WO2018199482A1

WO2018199482A1 - Composition for cryopreservation of cells or tissues including mesenchymal stem cell culture medium

Info

Publication number: WO2018199482A1
Application number: PCT/KR2018/003579
Authority: WO
Inventors: Jang Ho Lee
Original assignee: Individual
Current assignee: Individual
Priority date: 2017-04-26
Filing date: 2018-03-27
Publication date: 2018-11-01
Anticipated expiration: 2019-10-26
Also published as: KR102018609B1; KR20180119924A

Abstract

Provided are a composition for cryopreservation of cells or tissues, including human mesenchymal stem cell-derived proteins, and a preparation method thereof. Since the composition of the present disclosure includes no serum, it may be safely used without the risk of infection, etc., and may be also applied to cryopreservation of all kinds of cells or tissues, irrespective of an origin or a kind thereof.

Description

COMPOSITION FOR CRYOPRESERVATION OF CELLS OR TISSUES INCLUDING MESENCHYMAL STEM CELL CULTURE MEDIUM

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of Korean Patent Application No. 10-2017-0053653, filed on April 26, 2017, in the Korean Intellectual Property Office, the disclosure of which is incorporated herein in its entirety by reference.

The present disclosure relates to a composition for cryopreservation of cells or tissues, including a human mesenchymal stem cell culture medium, and a preparation method thereof.

Cryopreservation of cells, tissues, or organs is very important in view of their various applications including development of new drugs such as cell therapeutic agents, etc., treatment of intractable diseases/incurable diseases, and organ transplantation. In particular, stem cells are expected to greatly contribute to the treatment of incurable diseases and intractable diseases which are impossible or difficult to treat until now, and cell therapy using stem cells is the most promising next-generation biopharmaceutical field, and many researches and developments and clinical trials are under way. Thus, it is essential to cryopreserve various types of cells as a source of cell therapy in a safe and efficient manner, and particularly, in order to minimize immune rejection, patient's own stem cells may be used after being preserved for a predetermined period of time. Therefore, cryopreservation of cells without damage is emerging as a more important issue.

However, a method of safely preserving a large amount of cells with enough quality to be used immediately for therapeutic purposes has not been clearly established. In particular, unlike other adult cells, stem cells tend to easily die or differentiate when they are thawed after cryopreservation, and it is known that stem cells thawed after cryopreservation have a property to easily differentiate during subculture. A previously established embryo preservation method is applied to a cryopreservation method of stem cells. However, when this method is applied to stem cells, viability of stem cells is only about 10% whereas viability of embryos reaches 80% or more. Therefore, it is urgent to develop a novel cryopreservation method suitable for human stem cells.

Meanwhile, a composition containing heterologous proteins such as fetal bovine serum, etc. is generally used in cell cryopreservation. However, use of animal-derived serum is associated with the risk of infection of cells with animal-derived viruses or infectious prions such as mad cow disease during culture, and they act as contaminants during cell culture to produce various toxins, which may inhibit cell culture or cause a variation of cell surface. Further, there are problems that expensive serums raise the price of the composition and it is difficult to secure bioequivalence due to difference of components in each production batch.

To overcome these problems, several attempts have been made to prepare a composition for cell cryopreservation by using recombinant human serum albumin, etc. (Korean Patent No. 10-1407355), and to use human serum instead of fetal bovine serum (Transplantation. 2000 Dec 27;70(12):1780-1787). However, these methods generate problems that the intrinsic characteristics of stem cells are modified, e.g., a proliferation or differentiation ability of the stem cells is reduced or even promoted.

Under this background, the present inventors have made many efforts to develop a safe composition for cryopreservation of cells or tissues which does not modify cell characteristics even after thawing, and as a result, they found that cells cryopreserved in a composition including a culture medium containing human mesenchymal stem cell-derived proteins exhibit excellent viability, proliferation rate, or protein secretion ability even after being thawed, thereby completing the present disclosure.

An object of the present disclosure is to provide a composition for cryopreservation of cells or tissues, the composition including a human mesenchymal stem cell culture medium.

Another object of the present disclosure is to provide a method of preparing the composition.

To achieve the above objects, an aspect of the present disclosure provides a composition for cryopreservation of cells or tissues, the composition including a human mesenchymal stem cell culture medium.

In the present disclosure, it was confirmed that when the human mesenchymal stem cell culture medium, specifically, the culture medium containing human mesenchymal stem cell-derived proteins is used, various kinds of human- or animal-derived cells or tissues may be cryopreserved without damage for a long period of time.

In a known method of cryopreserving cells or tissues, fetal bovine serum (FBS) is generally used, but use of serum components are disadvantages in that there is the risk of infection with animal-derived viruses or infectious prions, and cells thawed after cryopreservation have low viabilities. The present disclosure confirmed that the human mesenchymal stem cell culture medium may replace FBS, thereby overcoming the above-described technical limitations.

The term "cryopreservation", as used herein, refers to a process by which cells or tissues are stably preserved via freezing for a long period time. About one out of ten thousand of cells generally mutate during culture, and cells are changed to different cell populations from the original cell populations by continuing cell subculture over a long period of time, in severe cases, specific functions of cells are lost by subculture. Further, cells may be infected with mycoplasma during subculture. Due to these problems, cells or tissues are frozen and preserved before loss of their own characteristics, and then taken out and used when needed. To use stem cells as therapeutic agents, healthy stem cells must be immediately available when needed, and therefore, methods of effectively cryopreserving stem cells are particularly needed.

The term "mesenchymal stem cell", as used herein, refers to a stem cell that is the origin of cartilages, bones, fats, bone marrow stroma, muscles, nerves, etc., and in adult, mesenchymal stem cells usually remain in the bone marrow, but also exist in other tissues such as cord blood, peripheral blood, fat, amniotic fluid, etc., and thus mesenchymal stem cells may be stem cells obtained therefrom. Specifically, the mesenchymal stem cells may be derived from fat, bone marrow, umbilical cord blood, amniotic fluid, or amniotic membrane, but are not limited thereto. In the present disclosure, the mesenchymal stem cells may be derived from humans or mammals including humans. Therefore, a culture medium of mesenchymal stem cells of mammals including humans may also be used for the same purpose as the culture of human mesenchymal stem cells of the present disclosure.

The term "culture medium", as used herein, refers to a medium that allows growth and survival of cells in vitro, and includes all kinds of media suitable for cell culture which are commonly used in the art. With respect to the objects of the present disclosure, the culture medium included in the composition of the present disclosure may be used in culturing human mesenchymal stem cells.

Further, the culture medium and culture conditions may be selected according to a kind of cells. For example, the culture medium may be one or more selected from the group consisting of a complete Dulbecco's modified eagle medium (cDMEM), a Dulbecco's modified eagle medium (DMEM), a minimal essential medium (MEM), an α-MEM, a McCoys 5A medium, an eagle's basal medium, Connaught Medical Research Laboratory (CMRL) medium, Glasgow MEM, Ham's F-12 medium, Iscove's modified Dulbecco's medium (IMDM), Liebovitz' L-15 medium, Roswell Park Memorial Institute (RPMI) 1640 medium, and mixed media thereof, and more specifically, cDMEM, but is not limited thereto.

Furthermore, with respect to the objects of the present disclosure, the culture medium may be a serum-free medium. Generally, serum is essentially added to a culture medium when cells are cultured ex vivo. As described above, since serum components have the risk of infection with animal-derived viruses or infectious prions, the composition of the present disclosure includes no serum.

In the present disclosure, the human mesenchymal stem cell culture may include human mesenchymal stem cell-derived proteins.

The term "human mesenchymal stem cell-derived proteins", as used herein, refer to proteins secreted from human mesenchymal stem cells. The proteins may include human mesenchymal stem cell-derived growth factors or cytokines, and a total concentration of the proteins may be 30 ㎍/ml to 70 ㎍/ml, as measured by a BCA assay.

Specifically, the proteins may include AR, BDNF, bFGF, BMP-4, BMP-5, BMP-7, b-NGF, EGF-R, FGF-4, FGF-7, GDF-15, GDNF, HGF, IGFBP-1, IGFBP-2, IGFBP-3, IGFBP-4, IGFBP-6, IGF-1, Insulin, MCSF, MCSF-R, NGF-R, NT-3, NT-4, OPG, PDGF-AA, PIGF, SCF, SCF-R, TGF-α, TGF-β1, VEGF, VEGF-R3, ICAM-1, G-CSF, IL-1α, IL-2, IL-5, IL-6, IL-8, IL-11, MCP-1, MIG, MIP-1a, MIP-b, MIP-d, TIMP-1, TIMP-2, TNFα, TNFβ, TNF-R1, or TNF-R11.

The term "androgen receptor (AR)", as used herein, is a kind of nuclear receptor that is activated by binding either of androgenic hormones, testosterone, or dihydrotestosterone. It is known that AR functions as a DNA-binding transcription factor that regulates gene expression, and is critical for the development and maintenance of the male sexual phenotype.

The term "brain-derived neurotrophic factor (BDNF)", as used herein, is a brain-derived neurotrophic factor that acts on certain neurons of the central nervous system and the peripheral nervous system, helps to support the survival of existing neurons, and encourages growth and differentiation of new neurons and synapses.

The term "bone morphogenetic protein 4 (BMP-4)", as used herein, is a kind of bone morphogenetic proteins belonging to the TGF-β superfamily. It is known that BMP-4 is involved in bone and cartilage development, specifically tooth and limb development and fracture repair.

The term "bone morphogenetic protein 5 (BMP-5)", as used herein, is a kind of the TGF-β superfamily. It is known that BMP-5 functions to induce bone and cartilage development.

The term "bone morphogenetic protein 6 (BMP-6)", as used herein, is a kind of the TGF-β superfamily. It is known that BMP-6 induces growth of bone and cartilage and induces expression of bone morphogenetic factor in mesenchymal stem cells.

The term "bone morphogenetic protein 7 (BMP-7)", as used herein, is a kind of the TGF-β superfamily. It is known that like other bone morphogenetic proteins (BMP family), BMP-7 also induces growth of bone and cartilage and plays a critical role in conversion of mesenchymal stem cells into bone or cartilage.

The term "beta-nerve growth factor (b-NGF)", as used herein, is a neurotrophic factor and neuropeptide. It is known that b-NGF is primarily involved in the regulation of growth, maintenance, proliferation, and survival of certain target neurons.

The term "fibroblast growth factor 2 (bFGF)", as used herein, is known to play an important role in the regulation of cell survival, cell division, angiogenesis, cell differentiation, and cell migration, and also in wound healing.

The term "epidermal growth factor receptor (EGF-R)", as used herein, is an epidermal growth factor receptor that functions as a tyrosine kinase.

The term "fibroblast growth factor 4 (FGF-4)", as used herein, is a protein belonging to the FGF family. It is known that FGF-4 acts as a developmental protein, a growth factor, and a mitogen, and plays an important role in the regulation of embryonic development, cell proliferation, and cell differentiation.

The term "fibroblast growth factor 7 (FGF-7)", as used herein, is a protein belonging to the FGF family. It is known that FGF-7 acts as a growth factor of keratinocytes and acts on normal epithelial cell proliferation, and also plays an important role in epithelialization, re-epithelialization of wound, hair development, and lung development.

The term "growth/differentiation factor 15 (GDF-15)", as used herein, is a protein belonging to the TGF-superfamily, and also known as TGF-PL, MIC-1, PDF, PLAB, or PTGFB. It is known that GDF-15 plays a role in the regulation of inflammatory and apoptotic pathways of disease processes in injured tissues.

The term "glial cell-derived neurotrophic factor (GDNF)", as used herein, is a protein promoting neuronal survival. It is known that GDNF plays a role in the promotion of survival of dopaminergic motor neurons.

The term "hepatocyte growth factor (HGF)", as used herein, refers to a protein that is secreted from various kinds of mesenchymal cells. It is known that HGF acts to promote cell proliferation, cell motility, and epithelial morphogenesis, and functions as a neurotrophic factor and an angiogenic factor.

The term "insulin-like growth factor-binding protein 1 (IGFBP-1)", as used herein, is an insulin-like growth factor-binding protein that prolongs the half-life of IGF to either inhibit or stimulate growth-promoting effects of IGFs on cell culture, and also promotes cell migration.

The term "insulin-like growth factor-binding protein 2 (IGFBP-2)", as used herein, refers to an insulin-like growth factor-binding protein.

The term "insulin-like growth factor-binding protein 3 (IGFBP-3)", as used herein, is an insulin-like growth factor-binding protein that circulates in the plasma as a complex with insulin-like growth factor acid-labile subunit (IGFALS). It is known that IGFBP-3 prolongs the half-life of IGF to stimulate growth promoting effects of IGF on cell culture.

The term "insulin-like growth factor-binding protein 4 (IGFBP-4)", as used herein, is known to bind with IGF to circulate in the plasma in both glycosylated and non-glycosylated forms, and to prolong the half-life of IGF to either inhibit or stimulate growth promoting effects of IGFs on cell culture.

The term "insulin-like growth factor-binding protein 6 (IGFBP-6)", as used herein, is known to bind by interaction of a cell surface receptor of the protein and IGF and to prolong the half-life of IGF to either inhibit or stimulate growth promoting effects of IGFs on cell culture.

The term "insulin-like growth factor 1 (IGF-1)", as used herein, is an insulin-like growth factor and also called somatomedin C. it is known that IGF-1 has a similar molecular structure to insulin and plays an important role in childhood growth and continues to have anabolic effects in adults.

The term "insulin", as used herein, refers to a hormone protein that promotes glucose uptake from blood to adipocytes, hepatocytes, and skeletal muscle cells to regulate carbohydrate, fat, and protein metabolisms.

The term "macrophage colony-stimulating factor (MCSF)", as used herein, refers to a protein that influences hematopoietic stem cells to differentiate into macrophages or other related cell types. It is known that MCSF is produced in eukaryotic cells against viral infection, and is associated with placental development.

The term "macrophage colony-stimulating factor receptor (MCSF-R)", as used herein, refers to a receptor protein of colony stimulating factor 1 (CSF1).

The term "nerve growth factor receptor (NGF-R)", as used herein, refers to a growth factor receptor protein specifically binding to neurotrophins.

The term "neurotrophin-3 (NT-3)", as used herein, refers to a neurotrophic factor that exists in brain and peripheral tissues and contributes to promotion and regulation of neurogenesis. Further, it is known that NT-3 enhances the survival of visceral sensory neurons and proprioceptive sensory neurons, and is expressed together with FGF5, TGF-1, etc. in the catagen phase of hair cycle.

The term "neurotrophin-4 (NT-4)", as used herein, is a neurotrophic factor that mediates signals through TrkB receptor tyrosine kinase.

The term "tumor necrosis factor receptor superfamily member 11B (OPG)", as used herein, is also known as osteoprotegerin (OPG) and osteoclastogenesis inhibitory factor (OCIF). It is known that OPG inhibits negatively regulates osteoclastogenesis (bone resorption) and inhibits the activation of osteoclasts and promotes osteoclast apoptosis ex vivo.

The term "platelet-derived growth factor subunit A (PDGF-AA)", as used herein, refers to a growth factor that plays an essential role in the regulation of embryonic development, cell proliferation, cell migration, survival and chemotaxis. It is known that PDGF-AA acts as a mitogen for cells of mesenchymal origin and plays an important role in wound healing.

The term "placenta growth factor (PIGF)", as used herein, is a placental growth factor that acts on angiogenesis and endothelial cell growth, and stimulation and activation of their proliferation and migration.

The term "stem cell factor (SCF)", as used herein, refers to a cytokine that binds to c-KIT receptor (CD117). It is known that SCF exists as a transmembrane protein or a soluble protein and plays a critical role in hematopoiesis (blood cell formation), spermatogenesis, and melanogenesis.

The term "stem cell factor receptor (SCF-R)", as used herein, is a stem cell factor receptor protein, also called KIT or C-kit receptor. It is known that SCF-R binds to a stem cell factor and is expressed in hematopoietic stem cells as well as in other cell types.

The term "transforming growth factor alpha (TGF-α)", as used herein, is a member of the epidermal growth factor (EGF) family and is a ligand of epidermal growth factor receptor activating signal transduction pathway for cell proliferation, differentiation, and development, and is also known as a mitogenic polypeptide.

The term "transforming growth factor-β1 (TGF-β1)", as used herein, refers to a multifunctional protein that positively and negatively regulates other growth factors, and controls proliferation, differentiation, and other functions in many cell types. TGF-β1 plays an important role in bone remodeling by stimulating osteoblastic bone formation, and also role in wound healing. It is also known that TGF-β1 plays an important role in controlling the immune system, and most immune cells secrete TGF-β1.

The term "vascular endothelial growth factor (VEGF)", as used herein, is a developmental protein, growth factor, or mitogen that functions to activate neovascularization, angiogenesis, and endothelial cell growth. Further, VEGF plays a role in inducing endothelial cell proliferation, promoting cell migration, inhibiting cell death, and promoting vascular permeability.

The term "transforming growth factor alpha receptor 3 (VEGF-R3)", as used herein, is a receptor protein of VEGF, and a complex of VEGF-R3 and VEGF is known to increase VEGF signaling activity in endothelial cells.

The term "intercellular adhesion molecule 1 (ICAM-1)", as used herein, is a kind of intercellular adhesion molecules and is a type of inflammatory protein involved in adhesion and migration of inflammatory cells. It is known that ICAM-1 induces structural changes of the vascular inner cells and converts cells of the immune system.

The term "granulocyte-colony stimulating factor (G-CSF)", as used herein, is a glycoprotein that stimulates the bone marrow to produce granulocytes and stem cells and to release them into the bloodstream. It is known that G-CSF stimulates survival, proliferation, differentiation, and function of neutrophil precursors and mature neutrophils.

The term "interleukin-1 alpha (IL-1α)", as used herein, refers to a cytokine produced by activated macrophages, neutrophils, epithelial cells, and endothelial cells. It is known that IL-1α has metabolic, physiological, and hematopoietic functions and plays a central role in the regulation of immune responses.

The term "interleukin-2 (IL-2)", as used herein, is a kind of cytokine signaling molecules in the immune system and is a protein that regulates the functions of leukocytes (lymphocytes). It is known that IL-2 is involved in the body's natural reaction against microbial infection and distinguishes between self and non-self.

The term "interleukin-5 (IL-5)", as used herein, functions to stimulate B cell growth and to increase secretion of immunoglobulin and acts as a major mediator of eosinophil activation.

The term "interleukin-6 (IL-6)", as used herein, is a protein that induces final differentiation of beta cells into antibody-producing cells, and functions on muscle cells and forms bone marrow cells.

The term "interleukin-8 (IL-8)", as used herein, is known to induce chemotaxis in target cells, primarily neutrophils or granulocytes, causing them to migrate toward the site of infection, and to induce phagocytosis.

The term "interleukin 11 (IL-11)", as used herein, is a protein that affects B lymphocyte differentiation, proliferation and differentiation of hematopoietic stem cells, proliferation and maturation of megakaryocytes, and action on hematopoiesis.

The term "monocyte chemoattractant protein-1 (MCP-1)", as used herein, is a CC chemokine that selectively induces monocytes, lymphocytes, and basophils, and plays an important role in the pathophysiology of inflammatory renal disease.

The term "gamma interferon (MIG)", as used herein, refers to a dimerized soluble cytokine which is a member of the type II class of interferons and plays a critical role in innate and adaptive immunity against viral, some bacterial and protozoal infections.

The term "macrophage inflammatory protein 1a (MIP-1a)", as used herein, is a macrophage inflammatory protein, also called CCL3, which play a crucial role in the immune response to infection and inflammation. It is known that MIP-1a induces the synthesis and release of other pro-inflammatory cytokines such as IL-1, IL-6, and TNF-α from fibroblasts and macrophages.

The term "macrophage inflammatory proteins b (MIP-b)", as used herein, is a kind of macrophage inflammatory proteins that plays a crucial role in the immune response to infection and inflammation.

The term "macrophage Inflammatory Proteins d (MIP-d)", as used herein, is a kind of macrophage inflammatory proteins that plays a crucial role in the immune response to infection and inflammation.

The term "TIMP metallopeptidase inhibitor 1 (TIMP-1)", as used herein, is a constitutive protein of tissue inhibitors of metalloproteinases (TIMP), which acts as an inhibitor of matrix metalloproteinases (MMP) and plays a role in preventing apoptosis.

The term "TIMP metallopeptidase inhibitor 2 (TIMP-2)", as used herein, is a constitutive protein of TIMP, which is known to regulate melanocytes by microphthalmia-associated transcription factor (MITF).

The composition of the present disclosure may include the human mesenchymal stem cell culture medium including the human mesenchymal proteins at a ratio of about 5%(v/v) to about 50%(v/v), specifically about 18%(v/v) to about 22%(v/v), and most specifically about 20%(v/v), but is not limited thereto. When the composition includes the culture medium at the above ratio, the cryopreservation effect of cells or tissues is improved. When the composition includes the culture medium at a ratio lower or higher than the above ratio, there are disadvantages that viability of the cells or tissues after thawing is reduced, and intrinsic characteristics of the cells are deteriorated.

The composition of the present disclosure may be applied to all kind of cells or tissues which are needed to be cryopreserved.

In other words, the composition is not limited to an origin or a kind of cells or tissues, and specifically, the composition may be applied to human- or animal-derived cells or tissues. In the case of cells, the composition may be applied to undifferentiated stem cells or differentiated cells, and applied to germ cells such as eggs, sperms, etc., or various somatic cells, but is not limited thereto. In the case of tissues, the composition may be applied to various tissues such as epithelial tissues, connective tissues, cartilage tissues, bone tissues, muscle tissues, nervous tissues, etc., and furthermore, applied to organs, but is not limited thereto.

In a specific embodiment of the present disclosure, the human mesenchymal stem cell culture medium including human mesenchymal stem cell-derived proteins containing many different growth factors or cytokines was obtained (Example 1). It was confirmed that when a composition including the culture medium was used to cryopreserve human amniotic fluid-derived mesenchymal stem cells or mouse-derived ovarian cells, the cells after thawing showed superior cell viability, proliferation rate, and protein secretion ability, as compared to those cryopreserved by a known method of using FBS or by using a commercially available cell freezing medium CRYO-GOLD, and when the composition including about 20% of the culture medium was used, cell preservation effects were maximized (FIGS. 1 to 7).

These results indicate that the composition including the human mesenchymal stem cell culture medium of the present disclosure may be usefully applied to cryopreservation of cells or tissues.

In another aspect provides a method of preparing the composition for cryopreservation of cells or tissues, the method including producing the human mesenchymal stem cell culture medium including the human mesenchymal stem cell-derived proteins.

With respect to the objects of the present disclosure, the producing of the culture medium may include (a) seeding human mesenchymal stem cells at a density of about 18,000 cells/㎠ to about 22,000 cells/㎠; (b) culturing the stem cells in a serum-free medium; and (c) collecting the stem cell culture medium at about 114 hours to about 126 hours after culture, thereby obtaining the human mesenchymal stem cell-derived proteins as much as possible.

In this regard, the above procedures are included in a method of producing a large amount of mesenchymal stem cell-derived proteins, which is described in Korean Patent No. 10-1566450. The method described in Korean Patent No. 10-1566450 is included in the scope of the present disclosure.

Specifically, (a) is a procedure of dispending and seeding the human mesenchymal stem cells at an appropriate density, particularly, at a density of about 18,000 cells/㎠ to about 22,000 cells/㎠. A kind and an amount of proteins secreted therefrom may vary depending on the density of the cells to be seeded, and therefore, in the present disclosure, a cell density at which production of the proteins may be maximized may be used.

In the present disclosure, the density of the cells to be seeded may be about 18,000 cells/㎠ to about 22,000 cells/㎠, specifically about 19,000 cells/㎠ to about 20,000 cells/㎠, and most specifically about 20,000 cells/㎠.

Next, (b) is a procedure of culturing the human mesenchymal stem cells in a serum-free medium. Animal-derived serum has risks of causing an immune response or a specific disease, and the possibility of side effects caused by application of cells cultured in a serum-containing medium or a cell culture medium thereof to humans has been pointed out, and there is also a difficulty in conducting clinical trials. Accordingly, in the present disclosure, human mesenchymal stem cells are cultured in a serum-free medium in order to obtain a cell culture medium safe enough to be applied to the human body.

Lastly, (c) is a procedure of collecting the stem cell culture medium at about 114 hours to about 126 hours after culturing the human mesenchymal stem cells in the serum-free medium. Depending on the time, a kind and an amount of the proteins secreted therefrom may vary and the content of the proteins in the culture medium may also vary, and therefore, in the present disclosure, the culture time for which production of the proteins may be maximized may be used.

In the present disclosure, the time for culturing the stem cells may be about 114 hours to about 126 hours, specifically about 117 hours to about 123 hours, and most specifically about 120 hours.

In a specific embodiment of the present disclosure, the human mesenchymal stem cells were seeded in a DMEM/F12 serum-free medium at a density of about 20,000 cells/㎠, and then cultured for about 120 hours, and human mesenchymal stem cell-derived proteins were collected from the stem cell culture medium (Example 1). Further, it was confirmed that when the composition including about 20% of the culture medium including the proteins was used to cryopreserve cells, preservation effects of human amniotic fluid-derived mesenchymal stem cells or mouse-derived ovarian cells were maximized after thawing (FIGS. 1 to 7).

Since the composition of the present disclosure includes no serum, it may be safely used without the risk of infection, and may be also applied to cryopreservation of all kinds of cells or tissues, irrespective of an origin or a kind thereof.

Reference will now be made in detail to embodiments, examples of which are illustrated in the accompanying drawings, wherein like reference numerals refer to like elements throughout. In this regard, the present embodiments may have different forms and should not be construed as being limited to the descriptions set forth herein. Accordingly, the embodiments are merely described below, by referring to the figures, to explain aspects of the present description.

These and/or other aspects will become apparent and more readily appreciated from the following description of the embodiments, taken in conjunction with the accompanying drawings in which:

FIG. 1 is a graph showing a comparison of cell viabilities after thawing of human mesenchymal stem cells cryopreserved in a cryopreservation composition prepared according to one embodiment of the present disclosure, in which Control group 1 represents a composition containing 20% FBS, Control group 2 represents a commercially available cryopreservation composition CYRO-GOLD. The composition of the present disclosure is prepared to include 5% to 50% of a culture medium containing human mesenchymal stem cell-derived proteins (human protein complex from human mesenchymal stem cell, hereinafter, referred to as hPC);

FIG. 2 is a graph showing a comparison of cell proliferation rates after thawing of human mesenchymal stem cells cryopreserved in a cryopreservation composition prepared according to one embodiment of the present disclosure, in which Control group 1 represents a composition containing 20% FBS, Control group 2 represents a commercially available cryopreservation composition CYRO-GOLD. The composition of the present disclosure is prepared to include 5% to 50% of a culture medium containing hPC;

FIG. 3 is a graph showing a comparison of protein secretion abilities after thawing of human mesenchymal stem cells cryopreserved in a cryopreservation composition prepared according to one embodiment of the present disclosure, in which Control group represents a composition containing 20% FBS, and the composition of the present disclosure is prepared to include 5% to 50% of a culture medium containing hPC;

FIG. 4 is a graph showing a comparison of cell viabilities after thawing of CHO-K1 cells cryopreserved at -80℃ in a cryopreservation composition prepared according to one embodiment of the present disclosure, in which Control group represents a composition containing 20% FBS, and the composition of the present disclosure is prepared to include 5% to 50% of a culture medium containing hPC;

FIG. 5 is a graph showing a comparison of cell proliferation rates after thawing of CHO-K1 cells cryopreserved at -80℃ in a cryopreservation composition prepared according to one embodiment of the present disclosure, in which Control group represents a composition containing 20% FBS, and the composition of the present disclosure is prepared to include 5% to 50% of a culture medium containing hPC;

FIG. 6 is a graph showing a comparison of cell proliferation rates after thawing of CHO-K1 cells cryopreserved at -196℃ in a cryopreservation composition prepared according to one embodiment of the present disclosure, in which Control group represents a composition containing 20% FBS, and the composition of the present disclosure is prepared to include 5% to 50% of a culture medium containing hPC; and

FIG. 7 is a graph showing a comparison of cell viabilities after thawing of CHO-K1 cells cryopreserved at -196℃ or -80℃ in a cryopreservation composition prepared according to one embodiment of the present disclosure, in which Control group represents a composition containing 20% FBS, and the composition of the present disclosure is prepared to include 5% to 50% of a culture medium containing hPC.

Hereinafter, the present disclosure will be described in more detail with reference to Examples. However, these Examples are for illustrative purposes only, and the scope of the present disclosure is not intended to be limited by these Examples.

Example 1. Preparation of human mesenchymal stem cell culture medium and human mesenchymal stem cell-derived proteins

Example 1-1. Establishment of culture conditions of stem cells

To prepare a composition for cryopreservation of cells or tissues, a human mesenchymal stem cell culture medium was used. The culture medium was prepared to include human mesenchymal stem cell-derived proteins (human protein complex from human mesenchymal stem cell, hereinafter, referred to as hPC).

First, human adipose-derived mesenchymal stem cells (STEMPRO, INVITROGEN, Cat.# R7788-110), human bone marrow-derived mesenchymal stem cells (SCIENCELL, Cat.# 7500), human umbilical mesenchymal stem cells (SCIENCELL, Cat.# 7530), human amniotic fluid-derived mesenchymal stem cells (ANGIOCRINE, cat.# hAmnio-01), or human amniotic mesenchymal stem cells (SCIENCELL, Cat.# 7501) were used.

Specifically, a method for mass production of proteins from mesenchymal stem cells described in Korean Patent No. 10-1566450 was used to prepare the mesenchymal stem cell-derived proteins of the present disclosure. The method of Korean Patent No. 10-1566450 is included in the scope of the present disclosure. Briefly, one kind of stem cell selected from the human mesenchymal stem cells was seeded in a DMEM/F12 serum-free medium at a density of about 20,000 cells/㎠, and cultured in a 5% CO₂ incubator at 37℃ for about 120 hours.

Thereafter, hPC was obtained from the stem cell culture medium, and a concentration thereof was quantified by a BCA assay. As a result, the concentration of hPC in the culture medium was about 30 ㎍/ml to about 70 ㎍/ml.

Example 1-2. Identification of kinds of proteins in culture medium

To identify kinds of the proteins in the human mesenchymal stem cell culture medium produced in Example 1-1, a qualitative analysis was performed by using an antibody array kit (Signaling Explorer Antibody Array, Fullmoon BioSystems, Cat. No. SET100).

As a result, proteins constituting the hPC were found to be growth factors or cytokines such as FGF-7, IGF-1, IGFBP-1, IGFBP-2, IGFBP-3, IGFBP-4, IGFBP-6, MCSF-R, NT-4, TGF-β1, bFGF, EGF-R, GFG-4, GDF-15, HGF, NT-3, OPG, PDGF-AA, PIGF, VEGF, AR, BDNF, BMP-4, BMP-5, BMP-7, b-NGF, GDNF, GH, insulin, NGFR, SCF, SCFR, TGF-α, VEGFR3, VEGF-D, G-CSF, ICAM-1, IL-1a, IL-2, IL-5, IL-6, IL-8, IL-11, MCP-1, MCSF, MIG, MIP-1a, MIP-1b, MIP-1d, RANTES, TIMP-1, TIMP-2, TNF-α, TNF-β, TNFR1, TNFRII, etc.

Example 2. Preparation of composition for cryopreservation of cells or tissues

A composition for cryopreservation of cells or tissues was prepared by using the culture medium including hPC produced in Example 1.

Specifically, as described in the following Table 1, the compositions were prepared to include 10% of DMSO; 40% to 85% of cDMEM; and 5% to 50% of the hPC-containing culture medium. As control groups, a general composition containing 10% DMSO, 20% FBS, and 70% cDMEM (Control group 1) and a commercially available cryopreservation composition "CRYO-GOLD" (Control group 2) were used. In this regard, a final volume of the composition was adjusted to 1 ml.

Kind	Cryopreservation composition
Control group
1	DMSO 10% + cDMEM 70% + FBS 20%
Control group
2	CRYO-GOLD (Revive Organtech, Cat. #10003)
Experimental group 1(5%)	DMSO 10% + cDMEM 85% + hPC-containing culture medium 5%
Experimental group 2(10%)	DMSO 10% + cDMEM 80% + hPC-containing culture medium 10%
Experimental group 3(15%)	DMSO 10% + cDMEM 75% + hPC-containing culture medium 15%
Experimental group 4(20%)	DMSO 10% + cDMEM 70% + hPC-containing culture medium 20%
Experimental group 5(30%)	DMSO 10% + cDMEM 60% + hPC-containing culture medium 30%
Experimental group 6(40%)	DMSO 10% + cDMEM 50% + hPC-containing culture medium 40%
Experimental group 7(50%)	DMSO 10% + cDMEM 40% + hPC-containing culture medium 50%

Example 3. Examination of human cell preservation effect of composition for cryopreservation of cells or tissues

Example 3-1. Examination of cell viability

To examine the human cell cryopreservation effect of the composition prepared in Example 2, cell viability was measured.

First, 1Х10⁶ human amniotic fluid-derived mesenchymal stem cells at the same passage were put in a vial containing the composition and preserved in a deep freezer at -80℃ for 2 weeks. Thereafter, the cells were thawed and the viability thereof was measured.

Specifically, the vial cryopreserved was placed in a water bath at 37℃ to thaw the cells, which were transferred to 10 ml of growth medium, followed by centrifugation at 1,000 rpm for 5 minutes. A supernant was removed to obtain a cell pellet. 10 ml of growth medium was added to the cell pellet, and 20 ㎕ thereof was added to a 96-well plate and mixed with 20 ㎕ of 0.4% trypan blue. Thereafter, 10 ㎕ of the solution was added to a hemocytometer, and the number of cells was measured under a microscope (40x magnification). In this regard, the number of dead cells stained with trypan blue was first counted, and then the number of unstained live cells was counted. A total number of cells in four grids of the counting chamber was counted, and then the number of cells per grid was averaged. Then, the number of live cells was calculated according to the following Equation: live cells = the number of live cells per grid x 2 x 10⁴ x volume of cell solution. Further, the cell viability was calculated according to the following Equation: the cell viability = the number of live cells/(the number of live cells + the number of dead cells) x 100.

As a result, as shown in FIG. 1, the mesenchymal stem cells preserved by the known cryopreservation method of using FBS (Control group 1) showed 86.45% of cell viability on average, whereas all the cells preserved in the compositions of the present disclosure showed high viability of about 89% to about 94%. Specifically, Experimental groups 1 to 7 including 5%, 10%, 15%, 20%, 30%, 40%, or 50% of the hPC-containing culture medium showed about 90.21%, about 89.51%, about 90.44%, about 93.21%, about 93.16%, about 92.68%, or about 92.19% of cell viability on average, respectively, which are similar to the commercially available cell cryopreservation solution CRYO-GOLD (Control group 2) showing about 93.97% of viability.

These results suggest that since the composition of the present disclosure shows the similar or superior cell viability to the known cell cryopreservation solution containing FBS or the commercially available cryopreservation solution CRYO-GOLD, the composition of the present disclosure may be used in place thereof.

Example 3-2. Examination of cell proliferation rate

To examine the human cell cryopreservation effect of the composition prepared in Example 2, a cell proliferation rate was measured.

First, 1Х10⁶ mesenchymal stem cells thawed after cryopreservation were seeded in a T75 flask containing DMEM (Low Glucose, Welgene) containing 10% fetal bovine serum (Gibco, Austria origin) and 100 ㎕/ml penicillin and streptomycin (Gibco). Thereafter, the cells were cultured in a 5% CO₂ incubator at 37℃ for 3 days. When cells proliferated and occupied about 70% to about 90% of the flask, subculture was performed at a ratio of 1:3 twice, and then cell proliferation rates were compared.

As shown in FIG. 2, the mesenchymal stem cells preserved in the commercially available cell cryopreservation solution CRYO-GOLD (control group 2), showed the cell proliferation rate of about 70% whereas the cells preserved in the composition of the present disclosure showed similar or higher cell proliferation rate. In particular, Experimental group 4 or 7 containing 20% or 50% of the hPC-containing culture medium showed the cell proliferation rate of about 105.80% and about 108.21%, respectively, indicating that the composition of the present disclosure exhibits the superior effect, as compared with the known cryopreservation method of using FBS (Control group 1) showing the cell proliferation rate of about 100%.

These results suggest that since the composition of the present disclosure shows the similar or superior cell proliferation effect to the known cell cryopreservation solution containing FBS or the commercially available cryopreservation solution CRYO-GOLD, the composition of the present disclosure may be used in place thereof.

Example 3-3. Examination of protein secretion ability

To examine the human cell cryopreservation effect of the composition prepared in Example 2, the protein secretion ability of cells was measured.

First, mesenchymal stem cells subcultured according to the method of Example 3-2 were seeded in a DMEM/F12 serum-free medium at a density of about 20,000 cell/㎠, and cultured for about 120 hours. Thereafter, a total amount of the proteins secreted by the mesenchymal stem cells in the culture medium was quantified and compared by a BCA assay.

As shown in FIG. 3, the total amount of the proteins secreted from the mesenchymal stem cells preserved in the known cell cryopreservation method of using FBS (Control group 1) was about 50.78 ㎍/ml whereas the cells preserved in the composition of the present disclosure showed similar or higher amount of the secreted proteins. In particular, the total amount of proteins secreted from Experimental group 4 containing 20% of the hPC-containing culture medium was about 53.91 ㎍/ml, indicating that the composition of the present disclosure exhibits a superior effect, as compared with the commercially available cryopreservation solution CRYO-GOLD (Control group 2) showing about 52.52 ㎍/ml of the secreted proteins.

These results suggest that since the composition of the present disclosure shows the similar or superior cell cryopreservation effect to the known cell cryopreservation solution containing FBS or the commercially available cryopreservation solution CRYO-GOLD, the composition of the present disclosure may be used in place thereof.

Example 4. Examination of animal cell preservation effect of composition for cryopreservation of cells or tissues

Example 4-1. Examination of cell viability

To examine the animal cell cryopreservation effect of the composition prepared in Example 2, the cell viability was measured.

First, about 1Х10⁶ CHO-K1 cells (Chinese Hamster Ovary Cell, KCLB Cat. No. #10061) at the same passage were put in a vial containing the composition, and preserved in a deep freezer at -80℃ for 2 weeks. Thereafter, the cells were thawed and viability thereof was measured according to the method of Example 3-1.

As a result, as shown in FIG. 4, CHO-K1 cells preserved by the known cryopreservation method of using FBS (Control group) showed about 68.66% of cell viability, whereas all the cells preserved in the compositions of the present disclosure showed high viability of about 84% to about 92%. Specifically, Experimental groups 1 to 7 including 5%, 10%, 15%, 20%, 30%, 40%, or 50% of the hPC-containing culture medium showed about 86.96%, about 84.82%, about 88.31%, about 91.95%, about 89.74%, about 89.49% or about 88.03% of cell viability on average, respectively.

These results suggest that since the composition of the present disclosure shows the superior cell viability to the known cell cryopreservation solution containing FBS, the composition of the present disclosure may be used in place thereof.

Example 4-2. Examination of cell proliferation rate

To examine the animal cell cryopreservation effect of the composition prepared in Example 2, the cell proliferation rate was measured.

First, 1Х10⁶ thawed mesenchymal stem cells were seeded in a T75 flask containing DMEM containing 10% fetal bovine serum and 100 ㎕/ml penicillin and streptomycin. Thereafter, the cells were cultured in a 5% CO₂ incubator at 37℃ for 5 days. When cells proliferated and occupied about 70% to about 90% of the flask, subculture was performed at a ratio of 1:5 twice and then cell proliferation rates were compared.

As a result, as shown in FIG. 5, the CHO-K1 cells preserved by the known cryopreservation method of using FBS (Control group 1) showed the cell proliferation rate of about 100% whereas the cells preserved in the composition of the present disclosure showed the high cell proliferation rates of about 110% or more. Specifically, Experimental groups 1 to 7 including 5%, 10%, 15%, 20%, 30%, 40%, or 50% of the hPC-containing culture medium showed the cell proliferation rate of about 124.88%, about 128.88%, about 119.39%, about 131.13%, about 108.37%, about 121.89%, or about 116.89% on average, respectively.

Particularly, as shown in FIG. 6, although CHO-K1 cells were preserved at an ultra-low temperature of -196℃, all the cells preserved in the composition of the present disclosure showed the high cell proliferation rates of about 110% or more, indicating that the composition of the present disclosure exhibits the superior effect, as compared with the known cryopreservation method (control group 1) showing the cell proliferation rate of about 100%.

These results suggest that since the composition of the present disclosure shows the superior cell proliferation effect to the known cell cryopreservation solution containing FBS, the composition of the present disclosure may be used in place thereof.

Example 5. Comparison of viability according to long-term cryopreservation

To demonstrate the effects of the cell cryopreservation composition prepared in Example 2, cells were preserved for a long period of time from 2 months to 6 months, and viability thereof was measured.

Specifically, since the cell cryopreservation composition containing 20% of hPC-containing culture medium showed the most excellent effect, as confirmed in Examples 3, 4-1 and 4-2, the composition was prepared to include 20% of hPC-containing culture medium. Thereafter, 1Х10⁶ CHO-K1 cells at the same passage were put in a vial for cell cryopreservation containing 1 ml of the composition, and preserved at -196℃ or -80℃ for 2 months to 6 months, respectively. Then, the cells were thawed and the viabilities thereof were measured and compared. In this regard, preservation conditions are shown in the following Table 2, respectively.

Group	Cryopreservation composition and Method
Control group(2 months)	DMSO 10% + cDMEM 70% + FBS 20% (-196℃)
Experimental group 1(2 months)	DMSO 10% + cDMEM 70% + hPC-containing culture medium 20% (-196℃)
Experimental group 2 (2 months)	DMSO 10% + cDMEM 70% + hPC-containing culture medium 20% (-80℃)
Control group(4 months)	DMSO 10% + cDMEM 70% + FBS 20% (-196℃)
Experimental group 1(4 months)	DMSO 10% + cDMEM 70% + hPC-containing culture medium 20% (-196℃)
Experimental group 2 (4 months)	DMSO 10% + cDMEM 70% + hPC-containing culture medium 20% (-80℃)
Control group(6 months)	DMSO 10% + cDMEM 70% + FBS 20% (-196℃)
Experimental group 1 (6 months)	DMSO 10% + cDMEM 70% + hPC-containing culture medium 20% (-196℃)
Experimental group 2 (6 months)	DMSO 10% + cDMEM 70% + hPC-containing culture medium 20% (-80℃)

As a result, as shown in FIG. 7, the cells preserved at -196℃ by the known cryopreservation method of using FBS (Control group) showed the cell viabilities of about 82.29%, about 81.62%, or about 80.16% on average after 2 months, 4 months, or 6 months, respectively. In contrast, the cells preserved at -196℃ in the cryopreservation composition containing 20% of the hPC-containing culture medium of the present disclosure (Experimental group 1) showed the similar cell viabilities of about 80.88%, about 80.36%, or about 80.16% on average after 2 months, 4 months, or 6 months, respectively. In particular, the cells preserved at -80℃ (Experimental group 2) showed the remarkably high cell viabilities of about 89.02%, about 88.12%, or about 87.36% on average after 2 months, 4 months, or 6 months, respectively.

These results suggest that since the composition of the present disclosure shows the similar or superior effect to the known cell cryopreservation solution containing FBS even upon long-term cryopreservation, the composition of the present disclosure may be used in place thereof.

It should be understood that embodiments described herein should be considered in a descriptive sense only and not for purposes of limitation. Descriptions of features or aspects within each embodiment should typically be considered as available for other similar features or aspects in other embodiments.

While one or more embodiments have been described with reference to the figures, it will be understood by those of ordinary skill in the art that various changes in form and details may be made therein without departing from the spirit and scope of the disclosure as defined by the following claims.

Claims

A composition for cryopreservation of cells or tissues, the composition comprising a human mesenchymal stem cell culture medium as an active ingredient.
The composition of claim 1, wherein the human mesenchymal stem cells are derived from fat, bone marrow, umbilical cord blood, amniotic fluid, or amniotic membrane.
The composition of claim 1, wherein the human mesenchymal stem cell culture medium comprises human mesenchymal stem cell-derived proteins.
The composition of claim 3, wherein the proteins comprise AR, BDNF, bFGF, BMP-4, BMP-5, BMP-7, b-NGF, EGF-R, FGF-4, FGF-7, GDF-15, GDNF, HGF, IGFBP-1, IGFBP-2, IGFBP-3, IGFBP-4, IGFBP-6, IGF-1, Insulin, MCSF, MCSF-R, NGF-R, NT-3, NT-4, OPG, PDGF-AA, PIGF, SCF, SCF R, TGF-α, TGF-β1, VEGF, VEGF-R3, ICAM-1, G-CSF, IL-1α, IL-2, IL-5, IL-6, IL-8, IL-11, MCP-1, MIG, MIP-1a, MIP-b, MIP-d, TIMP-1, TIMP-2, TNFα, TNFβ, TNF-R1, or TNF-R11.
The composition of claim 3, wherein a total concentration of the proteins is 30 ㎍/ml to 70 ㎍/ml, as measured by a BCA assay.
The composition of claim 1, wherein the composition comprises 5%(v/v) to 50%(v/v) of the culture medium.
The composition of claim 1, wherein the cells or tissues are derived from humans or animals.
A method of preparing a composition for cryopreservation of cells or tissues, the method comprising producing the human mesenchymal stem cell culture medium comprising human mesenchymal stem cell-derived proteins.
The method of preparing the composition for cryopreservation of cells or tissues of claim 8, wherein the producing of the human mesenchymal stem cell culture medium comprises (a) seeding human mesenchymal stem cells at a density of 18,000 cells/㎠ to 22,000 cells/㎠; (b) culturing the stem cells in a serum-free medium; and (c) collecting the stem cell culture medium at 114 hours to 126 hours after culture.