Composition for preserving cells, containing, as active ingredients, plant-derived recombinant human serum albumin and plant peptides

Abstract

The present invention relates to a composition for preserving cells and a method for preserving cells and, more specifically, to: a composition for preserving cells, containing, as active ingredients, plant-derived recombinant human serum albumin and plant peptides, wherein the composition maintains a high cellular survival rate while maintaining animal-free and xeno-free properties and is stable without changes in cellular morphology or surface expression factors in the short-term preservation of cells such as stem cells or primary cultured cells; and a method for preserving cells by using the same.

Claims

1. A method for preserving animal stem cells or animal primary cells, the method comprising: contacting the cells with a composition for cell preservation, wherein the composition contains plant-derived recombinant human serum albumin and plant peptide as active ingredients, the plant-derived recombinant human serum albumin being contained at 0.5-5 parts by weight, and the plant peptide being contained at 1-5 parts by weight, on a basis of the total 100 parts by weight of the composition, and wherein the composition further contains CaCl.sub.2KCl, MgSO.sub.4NaCl, NaH.sub.2PO.sub.4H.sub.2O, L-alanine, L-asparagine-H2O, L-aspartic acid, L-glutamic acid, glycine, L-lysine-HCl, L-proline, L-serine, L-valine, L-ascorbic acid, D-Ca pantothenate, choline chloride, folic acid, i-inositol, niacinamide, pyridoxal HCl, thiamine HCl, adenosine, cytidine, guanosine, uridine, 2′deoxyadenosine, 2′deoxycytidine-HCl, 2′deoxyguanosine and thymidine; and preserving the cells in the composition at 4° C. to 28° C. for 1 day to 6 days without freezing.

2. The method of claim 1, wherein the stem cells comprise umbilical cord mesenchymal stem cells, adipose-derived mesenchymal stem cells, or bone marrow-derived mesenchymal stem cells.

3. The method of claim 1, wherein the composition is xeno-free and animal-free.

4. The method of claim 1, wherein the plant-derived recombinant human serum albumin is derived from rice and the plant peptide is derived from soybean.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is a graph confirming, through cell counting, the viability of UCMSCs over storage days after storage at a low temperature.

(2) FIG. 2 is a graph confirming, through cell counting, the viability of ADMSCs over storage days after storage at a low temperature.

(3) FIG. 3 is a graph confirming, through cell counting, the viability of BMMSCs over storage days after storage at a low temperature.

(4) FIG. 4 is a graph confirming, through cell counting, the viability of human diploid fibroblasts (HDFs), as primary cells, over storage days after storage at a low temperature.

(5) FIG. 5 shows images confirming, through a microscope, the viability and adhesive degree of UCMSCs over storage days after storage at a low temperature and re-culture for 24 hours.

(6) FIGS. 6 and 7 show diagrams confirming, through a flow cytometer, the change in cell surface factor CD31 of UCMSCs over storage days after storage at a low temperature while UCMSCs were treated with the composition for cell preservation (CEFO-vive) of an example of the present invention.

(7) FIGS. 8 and 9 show diagrams confirming, through a flow cytometer, the change in cell surface factor CD73 of UCMSCs over storage days after storage at a low temperature while UCMSCs were treated with the composition for cell preservation (CEFO-vive) of an example of the present invention.

(8) FIGS. 10 and 11 show diagrams confirming, through a flow cytometer, the change in cell surface factor CD105 of UCMSCs over storage days after storage at a low temperature while UCMSCs were treated with the composition for cell preservation (CEFO-vive) of an example of the present invention.

(9) FIGS. 12 and 13 show diagrams confirming, through a flow cytometer, the change in cell surface factor CD146 of UCMSCs over storage days after storage at a low temperature while UCMSCs were treated with the composition for cell preservation (CEFO-vive) of an example of the present invention.

(10) FIG. 14 shows diagrams confirming, through a flow cytometer, the change in cell surface factor CD31 of UCMSCs over storage days after storage at room temperature while UCMSCs were treated with the composition for cell preservation (CEFO-vive) of an example of the present invention.

(11) FIG. 15 shows diagrams confirming, through a flow cytometer, the change in cell surface factor CD73 of UCMSCs over storage days after storage at room temperature while UCMSCs were treated with the composition for cell preservation (CEFO-vive) of an example of the present invention.

(12) FIG. 16 shows a view confirming, through a flow cytometer, the change in cell surface factor CD105 of UCMSCs over storage days after storage at room temperature while UCMSCs were treated with the composition for cell preservation (CEFO-vive) of an example of the present invention.

(13) FIG. 17 shows a view confirming, through a flow cytometer, the change in cell surface factor CD146 of UCMSCs over storage days after storage at room temperature while UCMSCs were treated with the composition for cell preservation (CEFO-vive) of an example of the present invention.

BEST MODE FOR CARRYING OUT THE INVENTION

(14) Hereinafter, the present invention will be described in detail with reference to the following examples. However, the present invention may be realized in various different forms, and therefore is not limited to embodiments to be described herein.

(15) In an aspect, the present invention is directed to a composition for cell preservation containing a plant-derived recombinant human serum albumin and a plant peptide as active ingredients.

(16) The composition for cell preservation of the present invention refers to a composition for protecting and storing cells, which are to be used before a certain experiment, operation, or treatment using cells, for several hours to several days. The composition of the present invention is different from ordinary animal cell culture media that have been used optimally for the culture and proliferation of particular cells in view of constituent ingredients, contents thereof, and a purpose of use. In other words, there is a medium optimized for each particular type of cells for the culture and proliferation of particular cells, and thus the composition for cell preservation of the present invention per se is not suitable for the use as a cell culture medium, but is merely suitable for the use as a preservation solution for increasing the cell viability when cells are stored at room temperature or at low temperatures and for preventing the deformation of cells.

(17) In an embodiment, the composition for cell preservation of the present invention may contain 0.1-20 parts by weight of a plant-derived recombinant human serum albumin on the basis of the total 100 parts by weight of the composition. When containing a plant-derived recombinant human serum albumin and a plant peptide as active ingredients, the composition for cell preservation of the present invention may contain, on the basis of the total 100 parts by weight of the composition, 0.1-10 parts by weight of a plant-derived recombinant human serum albumin and 0.1-10 parts by weight of a plant peptide. More preferably, the composition for cell preservation of the present invention may contain 0.5-5 parts by weight of a plant-derived recombinant human serum albumin and 1-5 parts by weight of a plant peptide. Most preferably, the composition for cell preservation of the present invention may contain 1 part by weight of a plant-derived recombinant human serum albumin and 5 parts by weight of a plant peptide.

(18) In an embodiment, the cells may be stem cells or primary cells, and the stem cells may be umbilical cord mesenchymal stem cells (UCMSCs), adipose-derived mesenchymal stem cells (ADMSCs), or bone marrow-derived mesenchymal stem cells (BMMSCs).

(19) In an embodiment, the composition for cell preservation of the present invention may be xeno-free and animal-free since it contains no animal-derived ingredient.

(20) The cells for which the composition for cell preservation of the present invention can be used are animal cells. The composition of the present invention is used for, preferably stem cells, and more preferably adult stem cells. The composition for cell preservation of the present invention uses no animal factor and contains a plant-derived recombinant human serum albumin and a plant peptide, thereby maintaining the xeno-free property thereof, so that the composition can be safely used because of the low infection risk of prion proteins and the like in the preservation of stem cells or primary cells.

(21) In an embodiment, the plant-derived recombinant human serum albumin may be derived from rice, and the plant peptide may be derived from soybean.

(22) The composition of the present invention may further contain inorganic salts, vitamins and nucleic acids, and the minerals and vitamins can perform functions (for example, antioxidative action) in association with the role of removing toxic substances produced by cells, thereby increasing the cell viability.

(23) As used herein, the term “plant-derived recombinant human serum albumin” refers to a protein that is obtained by the transformation of plant cells using the whole amino acid sequence of a human serum albumin or an amino acid sequence of a portion of a human serum albumin having biological activity. The human serum albumin can be used as an animal-free alternative that is equivalent or superior to the serum in the preservation of stem cells or primary cells, and can be added to the composition for the preservation of stem cells or primary cells to stabilize cells. Since an embodiment of the present invention has an advantage in that the risk of an animal or viral infectious factor can be minimized by using a recombinant human serum albumin produced in a plant (rice), the composition of the present invention has a usefulness in view of the use for stem cells or a primary cells, especially, the use as a therapeutic agent. In addition, the plant-derived recombinant human serum albumin contained in the composition of the present invention has an advantage in that it rarely exhibits lot to lot variation, unlike human serum albumins derived from animal cells or serum.

(24) As used herein, the term “plant peptide” refers to a peptide extracted from a plant. Considering the purpose of the present invention, the plant peptide is not particularly limited as long as it contains an amino acid capable of reducing cell damage in cell preservation. In one example of the present invention, a plant peptide extracted from soybean was used. The plant peptide has a low risk of infection with prion proteins due to animal-free property thereof, maintains the xeno-free property thereof, and contains essential amino acids and/or non-essential amino acids, which can be used as basic energy sources for cells, and therefore, at the time of cell preservation, the plant peptide contributes to the increase in the cell viability by supplying nutrients to stem cells or primary cells and enhancing activity of the cells.

(25) As used herein, the term “stem cells” refers to undifferentiated cells having self-renewal and differentiation potency. Stem cells include sub-groups of pluripotent stem cells, multipotent stem cells, and unipotent stem cells, according to their differentiation potency. Pluripotent stem cells mean cells that have the potency to differentiate into all tissues or cells constituting a living organism, and multipotent stem cells mean cells that do not have the potency to differentiate into all kinds but into plural kinds of tissues or cells. Unipotent stem cells mean cells that have potency to differentiate into particular tissues or cells. The pluripotent stem cells may include embryonic stem cells (ES cells), embryonic germ cells (EG cells), induced pluripotent stem cells (iPS cells), and the like. The multipotent stem cells may include adult stem cells, such as mesenchymal stem cells (derived from adipose, bone marrow, umbilical cord blood, or umbilical cord, etc.), hematopoietic stem cells (derived from bone marrow or peripheral blood), neural stem cells, germ stem cells, etc. The unipotent stem cells may include committed stem cells for hepatocytes, which are usually quiescent with low self-renewal capacity but vigorously differentiate into only hepatocytes after activation. In an example of the present invention, it was verified that, for bone marrow-derived mesenchymal stem cells, adipose-derived stem cells, and umbilical cord derived stem cells, the composition of the present invention can be safely and stably used in the preservation of the stem cells at room temperature or low temperatures.

(26) As used herein, the term “primary cells” refers to cells that are isolated from a tissue extracted from an individual, without any genetic manipulation or the like, and represent functions of an organ/tissue of a living organism. Primary cells are isolated from skin, vascular endothelium, bone marrow, adipose, cartilage, or the like, and are used for studying functions of corresponding tissues and cells or as cell therapeutic agents for restoring lost tissues.

(27) The origin of stem cells or primary cells is not particularly limited as long as the cells can be stably preserved by the composition of the present invention at room temperature or at low temperatures. Examples thereof may include cells derived from human, monkey, pig, horse, cow, sheep, dog, cat, mouse, or rabbit. The stem cells or primary cells are preferably human stem cells or primary cells, but are not limited thereto.

(28) In an aspect, the present invention is directed to a method for preserving cells, the method comprising treating cells with the composition for cell preservation of the present invention.

(29) In an embodiment, the cells treated with the composition for cell preservation of the present invention may be preserved at 30° C. or lower, preferably at room temperature or at a low temperature of 0° C. to 10° C., and most preferably at 4° C.

(30) As used herein, the term “room temperature” refers to 18° C. to 27° C.

(31) The present invention will be described in more detail through the following examples. However, the following examples are provided merely to illustrate the present invention and not to restrict the scope of the present invention.

MODE FOR CARRYING OUT THE INVENTION

Example

Preparation of Animal-Free and Xeno-Free Composition for Cell Preservation

(32) In order to prepare a composition for preserving cells at room temperature or at low temperatures, the composition being capable of stably preserving stem cells or primary cells in a xeno-free manner, a human recombinant albumin extracted from rice (rAlbumin ACF, Sheffield Bio-Science, USA) and a plant peptide extracted from soybean (UltraPep™Soy, Sheffield Bio-Science, USA) used as active ingredient were mixed with a basal medium not containing Phenol Red to prepare a composition for cell preservation, which was called CEFO-vive. In the prepared composition for cell preservation, the human recombinant albumin was used at 1 wt % and the plant peptide was used at 5 wt %. The plant peptide had an amino acid composition shown in table 1. In addition, the specific contents of inorganic salts, amino acids, vitamins, nucleic acid ingredients, and other ingredients of the composition for cell preservation, CEFO-vivo, were tabulated in tables 2, 3, 4, 5, and 6.

(33) TABLE-US-00001 TABLE 1 Amino acid composition of plant peptide Ala Arg Asp Glu Gly His Ile Lys Met Phe Pro Ser Thr Tyr Val mg/L 475 750 1,275 137 550 250 600 1,000 200 625 525 725 600 400 1,800

(34) TABLE-US-00002 TABLE 2 Inorganic salt mg/L CaCl.sub.2 (anhyd.) 200.0 KCl 400.0 MgSO.sub.4 (anhyd.) 98.0 NaCl 15,300.0 NaH.sub.2PO.sub.4H.sub.2O 140.0

(35) TABLE-US-00003 TABLE 3 (  a.a) mg/L L-Alanine 1,390.0 L-arginine•HCl 877.0 L-asparagine•H.sub.2O 2,645.0 L-aspartic acid 1,360.0 L-cysteine•HCl 31.0 L-cysteine•HCl•H.sub.2O 100.0 L-glutamic acid 1,863.0 L-glutamine 292.0 Glycine 1,350.0 L-histidine•HCl•H.sub.2O 292.0 L-isoleucine 652.0 L-leucine 52.0 L-lysine•HCl 1,073.0 L-methionine 215.0 L-phenylalanine 657.0 L-proline 1,715.0 L-serine 1,800.0 L-tryptophan 610.0 L-tyrosine (disodium Salt) 452.0 L-valine 1,8460.0

(36) TABLE-US-00004 TABLE 4 Vitamin mg/L L-ascorbic acid 100.0 Biotin 0.1 D-Ca pantothenate 101.0 Choline chloride 101.0 Folic acid 101.0 i-inositol 202.0 Niacinamide 101.0 Pyridoxal HCl 101.0 Riboflavin 10.1 Thiamine HCl 101.0 Vitamin B12 1.4

(37) TABLE-US-00005 TABLE 5 Nucleoside mg/L Adenosine 10.0 Cytidine 10.0 Guanosine 10.0 Uridine 10.0 2’Deoxyidenosine 10.0 2’Deoxycytidine•HCl 11.0 2’Deoxyguanosine 10.0 Thymidine 10.0

(38) TABLE-US-00006 TABLE 6 Other ingredients mg/L Human recombinant 10,000.0 albumin (Plant derived) D-Glucose 1,000.0 Lipoic acid 0.2 Sodium pyruvate 110.0

Test Example 1

Verification on Cell Viability of Composition for Cell Preservation

(39) Preparation of Stem Cells or Primary Cells

(40) Umbilical cord mesenchymal stem cells (UCMSCs), adipose-derived mesenchymal stem cell (ADMSCs), and bone marrow-derived mesenchymal stem cells (BMMSCs) were directly extracted from the umbilical cord, adipose, and bone marrow that have been donated by patients, respectively. CEFOgro™UCMSC, CEFOgro™ADMSC, and CEFOgro™BMMSC media (basal media) were used as culture media for the stem cells. Human diploid fibroblasts (HDFs) were used as primary cells, and cultured in CEFOgro™HF media. These cells were cultured in an incubator under conditions of 37° C. and 5% CO.sub.2, and the culture media were sustained through the replacement every 3 days. All tests of the present invention were approved by the Institutional Bioethics Review Board at CEFO Co., Ltd., and performed by procedures and methods in compliance with Bioethics and Safety Act.

(41) Cell Counting After Low-Temperature Preservation

(42) Stem cells UCMSCs, ADMSCs, and BMMSCs were cultured in their respective basal media, followed by treatment with trypsin, and then the cells were individually collected. The individually collected cells were put in the composition for cell preservation of the above example (CEFO-vive), a basal medium, or a saline solution, and stored in a refrigerator (4° C.). Then, cell viability was monitored by taking three vials each day to count viable cells until the 6th day of storage. The results verified that the CEFO-vive showed an average cell viability of 70% or more even on the 6th day of storage, unlike the basal medium or saline solution (FIGS. 1, 2, and 3). In addition, human diploid fibroblasts (HDFs) as primary cells were cultured in the CEFOgro™HF medium, and then treated with trypsin, followed by collection. The collected cells were put in the composition for cell preservation of the above example (CEFO-vive), a basal medium, or a saline solution, respectively, and stored in a refrigerator (4° C.). Then, cell viability was monitored by taking three vials each day to count viable cells until the 6th day of storage. The results verified that the CEFO-vive showed an average cell viability of 90% or more even on the 5th day of storage, but the cell viability was rapidly reduced from the 2nd day of storage in the basal medium or saline solution (FIG. 4).

(43) Observation of Cells Cultured After Low-Temperature Cell Preservation

(44) UCMSCs were cultured in CEFOgro™UCMSC, and then treated with trypsin, followed by individual collection. The collected cells were put in the composition for cell preservation of the above example (CEFO-vive), a basal medium, or a saline solution, respectively, and stored in a refrigerator (4° C.). Then, three vials were taken each day, and the cells were inoculated in culture dishes and cultured in CEFOgro™UCMSC media. After cell culture for 24 hours, the cells were observed using a microscope. As a result, it was verified that, as for CEFO-vivo, the cells were favorably attached on the culture dishes at the cell re-culture even at the 5th day of storage in the refrigerated storage, but as for the basal medium or saline solution, the cells adapted poorly to the culture dishes and died from the 2nd day of the refrigerated storage (FIG. 5).

(45) Verification on Cell Surface Factors After Low-Temperature Preservation

(46) UCMSCs were cultured in CEFOgro™UCMSC, and then treated with trypsin, followed by individual collection. The collected cells were put in the composition for cell preservation of the above example (CEFO-vive), a basal medium, or a saline solution, respectively, and stored in a refrigerator (4° C.). Then, three vials were taken each day, and the cells were inoculated in culture dishes and cultured in CEFOgro™UCMSC media for 4 days. Thereafter, the respective cells were collected, and a flow cytometer was used to investigate the changes of CD markers (CD31, CD73, CD105, and CD146) that have been known as cell surface factors for mesenchymal stem cells. However, in the case of the cells using the basal medium or saline solution, excluding the cells preserved in CEFO-vive, the culture for 4 days could not give as many cells as can be used for analysis. Therefore, only the cell groups preserved in CEFO-vive were subjected to flow cytometry. As a result, CD31(−), CD73(+), CD105(+), and CD146(+) were shown on Day 1, Day 2, Day 3, Day 4, and Day 5, and these results indicate that there was no change in cell morphology or cell surface protein expression in the refrigerated storage of the stem cells in CEFO-vive (FIGS. 6 and 7, 8 and 9, 10 and 11, and 12 and 13).

(47) Verification on Cell Surface Factors After Room-Temperature Preservation

(48) UCMSCs were cultured in CEFOgro™UCMSC, and then treated with trypsin, followed by individual collection. The collected cells were put in CEFO-vive of the above example, a basal medium, or a saline solution, respectively, and stored at room temperature (25° C.). Then, three vials were taken on Day 1 or 3, and the cells were inoculated in culture dishes and cultured in CEFOgro™UCMSC media for 4 days. Thereafter, the respective cells were collected, and a flow cytometer was used to investigate the changes of CD markers (CD31, CD73, CD105, and CD146) that have been known as cell surface factors for mesenchymal stem cells. However, in the case of the cells using the basal medium or saline solution, excluding the cells preserved in CEFO-vive, the culture for 4 days could not give as many cells as can be used for analysis, in spite of room-temperature preservation. Therefore, only the cell groups preserved in CEFO-vive were subjected to flow cytometry. As a result, CD31(−), CD73(+), CD105(+), and CD146(+) were shown on Day 1 and Day 3, and these results indicate that there was no change in cell morphology or cell surface protein expression stored at the room temperature of the stem cells in CEFO-vive (FIGS. 14, 15, 16, and 17).

(49) It can be seen from the above results that the animal-free and xeno-free composition for cell preservation of the present invention containing a plant-derived recombinant human serum albumin and a plant peptide as active ingredients ensures the cell survival and maintains the stability of cells without changing cell morphology or surface expression factors in the preservation of cells at room temperature or at low temperatures.