KR20110121492A - Composition for cryopreservation of human embryonic stem cell-derived cardiomyocytes - Google Patents

Abstract

PURPOSE: A composition containing ROCK(Rho-associated kinase) inhibitors for cryopreservation of cardiac myocytes from human embryonic stem cells is provided to develop a cell therapeutic agent. CONSTITUTION: A composition for cryopreservation of cardiac myocytes differentiated from human embryonic stem cells contains ROCK(Rho-associated kinase) inhibitor. The human stem cells are human embryonic stem cells, human embryonic stem cell-derived embryoid body, or human pluripotent stem cells. The ROCK inhibitor is Y-27632, HA-1077, Y-39983, or Wf-536. A method for cryopreservation of differentiated cardiac myocytes comprise: a step of treating the composition to the differentiated cardiac myocytes for 0.25-5 hours; and a step of freezing.

Description

Composition for cryopreservation of human embryonic stem cell-derived cardiomyocytes}

The present invention relates to a composition for cryopreservation of cardiomyocytes induced by differentiation from human stem cells. Specifically, the composition for cryopreservation of cardiomyocytes induced from differentiation from human embryonic stem cells, including ROCK (Rho-associated kinase) inhibitor, And it relates to a method for cryopreserving cardiomyocytes comprising the step of treating the composition to cardiomyocytes induced differentiation from human embryonic stem cells.

The number of deaths from cardiovascular diseases, including myocardial infarction, is increasing rapidly due to the influence of western eating habits, lack of exercise, and the increasing number of older people due to the aging society. Drug therapy and cardiac transplantation are mainly used as the ultimate treatment methods, but new alternative treatments are urgently needed due to cost and limitation of donors.

The use of stem cells is in the spotlight as such alternative treatments. Stem cells have the potential to be widely applied in the fields of regenerative medicine, pharmacology and basic science research, and are expected to serve as cell sources for clinical applications such as cell transplantation and tissue engineering. In particular, cardiomyocytes derived from human embryonic stem cells are of interest as a suitable cell source to provide new cells to patients with cardiovascular disease.

On the other hand, cardiomyocytes derived from human embryonic stem cells need to be effectively and safely cryopreserved in order to use for cardiovascular disease research, clinical, and therapeutic purposes. Safe freezing methods are also essential for the establishment of cell banks that require the storage and transportation of stem cells or differentiated cardiomyocytes. However, many studies have reported changes in stem cell characteristics or genetically normal karyotypes when long-term cultured cardiomyocytes differentiated from stem cells. However, to date, human embryonic stem cell-derived cardiomyocytes have been lowly evaluated for research and treatment purposes due to the lack of optimized cryopreservation methods.

In previous studies, human embryonic stem cells were cryopreserved using vitrified freezing or mass cryopreservation, which mainly treated with DMSO. However, this method has a limitation in that the survival rate of thawed cells is very low. Overcoming the low survival rate after cryopreservation of human embryonic stem cell-derived cardiomyocytes is a top priority for the use of differentiated cardiomyocytes for clinical applications or therapeutic purposes. Thus, cryopreservation methods to increase survival rate after cryopreservation have been studied by many researchers, and the results of successful cryopreservation of human embryonic stem cells have been published, but long-term preservation of cardiomyocytes derived from human embryonic stem cells Very little research has been reported on cryopreservation. The only reported literature only shows the basic properties of the cells after thawing in which the heartbeat activity of the cell cluster is not restored.

Under these circumstances, the present inventors have made diligent efforts to develop a method for successfully cryopreserving cardiomyocytes derived from human embryonic stem cells and optimal conditions for survival after thawing, and thus induce differentiation from human embryonic stem cells including a ROCK inhibitor. A cryopreserved composition of the cardiomyocytes was developed and thawed after the cryopreservation of the differentiation-induced cardiomyocytes by treatment of the composition. It confirmed and completed this invention.

One object of the present invention to provide a composition for cryopreservation of cardiomyocytes induced differentiation from human embryonic stem cells, including a ROCK inhibitor.

Another object of the present invention is to provide a method for cryopreserving differentiation-induced cardiomyocytes, comprising treating the composition with cardiomyocytes differentiated from human embryonic stem cells.

As one embodiment for achieving the above object, the present invention relates to a composition for cryopreservation of cardiomyocytes induced differentiation from human embryonic stem cells, including a ROCK inhibitor.

As used herein, the term "human embryonic stem cells" refers to cells having a pluripotency capable of differentiating into cells derived from endoderm, mesoderm, and ectoderm, or muscle, bone, brain, skin, It refers to a cell having a multipotency capable of differentiating into closely related cells in different tissues or functions such as the heart. Examples of human stem cells include pluripotent stem cells formed when the fertilized egg first divides, embryonic stem cells that can be made using human embryos, and adult stem cells that exist in mature tissues and organs. The type of cell is not limited. Preferably, the human stem cells in the present invention are human embryonic stem cells, human embryonic stem cell-derived embryoid bodies, or human induced pluripotent stem cells, more preferably human embryonic stem cells.

In the present invention, the term "human embryonic stem cells" refers to cells having a pluripotency capable of differentiating into all cells of the human body by extracting internal cell masses from blastocyst embryos in which the fertilized eggs are implanted in the mother's womb. In a broad sense, it includes similar stem cells such as embryonic bodies derived from embryonic stem cells.

In the present invention, the term "human induced pluripotent stem cells" is made by inserting a reverse differentiation gene into human somatic cells to establish an undifferentiated stem cell having a differentiation capacity similar to that of embryonic stem cells. It means a level of stem cells similar to the. In addition, the human embryonic stem cells or human induced pluripotent stem cells can be easily constructed by those skilled in the art by a known method.

As used herein, the term "differentiation" refers to a phenomenon in which each structure or function is specialized during cell division and proliferation, that is, a change in form or function of a cell or tissue of an organism to perform a task given to each. do. Specifically, in the present invention, it means differentiation from human embryonic stem cells to cardiomyocytes.

In the present invention, the term "cardiomyocytes" refers to all differentiation such as myocardial progenitor cells or fetal cardiomyocytes, adult cardiomyocytes, cardiomyocytes before beats, and cardiomyocytes after beats, which have the ability to become functional cardiomyocytes in the future. It means a cell which includes the cells of the step without limitation and can be identified by at least one, preferably a plurality of markers or criteria by at least one, preferably a plurality of methods described below. Preferably in the present invention means cardiomyocytes before the beat. Expression of various cardiomyocyte specific markers can be detected by known biochemical or immunochemical methods, and such methods can be used without limitation. In this method, marker specific polyclonal antibodies or monoclonal antibodies that bind to myocardial progenitor cells or cardiomyocytes can be used. Antibodies that target individual specific markers can be used commercially or without limitation, those prepared by known methods. Examples of markers specific for myocardial progenitor cells or cardiomyocytes are myosin heavy chain / light chain (MHC / MLC), α-actinin, troponin I (cTn I), ANP, GATA4, Nkx2.5 and MEF -2c, and the like. Alternatively, the expression of myocardial progenitor or cardiomyocyte specific markers is not limited to specific methods, but amplifies mRNA encoding any marker protein, which is reverse transcriptase mediated polymerase chain reaction (RT-PCR) or hybridization assay, It can be confirmed by molecular biological methods commonly used for detection and interpretation. Nucleic acid sequences encoding proteins specific for myocardial progenitor cells or cardiomyocytes (eg myosin heavy / light chain, α-actinine, troponin I, ANP, GATA4, Nkx2.5 and MEF-2c) proteins are already known. Obtained from public databases such as GenBank, the marker specific sequences required for use as primers or probes can be readily determined. In addition, physiological criteria can be additionally used to confirm the differentiation of pluripotent cells into cardiomyocytes. In other words, cells derived from pluripotent cells may be used as useful indicators, such as having independent pulsatile rhythms, expressing various ion channels, and responding to electrophysiological stimuli.

Cardiomyocytes frozen using the cryopreservation composition of the present invention can be used as a cell composition for myocardial regeneration or cell replacement therapy to treat heart-related diseases. Heart-related diseases can be included without limitation as long as the disease is caused by abnormal or degenerative heart failure, for example, myocardial infarction, ischemic heart disease, congestive heart failure, hypertrophic cardiomyopathy, enlarged cardiomyopathy, myocarditis, chronic heart failure, etc. There is this. If the cell composition for cardiomyocyte regeneration or cell replacement therapy contains the cardiomyocytes prepared according to the present invention in high purity, the cells are suspended in an aqueous carrier such as a medium, and the cells are contained in a support such as a biodegradable substrate. It can be used in a variety of forms, such as one, or a single or multilayer cardiomyocyte sheet (Shimizu et al., Circ. Res. 90: e40, 2002). The therapeutic agent may be transported to the injured site by injecting into the heart directly by using a syringe, surgically dissecting a part of the heart, and implanting by a catheterized or transvascular method. However, it is not limited to this. Therefore, in the above-mentioned cardiac-related diseases due to the decline and loss of cardiomyocytes, cardiomyocytes treated by the cryopreservation method of the present invention can be supplemented with diseased heart tissue, thereby promoting improvement of heart function.

As a method for producing differentiation-induced cardiomyocytes from human embryonic stem cells for use of the cryopreservation composition of the present invention, direct differentiation induction and via embryonic body method can be used, and known floating flocculation culture methods are generally used. In addition, the method of inducing differentiation from human embryonic stem cells to cardiomyocytes may be used in combination with a hanging drop culture method and a co-culture method using a feeder. Does not. Preferably, a direct differentiation method using BMP2 may be used for cryopreservation of the survival rate of differentiated induction cardiomyocytes.

As used herein, the term "freezing preservation" refers to the act of keeping the cell line stable over a long period of time through freezing. Cells are usually mutated mutagenesis at a rate of 10,000 to 10,000 in culture. In other words, if the cell passage is continued for a long time, the cell group is completely different from the original cell group, and in the worst case, some kind of function possessed by the cell may be lost by the long-term passage. Therefore, the act of keeping the cell line through freezing and preventing the loss of cells is called cryopreservation. Cryopreservation methods in the present invention may include vitrified freezing or bulk cryopreservation, but the cryopreservation method is not limited by the above examples. Preferably cryopreservation in the present invention means the mass cryopreservation of cardiomyocytes induced differentiation from human embryonic stem cells. Cryopreservation of differentiation-induced cardiomyocytes from human embryonic stem cells using the cryopreservation composition of the present invention maintains the characteristics of cardiomyocytes for a long time and enables preservation without alteration of genotype or karyotype, thereby enabling proliferation after thawing.

In the present invention, the term "ROCK (Rho-associated kinase)" means a substance capable of inhibiting ROCK, a serine / threonine kinase acting downstream of Rho, which is a low molecular GTP-binding protein. Although not limited to, Y-27632, HA-1077, Y-39983, and Wf-536 may be included. Preferably Y-27632 can be used.

Preferably the ROCK inhibitor in the composition of the present invention may be at a concentration of 1 μM to 20 μM, more preferably at a concentration of 10 μM.

According to one specific embodiment, the treated cells were treated with a ROCK inhibitor, the cells were separated, and cryopreserved by mass cryopreservation, and the survival rate of thawed cardiomyocytes was confirmed to be high. In the case of mass cryopreserved cells, pulsation was observed in cells treated with ROCK inhibitor from day 5 after thawing, with a rate of 10%.

In addition, it was confirmed that the number of surviving cells was the highest in the cell group treated with the ROCK inhibitor in both the 12th day of differentiation and 16th day of differentiation. Survival was about four times higher in the ROCK inhibitor-treated cell group than in the control group, and two-fold higher in the NT 4 (Neurotrophin 4) -treated cell group. This suggests that the use of a ROCK inhibitor is better than the use of NT 4 (Neurotrophin 4) survival and recovery of function, suggesting that the effect of the ROCK inhibitor of the freezing composition of the present invention is good.

In another aspect, the present invention relates to a method for cryopreserving differentiation-induced cardiomyocytes comprising treating a cryopreservation composition comprising a ROCK inhibitor to cardiomyocytes differentiated from human embryonic stem cells. .

Cardiomyocytes and embryonic stem cells are as described above.

Preferably, the composition for cryopreservation of cardiomyocytes differentiated from human embryonic stem cells, including the ROCK inhibitor of the present invention, may be treated for 0.25 hours to 5 hours , and more preferably for 1 hour.

According to one specific embodiment, after treatment with the ROCK inhibitor, and the cells reattached after thawing, it was confirmed that the number grows. In addition, in order to confirm the proliferation of the thawed cells, immunocytostaining of BrdU and α-actinine was performed 5 days after thawing, and cells expressing BrdU and α-actinin were identified. These results suggest that the cardiomyocytes after thawing have the ability to proliferate while maintaining the cardiomyocyte characteristics.

In a preferred embodiment the cryopreservation method may further comprise a mass cryopreservation step of freezing in a composition further comprising DMSO and FBS. In the mass cryopreservation step, the treated cells were treated with ROCK inhibitor Y-27632, then treated with DMSO and FBS in the cryopreservation vessel containing Y-27632, and then stored at -70 ° C for at least 12 hours, and then liquid nitrogen. Long-term preservation at. After thawing slowly in a 37 ° C. constant temperature water bath, the cryopreservation solution was removed by centrifugation, and then suspended in a culture solution containing Y-27632 and attached to a culture dish. Two days after thawing, activity can be restored by culturing in a culture medium that does not contain Y-27632.

In the cryopreservation method according to one preferred embodiment, the timing of treatment of the ROCK inhibitor to differentiated cardiomyocytes may be cells prior to myocardial pulsation, stably cryopreservation and thawing using the composition of the present invention. The cardiomyocytes can be used without limitation to the cells at the stage of restoring the characteristics of the cardiomyocytes, but preferably the cells before the myocardial pulsation. have.

In a preferred embodiment, the cryopreservation method comprises the steps of: treating the cryopreservation composition of cardiomyocytes differentiated from human embryonic stem cells; Bulk cryopreservation step of freezing in a composition comprising a ROCK inhibitor, DMSO and FBS; And treating the thawed cells with a composition comprising a ROCK inhibitor after thawing.

According to one preferred specific embodiment, a mass cryopreservation method after reacting the ROCK inhibitor of the present invention with cells of 12 days (before myocardial heartbeat) and 16 days (after myocardial heartbeat) after induction of differentiation from human embryonic stem cells or After thawing after freezing by vitrification freezing method, the cryopreservation composition containing the ROCK inhibitor was treated to the cardiomyocytes prior to myocardial pulsation. It was confirmed that the cell death inhibitory effect and also proliferative effect.

A cryopreservation composition of differentiation-induced cardiomyocytes from human embryonic stem cells, comprising the ROCK inhibitor according to the present invention, and the step of treating the composition, using the cryopreservation method to maintain the viability of cardiomyocytes Therefore, since it is possible to preserve for a long time, it can contribute to the development of cell therapeutics for the treatment of heart-related diseases. In addition, the cardiomyocytes cryopreserved by the present invention is thawed at an appropriate time, and the biological efficacy verification system using stem cells required in the new drug development process, in particular, the drug validation system for developing cardiomyocyte differentiation inducing substance and cardiac function enhancing substance. It can be used for the commercialization of new drugs.

1 is a study flow diagram for cryopreservation of cardiomyocytes differentiated from human embryonic stem cells.
2 is a flow chart of induction of differentiation from human embryonic stem cells into cardiomyocytes.
Figure 3 is a microstructure analysis results before and after cryopreservation of human embryonic stem cell-derived cardiomyocytes using a scanning electron microscope.

Hereinafter, the present invention will be described in more detail with reference to Examples. These embodiments are only for illustrating the present invention, and the scope of the present invention is not construed as being limited by these embodiments.

Example 1 Human Embryonic Stem Cell Culture

Three human embryonic stem cell lines, SNUhES3, SNUhES16 and SNUhES12, were placed on Oh, et al. (Methods for expansion of human embryonic stem cells, Stem , on STO support cells (ATCC) treated with mitomycin (Mitomycin C, Sigma). Cells , 2005 May; The culture was carried out according to the method of 23 (5): 605-9). 20% knockout serum replacement (KO-SR, Invitrogen), 1% non-essential amino acids (Invitrogen), 0.1 mM beta-mercapto with DMEM / F12 (Invitrogen) as a base culture Human embryonic stem by adding ethanol (β-mercaptoethanol, Sigma), 4 ng / ml basic fibroblast growth factor (bFGF, Invitrogen), 50 U / ml penicillin (Invitrogen) and 50 μg / ml streptomycin (Invitrogen) Used as cell culture.

Example  2: using direct differentiation Cardiomyocyte  formation

Undifferentiated human embryonic stem cells obtained in Example 1 were treated with 0.025% trypsin-EDTA (Invitrogen) at 37 ° C. for 3 minutes. Then, the support cells were removed and washed with PBS (Invitrogen). After washing, the culture medium containing B27 (Invitrogen) was added to the RPMI 1640 (Invitrogen) medium, and 100 ng / ml of Actinin A (Activin A, R & D Systems) was treated for 24 hours, and then Actinbin A was removed. ng / ml BMP2 (R & D Systems) was treated for 4 days. During the differentiation period, starting from day 5, growth factors were not added and cultures were changed every two days.

The overall overview of the differentiation process as described above is shown in FIG.

Example  3: derived from human embryonic stem cells Cardiomyocyte  Cryopreservation and Thawing

The cardiomyocytes differentiated in Example 2 were 10 μM Y-27632 (Calbiochem), 50 μg / ml Neurotrophin 3 (PeproTech), or 50 μg / ml neurons, respectively, on days 12 and 16 after differentiation. Tropin 4 (PeproTech) was added and reacted at 37 ° C for 1 hour. After the reaction, accutase (Chemicon) was treated at 37 ° C. for 10 minutes to separate into small cell masses.

For mass cryopreservation, the isolated cardiomyocytes were suspended in 500 μl of culture medium, into a cryopreservation container containing 500 μl of a solution consisting of 20% DMSO (Sigma) and 60% FBS (HyClone). It was transferred and Y-27632, neurotrophin 3 or 4 mentioned above was added. Cryopreservation containers were transferred to a freezing container and stored at -70 ° C for at least 12 hours. After storage, cryopreservation vessels were immersed in liquid nitrogen for long term preservation. After several days, cryopreserved human embryonic stem cell-derived cardiomyocytes were thawed in a 37 ° C. water bath. After slowly adding 2 ml of the culture solution to the cryopreservation solution, the cardiomyocyte mass was gently washed. Using centrifugation, the cryopreservation solution was removed and suspended in a culture solution containing each of Y-27632, Neurotropin 3, or 4 and attached to the culture dish. Two days after thawing, a culture medium containing no Y-27632, neurotrophin 3, or 4 was added thereto.

Two kinds of solutions were used for vitrification. Vitrification solution I and Vitrification solution II, solution I is a solution in which 10% ethylene glycol (EG; Sigma) and 10% DMSO are added to the culture, and solution II is 20% ethylene glycol, 20 in the culture % DMSO and 0.5 M sucrose (Sigma) are added solutions. For thawing, two kinds of solutions were used. As thawing solution I and thawing solution II, solution I is a solution in which 0.2 M sucrose (Sigma) is added to the culture, and solution II is a solution in which 0.1 M sucrose is added to the culture. The isolated cardiomyocytes were placed in a cell strainer (BD Biosciences) and then immersed in vitrified freezing solution I for 1 minute at 37 ° C, and then immersed in solution II for 30 seconds at 37 ° C. The cell strainer was then stored in liquid nitrogen. After several days, the cell strainer was collected, soaked in thawing solution I for 1 minute, then transferred to thawing solution II, and soaked for 5 minutes. Thawed cells were transferred to a culture dish in which Y-27632, neurotropin 3, or 4 was added to the culture, respectively. Two days after thawing, a culture medium containing no Y-27632, neurotrophin 3, or 4 was added.

Survival and recovery of function were confirmed by microscopy at two-day intervals. The survival rate of thawed cardiomyocytes was significantly higher in the frozen cells using mass cryopreservation than in thawed cells by vitrification freezing. In the case of vitrified frozen cells, most thawed cells did not adhere to the culture dish and did not recover morphological shape or beats. In the case of mass cryopreserved cells, pulsation was observed in cells treated with ROCK inhibitor from day 5 after thawing, with a rate of 10%.

The number of surviving cells was highest in cells treated with ROCK inhibitor in both 12th day of differentiation and 16th day of differentiation. Survival was about four times higher in the ROCK inhibitor-treated cell group than in the control group, and two-fold higher in the NT 4 (Neurotrophin 4) -treated cell group. In addition, the beat rate of thawed cell groups treated with ROCK inhibitor was between 58 and 108 per minute, which was faster than non-freezing cardiomyocytes.

These results suggest that the survival rate after thawing of the method of freezing with the composition containing the ROCK inhibitor of the present invention is high.

Example  4: after freezing and thawing Cardiomyocyte  Property retention confirmation

<4-1> After thawing by scanning electron microscope analysis Cardiomyocyte  Microstructure Change Analysis

Differentiated cells were washed with PBS and reacted with 2.5% glutaraldehyde (Sigma) solution at room temperature for 20 minutes. Thereafter, the cells were reacted at 4 ° C. for 10 minutes by adding 1% osmium tetroxide (Sigma) to which 0.14 M sucrose was added. Epoxy resin was added and then transferred to a beam capsule for polymerization. Polymerized cells were separated from the resin using a razor. Samples were cut using an ultramicrotome (Ultramicrotome, MT-6000, DuPont Instruments-Sorvall Biomedical Div.) And observed with a scanning electron microscope (JEM-1400; JEOL Ltd.).

In addition, scanning electron microscopic analysis was performed to analyze microstructural changes in human embryonic stem cell-derived cardiomyocytes after cryopreservation. As a result, cell structure destruction was confirmed in both the 12th and 16th day of differentiation. The destruction of cell structure was observed in the cell population at 16 days of differentiation. Cellular organelles, such as the nucleus and cell membrane, were also partially destroyed, and lipid droplets and expanded vesicles were observed. Mitochondria were present in large numbers in all cells, most of which were perinuclear mitochondria.

This suggests that there is little destruction of the cell structure in the cell population of the group treated with the ROCK inhibitor at day 12 of differentiation.

Figure 3 relates to the results of microstructural analysis of human embryonic stem cell-derived cardiomyocytes using a scanning electron microscope, confirming the presence of a large number of mitochondria and Z band precursors of the cardiomyocyte microstructural characteristics.

<4-2> Annexin  Through V staining ROCK  Investigation of anti-apoptotic effect of inhibitor

Annexin V staining was performed to confirm the anti-apoptotic effect of the ROCK inhibitor.

The cells were reacted for 10 minutes at 37 ° C. in 0.25% trypsin-EDTA, and then separated into single cells. Annexin V staining was performed using FITC Annexin V apoptosis detection kit I (BD Biosciences). Briefly describing the procedure, cells were washed twice with cold PBS and then suspended by addition of 1 × binding buffer. Thereafter, 5 μl of FITC-bound Annexin V and PI (Sigma) were added and reacted for 15 minutes at room temperature under dark conditions. Cells BD FACS Calibur ^TM (BD Biosciences).

As a result, Annexin V staining showed that the treatment of ROCK inhibitors before and after cryopreservation significantly inhibited apoptosis in cardiomyocytes at 12 days of differentiation. In contrast, treatment with ROCK inhibitors did not have a significant effect on cryopreservation of cardiomyocytes at 16 days of differentiation. These results indicate that treatment with ROCK inhibitors before and after cryopreservation increased the survival and recovery of function of human embryonic stem cell-derived cardiomyocytes. More appropriate.

<4-3> BrdU  Confirmation of proliferation of thawed cells using label

In order to confirm the proliferation of thawed cells, immunocytostaining of BrdU and α-actinine was performed 5 days after thawing.

Thawed cells were reacted for 24 hours with the addition of 10 μM 5-bromo-2-dioxyuridine (BrdU; Roche Diagnostics, Basel, Switzerland) and washed with PBS. Thereafter, the mixture was fixed with 4% PFA and then reacted with 3% BSA. Subsequent procedures were performed as described in the section on investigation of marker expression by immunocytostaining with the addition of primary antibody, mouse anti-BrdU (Chemicon) and goat anti α-actinin.

After reacting at 4 ° C. overnight, the mixture was washed three times with PBST (PBS containing 0.05% Tween 20), and a secondary antibody was added thereto to react at room temperature for 1 hour. After washing three times using PBST, stained with a solution containing DAPI, and observed using a confocal fluorescence microscope (BioRad).

As a result, cells expressing BrdU and α-actinin were identified. These results show that the cardiomyocytes after thawing have the ability to proliferate even after cryopreservation and thawing if the cardiomyocytes retain their properties. Human embryonic stem cells using the cryopreservation composition comprising the ROCK inhibitor of the present invention. This suggests that cryopreservation of differentiation-induced cardiomyocytes may restore the cardiomyocyte characteristics and proliferative capacity.

Claims (11)

A composition for cryopreservation of differentiation-induced cardiomyocytes from human embryonic stem cells, comprising a ROCK (Rho-associated kinase) inhibitor.
The composition of claim 1, wherein the human stem cells are human embryonic stem cells, human embryonic stem cell derived embryoid bodies, or human induced pluripotent stem cells.
The composition of claim 1, wherein the ROCK inhibitor is at least one substance selected from the group consisting of Y-27632, HA-1077, Y-39983, and Wf-536.
The composition of claim 3, wherein the ROCK inhibitor is Y-27632.
The composition of claim 4 wherein the concentration of Y-27632 is comprised between 20 μM in 1 μM.

A method for cryopreserving differentiation-induced cardiomyocytes comprising the step of treating the composition of any one of claims 1 to 5 to differentiation-induced cardiomyocytes from human stem cells.
The method of claim 6, wherein the stem cells are human embryonic stem cells, human embryonic stem cell-derived embryoid bodies, or human induced pluripotent stem cells.
The method of claim 6, wherein the step of treating the composition is for 0.25 hours to 5 hours.
7. The method of claim 6, further comprising a mass cryopreservation step of freezing in a composition comprising a ROCK inhibitor, DMSO, and FBS.

The method of claim 6, wherein the differentiation-induced cardiomyocytes are cells prior to myocardial pulsation.
The method of claim 6, further comprising: i) treating the cardiomyocytes induced by differentiation from human stem cells with the composition of any one of claims 1 to 5; ii) a mass cryopreservation step of freezing in a composition comprising a ROCK inhibitor, DMSO and FBS; And iii) treating the thawed cells with a composition comprising a ROCK inhibitor after thawing.

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