WO2011105658A1

WO2011105658A1 - Cell protectants comprising placenta extracts

Info

Publication number: WO2011105658A1
Application number: PCT/KR2010/002457
Authority: WO
Inventors: Sun Ray Lee; Hyun Sook Park; Kyung Hye Kim; Tae-Hoon Kang
Original assignee: Ark Stem
Priority date: 2010-02-26
Filing date: 2010-04-20
Publication date: 2011-09-01
Also published as: KR20110098478A; KR101169701B1; CN102791278A

Abstract

Disclosed is a cell protectant comprising a placenta extract, more specifically a cell protectant for storing and protecting animal cells, including amino acids, minerals, vitamins, growth factors and placenta extract.

Description

[DESCRIPTION]

[invention Title]

CELL PROTECTANTS COMPRISING PLACENTA EXTRACTS

[Technical Field]

The present invention relates to a cell protectant comprising a placenta extract, more specifically to a cell protectant for storing and protecting animal cells, comprising an amino acid, a mineral, a vitamin, a growth factor and a placenta extract.

[Background Art]

In general, cells are preserved by freezing at extremely low temperature of -196 °C and thawed rapidly to obtain living cells when they are needed. Although the survival rate of the frozen-and-then-thawed cells varies depending on the cell type or the proficiency of the operator, the survival rate of normal and useful cells, not cancer cells, is very low. And, in case transportation or storage is required only for hours or days, not for a long period of time, the cryopreservation method is impractical .

Recently, cell therapy techniques of using mature cells or stem cells isolated from animal tissue to treat various diseases are being developed. Thus, development of a protectant that can maintain the viability of the mature cells and stem cells acquired from the tissue for a long time and provide better preservation is stronglyneeded. At present, culture media used to culture animal cells are provisionally used as cell protectant and there are few studies about useful protectant specialized for preservation of cells .

For cultivation of human stem cells, media including animal serum ( fetal bovine serum) are used. Since the cells cultured using fetal bovine serum are associated with the risk of infection of mad cow disease or other diseases even after the cells are washed with serum-free solution, transplantation of the cells cultured using fetal bovine serum into humans is prohibited in the United States. Techniques of isolating mesenchymal stem cells from human bone marrow and from adipose tissue are disclosed respectively in Pittenger et al. [Science 284: 143-147, 1997] and Van et al. [J. Clin. Invest. 58: 699-704, 1976], where a-MEM or DMEM and 10-20% fetal bovine serum are used to culture the cells. However, the use of the animal serum (fetal bovine serum) is controversial with regard to safety in transplantation into human body.

To overcome this problem, there was an attempt to use human serum instead of fetal bovine serum [Kuznetsov SA, et al., Transplantation.2000 Dec 27; 70 (12) : 1780-7] . However, when human serum is used in the cell culture medium without any treatment, the stem cells tend to lose their characteristics, e.g., decreased ability to proliferate and differentiate, facilitated differentiation into bone cells, or the like. Further, after transplantation, the stem cells have very low viability and do not fully exert their abilities. In order to optimize tissue regeneration of adult stem cells, researches on techniques to improve viability of the transplanted stem cells and optimize and improve the effect of transplantation are required.

[Disclosure]

[Technical Problem]

Accordingly, an object of the present invention is to provide a cell protectant capable of improving viability of cells stored at low temperature as compared to existing animal cell culture media and improving proliferation and capability of stem cells.

[Technical Solution]

The inventors of the present invention have worked to solve the aforesaid problems of the related art. As a result, they have developed a cell protectant comprising an amino acid, a mineral, a vitamin, a growth factor and a placenta extract , and have confirmed that it can improve viability of animal cells without using an animal serum and can enhance the capability of the stem cells.

[Advantageous Effects]

The cell protectant of the present invention may be used to store cells safely, not only stem cells but also various other animal cells. It may be used to protect cells used for cell therapy when they are stored and transported at low temperature. When animal cells are treated with the cell protectant of the present invention before cultivation, improved cell proliferation rate and stem cell capability may be attained. Accordingly, the cell protectant of the present invention is very useful as a protectant to preserve cells and improve stem cell capability.

[Description of Drawings]

The above and other objects, features and advantages of the present invention will become apparent from the following description of preferred embodiments given in conjunction with the accompanying drawings, in which:

Fig. 1 compares cell survival rate of fat cells isolated from the adipose tissue stored at low temperature (4 °C) for 72 hours in^' a control culture medium or in the CeLLGUARD solution of the present invention without culturing; Fig .2a compares stem cell capability of mesenchymal stem cells derived from the adipose tissue treated with a control culture medium or CeLLGUARD;

Fig. 2b compares cell proliferation rate of mesenchymal stem cells derived from the adipose tissue treated with a control culture medium or CeLLGUARD;

Fig. 3a compares cell proliferation rate of mesenchymal stem cells derived from the adipose tissue treated with a control culture medium or CeLLGUARD for 7, 10 and 14 days;

Fig. 3b shows a result of identifying a surface expression marker after treating mesenchymal stem cells derived from the adipose tissue with a control culture medium or CeLLGUARD;

Figs. 4a and 4b compare wound healing effect by mesenchymal stem cells treated with a control culture medium or CeLLGUARD;

Fig.5 shows a result of identifying a surface expressionmarker after treating keratinocytes isolated from the skin tissue with a control culture medium or CeLLGUARD and then culturing; and

Fig. 6 compares stem cell capability of keratinocytes isolated from the skin tissue cultured after treating with a control culture medium or CeLLGUARD.

[Best Mode]

Hereinafter, the embodiments of the present invention will be described in detail with reference to accompanying drawings.

In an aspect, the present invention provides a cell protectant comprising an amino acid, a mineral, a vitamin, a growth factor and a placenta extract.

In the present invention, the cell protectant referstoa medium composition for storing animal cells at low temperature or room temperature for hours to days until they are used in experiment, operation or treatment. It is different from the general animal cell culture media optimized for growth and proliferation of particular cells in compositions, contents and uses . Although the cell protectant of the present invention may be added as an additive for culturing cells, it is inadequate to use itself as a culture medium. That is to say, for cultivation and growth of particular cells, it is recommended to use the media optimized for them, and to use the cell protectant of the present invention itself as a cell culture medium is not recommended. The cell protectant may be used to preserve cells, added to a culture medium in order to improve cell viability or growth, or treated to cells before growing in them in an adequate culture medium.

The cell protectant of the present invention may be used for animal cells, preferably stem cells, more preferably adult stem cells. In general, animal cells need amino acids, minerals, vitamins, or the like to survive. Commonly used animal cell culture media include these components. Although the cell protectant of the present invention also includes those components such as amino acids, minerals, vitamins, etc., their compositions and contents are different from those of animal cell culture media since the purpose is preservation of various animal cells rather than cultivation or proliferation of them (see Tables 1 to 4) .

In Example 1 of the present invention, cell viability test was performed for fat cells. When they were stored at low temperature 72 hours, a viability of at least 70% was attained (see Fig. 1) . Further, test result of cell proliferation rate and stem cell capability for keratinocytes and adipose-derived stem cells was significantly better than that of an existing animal cell culture medium (see Figs. 2 and 6) .

The existing animal cell culture media include animal serum as a nutritional component. Although it helps the cells to grow stably, it may lead to decreased cell viability when included in a cell protectant and kept for a long time at room temperature or low temperature, not ultralow temperature . However, since the cell protectant of the present invention is not for culturing or proliferating of cells but for improving cell viability until the cells are used for experiment or operation, it is serum-free and contains the ingredients required for survival in larger amounts.

Especially, the cell protectant of the present invention may be usefully used to preserve stem cells . In regard to cell therapy, which is recently researched a lot, it is useful as a protectant capable of storing and transporting stem cells. Further, by treating stem cells with the cell protectant of the present invention, the stem cell capability may be improved. When treated to stem cells, the cell protectant of the present invention decreases cell death and activates the cells. Thus, the cell protectant of the present invention is very useful to stabilize the stem cells.

Preferably, in the cell protectant of the present invention, the amino acid comprises L-cystine and L-hydroproline in addition to the 20 basic amino acids constituting proteins.

L-Hydroproline serves to defend against oxidative attack by reduced DNA, repair damaged DNA and produce collagen. It is an essential cofactor in collagen biosynthesis as a donor of a hydroxyl group to hydroxyproline and hydroxylycine, which are essential amino acids in biosynthesis and maintenance of collagen. Accordingly, L-hydroproline is expected to act as antioxidant to remove reactive oxygen species and to prevent cell death.

L-Cystine contains sulfur and is a constituent of hair or horn.

Especially, it is contained in keratin in an amount of about 10% and is easily reduced to L-cysteine. It plays an important role in determination of the tertiary structure of polypeptides and activity of enzymes or hormones. Accordingly, L-cystine has an important effect on cell activity.

Preferably, in the cell protectant of the present invention, the mineral comprises trace elements zirconium, germanium and vanadium in addition to general elements required for cell growth, such as calcium, sodium, potassium, copper, iron, magnesium and zinc. The mineral included in the cell protectant of the present invention may be in the form of an inorganic salt , e.g., ZrOCl₂"8H₂0, Ge0₂ and NaV0₃.

Preferably, in the cell protectant of the present invention, the vitamin comprises calciumpantothenate, choline chloride, folic acid, inositol and ascorbic acid, which are capable of inhibiting reactive oxygen species.

The mineral and the vitamin serve to remove toxic substances produced by cells (e.g., antioxidative action) and facilitate cell metabolism, thereby improving cell viability. These functions are considered to be attained by the interactions of the components included in the cell protectant of the present invention.

Preferably, in the cell protectant of the present invention, the growth factor comprises basal fibroblast growth factor (bFGF) , epithelial growth factor (EGF) and platelet-derived growth factor (PDGF) . These growth factors are necessary for cell survival and also affect cell activation. The cell protectant of the present invention may further comprise a serum replacement such as albumin, in addition to the growth factor.

Preferably, the aforesaid components of the cell protectant are included in the following contents, based on the entire protectant : amino acid 0.5 to 20 g/L, vitamin 0.01 to 1.0 g/L, mineral 1.0 to 100 g/L, and growth factor 0.001 to 0.1 mg/L.

In the cell protectant of the present invention, the placenta extract is included to keep cells alive during storage and, if necessary, to improve cell activity during culturing after the storage. It may be an extract of the placenta or a hydrolysate of the placenta. The placenta extract or the placenta hydrolysate may be included in the cell protectant in an amount of 1 to 20 wt%, preferably 5 to 20 w% based on the total weight of the protectant.

The placenta extract may be prepared by hydrochloric acid hydrolysis of the placenta from a healthy person who is negative for hepatitis B virus (HBV) , hepatitis C virus (HCV) and human immunodeficiency virus (HIV) . Specifically, in order to deactivate viruses or the like, the placenta is treated with hydrochloric acid over 101 °C for at least 1 hour, and then subjected to sterilization by high pressure steam at about 121 °Cfor 60 minutes . The placenta hydrolysate may be prepared by treating the placenta with acetone to remove fat and performing hydrolysis sufficiently with pepsin and hydrochloric acid. In the example of the present invention, the placenta extract and the placenta hydrolysate commercially available from Hwa Sung Bio Pharm were used.

In a preferred embodiment of the present invention, the cell protectant comprises the components listed in Tables 1 to 4. The compositions and contents of the cell protectant developed by the inventors of the present invention, named "CeLLGUARD", are listed in Tables 1 to .

The cell protectant of the present invention may be used to improve viability of cells stored at 0 to 37 °C, preferably at low temperature of 0 to 10 °C, more preferably at low temperature of 0 to 4 °C, most preferably at low temperature of 4 °C.

Unlike existing stem cell culture media for proliferation and differentiation of cells, the cell protectant of the present invention improves stem cell capability and cell proliferation rate of stem cells without changing cell characteristics. When stem cells are treated with the cell protectant of the present invention and then cultured, they proliferate well and have improved stem cell capability. In Examples 2 and 3 of the present invention, stem cell capability and cell proliferation rate of adipose-derived mesenchymal stem cells were measured. The result revealed that the cell protectant of the present invention had a remarkable effect as compared to the existing stem cell culture medium.

The cell protectant of the present invention may be used to store and transport a cell therapy agent in order to enhance therapeutic effect of the cell therapy agent . The term "cell therapy agent" refers to a new concept medicine used for the purpose of treatment, diagnosis or prevention of diseases by proliferating or screening autologous, allogeneic or xenogenic living cells in vitro or otherwise changing biological characteristics of cells in order to restore or improve the functions of the cells and tissues . Specifically, the patient's own or other animal's somatic cells may be proliferated or stem cells may be differentiated into desired cells in order to treat burns or various incurable such as cancer, dementia, etc. In this regard, hundreds of clinical trials are under way in the US and researches are actively carried out in Korea, too .

To store and transport the cells used for the cell therapy safe until they are applied are very important. A large portion of animal cells, particularly stem cells, die or are deactivated after they are isolated from their tissue. In Example 1 of the present invention, fat cells isolated from the adipose tissue were stored at low temperature of 4 °C for 72 hours using either the existing fat cell culturemediumor the cell protectant of the present invention, and then cell viability was measured. The cells stored using the cell protectant of the present invention showed a cell survival rate of 70% or higher, whereas those stored using the existing fat cell culture medium showed a cell survival rate of

50% or lower. Therefore, the cell protectant of the present invention is very useful to keep cells alive until they are applied to cell therapy.

[Mode for Invention]

The examples and experiments will now be described. The following examples and experiments are for illustrative purposes only and not intended to limit the scope of this disclosure.

Preparation Example 1: Preparation of cell protectant

The cell protectant according to the present invention was prepared by combining the amino acids listed in Table 1 , the minerals listed in Table 2, the vitamins listed in Table 3 and the other constituents listed in Table 4, and was named "CeLLGUARD". As controls, Invitrogen' s DMEM/F12 (1:1) medium further containing 10% fetal bovine serum was used for adipose-derived mesenchymal stem cells and Lonza's KGM medium was used for keratinocytes .

[Table 1] Amino acids

L-Leucine 59.05

L-Lysine · HC1 91. 25

L-Methionine 75. 91

L-Phenylalanine 448 .00

L-Proline 835 .48

L-Hydroproline 20

L-Serine 242 .25

L-Threonine 620 .12

L-Tryptophan 118 .35

L-Tyrosine- 2Na- 2H₂0 167 .79

L-Valine 658 .18

[Table 2] Minerals

[Table 3] Vitamins

Vitamins mg/L

Biotin 0.0035

D-Calicum pantothenate 2.24

Choline chloride 8.98

Folic acid 2.65 i-Inositol 12.60

Niacinamide 2.02

Pyridoxine HC1 2.031

Riboflavin 0.219

Thiamine HC1 14.17

Thymidine 0.365

Vitamin Bi₂ 0.68

Ascorbic acid-2P0₄ (Mg salt) 66.67

[Table 4] Other constituents

Other constituents mg/L

D-Glucose 3151.00

HEPES -

Sodium hypoxanthine 2.39

Linoleic acid 0.042

Lipoic acid 0.105

Phenol red -

Sodium putrescine · 2HC1 0.081

Sodium pyruvate 55

AlbuMAX 1 16666.67

Sodium selenite 0.00001333

Insulin 13.33

Reduced glutathione 2.00 Transferrin (iron salt) 10.67

bFGF 0.01

EGF 0.01

PDGF-AB 0.01

Placenta extract type A 10%

Placenta extract type B 10%

Example 1: Comparison of viability of fat cells

lxlO⁶ fat cells isolated from the adipose tissue were put in vials each containing a control culture medium and the cell protectant of the present invention (hereinafter, "CeLLGUARD") . After keeping at 4 °C, cell viability was measured by counting the number of living cells at predetermined time intervals. The cell counting was carried out using a hemocytometer under a microscope after staining the cells with trypan blue.

Fig. 1 compares cell survival rate of the fat cells isolated from the adipose tissue stored at low temperature (4 °C) for 72 hours in the control culture medium or in the CeLLGUARD solution without culturing. The cell viability was measured by counting living cells. As seen in Fig. 1, cell survival rate of the fat cells stored in CeLLGUARD was 72%, whereas that of the fat cells stored in the fat cell culture medium was only 45%. That is, the CeLLGUARD resulted in 60% increase of cell survival rate as compared to the control culture medium.

Example 2: Confirmation of stem cell capability of adipose-derived mesenchymal stem cells

1) CFU analysis to confirm stem cell capability

Mesenchymal stem cells derived from the adipose tissue were treated with the control culture medium or CeLLGUARD for 1 hour and 500 cells were cultured for 7 days after being inoculated on a 6-well plate. In order to measure colony-forming units (CFU) , the medium was completely removed from the plate. After staining the cells for 30 minutes by adding a 1 : 1 mixture solution of Rhodamine Red and Nile blue, the number of colonies was counted.

Fig. 2a compares stem cell capability of the mesenchymal stem cells derived from the adipose tissue. The cells derived from the adipose tissue were treated with the control culture medium or CeLLGUARD for 1 hour and then stem cell capability was determined by measuring CFU . As seen in Fig. 2a, CeLLGUARD resulted in a colony forming efficiency (CFE) of 10%, which was more than 3 times superior to that of the control culture medium (about 3%) .

2) Confirmation of cell proliferation rate

The mesenchymal stem cells derived from the adipose tissue were treated with the control culture medium or CeLLGUARD for 1 hour and were cultured for 7 days after being inoculated on a culture plate at 25 cells per unit area (cm²) . The cells were collected, stained with trypan blue, and counted under a microscope using a hemocytometer .

Fig. 2b compares cell proliferation rate of the mesenchymal stem cells isolated from the adipose tissue . As seen in the figure , CeLLGUARD resulted in a cell number of 1.5><10⁵, which was about 60% more than that of the control culture medium (9.5^χ10⁴) .

Example 3: Confirmation of cell proliferation rate of adipose-derived mesenchymal stem cells and identification of surface expression marker

1) Cell counting

The mesenchymal stem cells isolated from the adipose tissue were treated with the control culture medium or CeLLGUARD for 1 hour and Ι^χΙΟ⁵ cells were inoculated on a culture plate. After culturing for 7, 10 and 14 days, the cells were collected, stained with trypan blue, and counted under a microscope using a hemocytometer .

Fig .3a compares cell proliferation rate of the adipose-derived mesenchymal stem cells treated with the control culture medium or CeLLGUARD. As seen in Fig. 3a, when the cells were cultured after treating with CeLLGUARD for 1 hour, the cell number increased continuously to 40,000 on the 7th day, 115,000 on the 10th day, and about 180,000 on the 14th day. Especially, the cell number on the on the 14th day was about 80% superior to that of the cells treated with the control culture medium (about 100,000).

2) Flow cytometry

The cells isolated from the adipose tissue were treated with control culture medium or CeLLGUARD for 1 hour and inoculated on a culture plate. After culturing for 7 days, the cells were collected, washed well with phosphate buffer saline (PBS) , washed once again with PBA (1% bovine serum albumin in PBS), and treated with primary antibodies of stem cell markers CD29 (Santa Cruz, USA) , CD34 (Millipore, USA) and CD105 ( illipore, USA) at 1:100 for 1 hour. Then, the cells were washed well 2 times with PBA, treated with FITC-conjugated secondary antibodies (Jackson ImmunoResearch Laboratory, USA) for 30 minutes, and then analyzed using CellQuest Pro software (BD, USA) .

Fig. 3b shows a result of identifying the surface expression marker after treating the mesenchymal stem cells derived from the adipose tissue with the control culture medium or CeLLGUARD. Flow cytometry analysis was performed to identify the cell surface expression markers. As a result, the cells treated with CeLLGUARD did not show significant change in the cell surface markers, which reveals that CeLLGUARD promotes cell growth without changing the basic characteristics of the cells. Example 4: Confirmation of stem cell capability using animal model

A CF1 mouse was anesthetized and the hair on the back was removed cleanly using a razor. After wounding using an 8 mm punch, Ι^χΙΟ⁶ mesenchymal stem cells isolated from the adipose tissue, which had been treated with the control culture medium or CeLLGUARD for 1 hour, were sprayed on the wound site. After dressing, the degree of wound healing was observed 6 days later.

Figs. 4a and 4b compare the wound healing effect by the mesenchymal stem cells treated with the control culture medium or CeLLGUARD. Fig. 4a shows the result of observing wound healing 6 days after the dressing, and Fig. 4b shows the result of measuring the wound size. As seen in the figures, the mesenchymal stem cells treated with CeLLGUARD resulted in faster and better wound healing than those treated with the control culture medium.

Example 5: Confirmation of surface expression marker of keratinocytes

Keratinocytes isolated from human skin tissue were treated with the control culture medium or CeLLGUARD for 1 hour. After culturing, the cells were collected, washed well with PBS, washed once again with PBA ( 1% bovine serum albumin in PBS ) , and then treated with primary antibodies of keratinocyte marker CD24 and stem cell markers CD29 (Santa Cruz, USA), CD34 (Millipore, USA) and CD90 (Millipore, USA) at 1:100 for 1 hour. Then, the cells were washed well 2 times with PBA, treated with FITC-conj ugated secondary antibodies ( Jackson ImmunoResearch Laboratory, USA) for 30 minutes , and then analyzed using CellQuest Pro software (BD, USA) .

Fig. 5 shows the result of identifying the surface expression markers of the keratinocytes. As seen in Fig. 5, the keratinocytes treated with CeLLGUARD did not show significant change in the cell surface expression markers, which reveals that CeLLGUARD does not change the basic characteristics of the cells.

Example 6: Confirmation of stem cell capability of keratinocytes

The keratinocytes isolated from the skin tissue were treated with the control culture medium or CeLLGUARD for 1 hour and 500 cells were cultured for 7 days after being inoculated on a 6-well plate. In order to measure CFU, the medium was completely removed from the plate. After staining the cells for 30 minutes by adding a 1:1 mixture solution of Rhodamine and Nile blue, the number of colonies was counted.

Fig. 6 compares stem cell capability of the keratinocytes isolated from the skin tissue cultured after treating with the control culture medium or CeLLGUARD. As seen in Fig. 6, CeLLGUARD resulted in a CFE more than 4 times superior to that of the control culture medium. This means that CeLLGUARD results in remarkably improved stem cell capability.

The present application contains subject matter related to Korean Patent Application No . 10-2010-0018122, filed in the Korean Intellectual Property Office on 26, Feb. 2010, the entire contents of which is incorporated herein by reference.

While the present invention has been described with respect to the specific embodiments, it will be apparent to those skilled in the art that various changes and modifications may be made without departing from the spirit and scope of the invention as defined in the following claims.

[industrial Applicability]

As described, the cell protectant of the present invention may be used to safely preserve cells and may be used to store not only stem cells but also various other animal cells. It may be also used as a cell protectant to store and transport the cells used for cell therapy at low temperature. Further, by treating animal cells with the cell protectant of the present invention before culturing, cell proliferation rate and stem cell capability may be improved. Accordingly, the cell protectant of the present invention isveryusefulasa protectant to preserve cells and improve stem cell capability.

Claims

[CLAIMS]

[Claim l]

A cell protectant comprising an amino acid, a mineral, a vitamin, a growth factor and a placenta extract.

[Claim 2]

The cell protectant according to claim 1, wherein the ammo acid comprises L-cystine and L-hydroproline in addition to basic amino acids constituting proteins.

[Claim 3]

The cell protectant according to claim 1, wherein the mineral comprises trace elements zirconium, germanium and vanadium in addition to general elements required for cell growth.

[Claim 4]

The cell protectant according to claim 1, wherein the vitamin comprises calcium pantothenate, choline chloride, folic acid, inositol and ascorbic acid, which are capable of inhibiting reactive oxygen species.

[Claim 5]

The cell protectant according to claim 1, wherein the growth factor comprises basal fibroblast growth factor (bFGF) , epithelial growth factor (EGF) and platelet-derived growth factor (PDGF) .

[Claim 6]

The cell protectant according to claim 1, wherein the placenta extract is an extract of the placenta or a hydrolysate of the placenta .

[Claim 7]

The cell protectant according to claim 1, which comprises the components listed in Tables 1 to 4.

[Claim 8]

The cell protectant according to claim 1, which improves viability of cells stored at 0 to 37 °C.

[Claim 9]

The cell protectant according to claim 1, which improves stem cell capability and cell proliferation rate without changing cell characteristics .

[Claim 10]

The cell protectant according to claim 1, which is used to store and transport a cell therapy agent in order to enhance therapeutic effect of the cell therapy agent.