozen by slow freezing method. Then, the cells were stored in an LN2 tank for 7 days and thawed to measure cell viability. The results are shown in Fig. In addition, thawed cells were inoculated on a 100 mm culture dish and cultured for 3 days. Cells were counted to determine cell growth rate. The results are shown in FIG.

As a result, as shown in Fig. 1, when the cryopreservation composition containing albumin further contains lipid, the survival rate of the cells remarkably increased from about 70% to 80% or more. In addition, as shown in FIG. 2, when the cell growth rate when the composition for cryopreservation containing only human serum albumin was 100, the cell growth rate of the composition for cryopreservation further including lipid was 130 or more.

This result shows that the cell viability can be remarkably increased when the lipid is contained as an active ingredient in the frozen preservative solution. In particular, when the lipid concentrate containing cholesterol is additionally contained, especially 0.1% to 1% The results suggest that the cryopreservation effect of stem cells can be significantly increased.

Example  5: Plant protein Hydrolyzate  Confirming the effect of freezing preservation

In order to examine whether the addition of the plant protein hydrolyzate in addition to the plant-derived recombinant human serum albumin protein and lipid could further reduce the damage caused by cold / thawing of the umbilical cord stem cells, the composition of the frozen stock solution is shown in Table 4 Respectively.

Amino acid composition of the plant protein hydrolyzate used herein was as shown in Table 5, and stem cells derived from umbilical cord were used as the cells.

Plant protein Hydrolyzate  The composition of the main active ingredient of the frozen storage liquid for confirming the effect Base badge
(Basal medium) Freezing inhibitor
(Cryoprotectant) Serum and nutrients
(Serum & Nutrients) Etc
(Other) MS-GM (CEFO., Korea) DMSO (dimethyl sulfoxide)
5-15% rhAlbumin and lipid 5-50% - rhAlbumin and lipid 5-50% Plant peptide I & II
1-20%

The frozen embryo-derived stem cells were thawed by the slow freezing method of Example 3, stained with a propidium iodide solution, and then the number of viable cells was measured using a cell counter to determine the amount of the plant protein hydrolyzate Cell cryopreservation. The results are shown in Fig.

As a result, as shown in FIG. 3, when freezing was performed using the gentle freezing method, which is generally used in a laboratory, and thawing was carried out, it was found that, in addition to plant-derived proteins and lipids, To about 10% higher than that of the plant protein hydrolyzate. Thus, it was shown that the cryopreservation effect can be further enhanced when the composition of the present invention, which further contains a plant protein hydrolyzate, is used in the slow freezing method.

These results suggest that the composition for cryopreservation according to the present invention, which further comprises a plant protein hydrolyzate, can be usefully used for lyophilization of cells. Based on these results, the inventors of the present invention produced a composition for cryopreservation further comprising lipid and plant protein hydrolyzate as well as plant-derived recombinant human serum albumin protein, and their cryopreservation effect on stem cells and primary cultured cells Was confirmed in the following examples.

Example  6: bone marrow derived Mesenchymal  Plant-derived recombinant human serum albumin, lipid and plant proteins on cryopreservation Hydrolyzate  Check the effect

To investigate the effect of plant-derived recombinant human serum albumin, lipid and plant protein hydrolysates contained in the composition of the present invention on the damage caused by cold / thawing of bone marrow mesenchymal stem cells, FIG. 4 shows the result of observing the state of stem cells using a microscope on a frozen, thawed bone marrow mesenchymal stem cell through a slow freezing method of FIG.

The number of viable cells was measured with a C-Chip Disposable Hemocytometer (Digital Bio., Korea) and a cell counter (Digital Bio., Korea) using a Pripidium Iodide solution (Sigma, USA) ), And the results are shown in Fig.

As a result, as shown in Fig. 4, in the case of cryopreservation using the plant-derived recombinant human serum albumin, lipid and plant protein hydrolyzate, the preserved cells were inoculated into a culture dish immediately after thawing When cultured, it was observed that healthy stem cells were cultured at higher density than frozen - thawed embryos containing recombinant human serum albumin derived from fetal calf serum or microorganism. As shown in FIG. 5, the survival rate of the stem cells was also cryopreserved using the cryopreservative of the present invention including plant-derived recombinant human serum albumin, lipid and plant protein hydrolyzate, and then thawed, The highest survival rate was about 80%.

The above results show that when the composition for cryopreservation comprising both plant-derived recombinant human serum albumin, lipid and plant protein hydrolyzate is used, the bone marrow-derived mesenchymal stem cells can be cryopreserved It is not only safe but also suggests that the recombinant human serum albumin derived from fetal bovine serum or microorganism, which has been conventionally used, can be stored more stably than when using recombinant human serum albumin.

Example  7: Derived from umbilical cord  Stem Cells( Umbilical cord matrix stem cell ) On the cryopreservation of plant-derived recombinant human serum albumin, lipid and plant proteins Hydrolyzate  Check the effect

In order to investigate the effect of plant-derived recombinant human serum albumin, lipid and plant protein hydrolyzate on damage caused by cold / thawing of umbilical cord stem cells, the embryo-derived stem cells subjected to cold / thawing through the method of Example 3 Was observed using a microscope, and the results are shown in Fig.

The frozen embryo-derived stem cells were stained with a propidium iodide solution (Sigma, USA), and the number of living cells was measured using a C-Chip Disposable Hemocytometer (Digital Bio., Korea) (Adam cell counter, Digital Bio., Korea). The results are shown in FIG.

As a result, as shown in FIG. 6, when the cryopreservation agent of the present invention including plant-derived recombinant human serum albumin, lipid and plant protein hydrolyzate was used to cryopreserve, fetal serum or microbial-derived recombinant human serum albumin The morphologically confirmed that healthy stem cells were cultured at the highest density when cultured on a culture dish immediately after thawing. As shown in FIG. 7, when the survival rate of stem cells was also frozen and thawed using the cryopreservative of the present invention including plant-derived recombinant human serum albumin, lipid and plant protein hydrolyzate, the fetal calf serum or microorganism Derived recombinant human serum albumin, the survival rate was about 80%.

In addition, the frozen cells were thawed by the vitrification-free method rather than the slow freezing method, and then the survival rate of the umbilical cord-derived stem cells was confirmed. The results are shown in FIG.

As a result, in the case of the cryopreservation product of the present invention including plant-derived recombinant human serum albumin, lipid and plant protein hydrolyzate, the survival rate is 92% or more higher than other cryopreservation products containing fetal bovine serum or microbial-derived human serum albumin And the results indicating high survival rate were confirmed (Fig. 8).

The above results show that when the composition for cryopreservation comprising the plant-derived recombinant human serum albumin, lipid and plant protein hydrolyzate of the present invention is used, the mesenchymal stem cells derived from cordocentesis can be cryopreserved without using animal-derived factors This suggests that not only is it safe but also that the recombinant human serum albumin derived from fetal bovine serum or microorganism used conventionally can be stored more stably than the case using recombinant human serum albumin.

In addition, in view of the results and the results of Example 6, the composition comprising plant-derived human serum albumin protein, lipid and plant protein hydrolyzate of the present invention can be used for the cryopreservation of stem cells Suggests that stem cells can be safely and stably cryopreserved for a long period of time in a xeno-free and an animal-free state.

Example  8: Human foreskin  Derived fibroblast ( Human Dermal Fibroblast ) On the cryopreservation of plant-derived recombinant human serum albumin, lipid and plant proteins Hydrolyzate  Check the effect

The effect of plant-derived recombinant human serum albumin, lipid and plant protein hydrolysates contained in the composition of the present invention on the damage caused by cold / thawing of human poppy-derived fibroblasts was examined by the slow freezing method of Example 3 After freezing, the thawed human poppy-derived fibroblasts were stained with a propidium iodide solution (Sigma, USA) and the number of living cells was measured using a C-Chip Disposable Hemocytometer (Digital Bio., Korea) (Adam cell counter; Digital Bio., Korea). The results are shown in FIG.

As a result, as shown in Fig. 9, when frozen storage was carried out using the cryopreservative of the present invention including plant-derived recombinant human serum albumin, lipid and plant protein hydrolyzate, when the human fetoprotein-derived fibroblasts were frozen, Or 93.8%, compared with the other experimental group including the microbial-derived human serum albumin protein.

These results indicate that when the composition for cryopreservation comprising the plant-derived recombinant human serum albumin, lipid and plant protein hydrolyzate of the present invention is used, it is possible to provide a method for the cryopreservation of a primary cell such as human poppy- ) Can be safely frozen and stored, as compared with a composition for cryopreservation comprising human fetal calf serum or microorganism-derived human serum albumin.

Taken together, the results of Examples 6 and 7 and the results show that the composition of the present invention comprising plant-derived recombinant human serum albumin, lipid and plant protein hydrolyzate can be useful for cryopreservation of stem cells as well as primary cultured cells , Indicating that long-term cryopreservation of stem cells and primary cultured cells can be achieved in pure, xeno-free and animal-free conditions.

From the above description, it will be understood by those skilled in the art that the present invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. In this regard, it should be understood that the above-described embodiments are to be considered in all respects as illustrative and not restrictive. The scope of the present invention should be construed as being included in the scope of the present invention without departing from the spirit and scope of the present invention as defined by the appended claims and their equivalents.

&Lt; 110 > CEFO Co., Ltd. <120> A composition for cryopreservation of stem cells or primary cells          comprising plant-derived human serum albumin, plant protein          hydrolyseate and lipid <130> PA120332KR <160> 2 <170> Kopatentin 2.0 <210> 1 <211> 609 <212> PRT <213> Homo sapiens <400> 1 Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala   1 5 10 15 Tyr Ser Arg Gly Val Phe Arg Arg Asp Ala His Lys Ser Glu Val Ala              20 25 30 His Arg Phe Lys Asp Leu Gly Glu Glu Asn Phe Lys Ala Leu Val Leu          35 40 45 Ile Ala Phe Ala Gln Tyr Leu Gln Gln Cys Pro Phe Glu Asp His Val      50 55 60 Lys Leu Val Asn Glu Val Thr Glu Phe Ala Lys Thr Cys Val Ala Asp  65 70 75 80 Glu Ser Ala Glu Asn Cys Asp Lys Ser Leu His Thr Leu Phe Gly Asp                  85 90 95 Lys Leu Cys Thr Val Ala Thr Leu Arg Glu Thr Tyr Gly Glu Met Ala             100 105 110 Asp Cys Cys Ala Lys Gln Glu Pro Glu Arg Asn Glu Cys Phe Leu Gln         115 120 125 His Lys Asp Asp Asn Pro Asn Leu Pro Arg Leu Val Arg Pro Glu Val     130 135 140 Asp Val Met Cys Thr Ala Phe His Asp Asn Glu Glu Thr Phe Leu Lys 145 150 155 160 Lys Tyr Leu Tyr Glu Ile Ala Arg Arg His Pro Tyr Phe Tyr Ala Pro                 165 170 175 Glu Leu Leu Phe Phe Ala Lys Arg Tyr Lys Ala Ala Phe Thr Glu Cys             180 185 190 Cys Gln Ala Ala Asp Lys Ala Ala Cys Leu Leu Pro Lys Leu Asp Glu         195 200 205 Leu Arg Asp Glu Gly Lys Ala Ser Ser Ala Lys Gln Arg Leu Lys Cys     210 215 220 Ala Ser Leu Gln Lys Phe Gly Glu Arg Ala Phe Lys Ala Trp Ala Val 225 230 235 240 Ala Arg Leu Ser Gln Arg Phe Pro Lys Ala Glu Phe Ala Glu Val Ser                 245 250 255 Lys Leu Val Thr Asp Leu Thr Lys Val His Thr Glu Cys Cys His Gly             260 265 270 Asp Leu Leu Glu Cys Ala Asp Asp Arg Ala Asp Leu Ala Lys Tyr Ile         275 280 285 Cys Glu Asn Gln Asp Ser Ile Ser Ser Lys Leu Lys Glu Cys Cys Glu     290 295 300 Lys Pro Leu Leu Glu Lys Ser His Cys Ile Ala Glu Val Glu Asn Asp 305 310 315 320 Glu Met Pro Ala Asp Leu Pro Ser Leu Ala Ala Asp Phe Val Glu Ser                 325 330 335 Lys Asp Val Cys Lys Asn Tyr Ala Glu Ala Lys Asp Val Phe Leu Gly             340 345 350 Met Phe Leu Tyr Glu Tyr Ala Arg Arg His Pro Asp Tyr Ser Val Val         355 360 365 Leu Leu Leu Arg Leu Ala Lys Thr Tyr Glu Thr Thr Leu Glu Lys Cys     370 375 380 Cys Ala Ala Ala Asp Pro His Glu Cys Tyr Ala Lys Val Phe Asp Glu 385 390 395 400 Phe Lys Pro Leu Val Glu Glu Pro Gln Asn Leu Ile Lys Gln Asn Cys                 405 410 415 Glu Leu Phe Glu Gln Leu Gly Glu Tyr Lys Phe Gln Asn Ala Leu Leu             420 425 430 Val Arg Tyr Thr Lys Lys Val Pro Gln Val Ser Thr Pro Thr Leu Val         435 440 445 Glu Val Ser Arg Asn Leu Gly Lys Val Gly Ser Lys Cys Cys Lys His     450 455 460 Pro Glu Ala Lys Arg Met Pro Cys Ala Glu Asp Tyr Leu Ser Val Val 465 470 475 480 Leu Asn Gln Leu Cys Val Leu His Glu Lys Thr Pro Val Ser Asp Arg                 485 490 495 Val Thr Lys Cys Cys Thr Glu Ser Leu Val Asn Arg Arg Pro Cys Phe             500 505 510 Ser Ala Leu Glu Val Asp Glu Thr Tyr Val Pro Lys Glu Phe Asn Ala         515 520 525 Glu Thr Phe Thr Phe His Ala Asp Ile Cys Thr Leu Ser Glu Lys Glu     530 535 540 Arg Gln Ile Lys Lys Gln Thr Ala Leu Val Glu Leu Val Lys His Lys 545 550 555 560 Pro Lys Ala Thr Lys Glu Gln Leu Lys Ala Val Met Asp Asp Phe Ala                 565 570 575 Ala Phe Val Glu Lys Cys Cys Lys Ala Asp Asp Lys Glu Thr Cys Phe             580 585 590 Ala Glu Glu Gly Lys Lys Leu Val Ala Ala Ser Gln Ala Ala Leu Gly         595 600 605 Leu     <210> 2 <211> 2264 <212> DNA <213> Homo sapiens <400> 2 agtatattag tgctaatttc cctccgtttg tcctagcttt tctcttctgt caaccccaca 60 cgcctttggc acaatgaagt gggtaacctt tatttccctt ctttttctct ttagctcggc 120 ttattccagg ggtgtgtttc gtcgagatgc acacaagagt gaggttgctc atcggtttaa 180 agatttggga gaagaaaatt tcaaagcctt ggtgttgatt gcctttgctc agtatcttca 240 gcagtgtcca tttgaagatc atgtaaaatt agtgaatgaa gtaactgaat ttgcaaaaac 300 atgtgttgct gatgagtcag ctgaaaattg tgacaaatca cttcataccc tttttggaga 360 caaattatgc acagttgcaa ctcttcgtga aacctatggt gaaatggctg actgctgtgc 420 aaaacaagaa cctgagagaa atgaatgctt cttgcaacac aaagatgaca acccaaacct 480 cccccgattg gtgagaccag aggttgatgt gatgtgcact gcttttcatg acaatgaaga 540 gacatttttg aaaaaatact tatatgaaat tgccagaaga catccttact tttatgcccc 600 ggaactcctt ttctttgcta aaaggtataa agctgctttt acagaatgtt gccaagctgc 660 tgataaagct gcctgcctgt tgccaaagct cgatgaactt cgggatgaag ggaaggcttc 720 gtctgccaaa cagagactca agtgtgccag tctccaaaaa tttggagaaa gagctttcaa 780 agcatgggca gtagctcgcc tgagccagag atttcccaaa gctgagtttg cagaagtttc 840 caagttagtg acagatctta ccaaagtcca cacggaatgc tgccatggag atctgcttga 900 atgtgctgat gacagggcgg accttgccaa gtatatctgt gaaaatcaag attcgatctc 960 cagtaaactg aaggaatgct gtgaaaaacc tctgttggaa aaatcccact gcattgccga 1020 agtggaaaat gatgagatgc ctgctgactt gccttcatta gctgctgatt ttgttgaaag 1080 taaggatgtt tgcaaaaact atgctgaggc aaaggatgtc ttcctgggca tgtttttgta 1140 tgaatatgca agaaggcatc ctgattactc tgtcgtgctg ctgctgagac ttgccaagac 1200 atatgaaacc actctagaga agtgctgtgc cgctgcagat cctcatgaat gctatgccaa 1260 agtgttcgat gaatttaaac ctcttgtgga agagcctcag aatttaatca aacaaaattg 1320 tgagcttttt gagcagcttg gagagtacaa attccagaat gcgctattag ttcgttacac 1380 caagaaagta ccccaagtgt caactccaac tcttgtagag gtctcaagaa acctaggaaa 1440 agtgggcagc aaatgttgta aacatcctga agcaaaaaga atgccctgtg cagaagacta 1500 tctatccgtg gtcctgaacc agttatgtgt gttgcatgag aaaacgccag taagtgacag 1560 cgctctgg 1620 agtcgatgaa acatacgttc ccaaagagtt taatgctgaa acattcacct tccatgcaga 1680 tatatgcaca ctttctgaga aggagagaca aatcaagaaa caaactgcac ttgttgagct 1740 cgtgaaacac aagcccaagg caacaaaaga gcaactgaaa gctgttatgg atgatttcgc 1800 agcttttgta gagaagtgct gcaaggctga cgataaggag acctgctttg ccgaggaggg 1860 taaaaaactt gttgctgcaa gtcaagctgc cttaggctta taacatcaca tttaaaagca 1920 tctcagccta ccatgagaat aagagaaaga aaatgaagat caaaagctta ttcatctgtt 1980 tttctttttc gttggtgtaa agccaacacc ctgtctaaaa aacataaatt tctttaatca 2040 ttttgcctct tttctctgtg cttcaattaa taaaaaatgg aaagaatcta atagagtggt 2100 acagcactgt tatttttcaa agatgtgttg ctatcctgaa aattctgtag gttctgtgga 2160 agttccagtg ttctctctta ttccacttcg gtagaggatt tctagtttct tgtgggctaa 2220 ttaaataaat cattaatact cttctaaaaa aaaaaaaaaa aaaa 2264

Claims (10)

A composition for cryopreservation of stem cells or primary cells, comprising plant-derived recombinant human serum albumin, lipids and plant protein hydrolysates as active ingredients.
The composition according to claim 1, wherein said composition comprises 10 to 40 parts by weight of plant-derived recombinant human serum albumin, 0.1 to 1 part by weight of lipid and 1 to 10 parts by weight of plant protein hydrolyzate, &Lt; / RTI &gt;
2. The composition of claim 1, wherein the lipid comprises cholesterol.
The composition of claim 1, wherein the composition comprises 5 to 50 parts by weight of plant-derived recombinant human serum albumin per 100 parts by weight of the total composition.
2. The composition of claim 1, wherein the composition comprises lipid in an amount of 0.1 to 5 parts by weight based on 100 parts by weight of the total composition.
The composition of claim 1, wherein the composition comprises plant protein hydrolyzate in an amount of 1 to 40 parts by weight per 100 parts by weight of the total composition.
The composition according to claim 1, wherein the stem cells are bone marrow-derived mesenchymal stem cells or umbilical cord mesenchymal stem cells, and the primary cultured cells are fibroblasts.
The composition of claim 1, wherein the composition further comprises a cryoprotectant.
9. The composition of claim 8, wherein the freeze inhibitor is selected from the group consisting of dimethylsulfoxide (DMSO), glycerol, propylene glycol, ethylene glycol, sucrose, carboxymethylcellulose salt, carboxymethylcellulose (CMC), polysaccharides and disaccharides Composition.
10. A method for cryopreserving stem cells or primary cultured cells, comprising the step of freezing stem cells or primary cultured cells treated with the composition of any one of claims 1 to 9.

Images