KR101473315B1 - A freezing medium composition for cryopreserving amniotic fluid-derived stem cells and a method for cryopreserving the same - Google Patents

Abstract

The present invention can greatly reduce the Me ₂ SO concentration and remove animal serum (fetal bovine serum) while using amylase, sucrose and catalase to cryopreserve long-term cryopreservation With the method, amniotic fluid stem cells can be cryopreserved using standard 1/4 Me ₂ SO concentration by adding disaccharides, antioxidants and a caspase inhibitor. As a result, the use of low concentration Me ₂ SO ₄ in cryopreservation solutions has the industrial effect of supporting clinical development of AFSC.

Description

[0001] The present invention relates to a freezing medium composition for cryopreservation of amniotic stem cells and a cryopreserving method for cryopreserving amniotic stem cells,

The present invention relates to a cryopreservation composition for cryopreservation of amniotic fluid stem cells and a method for cryopreserving amniotic fluid stem cells. More particularly, the present invention relates to a method for cryopreservation of amniotic stem cells, which comprises the steps of significantly reducing the concentration of Me ₂ SO and removing fetal bovine serum, The present invention relates to a method for cryopreservation of amniotic stem cells for cryopreservation for a long period without cryopreservation using amylase, trehalose, sucrose and catalase, and a method for cryopreservation of amniotic stem cells.

Recently, amniotic fluid (AF) has emerged as a source of well-characterized mesenchymal stem cells that can be obtained without causing ethical problems associated with human embryonic stem cell research. Amniocentesis stem cells (AFSCs) have been shown to differentiate into a number of cell lines, including fat, bone, muscle and nerve cells.

Amniotic fluid, which is generally discarded after birth, can provide a richer source of stem cells than other parts of the body. AFSC exhibits better proliferation rate and increased differentiation compared to adult bone marrow stem cells. In addition, AFSC exhibits immunomodulatory properties and thus may be used in immune mediated diseases as well as in the treatment of graft-versus-host disease. Thus, it is relatively easy to supply stem cells from AF, and long term banking of AFSC will have a significant impact on future regenerative medicine technology. However, one of the main stumbling blocks in the production of clinical stem cells is that there is currently no good manufacturing method for cell processing, cryopreservation, storage and distribution.

Developing effective techniques for cryopreservation of AFSC is important in stem cell savings. The rate of freezing is an important factor in determining cell viability after cryopreservation and storage. Cooling the cells at a controlled gentle rate prevents intracellular ice accumulation that can cause cell membrane rupture. However, even in gentle freezing, cells can be dehydrated by extracellular ice formation, and a cryopreservative to prevent this is generally added to the freezing medium.

The most widely used cryopreservative (CPA) is dimethyl sulfoxide (Me ₂ SO), a polar compound with hygroscopicity that can be toxic to cells. For example, the most commonly used method AFSC cryopreservation medium comprises a freezing medium consisting of 10% Me ₂ SO as a CPA in the presence of animal or human serum. However, transplantation of Me ₂ SO- cryopreserved hematopoietic stem cell products has been associated with cryopreservation used in AFSC, despite the frequent adverse effects associated with severe side effects such as vomiting, hypotension, acute abdominal pain, dyspnea, cardiac arrhythmia and hemoglobinuria There were no studies on the clinical effect of Me ₂ SO in the medium.

Addition and exclusion of CPA is a complex process associated with osmotic shocks harmful to these cells. Therefore, it is important to develop CPA-free medium or non-toxic CPA for cryopreservation and storage of stem cells. To do this, we must know how the decrease in Me ₂ SO concentration affects the viability and performance of frozen AFSC in the absence of allogeneic / autologous serum or serum.

Disaccharides such as sucrose and trehalose have been widely used as excipients for freeze-drying and as stabilizers in dehydration processes as well as natural cryopreservation agents. In fact, many organisms can survive in nearly complete dehydration. This is a phenomenon known as dehydration and is similar to dehydration that occurs during cryopreservation. Dewatered lice are associated with the accumulation of large amounts of disaccharides such as trehalose in some cases, which has generated great interest in using trehalose as a non-toxic CPA. See, for example, L.S. Limaye, V. P. Kale, Cryopreservation of human hematopoietic cells with membrane stabilizers and bioantioxidants as additives in the conventional freezing medium, J. Hematother, Stem Cell Res. 10 (2001) 709-718) teaches that trehalose is effective in preserving hematopoietic progenitor cells. Scheinkonig, S. Kappicht, H.J. Kolb, M. Schleuning, Adoption of long-term cultures to evaluate the cryoprotective potential of trehalose for freezing hematopoietic stem cells, Bone Marrow Transplant. 34 (2004) 531-536] evaluated insulin and trehalose in the preservation of colony forming ability of bone marrow and peripheral blood stem cells.

In addition to dehydration, the formation of oxygen free radicals is another cause for decreased cell viability during or immediately after cryopreservation. Limaye et al. Describe that the addition of a bio-antioxidant to a cryopreservation solution increases recovery after cell thawing. The use of membrane stabilizers and bio-antioxidants has been shown to improve cryopreservation of human hematopoietic cells.

Another prior art technique is that serum proteins used in cryopreservation media are difficult to remove during washing, and residues left in the cell solution can cause side effects in patients who have undergone cell implantation or transplantation. Therefore, it is important to develop a serum-free medium for the storage of stem cells for clinical use.

Korean Patent Application No. 10-2006-7004375 " Biodegradable polymer-ligand conjugate and separation of cell subpopulations, cryopreservation of cells, use thereof in culture and transplantation " in the prior patent documents includes (a) at least one biodegradation (B) freezing the mixture, and (c) thawing and recovering the cells from the cell-polymer particle conjugate, wherein the anchorage-dependent ) Cell is cryopreserved,

Korean Patent Application No. 10-2009-7007620 'Method of cryopreserving tissue-derived cells' includes a method of' fragmenting a tissue containing a desired cell, culturing the fragmented tissue piece in a predetermined medium, recovering the tissue piece, And suspending the suspension in a protective solution and freezing the suspension of the tissue slice at a temperature of -70 ° C or lower.

Korean Registration No. 1005342150000 'Isolation and Culture of Mesenchymal Stem Cells from Cryopreserved Umbilical Cord Blood' includes thawing cryopreserved cord blood and diluting with aMEM (alpha-minimum essential medium) medium and centrifuging to harvest mononuclear cells; Separating CD133-positive cells from the monocytes obtained; The isolated cells were cultured in the presence of Stem Cell Factor, granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF), interleukin-3 Quot;) and a method of suspending and culturing mesenchymal stem cells from cryopreserved umbilical cord blood, comprising the step of suspending the cells in an aMEM culture medium,

None of the patent documents describe a composition for cryopreservation of amniotic fluid stem cells and a cryopreservation method thereof.

Conventionally, the greatest obstacle in the production of clinical stem cells is that there is no suitable composition and preparation method for cryopreservation, storage and distribution of these cells. The most recent cryopreservation compositions and methods used in stem cells include toxic cold protection (CPA) dimethyl sulfoxide (Me ₂ SO) in the presence of animal serum proteins in human therapy applications. To prevent complications associated with cryoprotectants, non-toxic CPAs in the used freeze medium should be developed or CPA concentration should be reduced.

The inventors have demonstrated that apoptosis induced by cryoinjury is a major cause of decreasing the viability of human embryonic stem cells after mild freezing. In addition, according to preclinical data, caspase activation during the freezing and thawing process can induce apoptosis, thus contributing to the freezing damage of the graft for transplantation. Therefore, the use of caspase inhibitors with other cytoprotective agents can provide protection during long-term storage of viable cells, which is important for tissue engineering success.

In the present invention, it has been found that a frozen medium containing a disaccharide, a bio-antioxidant and a caspase inhibitor may be useful for preserving AFSC. To prove this, various frozen media containing these components were prepared, The test confirms that the AFSC degree of protection is increased, thereby reducing the final concentration of Me ₂ SO present in the final AFSC-containing injection product.

Accordingly, the present invention provides a freeze-dried medium composition for cryopreservation of amniotic fluid stem cells, which comprises trehalose, catalase and zVAD-fmk together with a low concentration of Me ₂ SO.

In the present invention, wherein in Me ₂ SO concentration of 2.5 to 5% (v / v), a trehalose concentration of 60 mmol / L, catalase concentration of 100 mg / mL and, zVAD-fmk a concentration of 30 lt; RTI ID = 0.0 > uM. < / RTI >

The present invention also provides a method for cryopreserving amniotic fluid stem cells using the above freeze-dried media composition.

According to the present invention, cell viability after cryopreserved thawing in a solution containing trehalose, catalase and zVAD-fmk with 5% Me ₂ SO was statistically significantly increased (p < 0.05). Solutions containing trehalose and catalase in combination with 5% or 2.5% (v / v) Me ₂ SO were used for culture proliferation, cell surface antigen expression and mRNA expression of stem cell markers in cryopreserved AFSC for a minimum of 3 weeks (10% (v / v) Me ₂ SO and 30% FBS) in the control group. Thus, AFSC can be cryopreserved using standard 1/4 Me ₂ SO concentration by adding disaccharides, antioxidants and a caspase inhibitor. The use of low concentrations of Me ₂ SO in cell freezing solutions has industrial benefits that support the clinical development of AFSC.

Figure 1 shows the different compositions of Me ₂ SO, FBS, trehalose, catalase and zVAD-fmk (carbobenzoxy-v-allyl-alanyl-aspartyl- (O-methyl) -fluoromethyl ketone) Figure 6 shows the MTT assay of AFSC after cryopreserved thawing.
Figure 2 shows the culture growth curve of AFSC after thawing cryopreserved in different compositions of Me ₂ SO, FBS, trehalose, catalase and zVAD-fmk.
Figure 3 shows the flow cytometric analysis of AFSC after cryopreserved thawing in a solution containing different compositions of Me ₂ SO, FBS, trehalose and catalase.
Figure 4 shows the RT-PCR analysis of AFSC after cryopreserved thawing with different compositions of Me ₂ SO, FBS, trehalose, catalase and zVAD-fmk. Here, Non-cryo represents AFSC not cryopreserved.
Figure 5a shows the addition of trehalose and catalase with 5% (v / v) Me ₂ SO (solution 3) and 2.5% (v / v) Me ₂ SO (solution 6) on plastic plates precoated with Matrigel Containing solution after cryopreserved thawing and then induced by 5-azaC of AFSC, followed by Western blot analysis, and Fig. 5B is a diagram showing the above by immunofluorescence staining.

AFSC is a number of lineage-differentiated pluripotent stem cells, including three representative embryonic germ layers (ectoderm, endoderm and mesoderm). These cells can be easily mass produced, cryopreserved, and delivered to the hospital for immediate use as a cell source for therapeutic use. However, large quantities of frozen stored cells may be required in clinical applications, and thus the development of stem cell savings is required. Such savings should ensure the quality and safety of these cell products, especially when stored cells are intended for clinical use in cell therapy and regenerative medicine.

During cryopreservation, cell membranes are greatly damaged by intracellular ice formation occurring during current refrigeration protocols. Therefore, the development of more effective techniques for stem cell cryopreservation is an important aspect to be considered for the realization of cell saving.

The addition of CPA is a common measure during cryopreservation to reduce or eliminate cell damage from freezing. Currently, most stem cell cryopreservation protocols use the plasma membrane-permeable molecule Me ₂ SO as CPA. However, clinical use of frozen-thawed stem cells treated with Me ₂ SO can lead to renal failure, cardiac arrhythmia, and other complications. Therefore, it is important to develop cryopreservation protocols involving low concentrations of Me ₂ SO or non-toxic CPA.

Disaccharides, antioxidants and caspase activity inhibitors are used in the present invention. In order to reduce the amount of Me ₂ SO in the infusion products and to remove the FBS, the frozen effect of low concentration of Me ₂ SO in cryopreservation of AFSC was analyzed.

Many plants and animals can survive even in a nearly complete dehydration state by the accumulation of large amounts of disaccharides, particularly sucrose and trehalose. Disaccharides can form highly viscous, low-migration glass, increasing the stability of the preserved material. This suggests that what is needed to preserve the structure and function of membranes, liposomes and proteins is merely the ability of the additive (sugar or polysaccharide) to form glass. Besides glass formation, direct interaction between polar groups and sugars in proteins and phospholipids appears to be essential for stabilizing biomaterials of various compositions during air drying or lyophilization.

Trehalose, a non-toxic disaccharide of glucoses, has been widely used as an excipient for lyophilization and as a cryopreservative as a stabilizer during dehydration. His cryopreservation ability was evaluated in various tissues and cells. The survival rate of pancreatic tissue cryopreserved by adding trehalose to the cryomedia of Ms ₂ SO-system is increased. This also umbilical cord blood, and as evidenced by the colony formation increase of human hematopoietic stem cells preserved frozen from a fetal liver, 10% Me ₂ OZ is used with 10% Me ₂ SO to SO trehalose compared to that used by itself Thereby providing excellent cryopreservation properties.

Another cause of injury due to freezing is the formation of free radicals, which has been pointed out as a cause of decreased cell viability. Free radicals increase under low humidity, and in the freezing point conditions oxidative damage such as lipid peroxidation, protein oxidation and DNA damage occur. The main defense mechanisms of cells against free radical-mediated damage include antioxidants such as ascorbic acid, alpha tocopheryl acetate, reduced glutathione, superoxide dismutase, catalase and peroxide.

Apoptosis has been reported to play an important role in cell cold injury. Apoptosis is induced by activation of both caspase-8 and caspase-9 during cryopreservation. In the present invention, the synthetic broad-spectrum irreversible caspase inhibitor zVAD-fmk is used together with other cryopreservatives to increase survival rate after thawing of AFSC.

Since the cryopreservation modes of action of trehalose, catalase and zVAD-fmk are totally different, the present invention uses 5% or 2.5% Me ₂ SO 4 for the purpose of reducing the amount of Me ₂ SO in conventional freezing media or to exclude FBS The above three compounds are used in combination with SO.

Preferably, the trehalose, catalase and zVAD-fmk are contained in a cryopreservation solution in the range of 60 mmol / L, 100 mg / mL and 30 μM, respectively.

In the present invention, the disaccharide (trehalose), the antioxidant (catalase) and the disaccharide in the serum-free solution of the low-concentration Me ₂ SO and the antioxidant And that the cryopreserved cells in the presence of the caspase inhibitor (zVAD-fmk) are more viable after thawing than the control solution.

The disaccharide may be an umbilical cord blood cell, or an umbilical cord blood, for example, as described in JP Rodrigues, FH Paraguassu-Braga, L. Carvalho, E. Abdelhay, LF Bouzas, LCPorto, Evaluation of trehalose and sucrose as cryoprotectants for hematopoietic stem cells of umbilical cord blood, Of hematopoietic stem cells in a cryopreservation solution to reduce the Me ₂ SO concentration.

In addition, L. Limaye et al., Supra, has demonstrated that antioxidants added to conventional freezing media improve the protection of mouse bone marrow cells and human adult bone marrow.

Also, Xu, S. Cowley, C.J.Flaim, W. James, L. Seymour, Z. Cui, The roles of apoptotic pathways in cryopreservation of dissociated human embryonic stem cells, Biotechnol. Prog. 26 (2010) 827-837) demonstrated successful cell preservation and cell recovery by using caspase inhibitors as cryopreservatives.

However, none of the prior art specifically describes the ideal combination of a cryopreservative solution of amniotic fluid stem cells.

The present invention evaluates the use of disaccharides, antioxidants and caspase inhibitors for cryopreservation of AFSC with reduced concentrations of Me ₂ SO 4. Thawed cells are subjected to a survival test by an MTT assay and a proliferation curve is prepared to measure the population doubling time. In addition, flow cytometry analysis for cell surface antigens, RT-PCR for mRNA expression of stem cell markers, and assays to determine the cell's ability to differentiate into origin are performed.

According to the present invention, after thawing, AFSC proliferously grows rapidly. The proliferation curve pattern and dwell time were 5% and 2.5% (v / v) compared to the control (5% (v / v) Me ₂ SO + 30% FBS and 2.5% (v / v) Me ₂ SO + / v) Me ₂ SO in a solution containing trehalose, catalase and zVAD-fmk.

All surviving cells in solutions containing trehalose and catalase with 5% and 2.5% (v / v) Me ₂ SO in immunophenotyping express markers similar to the pluripotent mesenchymal lineage. According to the RT-PCR analysis, an Oct-4 gene encoding a transcription factor unique to differentiating pluripotent stem cells is expressed in both test solutions of the present invention. After thawing, AFSC can also be differentiated into myogenic cells.

According to the present invention, the disaccharide (trehalose), antioxidant (catalase) and caspase inhibitor (zVAD-fmk) are used in cryopreservation solutions to maintain the Me ₂ SO concentration at 10% (v / v) 5% (v / v) or 2.5% (v / v) and can be used to exclude FBS.

Further, according to the present invention, AFSC in a solution containing trehalose, catalase and zVAD-fmk together with a low concentration of Me ₂ SO can be kept frozen while retaining its identity and proliferating rapidly.

Hereinafter, the present invention will be described in more detail by way of examples, but it is for the purpose of illustration and it is obvious to those skilled in the art that the scope of the present invention is not limited thereto.

Specimen collection

AF samples were taken from amniocentesis performed on the second trimester for routine prenatal diagnosis. The samples were taken after each patient gave written prior consent.

Isolation and primary expansion of AFSC

AF samples were centrifuged at 1,200 rpm for 10 minutes. (HyClone, USA, Logan, Utah), Chang's B (Irvine Scientific, Santa Ana, CA), Chang's C (Irvine Scientific, Santa Ana, CA), Penicillin / Streptomycin The pellet was resuspended in Chang media consisting of L-glutamine (HyClone, Logan, Utah, USA), embryonic stem (ES) - fetal calf serum (FBS) (HyClone, , A humidified atmosphere containing 5% CO ₂ , and cells were cultured at 37 ° C. After 7 days, unattached cells were removed and fresh medium was added. The cells were then incubated for 14 days. Cells were harvested at 37 ° C for 3 minutes using 0.05% trypsin-EDTA (HyClone, Logan, Utah, USA) and replated under the same culture conditions. The adherent cells were then subcultured when they reached 70% confluency at weekly intervals.

Preparation of cryopreservation solution

Since Me ₂ SO is toxic to cells at room temperature, solutions were prepared at 4 ° C under sterile conditions. All freeze vials contained 1 mL of final cryopreservation medium after cell addition. (Sigma-Aldrich, St. Louis, Mo.) and zVAD-fmk (R & D Systems, Inc., Minnesota, USA) in all cases, Minneapolis) were added to cell freezing cryovials at concentrations of 60 mmol / L, 100 [mu] g / mL and 30 [mu] M, respectively. However, Me ₂ SO was added to cell freezing vials in different amounts (2.5%, 5% and 10% (v / v)) (see Table 1 below).

Preparation of different cryopreservative solutions solution Me ₂ SO
(% v / v) FBS
(% v / v) Trehalose
(mmol / L) Catalase
(쨉 g / mL) zVAD-fmk
(μM) One 10 30 0 0 0 2 5 30 0 0 0 3 5 0 60 100 0 4 5 0 60 100 30 5 2.5 30 0 0 0 6 2.5 0 60 100 0 7 2.5 0 60 100 30

Cryopreservation of AFSC

Seven cryopreservants with different combinations were tested by AFSC (see Table 1). A solution of 10% (v / v) Me ₂ SO + 30% FBS was used as a standard cryopreservation solution (100% viability). In all groups, 1 x 10 &lt; ⁶ &gt; cell aliquots of 100 [mu] l were transferred to 1 mL cell freezing vials containing each cryopreservation solution. Immediately after addition of the cells, cell freezing vials were frozen using controlled-rate freezing (Cyro, Rockville, Md.). All samples were then stored in a liquid nitrogen tank for at least 3 weeks prior to thawing and further analysis.

Thawing and Secondary Expansion of Cells

Cryopreserved cells were thawed by quickly placing the cell freezing vials in a water bath set at 37 占 폚, and the cells were diluted in the same growth medium as described for isolation and primary expansion. Cells were cultured for 2 passages until the culture reached the desired number of cells. After thawing, cell viability was measured by MTT assay. Viable cells were further characterized by flow cytometric analysis of specific surface antigens, generation of proliferation curves, RT-PCR and myogenic differentiation assay.

MTT assay

The cell solution fraction (1 x 10 ⁴ cells / mL) was plated with 10 μl MTT solution of each well of a 96-well tissue culture plate and incubated at 37 ° C for 4 hours. The absorbance of the purple formazan dye formed by reducing the MTT reagent was then measured at 570 nm using an ELISA plate reader (Molecular Devices, Sunnyvale, Calif.).

This MTT assay was used to measure cell viability after various cryopreservation conditions. The test solution was equivalent or superior to the control (5% (v / v) Me ₂ SO + 30% FBS and 2.5% (v / v) Me ₂ SO + 30% FBS). When the cells were stored in a solution containing trehalose, catalase and zVAD-fmk with 5% Me ₂ SO, cell viability after thawing was statistically significantly increased (p <0.05) (see FIG.

Growth Curve (Doubles)

AFSC was trypsinized and inoculated into 24-well plates. The cells were cultured at 5% humidified CO ₂ and 37 ° C, and the medium was changed three times a week. Cells were counted every 24 hours and the average cell number was recorded as the number of individual cells of the day. Doubling times of each culture were calculated using three observations.

As a result, AFSC was found to multiply rapidly. The proliferation curves of AFSC after thawing in all test solutions had the following characteristics: (i) on the first two days after inoculation the cells were attached to tissue culture plates; (ii) On day 3, the cells entered the growth phase logarithmic (logarithmic); (iii) there was peak proliferation on day 6; (Iv) Doubling times of the cells in the test solution were 29.0 to 31.9 hours (see FIG. 2). (5% (v / v) Me ₂ SO + 30% FBS and 2.5% (v / v) DMSO when solutions containing trehalose, catalase and zVAD- v / v) Me ₂ SO + 30% FBS) (Fig. 2).

Flow cytometry analysis

The specific surface antigens expressed by the cells were characterized by flow cytometric analysis. After thawing, cells were trypsinized and stained with phycoerythrin (PE) -binding human monoclonal antibodies against CD44, CD45, CD73, CD90, CD105, SSEA-4, HLA-ABC and HLA-DR. The cells were analyzed using a flow cytometric analyzer (Becton, Dickinson and Co., San Jose, CA).

Cells in all test solutions expressed markers corresponding to pluripotent mesenchymal precursor cell lines, including CD73, CD90, CD44 and CD105. As expected, these cells were positive for SSEA- and HLA-ABC (MHC class I) but negative for CD45 and HLA-DR (MHC class II). There was no difference in the surface antigen expression of cells after thawing in solutions containing 5% and 2.5% (v / v) Me ₂ SO 3 in trehalose and catalase. A representative phenotype profile of AFSC after thawing is shown in FIG.

Reverse transcription-polymerase chain reaction (RT-PCR)

Total RNA was extracted from the cultured cells using Trizol reagent (TaKaRa Bio Inc., Shiga, Japan) according to the manufacturer's instructions. RT-PCR was performed using specific DNA primers (see Table 2). The amplified DNA fragment was isolated using 1% agarose gel electrophoresis, the band was stained and photographed under UV light.

The primers used for RT-PCR gene order CK-18 F GAGATCGAGGCTCTCAAGGA
R CAAGCTGGCCTTCAGATTTC Vimentin F CCTTCGTGAATACCACGACCTGC
R TAATATATCGCCTGCCACTGAG FGF-5 F GCTGTGTCTCAGGGGATTGTAGGAATA
R TATCCAAAGCGAAACTTGAGTCTGTA SCF F CCATTGATGCCTTCAAGGAC
R CTTCCAGTATAAGGCTCCAA Oct-4 F ACATGTGTAAGCTGCGGCC
R GTTGTGCATAGTCGCTGCTTG GAPDH F GCTTGTCATCAATGGAAATCCC
R TCCACACCCATGACGAACATG Abbreviation:
CK-18: cytokeratin 18
FGF-5: fibroblast growth factor 5
SCF: Stem Cell Factor
Oct-4: Octamer-Binding Transcription Factor 4
GAPDH: Glyceraldehyde-3-phosphate dehydrogenase

According to RT-PCR analysis, mRNA expression of stem cell markers such as Oct-4, FGF-5, SCF, Vimentin and CK18 was detected in AFSC after thawing in all test solutions.

Root differentiation and its confirmation

(Gibco / BRL, Carlsbad, Calif., USA) and 1% penicillin / streptomycin (HyClone, USA) supplemented with 10% horse serum (Gibco / BRL, Carlsbad, CA), 0.5% chick embryo extract AFSC was seeded onto a plastic tissue culture plate precoated with Matrigel (BD Biosciences, Bedford, Mass.) Of DMEM (low glucose combination) containing a polyacrylamide gel. After 12 hours of seeding, 5 μM 5-aza-2'-deoxycytidine 3 (5-azaC; Sigma-Aldrich, St. Louis, Mo.) was added for 24 hours. Then, the culture was continued in a culture medium containing no 5-azaC, and the medium was replaced every 3 days.

(Western blot analysis)

After treating the plates with 100 μl of lysis buffer (150 mM NaCl, 20 mM TRIS, 1% Triton X-100 and 400 U / mL RNase inhibitor, pH 8), protein extracts were obtained and precipitated with methanol. 40 [mu] g of the protein was separated on SDS-polyacrylamide gel and transferred to a nitrocellulose membrane. (1: 500, Cell signaling, Danvers, Mass.), Secondary peroxidase-conjugated anti-human MyoD (1: 500, Santa Cruz Biotechnology, Santa Cruz, CA) Followed by incubation with antibody (1: 1000, Amersham Bioscience, Uppsala, Sweden) and a chemiluminescence kit (ECL, Amersham Bioscience, Uppsala, Sweden) was used for specificity protein identification.

(Immunofluorescent staining)

AFSC was plated on a 24-well plate containing the culture medium. Cells were incubated overnight at 37 ° C in a 5% humidified CO ₂ incubator. The next day, the cells were washed with 10 mM phosphate buffered saline (PBS), pH 7.4 and fixed in 2% paraformaldehyde (Sigma-Aldrich, St. Louis, Mo.) for 30 min at 4 ° C. Cells were infiltrated with 0.2% Triton-X (Sigma-Aldrich, St. Louis, Mo.) and incubated for an additional 20 minutes in blocking buffer (10% FBS in PBS). The cells were then stained with MyoD or a primary antibody against desmin (mouse anti-human monoclonal antibody, Santa Cruz, Biotechnology, Santa Cruz, CA) for 1 h in PBS. Subsequently, 0.5 μg / ml fluorescent isothiocyanate (FITC) -based goat anti-mouse (Santa Cruz, Biotechnology, Santa Cruz, CA, USA) was used as secondary antibody in PBS for 45 min.

When cultured in source property medium for the AFSC 6 days after thawing, Western according to blot analysis, 5% or 2.5% (v / v) the AFSC kept in a solution containing the odds and catalase as trehalose with Me ₂ SO Expressed Myo D and desmin protein (see Fig. 5A). Similarly, according to immunofluorescence staining, AFSC expressed Myodo and desmin proteins (in a solution containing trehalose and catalase with 5% or 2.5% (v / v) Me ₂ SO) 5B). Taken together, the data made it clear that AFSC can differentiate along the source pathway after thawing.

Statistical analysis

Data are presented as mean ± standard deviation. Averages were compared using Student's two-tailed test. P values less than 0.05 were considered significant.

Claims (2)

Regarding the gross weight of the cryopreservation medium,
(FBS) was removed, containing 2.5 to 5% (v / v) Me ₂ SO, 60 mmol / L trehalose, 100 ug / mL catalase and 30 μM zVAD-fmk Amniotic stem cell (AFSC) cryopreservation medium composition. delete

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