WO2014112675A1 - Natural cryopreservative agent for bone marrow-derived stem cells and cryopreservation method for bone marrow-derived stem cells using same - Google Patents

Abstract

The present invention relates to a cryopreservative agent for bone marrow-derived stem cells, and the present invention is achieved by the steps of: preparing a cryopreservative agent of various compositions of DMSO, FBS, trehalose, catalase and zVAD-fmk; cryopreserving for three weeks a bone marrow-derived mesenchymal stem cell introduced into the cryopreservative agent; after thawing, measuring and evaluating the cell viability and cell proliferation; and measuring the expression of a specific surface marker and stem cell marker according to the conditions, and by confirming and evaluating the differentiation ability after inducing the myocyte differentiation according to the conditions, where the present invention has an effect of providing a cryopreservative agent which protects cells when subjecting bone marrow-derived mesenchymal stem cells to cryopreservation, allows a long-term cryopreservation of stem cells and exhibits excellent effects such as maintaining high viability and achieving differentiation into tissue-specific cells.

Description

Natural cryopreservation agent of bone marrow-derived stem cells and cryopreservation method of bone marrow-derived stem cells

The present invention relates to a natural cryopreservative used for cryopreservation of bone marrow-derived stem cells, and more particularly to cryopreservatives configured to protect cells and increase cell viability during cryopreservation of bone marrow-derived stem cells.

Bone marrow mesenchymal stem cells (BMSCs) are self-renewal and have been proven to be able to differentiate into bone cells, chondrocytes, adipocytes, myoblasts and neurons, depending on the culture environment. It is applied in tissue engineering.

Cell therapy is applied to the treatment of many diseases and the use of mesenchymal stem cells (MSCs) in tissue regeneration has been used as cell therapy because it has no carcinogenic properties for autologous transplantation. In animal experiments, MSCs derived from bone marrow and adipose tissue were used to treat bone and cartilage. These cells have also been reported to be able to treat a wide range of diseases, including spinal cord injury and myocardial infarction. However, despite the regeneration research and development in cell and animal experiments of MSCs, cryopreservation of MSCs has not been developed for proper use in various clinical conditions, and large numbers of autologous cells, especially for cell therapies requiring repeated transplantation, have been developed. As it needs to be secured, clinically applicable cryopreservation standards are needed.

Cryopreservation is a process that freezes and stores below -80 ° C to maintain biological functions in liquid nitrogen (-196 ° C) for various uses of tissues and cells. Enable long term storage. Cryopreservation is increasing in importance in recent years due to the development of cell and tissue transplantation, genetic technology, tissue engineering, in vitro fertilization, and preservation of biological data. Therefore, if long-term cryopreservation of stem cells is possible, high viability and differentiation into tissue-specific cells can be diversified in clinical applications, and autologous transplantation can prevent transplant rejection as well as homogeneous cell therapy. It will be important to

Cryopreservatives improve the survival rate of cells after thawing by reducing the physical and chemical cell damage that occurs during freezing and thawing. Cryopreservatives fall into two categories. Intracellular drugs are DMSO, glycerol, and ethylene glycol, which penetrate into the cell to prevent ice crystal formation and membrane rupture. Extracellular mixtures are sucrose, trehalose, dextrose and polyvinylpyrrolidone that cannot penetrate the cell membrane and serve to reduce the high osmotic effect during the freezing process. The most widely used cryopreservative is DMSO, first described by Lovelock et al. In 1959. It is a hygroscopic polar compound and is known to be toxic at room temperature. DMSO stabilizes cellular proteins, phospholipid bilayers and plasma membranes at high concentrations of 10% (v / v) and is known to be associated with neurotoxicity in the brain (Y Liu, X Xu, X Ma et.al., 2010). ).

Injecting thawed cells into patients without removal of DMSO causes many side effects and toxic reactions. Examples of side effects include sedation, headache, nausea, vomiting, high blood pressure, adycardia, hypotension, and anaphylactic shock. Toxicity is known to be correlated with the amount of DMSO. DMSO should be removed or the concentration reduced. While removal of DMSO prior to infusion is most reasonable for patients, it may be costly, time consuming, or loss of cells in the removal process, so if cryopreservation reduces DMSO concentrations, there is no need to remove cryopreservatives. Thawed cells can also be injected directly.

Meanwhile, FBS (Fetal Bovine Serum) is known to protect cells during cryopreservation because it stabilizes biological membranes and adjusts osmotic pressure as cryopreservative. However, since FBS is not a component of the human body, it is not preferable to use for preservation of cells for therapeutic purposes, and there is a risk of infecting proteins causing viral transmission, prion disease, and early heterogeneous immune responses. The use of autologous serum instead of FBS can solve ethics and safety issues but is time consuming and very limited in production.

Commonly formulated formulations of cryopreservatives commonly used are 10% DMSO (v / v) and up to 90% FBS (v / v), which are widely used in various types of stem cells, including human bone marrow-derived stem cells However, since both DMSO and FBS have a number of disadvantages, cryopreservatives containing approved materials must be used for the clinical application of cryopreserved cells, and cryopreservatives need to be removed from animal-derived biological materials or components of infectious diseases. .

Disaccharides such as sucrose and trehalose are widely used as non-toxic natural cryopreservatives and act as cell membrane stabilizers when dehydration occurs during freezing. In general, trehalose is commonly found in organisms such as yeasts and nematodes that maintain life even in frozen or dehydrated plants, and these characteristics are being studied as alternative cryopreservatives. The usefulness of trehalose as a cryopreservative interacts with lipid membranes, stabilizes unstable proteins during freeze-thaw, inherent chemical safety, and also enables stable glasses formation during cryopreservation.

Another cause of loss of cell viability during or after freezing is the oxidation of oxygen free radicals. Peroxide radicals cause oxidative damage such as lipid peroxidation, protein oxidation, and DNA damage in cells, and have a protective effect by adding catalase. Therefore, the addition of antioxidants such as catalase to cryopreservatives can be expected to have cell regeneration after thawing bone marrow-derived mesenchymal stem cells. Despite improved cryopreservation, cell viability may be reduced within 24 hours after thawing. The mechanism of cell death during apoptosis by cryopreservation is known to be initiated by caspase activity. Protease inhibition of caspase strains in frozen injury inhibits apoptosis in various experimental environments, and zVAD-fmk is known as a caspase inhibitor.

As a domestic patent for a cryopreservation method of stem cells, a stem cell cryopreservation solution containing sucrose, ethylene glycol and polyvinylpyrrolidone in PBS (phosphate buffered saline) solution is disclosed in Korean Patent Publication No. 10-2012-0117209. There is a bar. Korean Patent No. 10-1101308 discloses a cryopreservation medium composition of human-derived mononuclear cells containing DMSO, hydroxyethyl starch, autologous plasma and animal-free serum medium. As a domestic patent for mesenchymal stem cell medium, Patent No. 10-0908481 discloses a mesenchymal stem cell culture medium including umbilical cord blood serum, insulin, hydrocortisone, EFG and LIF.

Accordingly, it is an object of the present invention to provide a natural cryopreservative that can protect cells and improve cell viability in cryopreservation of bone marrow-derived mesenchymal stem cells.

Another object of the present invention is to provide an effective cryopreservation method for reducing physical and chemical cell damage in cryopreservation process.

The object of the present invention is to prepare a cryopreservative with various compositions of DMSO, FBS, trehalose, catalase and zVAD-fmk; Cryopreserving the bone marrow-derived mesenchymal stem cells in the cryopreservative for three weeks; Measuring and evaluating cell viability and proliferation after thawing; Measuring expression of specific surface markers and stem cell markers according to the conditions; It was achieved by identifying and evaluating the differentiation capacity after myocyte differentiation induction by the above conditions.

The present invention has the effect of providing a cryopreservative to protect the cells in cryopreservation of bone marrow-derived mesenchymal stem cells, it is possible to maintain the long-term cryopreservation of the stem cells and to maintain a high survival rate and to differentiate into tissue-specific cells.

1 is a graph showing the cell viability after thawing of cryopreserved BMSCs with various compositions of DMSO, FBS, trehalose, catalase and zVAD-fmk,

Figure 2 is a graph showing the cell growth curve after thawing of cryopreserved BMSCs with various compositions of DMSO, FBS, trehalose, catalase and zVAD-fmk,

3 is a diagram showing the results of flow cytometry after thawing of BMSCs cryopreserved with various compositions of DMSO, FBS, trehalose, catalase and zVAD-fmk, (A) is solution 1 of Table 1, (B) is Solution 4, (C) shows solution 7.

4 is a diagram showing RT-PCR results after thawing of cryopreserved BMSCs with various compositions of DMSO, FBS, trehalose, catalase and zVAD-fmk,

FIG. 5 shows the differentiation capacity of BMSCs cryopreserved with a solution of trehalose, catalase and zVAD-fmk mixed with 5% (4th solution) DMSO or 2.5% (7th solution) DMSO, followed by differentiation into myocytes. (A) MyoD and desmin protein expression in Western blot of myocytes and (B) MyoD and desmin protein expression in immunofluorescence staining of myocytes.

Hereinafter, specific examples will be described in detail with reference to Examples.

Material Preparation

1-1: Preparation of Bone Marrow-derived Mesenchymal Stem Cells

Human bone marrow-derived mesenchymal stem cells supplied from the Department of Oral Pathology, College of Dentistry, Kyungpook National University, Korea, were used as experimental materials.

1-2: Cryopreservative Preparation

All frozen vials were prepared so that the final medium was 1 mL after cell addition. DMSO was added at a rate of 10%, 5%, 2.5% (v / v), trehalose (Sigma-Aldrich, St. Louis, MO, USA), catalase (Sigma-Aldrich, St. Louis, MO, USA), zVAD-fmk (R & D systems, Minneapolis, MN, USA) were added to the cell freeze vials at concentrations of 60 mmol / L, 100 μg / mL and 30 μM, respectively (Table 1).

Statistical analysis

The experimental results were expressed as mean ± standard error, and the difference between the means was obtained by t test calculator. The statistics were analyzed using GraphPad software, and statistical significance was set at p <0.01 and p <0.05.

Example 1 Cryopreservation and Thawing of Bone Marrow-derived Mesenchymal Stem Cells

Cryopreservation of bone marrow-derived stem cells was tested in cryopreservatives with different combinations of ingredients, as shown in Table 1. 100% viability). All groups were allowed to contain 1 × 10 ⁶ cells in 1 mL of the final freezing medium and frozen using a controlled-rate freezer (Cryo, Rockville, MD, USA). Each experimental group was stored in a liquid nitrogen tank for three weeks.

The thawing of bone marrow-derived mesenchymal stem cells was rapidly thawed in a water tank set at 37 ° C., and the thawed cells were cultured for two passages until the required cell number was reached.

Example 2: Survival analysis of bone marrow-derived mesenchymal stem cells

In order to confirm the survival rate after thawing the bone marrow-derived mesenchymal stem cells stored in the seven cryopreservatives obtained as a result of the experiment according to Example 1, cck-8 assay was performed on the 1st, 2nd and 3rd days, respectively. Cell viability was tested by the manufacturer's method using Cell Counting Kit-8 (Dojindo Molecular Technologies, Rockville, USA).

Experimental results showed that 5% DMSO added trehalose and catalase to the control group (10% DMSO + 30% FBS) showed statistically significant difference on the 1st and 2nd day compared to the control group (p <0.05). In addition, trehalose, catalase and zVAD-fmk in 5% DMSO group showed statistically significant difference (p <0.01), and the highest survival rates were seen in 1st, 2nd and 3rd day. On the other hand, in the group containing 2.5% DMSO showed a similar or lower survival rate compared to the control (Fig. 1).

Example 3: Analysis of proliferation capacity of bone marrow-derived mesenchymal stem cells

Growth curve experiments of bone marrow-derived mesenchymal stem cells after 3 weeks of freezing were performed by the hand method.

As a result of culturing thawed bone marrow-derived mesenchymal stem cells for 8 days and analyzing the proliferative capacity in each group, it was confirmed that the overall growth started from day 2 and gradually proliferated until day 6 and then increased rapidly from day 7.

 Compared with the control group, the growth rate was similar in the 5% DMSO group containing 30% FBS, and the growth rate was higher in the group containing 5% DMSO, trehalose, catalase and zVAD-fmk. When the population doubling time (PDT) was calculated, the control group was 47.6 hours, and the 5% DMSO group added with trehalose and catalase and the group added with catalase inhibitors were 45.4 and 45.7 hours, respectively (FIG. 2).

Example 4 Analysis of Expression of Specific Surface Markers According to Culture Conditions

According to Example 2, the cryopreserved bone marrow-derived mesenchymal stem cells were thawed under various cryopreservative conditions, and the expression of specific surface markers was analyzed by flow cytometry. To analyze cell specific surface antigen expression, FACS antibodies were analyzed by flow cytometry using PE-conjugated monoclonal antibodies control, CD44, CD45, CD73, CD90, CD105, SSEA4, HLA ABC and HLA DR (FACSCalibur, BD biosciences, San Jose, CA, USA) to analyze the expression.

As a result, CD44 (hyaluronan receptor), CD73 (marker of SH-3 and SH-4), CD90 (marker of Thy-1), CD105 (endoglin) were expressed, and CD45 (marker of hematopoietic lineage), SSEA-4 ( stage-specific embryonic antigen) was not expressed. Class I major histocompatibility (MHC) antigens (HLA-ABC) were expressed, whereas MHC Class II (HLA-DR) was not expressed. As with the control, trehalose, catalase and zVAD-fmk were added to 5% and 2.5% DMSO, showing similar results for the expression of specific surface markers under all cryopreserved conditions. Therefore, even after cryopreservation, human bone marrow-derived mesenchymal stem cells were confirmed to have characteristics (FIG. 3).

Example 5: Expression Analysis of Stem Cell Markers

Reverse transcription-polymerase chain reaction (RT-PCR) was performed to determine whether stem cell markers were expressed even when thawed after cryopreservation in bone marrow-derived mesenchymal stem cells cryopreserved for three weeks. Primers used for PCR refer to Moon et al. (Table 2). In all groups, expression of the stem cell markers GATA-4, SCF, Vimentin, FGF-5, CK-18, and Oct-4 was clearly observed, and there was no difference between the conditions (FIG. 4).

Example 6: Analysis of Differentiation Capacity into Myocytes

Each bone marrow-derived mesenchymal stem cells stored in the cryopreservative of the present invention were thawed and dispensed into cell culture plates. To induce differentiation into myocytes after 24 hours, 10% horse-serum (Gibco / BRL, Carlsbad, CA, USA), 0.5% chick embryo extract (Gibco / BRL, Carlsbad, CA, USA), 1% penicillin / streptomycin A low-glucose formulation (DMEM) differentiation medium (Hyclone, Logan, UT, USA) was added and the medium was changed every 2-3 days to induce differentiation for 7 days. Thereafter, Western blotting was performed to compare the differentiation ability to myocytes.

Differentiation-induced bone marrow-derived mesenchymal stem cells were washed with PBS after removal of medium and RIPA lysis buffer (15 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 50 mM Tris-HCl pH 7.5, 2 mM) Protein was extracted by adding EDTA pH 8.0, 1 × protease inhibitor cocktail). After quantification, the extracted protein was isolated from 10% SDS-poly acrylamide gel, and polyclonal anti-human MyoD (1: 1000, BD Pharmingen, San Diego, CA) was used as the primary antibody and secondary antibody was used as secondary antibody. Using a peroxidase-conjugated antibody (1: 1000, Amersham Bioscience, Uppsala, Sweden), photosensitize X-ray films using SuperSignal West Pico fluorescence reagents (Therm scientific, Inc., Bremen, Germany) to express specific protein expression. Confirmed.

Western blotting revealed that myoD and desmin, myocyte markers, were expressed in all groups. Among them, the addition of trehalose, catalase, and zNAD-fmk in 5% DMSO increased the expression of MyoD and desmin in 2.5% DMSO than in the same composition (FIG. 5 (A)).

In addition, immunofluorescence staining was performed after Western blotting in order to compare the differentiation potential into myocytes.

Each solution was seeded on 4-chamber slides (BD Biosciences, Bedford, MA, USA) to differentiate derivatized bone marrow-derived mesenchymal stem cells for 7 days from desmin (BD Pharmingen, San Diego, CA) and MyoD (BD Pharmingen, San Diego, CA) was diluted with PBS at 1: 400 and 1: 200, respectively, and reacted at 37 ° C. for 1 hour, followed by 0.5 mg / ml of fluorescein isothiocyanate (FITC) conjugated goat anti-mouse (Santa). cruz Biotechnology, Santa Cruz, CA, USA) was reacted for 1 hour at room temperature at a ratio of 1: 800, 1: 400, and nuclear staining was performed using DAPI (PI, Sigma, USA), and a fluorescence microscope (Nikon ECLIPSE Ti). -S, Tokyo, Japan).

As a result of immunofluorescence staining, it was confirmed that the groups in which trehalose, catalase, and zVAD-fmk were added in 5% and 2.5% DMSO, as in the control group, expressed myoD and desmin, myocyte markers (FIG. 5 (B)). Therefore, it can be seen that bone marrow-derived mesenchymal stem cells thawed after cryopreservation can differentiate into myocytes when cultured in differentiation medium.

Claims (2)

1. Contain 1 × 10 ⁶ cells per 1 mL of total volume of cryopreservative and consist of 5% (v / v) DMSO, 60 μg trehalose, 100 μg catalase, 0.03 μg zVAD-fmk Cryopreservative of bone marrow-derived stem cells characterized in that.
2. A cryopreservation method of bone marrow-derived stem cells according to claim 1, wherein the cryopreservative of the bone marrow-derived stem cells is placed in a frozen vial and frozen using a speed-controlled freezer and stored in a liquid nitrogen tank.

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