WO2023068624A1 - Method for cryopreservation of stem cells using elastin-like artificial extracellular matrix - Google Patents

Method for cryopreservation of stem cells using elastin-like artificial extracellular matrix Download PDF

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WO2023068624A1
WO2023068624A1 PCT/KR2022/015290 KR2022015290W WO2023068624A1 WO 2023068624 A1 WO2023068624 A1 WO 2023068624A1 KR 2022015290 W KR2022015290 W KR 2022015290W WO 2023068624 A1 WO2023068624 A1 WO 2023068624A1
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stem cells
cryopreservation
elastin
extracellular matrix
cells
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French (fr)
Korean (ko)
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김용준
전원배
이윤일
이영삼
김정희
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경희대학교 산학협력단
재단법인대구경북과학기술원
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Publication of WO2023068624A1 publication Critical patent/WO2023068624A1/en

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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
    • A01N1/02—Preservation of living parts
    • A01N1/0205—Chemical aspects
    • A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
    • A01N1/02—Preservation of living parts
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/15—Cells of the myeloid line, e.g. granulocytes, basophils, eosinophils, neutrophils, leucocytes, monocytes, macrophages or mast cells; Myeloid precursor cells; Antigen-presenting cells, e.g. dendritic cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0603—Embryonic cells ; Embryoid bodies
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0656—Adult fibroblasts
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/50—Proteins

Definitions

  • the present invention relates to a method for cryopreservation of stem cells using an artificial elastin-like extracellular matrix.
  • the use of the artificial elastin-like extracellular matrix promotes the increase in viability of human stem cells in the process of cryopreservation and subsequent cultivation, thereby providing a cell therapeutic agent. And the efficiency of stem cell research can be secured.
  • Stem cells have the potential to be widely applied in regenerative medicine, pharmacology, and basic science research fields, and are expected to play a role as a cell source for clinical applications such as cell transplantation and tissue engineering.
  • the cryopreservation method is to suspend the cells in a freezing medium to lower the temperature of the cells to a certain level and finally freeze them at -196°C. Freezing inhibitors such as , glycerol, and fetal bovine serum (FBS) or a cell culture medium containing the same are used as main components.
  • Freezing inhibitors such as , glycerol, and fetal bovine serum (FBS) or a cell culture medium containing the same are used as main components.
  • the vitrification freezing or mass cryopreservation method using DMSO has a limitation that the survival rate of thawed cells is very low, the low survival rate after cryopreservation of these stem cells is the highest priority for use for clinical application or treatment purposes. It is a challenge that needs to be tackled critically.
  • An object of the present invention is to secure the efficiency of cell therapy products and stem cell research by promoting an increase in viability in the process of culturing human stem cells after cryopreservation using an artificial elastin-like extracellular matrix.
  • the present invention provides a composition for cryopreservation of stem cells comprising an artificial elastin-like extracellular matrix.
  • the present invention provides a method for cryopreservation of stem cells comprising the step of cryopreserving the stem cells by treating the stem cells with the composition.
  • the present invention comprises the steps of cryopreserving the stem cells by treating the stem cells with the composition; removing the non-stem cell solution during the process of thawing the cryopreserved stem cells; And it provides a method for thawing and culturing stem cells comprising the step of raising the stem cells from which the solution is removed and then transferring them to a plate and culturing them again.
  • the present invention provides a cell therapy agent comprising the stem cells and the composition.
  • the stem cell cryopreservation technology including an artificial elastin-like extracellular matrix simulates the in vivo environment of human embryonic stem cells by using the RGD motif attached to integrin expressed by human embryonic stem cells, and at the same time, the initial By using the elastin protein structure, an extracellular matrix that does not exist in the human embryonic stem cell development environment, it shows the advantage of not stimulating the differentiation mechanism of unnecessary embryonic stem cells, so development of products for cryopreservation for scientific and technological research using stem cells, It can be usefully used for the development of cell therapy products and product development for cryopreservation.
  • A is a result of analyzing the integrin mRNA expression pattern of human embryonic stem cells (H1, H9) and fibroblasts (CRL, BJ), a number of integrins that human embryonic stem cells can bind to the RGD motif
  • B is the result of verifying protein expression in human embryonic stem cells of integrins capable of binding to the RGD motif using antibody staining and a flowcytometer.
  • A is to confirm the molecular weight by purifying REP containing the RGD motif
  • B is to confirm the conditions for forming aggregates by concentration for applying REP to cells
  • C is the cell culture temperature when mixed with the culture medium of high concentration REP Aggregates were formed, but it was confirmed that it was difficult to mix with the culture medium
  • D confirmed the conditions for inhibiting cell sedimentation using rotation after mixing with the cell culture medium of low concentration REP
  • E was cultured for 24 hours in the mixture of culture medium and REP concentration.
  • the survival rate of human embryonic stem cells is measured according to the method
  • F is a quantitative analysis of the E results
  • G is a measurement of the stem cell marker expression ratio of surviving embryonic stem cells following 24-hour culture in a mixture of culture medium and REP concentration.
  • H is the quantitative analysis of the G result
  • I is the result of re-separating and culturing the stem cell marker expressing cells under the REP 1 ⁇ M treatment condition of G, and confirms the continuous expression of the stem cell marker.
  • the stem cell growth rate of I was followed up for 7 days.
  • A is a schematic diagram of the process of reculturing after freezing and thawing after collection of stem cells during cryopreservation of human stem cells, resuspension into single cells or clumps using cryopreservative containing 0.5 to 10 ⁇ M of REP, B, as a result of confirming the ratio of apoptotic cells after thawing of stem cells preserved using cryopreservative containing REP and the number of viable cells 24 hours after plate attachment, the number of apoptotic cells and high survival rate of cells treated with cryopreservative containing REP were low.
  • C confirmed that there was no difference in the expression ratio of the stem cell marker (OCT4) of the cells cultured for 24 hours after plate attachment after thawing
  • D was the cell growth rate after thawing of stem cells using cryopreservation solution containing REP.
  • A confirms whether ERK and AKT signals are activated for identifying the mechanism for increasing the survival rate of REP for human embryonic stem cells
  • B is a quantification analysis result of FAK signal activation, and FAK signal is activated
  • C is ERK signal ERK signal was not activated as a result of quantification analysis of the activation result
  • AKT signal was activated as a result of quantification analysis of AKT signal activation result in D.
  • A is to verify the inhibition of ERK or AKT signal upon freezing of embryonic stem cells in REP addition conditions
  • B is to confirm the change in viability due to ERK or AKT signal inhibition when embryonic stem cells are frozen in REP addition conditions
  • C is When AKT was inhibited, it was confirmed that surviving embryonic stem cells were differentiated by REP treatment, and D confirmed that FOXO3a activation was inhibited and apoptosis factor BIM expression was suppressed when REP treatment was performed on embryonic stem cells, and E was a quantitative result of D
  • F is a schematic diagram of the molecular mechanism of increasing viability by the addition of REP including RGD-motif during cryopreservation and thawing of human embryonic stem cells and subsequent cultivation.
  • the inventors freeze stem cells by adding an artificial elastin-like extracellular matrix (REP) according to the present invention.
  • Preservation technology is to prevent anoikis of stem cells dissociated from the extracellular matrix by using an artificial extracellular matrix that contains the RGD motif of the extracellular matrix to which stem cells attach in vivo but does not activate unnecessary signaling mechanisms of human embryonic stem cells.
  • the present invention was completed by clarifying that stem cell viability increases in the re-culture process after thawing by preventing unnecessary stimulation of differentiation.
  • the present invention provides a composition for cryopreservation of stem cells comprising an artificial elastin-like extracellular matrix.
  • the elastin-like artificial extracellular matrix may be arginine-glycine-aspartate (RGD)-elastin like polypeptide (ELP).
  • the elastin-like artificial extracellular matrix does not activate ERK signal, activates AKT signal, inhibits FOXO3a activation, and inhibits the expression of apoptosis factor BIM, thereby improving the survival rate of human embryonic stem cells during cryopreservation and cultivation after thawing.
  • the stem cells are composed of embryonic stem cells, induced pluripotent stem cells, adult stem cells/tissue-specific stem cells, and mesenchymal stem cells. It may be selected from the group, but is not limited thereto.
  • composition for cryopreservation of the present invention may further include a buffer, an isotonic agent or an apoptosis inhibitor.
  • the buffering agent may be any one selected from the group consisting of citrate, phosphate, succinate, tartrate, fumarate, gluconate, oxalate, lactate, acetate, histidine and tris.
  • the isotonic agent is any one selected from the group consisting of sodium chloride, potassium chloride, boric acid, sodium borate, mannitol, glycerin, propylene glycol, polyethylene, glycol, maltose, sucrose, erythritol, arabitol, xylitol, sorbitol trihalose and glucose.
  • the apoptosis inhibitor may be any one selected from the group consisting of a Rho-associated kinase (ROCK) inhibitor, catalase, and zVAD-fmk.
  • ROCK Rho-associated kina
  • the present invention provides a method for cryopreservation of stem cells comprising the step of cryopreserving the stem cells by treating the stem cells with a composition for cryopreservation comprising an artificial elastin-like extracellular matrix.
  • the elastin-like artificial extracellular matrix may be added at a concentration of 0.5 to 10 ⁇ M.
  • the method for cryopreservation of stem cells according to the present invention is to contact or add a composition for cryopreservation of stem cells containing an effective amount of an artificial elastin-like extracellular matrix according to the present invention to a subject to be cryopreserved, and to obtain a temperature for cryopreservation. It may include a step of cooling, and may be performed using a known technique in the art, for example, a quick cooler or liquid nitrogen may be used, but is not limited thereto.
  • the present invention includes the steps of cryopreserving the stem cells by treating the stem cells with a composition for cryopreservation of stem cells comprising an artificial elastin-like extracellular matrix; removing the non-stem cell solution during the process of thawing the cryopreserved stem cells; And it provides a method for thawing and culturing stem cells comprising the step of raising the stem cells from which the solution is removed and then transferring them to a plate and culturing them again.
  • cryopreservation means stably maintaining cells over a long period of time through freezing.
  • Cells generally mutate at a rate of about one in 10,000 in culture, and if cell passages continue for a long period of time, they change into a cell population different from the original cell population, and in severe cases, the specific function of the cell is lost. In some cases, it is lost by subculture. In addition, it may be infected with mycoplasma or the like during subculture. Due to these problems, cryopreservation of the cells is performed so that the cells are frozen and preserved before the unique characteristics of the cells are lost, and taken out and used when necessary.
  • cryopreservation may be performed through a conventional method in the art of freezing and preserving cells, examples of which may include vitrification freezing (Vitrification method), slow freezing (Slow freezing method) or mass cryopreservation, but are limited thereto It is not.
  • the present invention provides a cell therapy agent comprising a composition for cryopreservation of stem cells including stem cells and an artificial elastin-like extracellular matrix.
  • the cell therapy agent may be a frozen product or a lyophilizate.
  • cell therapy product refers to cells and tissues manufactured by isolation, culture, and special manipulation from humans and animals, and is a drug (US FDA regulation) used for the purpose of treatment, diagnosis, and prevention. It refers to medicines used for the purpose of treatment, diagnosis, and prevention through a series of actions, such as proliferating and selecting living autologous, allogeneic, or heterogeneous cells in vitro or changing the biological characteristics of cells in other ways to restore them.
  • the cell therapy agent may further include a pharmaceutically acceptable carrier.
  • a pharmaceutically acceptable carrier means that the composition is not toxic to cells or humans exposed to the composition.
  • the carrier may be used without limitation as long as it is known in the art such as a buffer, a preservative, a pain reliever, a solubilizer, an isotonic agent, a stabilizer, a base, an excipient, a lubricant, and a preservative.
  • the cell therapy agent can be prepared in the form of various formulations according to commonly used techniques, and can be administered through any route as long as it can induce migration to a disease site. In some cases, loading the stem cells into a vehicle equipped with a means to direct them to the lesion may be considered. Accordingly, the composition of the present invention may be administered topically (including buccal, sublingual, dermal and intraocular administration), parenteral (including subcutaneous, intradermal, intramuscular, instillation, intravenous, intraarterial, intraarticular and intracerebrospinal fluid) or transdermal administration. It can be administered through several routes, including, preferably parenterally, and most preferably directly to the affected area.
  • Example 1 Analysis of integrin mRNA expression pattern in human embryonic stem cells
  • integrin mRNA expression of integrin mRNA by culturing human embryonic stem cells (H1, H9) and fibroblasts (CRL, BJ) with laminin-511 coating under feeder-free conditions and Essential 8 culture medium or DMDM containing 10% bovine serum, respectively Patterns were analyzed by mRNA sequencing. Among the entire mRNA sequencing analysis data, components of the integrin A and B groups capable of binding to the RGD motif were selected and the expression levels of each cell were compared for each integrin gene subtype. PolyA+ cells were isolated from two replicate samples for each cell, and then the prepared library was data-generated using an Illumina HiSeq2500 sequencer device. The generated FASTQ file was mapped based on hg38 human transcriptome release 29, and the transcript expression level was analyzed using RStudieo (R version 3.5.3) and package "tximport".
  • human embryonic stem cells express a plurality of integrins capable of binding to the RGD motif.
  • integrins capable of binding to the RGD motif in human embryonic stem cells was analyzed using antibody staining and a flowcytometer.
  • Human embryonic stem cells in adherent culture were collected using accutase treatment and centrifugation, and 1,000,000 cells per 100 ⁇ l of PBS containing 1% bovine serum albumin and 1 ⁇ g/ml DNaseI and 1 ⁇ g of each integrin protein were collected. After mixing the antibodies and refrigerating for 1 hour, the cells stained with the antibody were measured in a flowcytometer.
  • the protein expression of the integrin of the combination capable of binding to the RGD motif in human embryonic stem cells was verified using antibody staining and a flowcytometer.
  • the artificial elastin-like extracellular matrix used in the present invention is an arginine-glycine-aspartate (RGD)-elastin-like polypeptide (ELP), and is named REP for short.
  • REP is a protein with a structure having a ligand having an amino acid sequence of RGD (arginine-glycine-aspartic acid) with ELP as a template.
  • REP was synthesized according to a previously known synthetic method [Jeon WB, Park BH, Wei J, Park RW (2011) J Biomed Mater Res A97(2): 152-7].
  • a DNA fragment encoding the amino acid of RGD was successfully cloned by recursive directional ligation (RDL), and transformed into a pET-based expression vector, E. coli BLR ( DE3) was expressed and purified. The purity was increased by performing reverse transfer cycling for 3 rounds, and it was confirmed that the purity was 95% or more by SDS-PAGE (FIG. 2A).
  • REP and PBS were mixed at various concentration conditions to confirm the conditions for forming aggregates at the cell culture temperature. For the evaluation of the effect on the cells, conditions for minimizing aggregate formation were established.
  • the survival rate of human embryonic stem cells and the expression rate of stem cell markers according to REP treatment were evaluated by the apoptotic cell staining method using 7AAD reagent and the expression rate of OCT4, a marker expressed when maintaining the characteristics of pluripotent stem cells. It was analyzed as a confirmation method.
  • Figure 2E is a measurement of the survival rate of human embryonic stem cells according to 24 hours of culture in the mixture of culture medium and REP concentration, and it was confirmed that the survival rate of human embryonic stem cells is greatly improved when treated with 1 ⁇ M REP.
  • 2F shows the quantitative analysis of the survival rate results according to FIG. 2E.
  • Figure 2G is the measurement of the stem cell marker expression ratio of surviving embryonic stem cells according to the 24-hour culture in the culture medium and the mixed solution for each REP concentration. It was confirmed that the ratio was significantly increased, and FIG. 2H shows a quantitative analysis of the stem cell marker expression ratio results according to FIG. 2G.
  • Example 4 Analysis of cell performance according to cultivation after freezing and thawing using REP-containing cryoprotectant
  • Figure 3A shows a schematic diagram of the process of re-culturing after freezing and thawing after collection of stem cells during cryopreservation of human stem cells, resuspension into single cells or clumps using cryopreservative containing 0.5 to 10 ⁇ M of REP, and adherence culture.
  • 500,000 - 1,000,000 embryonic stem cells precipitated by centrifugation after being collected by treating human embryonic stem cells with accutase or versene were reconstituted in a cell cryopreservation solution composed of 1 ⁇ M REP, 900 ⁇ l of fetal bovine serum, and 100 ⁇ l of DMSO. Flotation and transfer to cryopreservation tubes and cryopreservation.
  • the thawed embryonic stem cells were centrifuged at 1,200 rpm for 3 minutes and moved to a Laminin-511 coated culture dish, and then, the REP was naturally washed by replacing the culture medium once a day, and the survival rate in the same manner as in Example 3 And stem cell marker expression ratio was analyzed using a flowcytometer.
  • Example 5 REP for human embryonic stem cells survival rate Identification of mechanism of action related to increase
  • the increase in the survival rate of REP for human embryonic stem cells was related to the activation of the FAK signal as shown in FIGS. 4A and 4B, and was not related to the activation of the ERK signal as shown in FIGS. 4A and 4C. As shown in Fig. 4D, it was related to the activation of AKT signaling.
  • Example 6 Identification of mechanism of action related to increase in viability by addition of REP during cryopreservation and cultivation of human embryonic stem cells after thawing
  • Figure 5A verifies the inhibition of ERK or AKT signal upon freezing of embryonic stem cells in REP-added conditions
  • Figure 5B confirms the change in survival rate by inhibition of ERK or AKT signal upon freezing of embryonic stem cells in REP-added conditions
  • FIG. 5C confirms that differentiation of embryonic stem cells surviving by REP treatment is promoted when AKT is inhibited.
  • Figure 5D confirms the inhibition of FOXO3a activation and inhibition of cell death factor BIM expression upon REP treatment of embryonic stem cells
  • Figure 5E shows a quantitative analysis of the inhibitory effect of Figure 5D.
  • Figure 5F shows a schematic diagram of the molecular mechanism of increasing viability by the addition of REP including RGD-motif during cryopreservation and cultivation of human embryonic stem cells after thawing.

Abstract

The present invention relates to a method for cryopreservation of stem cells using an elastin-like artificial extracellular matrix. The stem cell cryopreservation technique using an elastin-like artificial extracellular matrix according to the present invention enjoys the advantages of using the RGD motif adherent to the integrin expressed by human embryonic stem cells to mimic the in vivo environment of human embryonic stem cells, and at the same time, employing an elastic protein structure, which is an extracellular substrate absent in the early human embryonic stem cell development environment, to make no unnecessary stimuli to the differentiation mechanism of embryonic stem cells, and thus can be advantageously used for developing cryopreservation products for research on science and technology using stem cells, cell therapy products, and products for cryopreservation.

Description

엘라스틴 유사 인공 세포외기질을 이용한 줄기세포의 동결보존 방법Stem cell cryopreservation method using artificial elastin-like extracellular matrix
본 발명은 엘라스틴 유사 인공 세포외기질을 이용한 줄기세포의 동결보존 방법에 관한 것으로, 상기 엘라스틴 유사 인공 세포외기질을 이용하면 인간 줄기세포의 동결보존 후 재배양 과정에서의 생존력 증가를 촉진함으로써 세포치료제 및 줄기세포 연구의 효율성을 확보할 수 있다.The present invention relates to a method for cryopreservation of stem cells using an artificial elastin-like extracellular matrix. The use of the artificial elastin-like extracellular matrix promotes the increase in viability of human stem cells in the process of cryopreservation and subsequent cultivation, thereby providing a cell therapeutic agent. And the efficiency of stem cell research can be secured.
줄기세포는 재생의학, 약리학 및 기초과학 연구 분야로 광범위하게 적용이 가능하다는 잠재력을 보유하고 있으며, 세포이식 및 조직공학과 같은 임상에의 적용을 위한 세포 공급원으로서의 역할이 기대되고 있다. Stem cells have the potential to be widely applied in regenerative medicine, pharmacology, and basic science research fields, and are expected to play a role as a cell source for clinical applications such as cell transplantation and tissue engineering.
세포 보존 기술은 세포 배양 기술의 발전과 더불어, 생물학적 연구를 기초로 한 생명공학 분야에 널리 이용되고 있으며, 그 중 동결보존(cryopreservation) 방법이 주로 사용되고 있다. 동결보존 방법은 세포를 동결배지에 현탁하여 세포의 온도를 일정하게 낮추어 최종적으로 -196℃에 냉동 보존하는 것으로 일반적으로 세포에 내동성을 부여하고 보호하는 역할로 디메틸설폭사이드(dimethyl sulfoxide, DMSO), 글리세롤(glycerol) 등의 동해 방지제와 소태아혈청(fetal bovine serum, FBS) 또는 이를 포함한 세포배양 배지가 주성분으로 이용되고 있다.Cell preservation technology is widely used in the field of biotechnology based on biological research along with the development of cell culture technology, and among them, a cryopreservation method is mainly used. The cryopreservation method is to suspend the cells in a freezing medium to lower the temperature of the cells to a certain level and finally freeze them at -196℃. Freezing inhibitors such as , glycerol, and fetal bovine serum (FBS) or a cell culture medium containing the same are used as main components.
그러나, 이와 같은 세포 동결보존 방법은 동해방지제 DMSO와 동물 유래 혈청 FBS가 갖는 세포 독성, 동물 병원성 오염 등의 세포 손상을 야기할 수 있으므로, 새로운 세포 보존 방법과 조성액에 대한 개발과 검증이 필요하다.However, since such a cell cryopreservation method may cause cell damage such as cytotoxicity and animal pathogenic contamination of the freeze-preservation agent DMSO and animal-derived serum FBS, it is necessary to develop and verify a new cell preservation method and composition solution.
특히, DMSO를 처리하는 유리화 동결 또는 대량 동결보존 방법은 해동된 세포의 생존률이 매우 낮다는 한계가 있기 때문에, 이러한 줄기세포의 동결보존 후의 낮은 생존율은 임상학적 적용 또는 치료의 목적으로 이용하기 위해 최우선적으로 해결해야 할 과제이다. In particular, since the vitrification freezing or mass cryopreservation method using DMSO has a limitation that the survival rate of thawed cells is very low, the low survival rate after cryopreservation of these stem cells is the highest priority for use for clinical application or treatment purposes. It is a challenge that needs to be tackled critically.
지금까지 동결보존 후의 생존율을 높이기 위한 동결보존 방법들이 다수 연구된 바 있지만, 엘라스틴 유사 인공 세포외기질을 이용한 줄기세포의 동결보존 방법은 아직까지 알려진 바 없다.Until now, many cryopreservation methods for increasing the survival rate after cryopreservation have been studied, but a method for cryopreservation of stem cells using an artificial elastin-like extracellular matrix has not yet been known.
본 발명은 엘라스틴 유사 인공 세포외기질을 이용하여 인간 줄기세포의 동결보존 후 재배양 과정에서의 생존력 증가를 촉진함으로써 세포치료제 및 줄기세포 연구의 효율성 확보를 그 목적으로 한다.An object of the present invention is to secure the efficiency of cell therapy products and stem cell research by promoting an increase in viability in the process of culturing human stem cells after cryopreservation using an artificial elastin-like extracellular matrix.
상기 목적을 달성하기 위하여, 본 발명은 엘라스틴 유사 인공 세포외기질을 포함하는 줄기세포의 동결보존용 조성물을 제공한다.In order to achieve the above object, the present invention provides a composition for cryopreservation of stem cells comprising an artificial elastin-like extracellular matrix.
또한, 본 발명은 상기 조성물을 줄기세포에 처리하여 줄기세포를 동결보존시키는 단계를 포함하는 줄기세포의 동결보존 방법을 제공한다.In addition, the present invention provides a method for cryopreservation of stem cells comprising the step of cryopreserving the stem cells by treating the stem cells with the composition.
또한, 본 발명은 상기 조성물을 줄기세포에 처리하여 줄기세포를 동결보존시키는 단계; 상기 동결보존된 줄기세포의 해동 과정에서 줄기세포 외 용액을 제거하는 단계; 및 상기 용액이 제거된 줄기세포의 부양 후 플레이트에 이동하여 재배양하는 단계를 포함하는 줄기세포의 해동 및 재배양 방법을 제공한다.In addition, the present invention comprises the steps of cryopreserving the stem cells by treating the stem cells with the composition; removing the non-stem cell solution during the process of thawing the cryopreserved stem cells; And it provides a method for thawing and culturing stem cells comprising the step of raising the stem cells from which the solution is removed and then transferring them to a plate and culturing them again.
또한, 본 발명은 상기 줄기세포 및 상기 조성물을 포함하는 세포치료제를 제공한다.In addition, the present invention provides a cell therapy agent comprising the stem cells and the composition.
본 발명에 따른 엘라스틴 유사 인공 세포외기질을 포함하는 줄기세포 동결보존 기술은 인간 배아줄기세포가 발현하는 인테그린에 부착하는 RGD 모티브를 이용하여 인간 배아줄기세포의 생체 내 환경을 모사함과 동시에, 초기 인간 배아줄기세포 발생 환경에 존재하지 않는 세포외기질인 엘라스틴 단백질 구조를 이용함으로써 불필요한 배아줄기세포의 분화 기전을 자극하지 않는 장점을 나타내므로, 줄기세포 활용 과학기술 연구를 위한 동결 보존용 제품 개발, 세포치료제 개발 및 동결 보존을 위한 제품 개발에 유용하게 활용될 수 있다.The stem cell cryopreservation technology including an artificial elastin-like extracellular matrix according to the present invention simulates the in vivo environment of human embryonic stem cells by using the RGD motif attached to integrin expressed by human embryonic stem cells, and at the same time, the initial By using the elastin protein structure, an extracellular matrix that does not exist in the human embryonic stem cell development environment, it shows the advantage of not stimulating the differentiation mechanism of unnecessary embryonic stem cells, so development of products for cryopreservation for scientific and technological research using stem cells, It can be usefully used for the development of cell therapy products and product development for cryopreservation.
도 1에서 A는 인간 배아줄기세포(H1, H9)와 섬유아세포(CRL, BJ)의 인테그린(integrin) mRNA 발현 패턴을 분석한 결과, 인간 배아줄기세포가 RGD 모티브와 결합할 수 있는 다수의 인테그린을 발현하고 있음을 확인하였고, B는 RGD 모티브와 결합할 수 있는 조합의 인테그린의 인간배아줄기세포에서의 단백질 발현을 항체 염색 및 flowcytometer를 이용하여 검증한 결과이다.In Figure 1, A is a result of analyzing the integrin mRNA expression pattern of human embryonic stem cells (H1, H9) and fibroblasts (CRL, BJ), a number of integrins that human embryonic stem cells can bind to the RGD motif , and B is the result of verifying protein expression in human embryonic stem cells of integrins capable of binding to the RGD motif using antibody staining and a flowcytometer.
도 2에서 A는 RGD 모티브를 포함하는 REP를 정제하여 분자량을 확인한 것이고, B는 REP를 세포에 적용하기 위한 농도별 aggregates 형성 조건을 확인하였고, C는 고농도 REP의 배양액과의 혼합시 세포배양 온도에서 aggregates가 형성되었으나 배양액과 혼합이 어려운 것을 확인하였고, D는 저농도 REP의 세포 배양액과의 혼합 후 rotation을 활용한 세포 침전 억제 조건을 확인하였고, E는 배양액과 REP 농도별 혼합액 내 24시간 배양에 따른 인간 배아줄기세포의 생존률을 측정한 것이고, F는 E 결과의 정량적 분석을 나타낸 것이고, G는 배양액과 REP 농도별 혼합액 내 24시간 배양에 따른 생존 배아줄기세포의 줄기세포 표지인자 발현 비율을 측정한 것이고, H는 G 결과의 정량적 분석을 나타낸 것이고, I는 G의 REP 1 μM 처리 조건에서 줄기세포 표지인자 발현 세포를 재 분리하여 배양한 결과 지속적인 줄기세포 표지인자 발현을 재확인한 것이고, J는 I의 줄기세포 성장률을 7일간 추적 관찰한 것이다.In Figure 2, A is to confirm the molecular weight by purifying REP containing the RGD motif, B is to confirm the conditions for forming aggregates by concentration for applying REP to cells, C is the cell culture temperature when mixed with the culture medium of high concentration REP Aggregates were formed, but it was confirmed that it was difficult to mix with the culture medium, D confirmed the conditions for inhibiting cell sedimentation using rotation after mixing with the cell culture medium of low concentration REP, and E was cultured for 24 hours in the mixture of culture medium and REP concentration. The survival rate of human embryonic stem cells is measured according to the method, F is a quantitative analysis of the E results, and G is a measurement of the stem cell marker expression ratio of surviving embryonic stem cells following 24-hour culture in a mixture of culture medium and REP concentration. H is the quantitative analysis of the G result, I is the result of re-separating and culturing the stem cell marker expressing cells under the REP 1 μM treatment condition of G, and confirms the continuous expression of the stem cell marker. The stem cell growth rate of I was followed up for 7 days.
도 3에서 A는 인간 줄기세포 동결 보존시 줄기세포 수집 후 0.5 내지 10 μM의 REP를 포함한 동결보존액을 이용하여 single cell 또는 clump로 재부양 후 동결 및 해동 후 재배양하는 과정의 모식도를 나타낸 것이고, B는 REP를 포함한 동결보존액을 이용하여 보존된 줄기세포의 해동 후 사멸 세포 비율 및 플레이트 부착 24시간 후 생존 세포의 수를 확인한 결과, REP를 포함한 동결보존액 처리 세포의 사멸 세포가 적고 생존율이 높음을 확인하였고, C는 해동 후 플레이트 부착 및 24시간 배양된 세포의 줄기세포 표지인자(OCT4) 발현 비율의 차이가 없음을 확인하였고, D는 REP가 포함된 동결보존액을 사용한 줄기세포의 해동 후 세포 성장률에 이상이 없음을 확인하였고, E는 REP가 포함된 동결보존액을 사용한 줄기세포의 해동 후 세포 성장률은 이를 사용하지 않은 세포와 차이가 없으나, 최초 생존율의 차이로 인해 REP가 포함된 동결보존액을 사용한 줄기세포가 재배양 7일 후 더 많은 세포의 양을 가짐을 확인하였고, F는 REP가 포함된 동결보존액을 사용한 줄기세포 외배엽, 중배엽, 내배엽 분화 후 각 분화 표지인자의 발현을 qRT-PCR을 이용하여 분석한 결과, 분화능력에 이상이 없음을 확인한 것이다.In Figure 3, A is a schematic diagram of the process of reculturing after freezing and thawing after collection of stem cells during cryopreservation of human stem cells, resuspension into single cells or clumps using cryopreservative containing 0.5 to 10 μM of REP, B, as a result of confirming the ratio of apoptotic cells after thawing of stem cells preserved using cryopreservative containing REP and the number of viable cells 24 hours after plate attachment, the number of apoptotic cells and high survival rate of cells treated with cryopreservative containing REP were low. It was confirmed, C confirmed that there was no difference in the expression ratio of the stem cell marker (OCT4) of the cells cultured for 24 hours after plate attachment after thawing, and D was the cell growth rate after thawing of stem cells using cryopreservation solution containing REP. It was confirmed that there was no abnormality, and in E, the cell growth rate after thawing of stem cells using cryopreservation solution containing REP was not different from that of cells without using it, but due to the difference in initial survival rate, It was confirmed that the stem cells had a larger amount of cells after 7 days of cultivation, and F was the expression of each differentiation marker using qRT-PCR after differentiation of stem cells into ectoderm, mesoderm, and endoderm using cryopreservation solution containing REP. As a result of the analysis, it was confirmed that there was no abnormality in differentiation ability.
도 4에서 A는 인간 배아줄기세포에 대한 REP의 생존률 증가 기전 규명을 위한 ERK, AKT 신호 활성화 여부를 확인한 것이고, B는 FAK 신호 활성화 결과의 정량화 분석 결과로서 FAK 신호가 활성화되었고, C는 ERK 신호 활성화 결과의 정량화 분석 결과로서 ERK 신호가 활성화되지 않았으며, D는 AKT 신호 활성화 결과의 정량화 분석 결과로서 AKT 신호가 활성화되었다.In FIG. 4, A confirms whether ERK and AKT signals are activated for identifying the mechanism for increasing the survival rate of REP for human embryonic stem cells, B is a quantification analysis result of FAK signal activation, and FAK signal is activated, C is ERK signal ERK signal was not activated as a result of quantification analysis of the activation result, and AKT signal was activated as a result of quantification analysis of AKT signal activation result in D.
도 5에서 A는 REP 첨가 조건에서 배아줄기세포 동결시 ERK 또는 AKT 신호 억제를 검증한 것이고, B는 REP 첨가 조건에서 배아줄기세포 동결시 ERK 또는 AKT 신호 억제에 의한 생존율 변화를 확인한 것이고, C는 AKT 억제시 REP 처리에 의해 생존한 배아줄기세포는 분화가 촉진됨을 확인하였고, D는 배아줄기세포에 대한 REP 처리시 FOXO3a 활성화 억제 및 세포사멸인자 BIM 발현 억제를 확인하였고, E는 D 결과의 정량적 분석을 나타낸 것이고, F는 인간 배아줄기세포 동결보존 및 해동 후 재배양 시 RGD-모티브를 포함한 REP 첨가에 의한 생존력 증가 분자기전에 대한 모식도를 나타낸 것이다.In Figure 5, A is to verify the inhibition of ERK or AKT signal upon freezing of embryonic stem cells in REP addition conditions, B is to confirm the change in viability due to ERK or AKT signal inhibition when embryonic stem cells are frozen in REP addition conditions, and C is When AKT was inhibited, it was confirmed that surviving embryonic stem cells were differentiated by REP treatment, and D confirmed that FOXO3a activation was inhibited and apoptosis factor BIM expression was suppressed when REP treatment was performed on embryonic stem cells, and E was a quantitative result of D The analysis is shown, and F is a schematic diagram of the molecular mechanism of increasing viability by the addition of REP including RGD-motif during cryopreservation and thawing of human embryonic stem cells and subsequent cultivation.
이하, 본 발명을 보다 상세히 설명한다.Hereinafter, the present invention will be described in more detail.
본 발명자는 줄기세포 동결보존 시 생존력 확보 및 재배양시 회복력 증가를 위한 동결보존액 첨가물로서의 재조합 단백질을 이용하는 기존 기술과 비교하여, 본 발명에 따른 엘라스틴 유사 인공 세포외기질(REP)을 첨가하는 줄기세포 동결보존 기술은 줄기세포가 생체 내에서 부착하는 세포외기질의 RGD 모티브를 포함하지만 인간 배아줄기세포의 불필요한 신호전달 기전을 활성화하지 않는 인공 세포외기질을 사용함으로써 세포외기질로부터 해리된 줄기세포의 anoikis를 방지하고 불필요한 분화 자극을 방지하여 해동 후 재 배양 과정에서의 줄기세포 생존력을 증가시키는 것을 규명함으로써 본 발명을 완성하였다.Compared to the existing technology using a recombinant protein as a cryopreservative additive for securing viability during cryopreservation of stem cells and increasing resilience during cultivation, the inventors freeze stem cells by adding an artificial elastin-like extracellular matrix (REP) according to the present invention. Preservation technology is to prevent anoikis of stem cells dissociated from the extracellular matrix by using an artificial extracellular matrix that contains the RGD motif of the extracellular matrix to which stem cells attach in vivo but does not activate unnecessary signaling mechanisms of human embryonic stem cells. The present invention was completed by clarifying that stem cell viability increases in the re-culture process after thawing by preventing unnecessary stimulation of differentiation.
본 발명은 엘라스틴 유사 인공 세포외기질을 포함하는 줄기세포의 동결보존용 조성물을 제공한다.The present invention provides a composition for cryopreservation of stem cells comprising an artificial elastin-like extracellular matrix.
상기 엘라스틴 유사 인공 세포외기질은 아르기닌-글리신-아스파르트산(Arginine-Glysine-Aspartate; RGD)-엘라스틴 유사 폴리펩타이드(elastin like polypeptide; ELP)일 수 있다.The elastin-like artificial extracellular matrix may be arginine-glycine-aspartate (RGD)-elastin like polypeptide (ELP).
상기 엘라스틴 유사 인공 세포외기질은 ERK 신호는 활성화하지 않고, AKT 신호는 활성화하며, FOXO3a 활성화를 억제하고, 세포사멸인자 BIM 발현을 억제함으로써 인간 배아줄기세포 동결보존 및 해동 후 재배양시 생존률을 향상시킬 수 있다.The elastin-like artificial extracellular matrix does not activate ERK signal, activates AKT signal, inhibits FOXO3a activation, and inhibits the expression of apoptosis factor BIM, thereby improving the survival rate of human embryonic stem cells during cryopreservation and cultivation after thawing. can
상기 줄기세포는 배아줄기세포(embyonic stem cell), 유도만능줄기세포(induced pluripotent stem cell), 성체줄기세포(Adult stem cell/Tissue-specific stem cell) 및 중간엽줄기세포(mesenchymal stem cell)로 이루어진 군으로부터 선택될 수 있지만, 이에 한정되는 것은 아니다.The stem cells are composed of embryonic stem cells, induced pluripotent stem cells, adult stem cells/tissue-specific stem cells, and mesenchymal stem cells. It may be selected from the group, but is not limited thereto.
또한, 본 발명의 동결보존용 조성물은 완충제, 등장화제 또는 세포사멸 억제제를 추가로 포함할 수 있다. 상기 완충제는 시트레이트, 포스페이트, 숙시네이트, 타르트레이트, 푸마레이트, 글루코네이트, 옥살레이트, 락테이트, 아세테이트, 히스티딘 및 트리스로 이루어진 군에서 선택된 어느 하나일 수 있다. 상기 등장화제는 염화나트륨, 염화칼륨, 붕산, 붕산나트륨, 만니톨, 글리세린, 프로필렌글리콜, 폴리에틸렌, 글리콜, 말토스, 자당, 에리쓰리톨, 아라비톨, 자일리톨, 소르비톨 트리할로즈 및 포도당으로 이루어진 군에서 선택된 어느 하나일 수 있다. 상기 세포사멸 억제제는 ROCK(Rho-associated kinase) 억제제, 카탈라아제(catalase) 및 zVAD-fmk로 이루어진 군에서 선택된 어느 하나일 수 있다.In addition, the composition for cryopreservation of the present invention may further include a buffer, an isotonic agent or an apoptosis inhibitor. The buffering agent may be any one selected from the group consisting of citrate, phosphate, succinate, tartrate, fumarate, gluconate, oxalate, lactate, acetate, histidine and tris. The isotonic agent is any one selected from the group consisting of sodium chloride, potassium chloride, boric acid, sodium borate, mannitol, glycerin, propylene glycol, polyethylene, glycol, maltose, sucrose, erythritol, arabitol, xylitol, sorbitol trihalose and glucose. can be one The apoptosis inhibitor may be any one selected from the group consisting of a Rho-associated kinase (ROCK) inhibitor, catalase, and zVAD-fmk.
또한, 본 발명은 엘라스틴 유사 인공 세포외기질을 포함하는 줄기세포의 동결보존용 조성물을 줄기세포에 처리하여 줄기세포를 동결보존시키는 단계를 포함하는 줄기세포의 동결보존 방법을 제공한다.In addition, the present invention provides a method for cryopreservation of stem cells comprising the step of cryopreserving the stem cells by treating the stem cells with a composition for cryopreservation comprising an artificial elastin-like extracellular matrix.
상기 조성물 중 엘라스틴 유사 인공 세포외기질은 0.5 내지 10 μM의 농도로 첨가할 수 있다.In the composition, the elastin-like artificial extracellular matrix may be added at a concentration of 0.5 to 10 μM.
본 발명에 따른 줄기세포의 동결보존 방법은 동결 보존할 대상에 본 발명에 따른 유효량의 엘라스틴 유사 인공 세포외기질을 포함하는 줄기세포의 동결보존용 조성물을 접촉시키거나 첨가하고, 동결 보존을 위한 온도로 냉각하는 단계를 포함할 수 있으며, 당해 기술 분야에서 공지의 기술을 이용하여 수행할 수 있으며, 예를 들어, 급속 냉각기, 또는 액체 질소를 사용할 수 있으나, 이에 한정되는 것은 아니다.The method for cryopreservation of stem cells according to the present invention is to contact or add a composition for cryopreservation of stem cells containing an effective amount of an artificial elastin-like extracellular matrix according to the present invention to a subject to be cryopreserved, and to obtain a temperature for cryopreservation. It may include a step of cooling, and may be performed using a known technique in the art, for example, a quick cooler or liquid nitrogen may be used, but is not limited thereto.
또한, 본 발명은 엘라스틴 유사 인공 세포외기질을 포함하는 줄기세포의 동결보존용 조성물을 줄기세포에 처리하여 줄기세포를 동결보존시키는 단계; 상기 동결보존된 줄기세포의 해동 과정에서 줄기세포 외 용액을 제거하는 단계; 및 상기 용액이 제거된 줄기세포의 부양 후 플레이트에 이동하여 재배양하는 단계를 포함하는 줄기세포의 해동 및 재배양 방법을 제공한다.In addition, the present invention includes the steps of cryopreserving the stem cells by treating the stem cells with a composition for cryopreservation of stem cells comprising an artificial elastin-like extracellular matrix; removing the non-stem cell solution during the process of thawing the cryopreserved stem cells; And it provides a method for thawing and culturing stem cells comprising the step of raising the stem cells from which the solution is removed and then transferring them to a plate and culturing them again.
본 발명에서 "동결보존(cryopreservation)"은 동결을 통해 장기간에 걸쳐 세포를 안정하게 유지시키는 것을 의미한다. 세포는 일반적으로 배양에서 일만 개 중에 한 개 정도의 비율로 돌연변이가 일어나고 있으며, 장기간에 걸쳐 세포의 계대를 계속하면, 본래의 세포집단과는 다른 세포집단으로 변하고, 심하게는 세포가 가진 특정 기능이 계대 배양에 의해 소실되는 경우도 있다. 또한, 계대배양 중 마이코플라스마 등에 감염될 수도 있다. 이러한 문제점으로 인하여 세포의 고유한 특성이 소실되기 전에 세포를 동결시키고 이를 보존하고, 필요 시에 꺼내어 사용할 수 있도록 세포의 동결 보존을 수행한다. 특히, 줄기세포의 경우 치료제로 이용하기 위해서는 필요한 상황에 건강한 줄기세포를 즉시 사용할 수 있어야 하므로, 효과적인 동결보존은 줄기세포에서 더욱 중요하게 여겨지고 있다. 동결 보존은 세포를 동결시켜 보존하는 당업계의 통상적인 방법을 통하여 수행될 수 있으며, 그 예로 유리화 동결(Vitrification method), 완만동결(Slow freezing method) 또는 대량 동결보존이 포함될 수 있으나, 이에 한정되는 것은 아니다. In the present invention, "cryopreservation" means stably maintaining cells over a long period of time through freezing. Cells generally mutate at a rate of about one in 10,000 in culture, and if cell passages continue for a long period of time, they change into a cell population different from the original cell population, and in severe cases, the specific function of the cell is lost. In some cases, it is lost by subculture. In addition, it may be infected with mycoplasma or the like during subculture. Due to these problems, cryopreservation of the cells is performed so that the cells are frozen and preserved before the unique characteristics of the cells are lost, and taken out and used when necessary. In particular, in the case of stem cells, since healthy stem cells must be immediately available for use as a therapeutic agent, effective cryopreservation is considered more important in stem cells. Cryopreservation may be performed through a conventional method in the art of freezing and preserving cells, examples of which may include vitrification freezing (Vitrification method), slow freezing (Slow freezing method) or mass cryopreservation, but are limited thereto It is not.
또한, 본 발명은 줄기세포 및 엘라스틴 유사 인공 세포외기질을 포함하는 줄기세포의 동결보존용 조성물을 포함하는 세포치료제를 제공한다.In addition, the present invention provides a cell therapy agent comprising a composition for cryopreservation of stem cells including stem cells and an artificial elastin-like extracellular matrix.
상기 세포치료제는 동결물 또는 동결건조물일 수 있다.The cell therapy agent may be a frozen product or a lyophilizate.
본 발명에서 "세포치료제"는 사람 및 동물로부터 분리, 배양 및 특수한 조작을 통해 제조된 세포 및 조직으로 치료, 진단 및 예방의 목적으로 사용되는 의약품(미국 FDA 규정)으로서, 세포 혹은 조직의 기능을 복원시키기 위하여 살아있는 자가, 동종, 또는 이종세포를 체외에서 증식 선별하거나 다른 방법으로 세포의 생물학적 특성을 변화시키는 등의 일련의 행위를 통하여 치료, 진단 및 예방의 목적으로 사용되는 의약품을 의미한다.In the present invention, "cell therapy product" refers to cells and tissues manufactured by isolation, culture, and special manipulation from humans and animals, and is a drug (US FDA regulation) used for the purpose of treatment, diagnosis, and prevention. It refers to medicines used for the purpose of treatment, diagnosis, and prevention through a series of actions, such as proliferating and selecting living autologous, allogeneic, or heterogeneous cells in vitro or changing the biological characteristics of cells in other ways to restore them.
상기 세포치료제는 약학적으로 허용되는 담체를 추가로 포함할 수 있다. 상기 "약학적으로 허용되는" 이란 상기 조성물에 노출되는 세포나 인간에게 독성이 없는 것을 의미한다. 상기 담체는 완충제, 보존제, 무통화제, 가용화제, 등장제, 안정화제, 기제, 부형제, 윤활제, 보존제 등 당업계에 공지된 것이라면 제한없이 사용할 수 있다.The cell therapy agent may further include a pharmaceutically acceptable carrier. The "pharmaceutically acceptable" means that the composition is not toxic to cells or humans exposed to the composition. The carrier may be used without limitation as long as it is known in the art such as a buffer, a preservative, a pain reliever, a solubilizer, an isotonic agent, a stabilizer, a base, an excipient, a lubricant, and a preservative.
상기 세포치료제는 각종 제형의 형태로 통용되는 기법에 따라 제조될 수 있으며, 질병 부위로 이동을 유도할 수 있다면 어떠한 경로를 통해서든지 투여 가능하다. 경우에 따라서는 줄기세포를 병변으로 향하게 하는 수단을 구비한 비히클에 로딩하는 방안을 고려할 수도 있다. 따라서 본 발명의 조성물은 국소(협측, 설하, 피부 및 안내 투여를 포함), 비경구(피하, 피내, 근육내, 점적, 정맥 내, 동맥 내, 관절 내 및 뇌척수액 내를 포함) 또는 경피성 투여를 포함한 여러 경로를 통해 투여할 수 있으며, 바람직하게는 비경구로, 가장 바람직하게는 발병부위에 직접 투여할 수 있다.The cell therapy agent can be prepared in the form of various formulations according to commonly used techniques, and can be administered through any route as long as it can induce migration to a disease site. In some cases, loading the stem cells into a vehicle equipped with a means to direct them to the lesion may be considered. Accordingly, the composition of the present invention may be administered topically (including buccal, sublingual, dermal and intraocular administration), parenteral (including subcutaneous, intradermal, intramuscular, instillation, intravenous, intraarterial, intraarticular and intracerebrospinal fluid) or transdermal administration. It can be administered through several routes, including, preferably parenterally, and most preferably directly to the affected area.
이하에서는, 본 발명을 한정하지 않는 실시예에 따라 본 발명을 상세히 설명한다. 본 발명의 하기 실시예는 본 발명을 구체화하기 위한 것일 뿐 본 발명의 권리범위를 제한하거나 한정하는 것이 아님은 물론이다. 따라서, 본 발명의 상세한 설명 및 실시예로부터 본 발명이 속하는 기술분야의 전문가가 용이하게 유추할 수 있는 것은 본 발명의 권리범위에 속하는 것으로 해석된다. Hereinafter, the present invention will be described in detail according to examples that do not limit the present invention. Of course, the following examples of the present invention are only intended to embody the present invention and are not intended to limit or limit the scope of the present invention. Therefore, what can be easily inferred by an expert in the technical field to which the present invention belongs from the detailed description and examples of the present invention is interpreted as belonging to the scope of the present invention.
실시예 1: 인간 배아줄기세포의 인테그린 mRNA 발현 패턴 분석Example 1: Analysis of integrin mRNA expression pattern in human embryonic stem cells
인간 배아줄기세포(H1, H9)와 섬유아세포(CRL, BJ)를 각각 feeder free 조건의 laminin-511 코팅 및 Essential 8 배양액 또는 10% 소 혈청이 포함된 DMDM으로 배양하여, 인테그린(integrin) mRNA 발현 패턴을 mRNA sequencing으로 분석하였다. 전체 mRNA sequencing 분석 자료 중 RGD 모티프에 결합 가능한 인테그린 A와 B 그룹의 구성요소를 선별하여 인테그린 유전자 subtype 마다 각 세포 간의 발현 양을 비교하였다. 세포 별로 두 번의 반복 시료로부터 polyA+ 세포를 분리하고, 이후 제작된 라이브러리는 Illumina HiSeq2500 sequencer 기기를 이용하여 데이터화되었다. 생성된 FASTQ 파일은 hg38 human transcriptome release 29를 기준으로 맵핑하였으며, RStudieo (R version 3.5.3)과 pakage "tximport"를 이용하여 전사체 발현양이 해석되었다.Expression of integrin mRNA by culturing human embryonic stem cells (H1, H9) and fibroblasts (CRL, BJ) with laminin-511 coating under feeder-free conditions and Essential 8 culture medium or DMDM containing 10% bovine serum, respectively Patterns were analyzed by mRNA sequencing. Among the entire mRNA sequencing analysis data, components of the integrin A and B groups capable of binding to the RGD motif were selected and the expression levels of each cell were compared for each integrin gene subtype. PolyA+ cells were isolated from two replicate samples for each cell, and then the prepared library was data-generated using an Illumina HiSeq2500 sequencer device. The generated FASTQ file was mapped based on hg38 human transcriptome release 29, and the transcript expression level was analyzed using RStudieo (R version 3.5.3) and package "tximport".
그 결과, 도 1A와 같이 인간 배아줄기세포는 RGD 모티브와 결합할 수 있는 다수의 인테그린을 발현하고 있음을 확인하였다.As a result, as shown in FIG. 1A, it was confirmed that human embryonic stem cells express a plurality of integrins capable of binding to the RGD motif.
그리고, RGD 모티브와 결합할 수 있는 조합의 인테그린의 인간배아줄기세포에서의 단백질 발현을 항체 염색 및 flowcytometer를 이용하여 분석하였다. 부착 배양 중인 인간 배아줄기세포를 accutase 처리와 원심분리기를 이용하여 수거하였으며, 1% bovine serum albumin과 1 ㎍/ml DNaseI을 포함하는 PBS 100 μl 당 1,000,000 개의 세포와 1 ㎍의 각각의 인테그린 단백질에 대한 항체를 혼합하여 1시간의 냉장 보관 후 Flowcytometer에서 항체에 염색된 세포를 측정하였다.In addition, the protein expression of integrins capable of binding to the RGD motif in human embryonic stem cells was analyzed using antibody staining and a flowcytometer. Human embryonic stem cells in adherent culture were collected using accutase treatment and centrifugation, and 1,000,000 cells per 100 μl of PBS containing 1% bovine serum albumin and 1 μg/ml DNaseI and 1 μg of each integrin protein were collected. After mixing the antibodies and refrigerating for 1 hour, the cells stained with the antibody were measured in a flowcytometer.
그 결과, 도 1B와 같이 RGD 모티브와 결합할 수 있는 조합의 인테그린의 인간배아줄기세포에서의 단백질 발현을 항체 염색 및 flowcytometer를 이용하여 검증할 수 있었다.As a result, as shown in FIG. 1B, the protein expression of the integrin of the combination capable of binding to the RGD motif in human embryonic stem cells was verified using antibody staining and a flowcytometer.
실시예 2: 엘라스틴 유사 인공 세포외기질(RGD-ELP; REP) 정제Example 2: Elastin-Like Artificial Extracellular Matrix (RGD-ELP; REP) Purification
본 발명에서 사용된 엘라스틴 유사 인공 세포외기질은 아르기닌-글리신-아스파르트산(Arginine-GlycineAspartate; RGD)-엘라스틴 유사 폴리펩타이드(elastin like polypeptide; ELP)로서, 줄여서 REP로 명명하였다. REP는 ELP를 주형으로 RGD(아르기닌-글리신-아스파르트산)의 아미노산 배열을 가지는 리간드를 가지는 구조의 단백질이다.The artificial elastin-like extracellular matrix used in the present invention is an arginine-glycine-aspartate (RGD)-elastin-like polypeptide (ELP), and is named REP for short. REP is a protein with a structure having a ligand having an amino acid sequence of RGD (arginine-glycine-aspartic acid) with ELP as a template.
REP는 이전에 알려진 합성방법[Jeon W B, Park B H, Wei J, Park R W (2011) J Biomed Mater Res A97(2): 152-7]에 따라 합성하였다. RGD(아르기닌-글리신-아스파르트산)의 아미노산을 코딩한 DNA 단편을 재귀적 방향성 라이게이션(recursive directional ligation: RDL)에 의해 성공적으로 클로닝되었으며, 이를 pET계 발현벡터로 형질전환시킨 E. coli BLR(DE3)에서 발현시켜 정제하였다. 역전이 사이클링을 3 라운드 실시하여 순도를 높였고, SDS-PAGE로 순도 95% 이상임을 확인하였다(도 2A).REP was synthesized according to a previously known synthetic method [Jeon WB, Park BH, Wei J, Park RW (2011) J Biomed Mater Res A97(2): 152-7]. A DNA fragment encoding the amino acid of RGD (arginine-glycine-aspartic acid) was successfully cloned by recursive directional ligation (RDL), and transformed into a pET-based expression vector, E. coli BLR ( DE3) was expressed and purified. The purity was increased by performing reverse transfer cycling for 3 rounds, and it was confirmed that the purity was 95% or more by SDS-PAGE (FIG. 2A).
실시예 3: 농도별 REP 처리 조건 및 세포 성능 분석Example 3: Analysis of REP treatment conditions and cell performance by concentration
도 2B와 같이 REP를 세포에 적용하기 위해 다양한 농도 조건별로 REP와 PBS를 혼합하여 세포배양 온도에서 aggregates 형성 조건을 확인하였고, REP의 aggregate가 형성된 조건에서는 배양액의 침투가 어려워 세포 배양이 불가능하므로 REP의 세포에 대한 효과 평가를 위해 aggregate 형성 최소화 조건을 수립하였다. 적정 농도의 REP와 rotation을 병행하여 REP 처리에 따른 인간 배아줄기세포의 생존률과 줄기세포 표지인자 발현 비율을 7AAD 시약을 이용한 사멸세포 염색법과 전분화능 줄기세포 성격 유지시 발현하는 표지인자인 OCT4 발현률을 확인하는 방법으로 분석하였다.As shown in FIG. 2B, in order to apply REP to cells, REP and PBS were mixed at various concentration conditions to confirm the conditions for forming aggregates at the cell culture temperature. For the evaluation of the effect on the cells, conditions for minimizing aggregate formation were established. In parallel with appropriate concentrations of REP and rotation, the survival rate of human embryonic stem cells and the expression rate of stem cell markers according to REP treatment were evaluated by the apoptotic cell staining method using 7AAD reagent and the expression rate of OCT4, a marker expressed when maintaining the characteristics of pluripotent stem cells. It was analyzed as a confirmation method.
그 결과, 도 2C와 같이 고농도 REP의 배양액과의 혼합시 세포배양 온도에서 aggregates가 형성되었으나 배양액과 혼합이 어려운 것을 확인하였고, 도 2D와 같이 저농도 REP의 세포 배양액과의 혼합 후 rotation을 활용한 세포와 REP의 응집 및 침전 억제 조건을 확인하였다.As a result, as shown in Figure 2C, aggregates were formed at the cell culture temperature when mixed with the high-concentration REP culture medium, but it was confirmed that it was difficult to mix with the culture medium, and cells using rotation after mixing with the low-concentration REP cell culture medium as shown in Figure 2D and REP aggregation and precipitation inhibition conditions were confirmed.
그리고, 도 2E는 배양액과 REP 농도별 혼합액 내 24시간 배양에 따른 인간 배아줄기세포의 생존률을 측정한 것으로, 1 μM의 REP 처리 시 인간 배아줄기세포의 생존률이 크게 향상되는 것을 확인할 수 있었고, 도 2F는 도 2E에 따른 생존률 결과를 정량적으로 분석하여 나타낸 것이다.And, Figure 2E is a measurement of the survival rate of human embryonic stem cells according to 24 hours of culture in the mixture of culture medium and REP concentration, and it was confirmed that the survival rate of human embryonic stem cells is greatly improved when treated with 1 μM REP. 2F shows the quantitative analysis of the survival rate results according to FIG. 2E.
그리고, 도 2G는 배양액과 REP 농도별 혼합액 내 24시간 배양에 따른 생존 배아줄기세포의 줄기세포 표지인자 발현 비율을 측정한 것으로, 역시 1 μM의 REP 처리 시 인간 배아줄기세포의 줄기세포 표지인자 발현 비율이 크게 증가되는 것을 확인할 수 있었고, 도 2H는 도 2G에 따른 줄기세포 표지인자 발현 비율 결과의 정량적 분석을 나타낸 것이다.And, Figure 2G is the measurement of the stem cell marker expression ratio of surviving embryonic stem cells according to the 24-hour culture in the culture medium and the mixed solution for each REP concentration. It was confirmed that the ratio was significantly increased, and FIG. 2H shows a quantitative analysis of the stem cell marker expression ratio results according to FIG. 2G.
실시예Example 4: REP 포함 동결보존액을 이용한 동결 및 해동 후 재배양에 따른 세포 성능 분석 4: Analysis of cell performance according to cultivation after freezing and thawing using REP-containing cryoprotectant
도 3A에서 인간 줄기세포 동결보존 시 줄기세포 수집 후 0.5 내지 10 μM의 REP를 포함한 동결보존액을 이용하여 single cell 또는 clump로 재부양 후 동결 및 해동 후 재배양하는 과정의 모식도를 나타내었고, 부착 배양중인 인간 배아줄기세포를 accutase 또는 versene 으로 처리하여 수거 한 후 원심분리하여 침전된 500,000 - 1,000,000 개의 배아줄기세포를 1 μM REP, 900 μl의 fetal bovine serum, 100 μl의 DMSO로 구성된 세포 동결보존액으로 재부양 및 동결보존 튜브에 이동하고 동결처리하였다. 해동 된 배아줄기세포는 1,200 rpm에서 3 분간 원심분리하여 Laminin-511이 코팅된 배양접시로 이동하였으며, 이후 일 1회 배양액 교체를 통해 REP의 자연 세척을 진행하였고, 실시예 3과 동일한 방법으로 생존률 및 줄기세포 표지인자 발현 비율을 flowcytometer를 이용하여 분석하였다.Figure 3A shows a schematic diagram of the process of re-culturing after freezing and thawing after collection of stem cells during cryopreservation of human stem cells, resuspension into single cells or clumps using cryopreservative containing 0.5 to 10 μM of REP, and adherence culture. 500,000 - 1,000,000 embryonic stem cells precipitated by centrifugation after being collected by treating human embryonic stem cells with accutase or versene were reconstituted in a cell cryopreservation solution composed of 1 μM REP, 900 μl of fetal bovine serum, and 100 μl of DMSO. Flotation and transfer to cryopreservation tubes and cryopreservation. The thawed embryonic stem cells were centrifuged at 1,200 rpm for 3 minutes and moved to a Laminin-511 coated culture dish, and then, the REP was naturally washed by replacing the culture medium once a day, and the survival rate in the same manner as in Example 3 And stem cell marker expression ratio was analyzed using a flowcytometer.
도 3B와 같이 REP를 포함한 동결보존액을 이용하여 보존된 줄기세포의 해동 후 사멸 세포 비율 및 플레이트 부착 24시간 후 생존 세포의 수를 확인한 결과, 엘라스틴 유사 인공 세포외 기질을 포함한 동결보존액 처리 세포의 사멸 세포가 적고 생존율이 높음을 확인하였다.As shown in FIG. 3B, as a result of confirming the percentage of dead cells after thawing of stem cells preserved using cryopreservative containing REP and the number of viable cells 24 hours after plate attachment, death of cells treated with cryopreservative containing elastin-like artificial extracellular matrix It was confirmed that the number of cells was small and the survival rate was high.
도 3C에서는 해동 후 플레이트 부착 및 24시간 배양된 세포의 줄기세포 표지인자(OCT4) 발현 비율의 차이가 없음을 확인하였고, 도 3D에서는 REP가 포함된 동결보존액을 사용한 줄기세포의 해동 후 세포 성장률에 이상이 없음을 확인하였고, 도 3E에서는 REP가 포함된 동결보존액을 사용한 줄기세포의 해동 후 세포 성장률은 이를 사용하지 않은 세포와 차이가 없으나, 최초 생존율의 차이로 인해 REP가 포함된 동결보존액을 사용한 줄기세포가 재배양 7일 후 더 많은 세포의 양을 가짐을 확인하였다.In Figure 3C, it was confirmed that there was no difference in the expression ratio of the stem cell marker (OCT4) in the cells cultured for 24 hours after thawing and plate attachment, and in Figure 3D, the cell growth rate after thawing of stem cells using cryopreservation solution containing REP It was confirmed that there was no abnormality, and in FIG. 3E, the cell growth rate after thawing of the stem cells using the cryopreservation solution containing REP was not different from that of the cells without using it, but due to the difference in the initial survival rate, the use of the cryopreservation solution containing REP It was confirmed that the stem cells had a larger amount of cells after 7 days of cultivation.
또한, 도 3F와 같이 REP가 포함된 동결보존액을 사용한 줄기세포 외배엽, 중배엽, 내배엽 분화 후 각 분화 표지인자의 발현을 qRT-PCR을 이용하여 분석한 결과, 분화능력에 이상이 없음을 확인하였다.In addition, as shown in FIG. 3F, after differentiation of stem cells into ectoderm, mesoderm, and endoderm using cryopreservation solution containing REP, the expression of each differentiation marker was analyzed using qRT-PCR, and it was confirmed that there was no abnormality in differentiation ability.
실시예Example 5: 인간 배아줄기세포에 대한 REP의 5: REP for human embryonic stem cells 생존률survival rate 증가 관련 작용기전 규명 Identification of mechanism of action related to increase
인간 배아줄기세포에 대한 REP의 생존률 증가 관련 작용기전을 규명하기 위하여, FAK, ERK 및 AKT 신호 활성화 여부를 immunoblotting 방법으로 분석하였다. REP 적용 조건 별 동결된 배아줄기세포의 해동 직후 cell lysis buffer를 이용하여 단백질을 추출하였으며, immunoblotting 이후 densitometer를 이용하여 결과를 해석하였다.In order to identify the mechanism of action related to the increase in survival rate of REP on human embryonic stem cells, activation of FAK, ERK and AKT signals was analyzed by immunoblotting. Immediately after thawing the frozen embryonic stem cells for each REP application condition, proteins were extracted using cell lysis buffer, and the results were analyzed using a densitometer after immunoblotting.
그 결과, 인간 배아줄기세포에 대한 REP의 생존률 증가는 도 4A 및 도 4B와 같이 FAK 신호의 활성화와 관련되었고, 도 4A 및 도 4C와 같이 ERK 신호의 활성화와는 관련되지 않았으며, 도 4A 및 도 4D와 같이 AKT 신호의 활성화와 관련되었다.As a result, the increase in the survival rate of REP for human embryonic stem cells was related to the activation of the FAK signal as shown in FIGS. 4A and 4B, and was not related to the activation of the ERK signal as shown in FIGS. 4A and 4C. As shown in Fig. 4D, it was related to the activation of AKT signaling.
실시예Example 6: 인간 배아줄기세포 동결보존 및 해동 후 재배양 시 REP 첨가에 의한 생존력 증가 관련 작용기전 규명 6: Identification of mechanism of action related to increase in viability by addition of REP during cryopreservation and cultivation of human embryonic stem cells after thawing
REP 첨가 조건 하 인간 배아줄기세포 동결 시 작용기전을 규명하기 위하여, ERK 및 AKT 신호 활성화 여부와 FOXO3a 활성화 억제 및 세포사멸인자 BIM 발현 억제 효과를 신호전달기전 억제제 처리 및 immunoblotting 방법으로 분석하였다. REP를 적용한 세포 동결과정 3시간 전부터 ERK 또는 AKT 신호 저해제를 세포에 처리하였으며, 세포 동결보존액 역시 각 신호 저해제를 첨가하였다. 이후 해동된 세포로부터 단백질을 추출 및 immunoblotting을 시행하였으며, 결과는 densitometer를 이용하여 해석되었다.In order to investigate the mechanism of action during freezing of human embryonic stem cells under the addition of REP, the activation of ERK and AKT signals, the inhibition of FOXO3a activation and the inhibition of the expression of the apoptosis factor BIM were analyzed by treatment with inhibitors of signal transduction mechanism and immunoblotting. Cells were treated with ERK or AKT signal inhibitors 3 hours before the REP-applied cell freezing process, and each signal inhibitor was also added to the cell cryopreservative. Afterwards, proteins were extracted from the thawed cells and immunoblotting was performed, and the results were interpreted using a densitometer.
그 결과, 도 5A는 REP 첨가 조건에서 배아줄기세포 동결시 ERK 또는 AKT 신호 억제를 검증한 것으로, 도 5B는 REP 첨가 조건에서 배아줄기세포 동결시 ERK 또는 AKT 신호 억제에 의한 생존율 변화를 확인하였고, 도 5C는 AKT 억제시 REP 처리에 의해 생존한 배아줄기세포는 분화가 촉진됨을 확인하였다.As a result, Figure 5A verifies the inhibition of ERK or AKT signal upon freezing of embryonic stem cells in REP-added conditions, and Figure 5B confirms the change in survival rate by inhibition of ERK or AKT signal upon freezing of embryonic stem cells in REP-added conditions. FIG. 5C confirms that differentiation of embryonic stem cells surviving by REP treatment is promoted when AKT is inhibited.
도 5D는 배아줄기세포에 대한 REP 처리시 FOXO3a 활성화 억제 및 세포사멸인자 BIM 발현 억제를 확인하였고, 도 5E는 도 5D의 억제 효과에 대한 정량적 분석을 나타낸 것이다.Figure 5D confirms the inhibition of FOXO3a activation and inhibition of cell death factor BIM expression upon REP treatment of embryonic stem cells, and Figure 5E shows a quantitative analysis of the inhibitory effect of Figure 5D.
도 5F는 인간 배아줄기세포 동결보존 및 해동 후 재배양 시 RGD-모티브를 포함한 REP 첨가에 의한 생존력 증가 분자기전에 대한 모식도를 나타낸 것이다.Figure 5F shows a schematic diagram of the molecular mechanism of increasing viability by the addition of REP including RGD-motif during cryopreservation and cultivation of human embryonic stem cells after thawing.
이상으로 본 발명의 특정한 부분을 상세히 기술한 바, 당업계의 통상의 지식을 가진 자에게 있어서 이러한 구체적인 기술은 단지 바람직한 구현 예일 뿐이며, 이에 본 발명의 범위가 제한되는 것이 아닌 점은 명백하다. 따라서, 본 발명의 실질적인 범위는 첨부된 청구항과 그의 등가물에 의하여 정의된다고 할 것이다.Having described specific parts of the present invention in detail above, it is clear that these specific techniques are only preferred embodiments for those skilled in the art, and the scope of the present invention is not limited thereto. Accordingly, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.
본 발명의 범위는 후술하는 특허청구범위에 의하여 나타내어지며, 특허청구범위의 의미 및 범위 그리고 그 균등개념으로부터 도출되는 모든 변경 또는 변형된 형태가 본 발명의 범위에 포함되는 것으로 해석되어야 한다.The scope of the present invention is indicated by the claims to be described later, and all changes or modifications derived from the meaning and scope of the claims and equivalent concepts thereof should be construed as being included in the scope of the present invention.

Claims (9)

  1. 엘라스틴 유사 인공 세포외기질을 포함하는 줄기세포의 동결보존용 조성물.A composition for cryopreservation of stem cells comprising an artificial elastin-like extracellular matrix.
  2. 제 1 항에 있어서, 상기 엘라스틴 유사 인공 세포외기질은 아르기닌-글리신-아스파르트산(Arginine-Glysine-Aspartate; RGD)-엘라스틴 유사 폴리펩타이드(elastin like polypeptide; ELP)인 것을 특징으로 하는 줄기세포의 동결보존용 조성물.The freezing of stem cells according to claim 1, wherein the artificial elastin-like extracellular matrix is Arginine-Glysine-Aspartate (RGD)-elastin-like polypeptide (ELP). composition for preservation.
  3. 제 1 항에 있어서, 상기 엘라스틴 유사 인공 세포외기질은 ERK 신호는 활성화하지 않고, AKT 신호는 활성화하며, FOXO3a 활성화를 억제하고, 세포사멸인자 BIM 발현을 억제하는 것을 특징으로 하는 줄기세포의 동결보존용 조성물.The cryopreservation of stem cells according to claim 1, wherein the artificial elastin-like extracellular matrix does not activate ERK signaling, activates AKT signaling, inhibits FOXO3a activation, and inhibits the expression of the apoptosis factor BIM. composition for.
  4. 제 1 항에 있어서, 상기 줄기세포는 배아줄기세포(embyonic stem cell), 유도만능줄기세포(induced pluripotent stem cell), 성체줄기세포(Adult stem cell/Tissue-specific stem cell) 및 중간엽줄기세포(mesenchymal stem cell)로 이루어진 군으로부터 선택된 것을 특징으로 하는 줄기세포의 동결보존용 조성물.The method of claim 1, wherein the stem cells are embryonic stem cells, induced pluripotent stem cells, adult stem cells/tissue-specific stem cells, and mesenchymal stem cells ( A composition for cryopreservation of stem cells, characterized in that selected from the group consisting of mesenchymal stem cells).
  5. 제 1 항 내지 제 4 항 중 어느 한 항의 조성물을 줄기세포에 처리하여 줄기세포를 동결보존시키는 단계를 포함하는 줄기세포의 동결보존 방법.A method of cryopreservation of stem cells comprising the step of cryopreserving the stem cells by treating the stem cells with the composition of any one of claims 1 to 4.
  6. 제 5 항에 있어서, 상기 조성물 중 엘라스틴 유사 인공 세포외기질은 0.5 내지 10 μM의 농도로 첨가한 것을 특징으로 하는 줄기세포 동결보존 방법.The method of claim 5, wherein the elastin-like artificial extracellular matrix is added at a concentration of 0.5 to 10 μM in the composition.
  7. 제 1 항 내지 제 4 항 중 어느 한 항의 조성물을 줄기세포에 처리하여 줄기세포를 동결보존시키는 단계; 상기 동결보존된 줄기세포의 해동 과정에서 줄기세포 외 용액을 제거하는 단계; 및 상기 용액이 제거된 줄기세포의 부양 후 플레이트에 이동하여 재배양하는 단계를 포함하는 줄기세포의 해동 및 재배양 방법.Cryopreservation of the stem cells by treating the stem cells with the composition of any one of claims 1 to 4; removing the non-stem cell solution during the process of thawing the cryopreserved stem cells; and thawing and culturing the stem cells, comprising raising the stem cells from which the solution is removed, moving them to a plate, and then culturing them again.
  8. 줄기세포 및 제 1 항 내지 제 4 항 중 어느 한 항의 조성물을 포함하는 세포치료제.A cell therapy agent comprising stem cells and the composition of any one of claims 1 to 4.
  9. 제 8 항에 있어서, 상기 세포치료제는 동결물 또는 동결건조물인 것을 특징으로 하는 세포치료제.The cell therapy agent according to claim 8, wherein the cell therapy agent is a frozen product or a lyophilisate.
PCT/KR2022/015290 2021-10-18 2022-10-11 Method for cryopreservation of stem cells using elastin-like artificial extracellular matrix WO2023068624A1 (en)

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