WO2023080281A1 - Cell cryopreservation composition using pectin and alanine and cell cryopreservation method using same - Google Patents

Cell cryopreservation composition using pectin and alanine and cell cryopreservation method using same Download PDF

Info

Publication number
WO2023080281A1
WO2023080281A1 PCT/KR2021/015995 KR2021015995W WO2023080281A1 WO 2023080281 A1 WO2023080281 A1 WO 2023080281A1 KR 2021015995 W KR2021015995 W KR 2021015995W WO 2023080281 A1 WO2023080281 A1 WO 2023080281A1
Authority
WO
WIPO (PCT)
Prior art keywords
cells
alanine
cell
pectin
composition
Prior art date
Application number
PCT/KR2021/015995
Other languages
French (fr)
Korean (ko)
Inventor
심태진
김지훈
홍인기
김종필
이유진
이경민
정정일
김문정
Original Assignee
주식회사 프롬바이오
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 주식회사 프롬바이오 filed Critical 주식회사 프롬바이오
Priority to PCT/KR2021/015995 priority Critical patent/WO2023080281A1/en
Publication of WO2023080281A1 publication Critical patent/WO2023080281A1/en

Links

Images

Classifications

    • 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
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/04—Preserving or maintaining viable microorganisms
    • 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
    • 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

Definitions

  • the present invention relates to a cell cryopreservation composition using pectin and alanine and a cell cryopreservation method using the same.
  • cells are separated from tissues such as fat, bone marrow, umbilical cord blood, and peripheral blood, cultured and proliferated in vitro, and manipulated by physical, chemical, and biological methods.
  • Cell therapy products are made from living cells with a short survival period. In order to preserve these cells, they can be subcultured over a long period of time. In this case, cell characteristics may change due to genetic mutation, and there is a risk of cell loss due to contamination. Therefore, there is a need for a preservation method for minimizing cell loss due to genetic mutation and contamination and stably maintaining cell characteristics.
  • stem cells are undifferentiated cells that exist between differentiated cells in tissues or organs, and have self-renewal ability to proliferate indefinitely while maintaining an undifferentiated state, and all tissue cells in the body under certain conditions. Since it has differentiation to specific tissue, it can be used as a cell therapy product to treat degenerative diseases or tissues damaged by trauma, so it is known that it has very high medical utilization. In order to use these stem cells as a cell therapy agent, it is essential to establish a cell cryopreservation method that guarantees cell viability or undifferentiation.
  • Cell cryopreservation methods are divided into slow freezing and rapid freezing.
  • Slow freezing is a method of preserving cells at an extremely low temperature of about -196 ° C by gently lowering the temperature by 1 to 2 ° C per minute with liquid nitrogen.
  • glycerol, DMSO (dimethyl sulphoxide), keratin or gelatin hydrolysate, acetamide, propylene glycol, polyethylene glycol, animal or human-derived serum or serum albumin, etc. of cryoprotectants are used. The generation of ice crystals inside and outside the cells is inhibited by the gentle cooling and use of cryopreservatives, thereby preventing cell damage.
  • the rapid freezing method (ie, vitrification freezing method) is a method of freezing by suppressing the formation of ice crystals inside and outside the cells by rapid freezing, and cryopreservatives such as DMSO, acetamide, propylene glycol, and polyethylene glycol are used.
  • cryopreservatives such as DMSO, acetamide, propylene glycol, and polyethylene glycol are used.
  • the rapid freezing method enables the cryopreservation of early mouse embryos and the cryopreservation of cow embryos and pig embryos, which were difficult to cryopreservate with the slow freezing method.
  • glycerol which has been used as a cryopreservative, does not have a complete buffering function during freezing and thawing, which often causes cell damage, and DMSO is expensive and has the potential to change the character of cells.
  • animal-derived serum such as fetal bovine serum (FBS) or human-derived serum can cause infections such as viruses and prions, and there is a risk of inducing an immune response, and for this reason, human administration There is a problem that is not suitable for preservation of cell therapy products for.
  • cryopreservatives capable of minimizing cell damage due to freezing and thawing while having excellent cell preservation effects is still required.
  • the present invention discloses the effect of pectin and alanine as cryopreservatives.
  • An object of the present invention is to provide a cell cryopreservation composition using pectin and alanine and a cell cryopreservation method using the same.
  • the present invention uses a DPBS (Dulbecco's phosphate-buffered saline) preservation solution containing pectin and alanine as cryopreservatives, in which pectin and alanine are not cytotoxic to human adipose-derived stem cells.
  • DPBS Denbecco's phosphate-buffered saline
  • pectin and alanine are not cytotoxic to human adipose-derived stem cells.
  • the present invention is provided based on the above experimental results, and the composition for cryopreservation of cells of the present invention is characterized in that it contains pectin and alanine.
  • Pectin is a component of the cell wall of dicotyledonous plants and has been widely used in the food field as a gelling agent, thickener, stabilizer, emulsifier, etc.
  • the structure is the main part and has a molecular weight of 200,000 to 400,000.
  • Pectin is generally classified into high methoxy pectin (DM>50) and low methoxy pectin (DM ⁇ 50) according to the degree of methylation (DM). It is toxy pectin, and low methoxy pectin is obtained by demethylation using acid, alkali, enzyme, ammonia, etc. further.
  • any pectin may be used regardless of the plant material from which it is obtained, the size of the molecular weight (preferably 100 to 300 g/mol), or the degree of methylation.
  • alanine may be L-alanine.
  • pectin and alanine are 1 to 30 ⁇ M of pectin, 3 to 90 ⁇ M of alanine, especially 5 to 15 ⁇ M of pectin, and 20 to 10 ⁇ M of alanine in order to exhibit sufficient cryopreservation effects (i.e., cell viability improvement after thawing, cell property maintenance effect). It can be included in the range of 40 ⁇ M.
  • composition for cryopreservation of the present invention may contain a preservative solution.
  • the preservative solution may be a cell culture medium, buffered or isotonic solution.
  • Cell culture media generally contain a carbon source, a nitrogen source, and trace elements.
  • carbon source monosaccharides, disaccharides, and the like may be preferably used. Specifically, glucose, fructose, mannose, galactose, ribose, sorbose, ribulose, lactose, maltose, sucrose, raffinose, or a mixture of one or more thereof may be used.
  • the nitrogen source may be an inorganic nitrogen compound, an organic nitrogen compound, or a complex compound containing these compounds.
  • inorganic nitrogen compounds include ammonia, ammonium salts (eg, ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate, ammonium nitrate, etc.), nitrate, and the like
  • organic nitrogen compounds include urea, amino acids, and the like.
  • trace elements include calcium, magnesium, sodium, cobalt, molybdenum, potassium, manganese, zinc, copper, iron, phosphorus, sulfur, and the like, and these trace elements may be added to the medium in the form of salt compounds.
  • the cell culture medium may also further contain vitamins, growth factors, and the like, and in particular, serum substitutes such as insulin, transferrin, and selenium may be included for the use of a serum-free medium.
  • DMEM Dulbecco's Modified Eagle's Medium
  • MEM Minimal Essential Medium
  • BME Base Medium Eagle
  • RPMI 1640 F-10, F-12, DMEM/F12
  • MEM- ⁇ Minimal Essential Medium- ⁇
  • G-MEM Glasgow's Minimal Essential Medium
  • IMDM Iscove's Modified Dulbecco's Medium
  • MacCoy's 5A badge AmnioMax complete badge, AminoMaxII complete badge, EBM (Endothelial Basal Medium) badge, Chang's Medium, MesenCult-XF , DMEM/HG (Dulbecco's Modified Eagle's Medium high glucose) medium, and MCDB+DMEM/LG (MCDB + Dulbecco's Modified Eagle's Medium low glucose) medium.
  • the preservative solution may be a buffer solution, which is a physiological saline solution containing citrate, phosphate, succinate, tartrate, fumarate, gluconate, oxalate, lactate, acetate, histidine, tris, etc. as a buffer.
  • a buffer solution which is a physiological saline solution containing citrate, phosphate, succinate, tartrate, fumarate, gluconate, oxalate, lactate, acetate, histidine, tris, etc.
  • PBS Phosphate Buffered Saline
  • TBS Tris Buffered Saline
  • HEPES Buffered Saline HEPES Buffered Saline
  • DPBS Dulbecco's phosphate-buffered saline
  • the preservative solution also contains sodium chloride, potassium chloride, boric acid, sodium borate, mannitol, glycerin, propylene glycol, polyethylene, glycol, maltose, sucrose, erythritol, arabitol, xylitol, sorbitol trihalose, glucose, etc. as an isotonic agent. It may be an isotonic solution. Such an isotonic solution may be Ringer's solution, lactate Ringer's solution, acetate Ringer's solution, bicarbonate Ringer's solution, or 5% glucose aqueous solution, and may be directly prepared and used or purchased commercially available.
  • the cell cryopreservation composition of the present invention may further include conventionally known cryopreservatives such as glycerol, keratin or gelatin hydrolysate, acetamide, DMSO, ethylene glycol, propylene glycol, polyethylene glycol, sericin, isomaltooligosaccharide, etc.
  • cryopreservatives such as glycerol, keratin or gelatin hydrolysate, acetamide, DMSO, ethylene glycol, propylene glycol, polyethylene glycol, sericin, isomaltooligosaccharide, etc.
  • EDTA, EGTA, citric acid, chelating agents such as salicylate, dissolution aids, preservatives, antioxidants, and amino acids such as proline and glutamine may be further included.
  • pectin and alanine which are cryopreservatives, have an equivalent level of effect on cell viability or pluripotency of stem cells compared to the case of using fetal bovine serum (FBS). It may not contain human-derived serum or serum-derived components (eg albumin).
  • composition for cryopreservation of the present invention can be used for cryopreservation of any animal cells, particularly mammalian cells.
  • Stem cells may be embryonic stem cells, adult stem cells, and dedifferentiated stem cells.
  • Adult stem cells may be stem cells originating from blood or tissues including fat, bone marrow, umbilical cord blood, and peripheral blood.
  • Stem cells may in particular be adipose-derived stem cells (ADSC).
  • ADSC adipose-derived stem cells
  • Adipose-derived stem cells have self-renewal ability and can be differentiated into various types of cells such as skin, cartilage, and bone, and are easier to collect and mass-cultivate than bone marrow-derived stem cells or cord blood-derived stem cells. However, because of its high content of stem cells, its utilization in cell therapy is high.
  • stem cells have self-renewal and differentiation capabilities, the type and origin are not particularly limited. For example, it may be from a mammal, human, monkey, pig, horse, cow, sheep, dog, cat, mouse or rabbit.
  • the stem cells may be derived from separated umbilical cord, placenta, fat, bone marrow, umbilical cord blood or amniotic fluid.
  • Various methods are known in the art for isolating, culturing, and proliferating stem cells or preparing embryonic stem cells, such as those described in [Curr Opin Chem Eng.
  • animal cells that can be used in the composition for cryopreservation of the present invention are islet cells administered intravenously to patients with type I diabetes, dendritic cells administered for cancer treatment, and natural killer cells.
  • NK cell islet cells administered intravenously to patients with type I diabetes, dendritic cells administered for cancer treatment, and natural killer cells.
  • T cell particularly cytotoxic T cell, cytotoxic T lymphocyte
  • CAR-T cell CAR-NK cell, etc.
  • animal cells may be isolated from living organisms or subcultured in vitro.
  • the animal cells are in a single cell state in which the cells do not aggregate with each other.
  • Mammalian cells in a single cell state can be obtained by enzymatically treating mammalian cells cultured in vitro with trypsin/EDTA, and then suspending the cells by a method known in the art, such as pipetting or tapping.
  • the cell ratio in a single cell state can be determined by dispersing the cells in PBS, observing them under a microscope, and examining the presence or absence of aggregation of a certain number of randomly selected cells.
  • the present invention provides a cell cryopreservation method.
  • the cell cryopreservation method of the present invention includes preparing a preservative solution containing pectin and alanine, suspending cells in the preservative solution, and freezing and preserving the preservative solution in which the cells are suspended.
  • pectin may be high methoxy pectin or low methoxy methine as described above, and alanine may be L-alanine.
  • the preservation solution may be a cell culture medium, buffer, or isotonic solution as described above.
  • the preservation solution may not contain animal-derived or human-derived serum or serum-derived components (eg, albumin).
  • the cells are stem cells, pancreatic islet cells, dendritic cells, natural killer cells, T cells, CAR-T cells, CAR-NK cells, CHO cells, HEK cells, MCF-7 cells, MDCK cells, Vero cells or the like.
  • the cell suspension is in the range of 1 ⁇ 10 3 cells to about 1 ⁇ 10 9 cells, particularly 1 ⁇ 10 5 to 1 ⁇ 10 8 cells, based on 1 ml of the preservation solution. It can be made by adding to the range.
  • freezing may be performed through a method known in the art, such as a slow freezing method or a rapid freezing method (vitrification freezing method).
  • Slow freezing is generally a method of freezing a preservative solution in which cells are suspended in a low-temperature freezer or an ultra-low-temperature freezer (usually in the range of -20 ° C to -150 ° C) by slowly dropping the temperature at a constant level for 12 to 24 hours.
  • the rapid freezing method is generally a method of rapidly freezing a preservative solution in which cells are suspended using equipment such as CRF (controlled rate freezing), usually using liquid nitrogen in a temperature range of -150 ° C to -196 ° C.
  • CRF controlled rate freezing
  • the cell viability when cells are cryopreserved and then thawed may differ depending on the cell type. Therefore, it may be necessary to adopt an appropriate cryopreservation method to increase the cell viability after thawing depending on the cells to be cryopreserved.
  • the preservation may be performed in a liquid nitrogen tank or a deep freezer.
  • the storage temperature may be -100 ° C to about -300 ° C, particularly -150 ° C to about -220 ° C, particularly preferably -150 ° C to -196 ° C.
  • the preservation period may be 1 day or 3 days or more, particularly 1 month, particularly preferably 3 months, 6 months or 1 year or more depending on the use of the cells, such as for research or drug development.
  • a cell cryopreservation composition using pectin and alanine and a cell cryopreservation method using the same can be provided.
  • the cell cryopreservation composition of the present invention and the cell cryopreservation method using the same have an effect of maintaining high cell viability after thawing and maintaining cell characteristics, similar to the case of using DMSO and FBS, which are conventional cryopreservatives, and also derived from animals such as cattle or It has an excellent cryopreservation effect without using human-derived serum or serum-derived components.
  • FIG. 2 confirms the cryopreservation effect of the cryopreservative of the present invention.
  • Adipose-derived stem cells are cryopreserved for 3 days using the cryopreservative and then thawed to measure the viability to confirm the cryopreservation effect.
  • Figure 3 is a photograph of the cells cultured in a culture dish after thawing and observing the cells cultured for 3 days under a microscope at a magnification of 4x over the entire upper and lower parts.
  • 5 is a result of confirming the expression level of adipose-derived stem cell-specific marker genes in cells cryopreserved using the cryopreservative of the present invention and then thawed.
  • the human adipose tissue used in this example was purchased from Goma Biotech Co., Ltd. (Seoul, Korea), and the method for isolating stem cells from adipose tissue was described by Zuk et al. (Mol Biol Cell. 2002 Dec; 13(12): 4279-4295) was used for reference and modification.
  • penicillin 100 U/ml
  • streptomycin 100 ug/ml
  • 10% heat-inactivated serum were added to DMEM (Dulbecco's Modified Eagle's Medium) in 95% air and 5% CO. 2 and cultured at 37 °C conditions. When the cells adhered to the culture dish and grew, the cells were collected using 0.25% trypsin/10 mM EDTA, divided at a ratio of 1:3, and maintained in 10% DMEM.
  • Pectin (194.14 g/mol) and alanine (L-alanine) used in the experiment were purchased from Sigma-Aldrich and used, and adipose-derived stem cells were the cells isolated in Example 1 above. was used.
  • CCK-8 The reagent used in the experiment, was purchased from Dongin Biotech Co., Ltd. (Seoul, Korea).
  • Adipose-derived stem cells were treated in 96-well plates at a concentration of 1 ⁇ 10 4 cells/well by 100 ul, and then cultured for 24 hours. When the cells adhered to the bottom, they were treated with the solvent DPBS (Dulbecco's phosphate-buffered saline), pectin and alanine up to 100 ⁇ M for up to 48 hours. After culturing for 3 hours under conditions of 10 ul of CCK-8 reagent and 100 ul of adipose-derived stem cell culture medium, absorbance was measured at 540 nm with a spectrophotometer.
  • DPBS Disbecco's phosphate-buffered saline
  • pectin as shown in A of FIG. 1 and alanine as shown in B of FIG. 1 can be safely used without showing toxicity up to 100 uM.
  • the present inventors used pectin and alanine as active ingredients in order to prepare a new composition for preserving adipose-derived stem cells.
  • alanine powder was mixed with 28.06 mL of DPBS by vortexing to completely dissolve to prepare a 100 mM alanine solution, and then mixed with DPBS to a final concentration of 300 ⁇ M.
  • the composition of the inventive cryoprotective prepared by the above manufacturing method is shown in Table 1 below, and was named FNCP (Frombio New Cryoprotective) for convenience.
  • composition of composition for cryopreservation FNCP solution concentration Capacity (1mL) final concentration Pectin 100 ⁇ M 100 ⁇ L 10 ⁇ M L-alanine 300 ⁇ M 300 ⁇ L 30 ⁇ M DPBS - 700 ⁇ L -
  • Adipose-derived stem cells cultured in a 75T flask were collected with 0.25% trypsin/10 mM EDTA, and then the cells at 1 ⁇ 10 6 cells/mL were suspended in a composition for cryopreservation prepared according to the composition of Table 1 above, using a Freezing Container, Frozen.
  • a group using 10 w/v% DMSO and 90 w/v% FBS was used as a control. Then, after 3 days of storage in LN2 tank and thawing, viable cells were counted using Trypan blue exclusion assay.
  • Example 4 It was cryopreserved according to Example 4, thawed, cultured for 3 days, and then the expression level of the stem cell pluripotency marker gene was examined.
  • Cells that were not cryopreserved were used as a negative control (NC), and cells cryopreserved with 10 w/v% DMSO and 90 w/v% FBS were used as a positive control (PC).
  • NC negative control
  • PC positive control
  • cDNA was synthesized from 1 ⁇ g total RNA using SuperscriptII reverse transcriptase (Invitrogen) and oligo dT.
  • Real-time PCR was performed by the SYBR green method using the synthesized cDNA and the primers of the stem cell pluripotency marker genes in Table 2 below.
  • SYBR Green I is an interchelator that exhibits fluorescence by binding to double-stranded DNA. The interchelator binds to the double-stranded DNA synthesized by the PCR reaction and emits fluorescence, and the amount of amplification product can be measured by detecting the fluorescence intensity.
  • the expression level of the stem cell pluripotency marker gene was not different from that of the negative control group and the positive control group. It was confirmed that the composition can be preserved without affecting the expression of the pluripotency marker gene of adipose-derived stem cells.
  • Example 5 Using the cDNA prepared in Example 5, the expression level of the specific marker gene of adipose-derived stem cells was investigated in the same manner.
  • CD73 known as a specific marker gene for adipose-derived stem cells.
  • the expression levels of CD90 and CD105 were confirmed using real-time PCR, and the primer sequences of each factor are shown in Table 3 below.
  • Primer sequences of specific marker genes for adipose-derived stem cells CD73-Forward AAGTGTCGAGTGCCCCAGTTA CD73-Reverse TGATCCGACCTTCAACTGCT CD90-Forward AGTACGAGTTCAGCCTGACC CD90-Reverse TCTGAGCACTGTGACGTTCT CD105-Forward TCCATTGTGACCTTCAGCCT CD105-Reverse CTTGGATGCCTGGAGAGTCA
  • the expression level of the adipose-derived stem cell-specific marker gene was not different from that of the negative control group and the positive control group. It was confirmed that the composition can be stably cryopreserved while preserving the unique characteristics of adipose-derived stem cells.

Landscapes

  • Engineering & Computer Science (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Biotechnology (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biomedical Technology (AREA)
  • Genetics & Genomics (AREA)
  • General Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Virology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Medicinal Chemistry (AREA)
  • Cell Biology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

Disclosed are: a cell cryopreservation composition using pectin and alanine as cryoprotectants, which has effects of high cell viability after thawing and maintenance of cell characteristics, and has an excellent cryopreservation effect without using serum derived from humans or animals such as cows, or serum-derived components; and a cell cryopreservation method using the composition.

Description

펙틴과 알라닌을 이용한 세포 동결 보존용 조성물 및 이를 이용한 세포 동결 보존 방법Cell cryopreservation composition using pectin and alanine and cell cryopreservation method using the same
본 발명은 펙틴과 알라닌을 이용한 세포 동결 보존용 조성물 및 이를 이용한 세포 동결 보존 방법에 관한 것이다.The present invention relates to a cell cryopreservation composition using pectin and alanine and a cell cryopreservation method using the same.
자가, 동종, 이종 유래의 살아있는 세포를 세포치료제 등으로 이용하기 위해서는 지방, 골수, 제대혈, 말초혈 등 조직으로부터 세포를 분리하여 체외에서 배양, 증식하고 물리적, 화학적, 생물학적 방법으로 조작하게 되는데, 이러한 세포치료제는 생존기간이 짧은 살아있는 세포를 원료로 한다. 이러한 세포를 보존하기 위해 장기간에 걸쳐 계대배양할 수 있는데, 이럴 경우 유전적인 변이에 의해 세포 특성이 변화할 수 있고, 오염으로 인한 세포 손실의 위험성이 있다. 따라서 이러한 유전적 변이와 오염에 따른 세포 손실을 최소화하고 세포 특성을 안정적으로 유지하기 위한 보존 방법이 필요하다. In order to use autologous, allogeneic, or xenogeneic living cells as cell therapy products, cells are separated from tissues such as fat, bone marrow, umbilical cord blood, and peripheral blood, cultured and proliferated in vitro, and manipulated by physical, chemical, and biological methods. Cell therapy products are made from living cells with a short survival period. In order to preserve these cells, they can be subcultured over a long period of time. In this case, cell characteristics may change due to genetic mutation, and there is a risk of cell loss due to contamination. Therefore, there is a need for a preservation method for minimizing cell loss due to genetic mutation and contamination and stably maintaining cell characteristics.
특히 줄기세포는 조직이나 기관의 분화된 세포들 사이에 존재하는 미분화 세포로서, 미분화 상태를 유지하며 무한 증식할 수 있는 자가 재생능(self-renewal)과, 일정 조건이 주어질 경우 신체 내의 모든 조직세포로 분화할 수 있는 다분화 능(differentiation to specific tissue)을 가지기 때문에 세포치료제로 퇴행성 질환이나 외상에 의해 손상된 조직 등의 치료에 이용될 수 있어 의학적 활용도가 매우 높다고 알려져 있다. 이러한 줄기세포를 세포치료제로 이용하기 위해서는 세포 생존율이나 미분화성을 보장하는 세포 동결 보존 방법의 확립이 필수적이다. In particular, stem cells are undifferentiated cells that exist between differentiated cells in tissues or organs, and have self-renewal ability to proliferate indefinitely while maintaining an undifferentiated state, and all tissue cells in the body under certain conditions. Since it has differentiation to specific tissue, it can be used as a cell therapy product to treat degenerative diseases or tissues damaged by trauma, so it is known that it has very high medical utilization. In order to use these stem cells as a cell therapy agent, it is essential to establish a cell cryopreservation method that guarantees cell viability or undifferentiation.
세포의 동결 보존 방법은 완만 동결법과 급속 동결법으로 구별되는데, 완만 동결법은 세포를 액체 질소와 함께 1분에 1~2℃ 정도로 온도를 완만하게 내려 -196℃ 정도의 극히 낮은 온도에서 보존하는 방법으로, 세포 동결이나 해동에 따른 세포막이나 세포질 단백질의 변성을 방지하기 위하여, 글리세롤, DMSO(dimethyl sulphoxide), 케라틴이나 젤라틴 가수분해물, 아세트아미드, 프로필렌 글리콜, 폴리에틸렌 글리콜, 동물이나 인간 유래 혈청이나 혈청 알부민 등의 동결 보존제(cryoprotectant)가 사용된다. 이러한 완만한 냉각과 동결 보존제 사용에 의해 세포 내부와 외부의 얼음 결정의 생성이 저해되어 세포 손상을 막을 수 있다. Cell cryopreservation methods are divided into slow freezing and rapid freezing. Slow freezing is a method of preserving cells at an extremely low temperature of about -196 ° C by gently lowering the temperature by 1 to 2 ° C per minute with liquid nitrogen. , To prevent cell membrane or cytoplasmic protein denaturation due to cell freezing or thawing, glycerol, DMSO (dimethyl sulphoxide), keratin or gelatin hydrolysate, acetamide, propylene glycol, polyethylene glycol, animal or human-derived serum or serum albumin, etc. of cryoprotectants are used. The generation of ice crystals inside and outside the cells is inhibited by the gentle cooling and use of cryopreservatives, thereby preventing cell damage.
급속 동결법(즉 유리화 동결법)은 급속 동결에 의해 세포 내부와 외부의 얼음 결정 생성을 억제하여 동결하는 방법으로, DMSO, 아세트아미드, 프로필렌 글리콜, 폴리에틸렌 글리콜 등의 동결 보존제가 사용된다. 급속 동결법에 의하여 마우스 초기 배아의 동결 보존이나, 완만 동결법으로는 동결 보존이 어려웠던 소 배아나 돼지 배아의 동결 보존이 가능해졌다. The rapid freezing method (ie, vitrification freezing method) is a method of freezing by suppressing the formation of ice crystals inside and outside the cells by rapid freezing, and cryopreservatives such as DMSO, acetamide, propylene glycol, and polyethylene glycol are used. The rapid freezing method enables the cryopreservation of early mouse embryos and the cryopreservation of cow embryos and pig embryos, which were difficult to cryopreservate with the slow freezing method.
그러나 동결 보존제로 사용되어 온 글리세롤은 동결과 해동 과정에서 완충 기능이 완전하지 않아 종종 세포 손상을 일으키는 문제점이 있고, DMSO는 고가이고 또 세포의 형질을 변화시킬 가능성을 가지고 있다. 또 우태아혈청(FBS: fetal bovine serum) 등의 동물 유래 혈청이나 인간 유래 혈청(human serum) 등은 바이러스, 프리온 등의 감염을 일으킬 수 있고 면역반응을 유발할 수 있는 위험성이 있으며, 이러한 이유로 인체 투여를 위한 세포치료제 등의 보존에는 적합하지 않은 문제가 있다.However, glycerol, which has been used as a cryopreservative, does not have a complete buffering function during freezing and thawing, which often causes cell damage, and DMSO is expensive and has the potential to change the character of cells. In addition, animal-derived serum such as fetal bovine serum (FBS) or human-derived serum can cause infections such as viruses and prions, and there is a risk of inducing an immune response, and for this reason, human administration There is a problem that is not suitable for preservation of cell therapy products for.
따라서 세포의 보존 효과가 우수하면서도, 동결과 해동에 의한 세포 손상을 최소화할 수 있는 동결 보존제의 개발이 여전히 요구된다고 할 수 있다. Therefore, it can be said that the development of cryopreservatives capable of minimizing cell damage due to freezing and thawing while having excellent cell preservation effects is still required.
본 발명은 펙틴과 알라닌의 동결 보존제로서의 효과를 개시한다.The present invention discloses the effect of pectin and alanine as cryopreservatives.
본 발명의 목적은 펙틴과 알라닌을 이용한 세포 동결 보존용 조성물 및 이를 이용한 세포 동결 보존 방법을 제공하는 데 있다.An object of the present invention is to provide a cell cryopreservation composition using pectin and alanine and a cell cryopreservation method using the same.
본 발명의 다른 목적이나 구체적인 목적은 이하에서 제시될 것이다.Other objects or specific objects of the present invention will be presented below.
본 발명은 아래의 실시예에서 확인되는 바와 같이, 펙틴과 알라닌이 인간 지방 유래 줄기세포에 대해 세포독성이 없고, 동결 보존제로 펙틴과 알라닌을 함유한 DPBS(Dulbecco's phosphate-buffered saline) 보존액을 이용하여 지방 유래 줄기세포를 동결시키고 3일간 동결보존 후 해동시켜 세포 생존율을 살펴봤을 때 기존 동결 보존제인 10 w/v% DMSO와 90 w/v% FBS를 사용한 대조군과 동등한 수준의 세포 생존율을 보였으며, 해동 후 배양한 후에도 기존 동결 보존제를 사용한 대조군과 같은 수준의 밀도로 부착, 배양되었을 뿐만 아니라, 줄기세포의 전분화능 표지 유전자로 알려진 Sox2, Oct4, c-myc, Klf4, Nanog의 발현량을 측정하였을 때와 지방 유래 줄기세포의 특이적 표지 유전자로 알려진 CD73. CD90, CD105의 발현량을 측정하였을 때에도, 동결 보존제를 사용하지 않은 대조군이나 기존 동결 보존제를 사용한 대조군과 같은 수준임을 확인함으로써 완성된 것이다. As confirmed in the following examples, the present invention uses a DPBS (Dulbecco's phosphate-buffered saline) preservation solution containing pectin and alanine as cryopreservatives, in which pectin and alanine are not cytotoxic to human adipose-derived stem cells. When adipose-derived stem cells were frozen and thawed after 3 days of cryopreservation, cell viability was examined, and the cell viability was equivalent to that of the control group using 10 w/v% DMSO and 90 w/v% FBS, which are conventional cryopreservatives. After thawing and culturing, not only were they adhered and cultured at the same density as the control group using the conventional cryopreservation agent, but also the expression levels of Sox2, Oct4, c-myc, Klf4, and Nanog, known as marker genes for pluripotency of stem cells, were measured. CD73, known as a specific marker gene for stain and fat-derived stem cells. Even when the expression levels of CD90 and CD105 were measured, it was completed by confirming that the levels were the same as those of the control group without cryopreservation or the control group using existing cryopreservation agents.
본 발명은 전술한 바의 실험 결과에 기초하여 제공되는 것으로, 본 발명의 세포 동결 보존용 조성물은 펙틴과 알라닌을 포함함을 특징으로 한다.The present invention is provided based on the above experimental results, and the composition for cryopreservation of cells of the present invention is characterized in that it contains pectin and alanine.
펙틴(pectin)은 쌍자엽 식물의 세포벽 구성성분으로 겔화제, 증점제, 안정제, 유화제 등으로서 식품 분야에 널리 이용되어 온 물질로서, 갈락투론산(galacturonic acid) 단위가 α-1, 4 결합으로 연결된 기본 구조가 주요 부분을 이루며 200,000~400,000의 분자량을 갖는다. 펙틴은 일반적으로 메틸화 정도(Degree of Methylation, DM)에 따라서 고메톡시 펙틴(DM>50)과 저메톡시 펙틴(DM<50)으로 분류되는데, 식물 재료로부터 고온 산성 조건하에서 추출되는 펙틴의 다수는 고메톡시 펙틴이고, 저메톡시 펙틴은 산, 알칼리, 효소, 암모니아 등을 더 사용하여 탈메틸화시켜 얻는다. 본 발명에서 펙틴은 그것이 얻어지는 식물 재료나, 분자량의 크기(바람직하게는 100 내지 300g/mol)나, 메틸화 정도에 상관없이 임의의 것을 사용할 수 있다. Pectin is a component of the cell wall of dicotyledonous plants and has been widely used in the food field as a gelling agent, thickener, stabilizer, emulsifier, etc. The structure is the main part and has a molecular weight of 200,000 to 400,000. Pectin is generally classified into high methoxy pectin (DM>50) and low methoxy pectin (DM<50) according to the degree of methylation (DM). It is toxy pectin, and low methoxy pectin is obtained by demethylation using acid, alkali, enzyme, ammonia, etc. further. In the present invention, any pectin may be used regardless of the plant material from which it is obtained, the size of the molecular weight (preferably 100 to 300 g/mol), or the degree of methylation.
본 발명에서 알라닌은 엘-알라닌(L-alanine)일 수 있다.In the present invention, alanine may be L-alanine.
본 발명의 조성물에서, 펙틴과 알라닌은 충분한 동결보존 효과(즉 해동 후의 세포 생존율 향상, 세포 특성 유지 효과)를 나타내기 위하여 펙틴 1 내지 30μM, 알라닌 3 내지 90μM , 특히 펙틴 5 내지 15μM, 알라닌 20 내지 40μM의 범위로 포함될 수 있다. In the composition of the present invention, pectin and alanine are 1 to 30 μM of pectin, 3 to 90 μM of alanine, especially 5 to 15 μM of pectin, and 20 to 10 μM of alanine in order to exhibit sufficient cryopreservation effects (i.e., cell viability improvement after thawing, cell property maintenance effect). It can be included in the range of 40 μM.
본 발명의 동결 보존용 조성물은 보존액을 포함할 수 있다. The composition for cryopreservation of the present invention may contain a preservative solution.
보존액은 세포 배양 배지이거나, 완충액이거나 등장액일 수 있다.The preservative solution may be a cell culture medium, buffered or isotonic solution.
세포 배양 배지는 일반적으로 탄소원, 질소원, 미량원소를 포함한다.Cell culture media generally contain a carbon source, a nitrogen source, and trace elements.
탄소원은 바람직하게는 단당류, 이당류 등이 사용될 수 있다. 구체적으로 글루코오스, 프럭토오스, 만노오스, 갈락토오스, 리보오스, 소르보오스, 리불로오스, 락토오스, 말토오스, 수크로오스, 라피노오스 또는 이들의 1종 이상의 혼합물이 사용될 수 있다.As the carbon source, monosaccharides, disaccharides, and the like may be preferably used. Specifically, glucose, fructose, mannose, galactose, ribose, sorbose, ribulose, lactose, maltose, sucrose, raffinose, or a mixture of one or more thereof may be used.
질소원은 질소원으로서는 통상적으로 무기 질소 화합물, 유기 질소 화합물 또는 이들 화합물들을 포함하는 복합 화합물일 수 있다. 예컨대 무기 질소 화합물로서는 암모니아, 암모늄염(예컨대, 암모늄 술페이트, 암모늄 클로라이드, 암모늄 포스페이트, 암모늄 카르보네이트, 암모늄 니트레이트 등), 니트레이트 등을 들 수 있고, 유기 질소 화합물로서는 우레아, 아미노산 등을 들 수 있다.The nitrogen source may be an inorganic nitrogen compound, an organic nitrogen compound, or a complex compound containing these compounds. Examples of inorganic nitrogen compounds include ammonia, ammonium salts (eg, ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate, ammonium nitrate, etc.), nitrate, and the like, and examples of organic nitrogen compounds include urea, amino acids, and the like. can
미량원소로서는 칼슘, 마그네슘, 나트륨, 코발트, 몰리브덴, 칼륨, 망간, 아연, 구리, 철, 인, 황 등을 포함하며 이들 미량원소는 염 화합물 형태로 배지에 첨가될 수 있다.Examples of trace elements include calcium, magnesium, sodium, cobalt, molybdenum, potassium, manganese, zinc, copper, iron, phosphorus, sulfur, and the like, and these trace elements may be added to the medium in the form of salt compounds.
세포 배양 배지는 또한 비타민, 생장인자 등을 추가 포함할 수 있으며, 특히 무혈청 배지의 사용을 위해서는 혈청 대체 물질 예컨대 인슐린, 트렌스페린(transferrin), 셀레늄(selenium)을 포함할 수도 있다.The cell culture medium may also further contain vitamins, growth factors, and the like, and in particular, serum substitutes such as insulin, transferrin, and selenium may be included for the use of a serum-free medium.
이러한 세포배양용 배지는 직접 제조하여 사용하거나 상업적으로 시판되는 것을 사용할 수 있다. 상업적으로 시판되는 배지는 예컨대, DMEM (Dulbecco's Modified Eagle's Medium), MEM (Minimal Essential Medium), BME (Basal Medium Eagle), RPMI 1640, F-10, F-12, DMEM/F12, MEM-α (Minimal Essential Medium-α), G-MEM (Glasgow's Minimal Essential Medium), IMDM (Iscove's Modified Dulbecco's Medium), MacCoy's 5A 배지, AmnioMax complete 배지, AminoMaxⅡ complete 배지, EBM (Endothelial Basal Medium) 배지, Chang's Medium, MesenCult-XF, DMEM/HG (Dulbecco's Modified Eagle's Medium high glucose)배지, 및 MCDB+DMEM/LG (MCDB +Dulbecco's Modified Eagle's Medium low glucose) 배지 등일 수 있다. These cell culture media can be prepared and used directly or commercially available ones can be used. Commercially available media include, for example, DMEM (Dulbecco's Modified Eagle's Medium), MEM (Minimal Essential Medium), BME (Basal Medium Eagle), RPMI 1640, F-10, F-12, DMEM/F12, MEM-α (Minimal Essential Medium-α), G-MEM (Glasgow's Minimal Essential Medium), IMDM (Iscove's Modified Dulbecco's Medium), MacCoy's 5A badge, AmnioMax complete badge, AminoMaxⅡ complete badge, EBM (Endothelial Basal Medium) badge, Chang's Medium, MesenCult-XF , DMEM/HG (Dulbecco's Modified Eagle's Medium high glucose) medium, and MCDB+DMEM/LG (MCDB + Dulbecco's Modified Eagle's Medium low glucose) medium.
보존액은 완충액일 수도 있는데, 완충액은 시트레이트, 포스페이트, 숙시네이트, 타르트레이트, 푸마레이트, 글루코네이트, 옥살레이트, 락테이트, 아세테이트, 히스티딘, 트리스 등을 완충제로 포함하는 생리 식염수(saline solution)일 수 있다. 특히 인산 완충 생리 식염수(Phosphate Buffered Saline, PBS), 트리스 완충 생리 식염수(Tris Buffered Saline, TBS), HEPES 완충 생리 식염수(HEPES Buffered Saline), DPBS(Dulbecco's phosphate-buffered saline) 등일 수 있다.The preservative solution may be a buffer solution, which is a physiological saline solution containing citrate, phosphate, succinate, tartrate, fumarate, gluconate, oxalate, lactate, acetate, histidine, tris, etc. as a buffer. can In particular, it may be Phosphate Buffered Saline (PBS), Tris Buffered Saline (TBS), HEPES Buffered Saline, or Dulbecco's phosphate-buffered saline (DPBS).
보존액은 또한 염화나트륨, 염화칼륨, 붕산, 붕산나트륨, 만니톨, 글리세린, 프로필렌글리콜, 폴리에틸렌, 글리콜, 말토스, 자당, 에리쓰리톨, 아라비톨, 자일리톨, 소르비톨 트리할로즈, 포도당 등을 등장화제로 포함하는 등장액일 수도 있다. 이러한 등장액은 링거액, 젖산 링거액, 아세트산 링거액, 중탄산 링거액, 5% 글루코스 수용액일 수 있으며, 직접 제조하여 사용하거나 상업적으로 시판되는 것을 구입하여 사용할 수 있다.The preservative solution also contains sodium chloride, potassium chloride, boric acid, sodium borate, mannitol, glycerin, propylene glycol, polyethylene, glycol, maltose, sucrose, erythritol, arabitol, xylitol, sorbitol trihalose, glucose, etc. as an isotonic agent. It may be an isotonic solution. Such an isotonic solution may be Ringer's solution, lactate Ringer's solution, acetate Ringer's solution, bicarbonate Ringer's solution, or 5% glucose aqueous solution, and may be directly prepared and used or purchased commercially available.
본 발명의 세포 동결 보존용 조성물은 기존 공지된 동결 보존제 예컨대 글리세롤, 케라틴이나 젤라틴 가수분해물, 아세트아미드, DMSO, 에틸렌 글리콜, 프로필렌 글리콜, 폴리에틸렌 글리콜, 세리신, 이소말토올리고당 등을 추가로 포함할 수도 있고, 또한 EDTA, EGTA, 구연산, 살리실레이트 등의 킬레이트제, 용해 보조제, 보존제, 산화 방지제, 프롤린, 글루타민 등의 아미노산을 추가로 포함할 수도 있다.The cell cryopreservation composition of the present invention may further include conventionally known cryopreservatives such as glycerol, keratin or gelatin hydrolysate, acetamide, DMSO, ethylene glycol, propylene glycol, polyethylene glycol, sericin, isomaltooligosaccharide, etc. EDTA, EGTA, citric acid, chelating agents such as salicylate, dissolution aids, preservatives, antioxidants, and amino acids such as proline and glutamine may be further included.
본 발명의 조성물에서, 동결 보존제인 펙틴과 알라닌은 우태아혈청(FBS)을 사용한 경우에 비해 세포 생존율이나 줄기세포의 전분화능 등에 있어 동등한 수준의 효과를 가진다는 점에서, 소 등의 동물 유래 또는 인간 유래의 혈청 또는 혈청 유래 성분(예컨대 알부민)을 포함하지 않을 수 있다. In the composition of the present invention, pectin and alanine, which are cryopreservatives, have an equivalent level of effect on cell viability or pluripotency of stem cells compared to the case of using fetal bovine serum (FBS). It may not contain human-derived serum or serum-derived components (eg albumin).
본 발명의 동결 보존용 조성물은 임의의 동물세포, 특히 포유동물세포의 동결 보존을 위하여 사용될 수 있다.The composition for cryopreservation of the present invention can be used for cryopreservation of any animal cells, particularly mammalian cells.
이러한 동물세포는 먼저 줄기세포일 수 있다. 줄기세포는 배아줄기세포, 성체줄기세포, 역분화줄기세포일 수 있다. 성체줄기세포는 지방, 골수, 제대혈, 말초혈액을 포함하는 혈액이나 조직의 기저에서 기원되는 줄기세포일 수 있다. 줄기세포는 특히 지방유래 줄기세포(adipose-derived stem cell, ADSC)일 수 있다. 지방 유래 줄기세포는 자가 재생능(self-renewal)을 가지고 피부,연골,뼈 등 다양한 계열의 세포로 분화가 가능할 뿐만 아니라 골수 유래 줄기세포나 제대혈 유래 줄기세포에 비해 채취하기가 쉽고 대량 배양이 가능하면서도 줄기세포 함유량이 많기 때문에 세포치료제서의 활용도가 높다.These animal cells may first be stem cells. Stem cells may be embryonic stem cells, adult stem cells, and dedifferentiated stem cells. Adult stem cells may be stem cells originating from blood or tissues including fat, bone marrow, umbilical cord blood, and peripheral blood. Stem cells may in particular be adipose-derived stem cells (ADSC). Adipose-derived stem cells have self-renewal ability and can be differentiated into various types of cells such as skin, cartilage, and bone, and are easier to collect and mass-cultivate than bone marrow-derived stem cells or cord blood-derived stem cells. However, because of its high content of stem cells, its utilization in cell therapy is high.
줄기세포는 자가재생능과 분화능을 갖는 것이면, 그 종류 및 유래는 특별한 제한이 없다. 예컨대 포유동물, 인간, 원숭이, 돼지, 말, 소, 양, 개, 고양이, 마우스 또는 토끼 등으로부터 유래한 것일 수 있다. 상기 줄기세포는 분리된 탯줄, 태반, 지방, 골수, 제대혈 또는 양수로부터 유래한 것일 수 있다. 줄기세포의 분리, 배양, 증식 방법이나 배아줄기세포이 제조 방법은 당업계에 다양한 방법이 공지되어 있으며, 예컨대 문헌[Curr Opin Chem Eng. 2013, 2(1): 3-7], 문헌[Stem Cell Research & Therapy, 2014, 5:51], 문헌[Stem Cell Research & Therapy, 2019, 10:68], WO96/22362, WO02/101057, US5,843,780, US6,200,806, US6,280,718 등을 참조할 수 있다.As long as stem cells have self-renewal and differentiation capabilities, the type and origin are not particularly limited. For example, it may be from a mammal, human, monkey, pig, horse, cow, sheep, dog, cat, mouse or rabbit. The stem cells may be derived from separated umbilical cord, placenta, fat, bone marrow, umbilical cord blood or amniotic fluid. Various methods are known in the art for isolating, culturing, and proliferating stem cells or preparing embryonic stem cells, such as those described in [Curr Opin Chem Eng. 2013, 2(1): 3-7], Stem Cell Research & Therapy, 2014, 5:51, Stem Cell Research & Therapy, 2019, 10:68, WO96/22362, WO02/101057, See US 5,843,780, US 6,200,806, US 6,280,718 and the like.
본 발명의 동결 보존용 조성물이 사용될 수 있는 동물세포는 줄기세포 이외에도 제I형 당뇨병 환자에게 정맥내 투여되는 췌도세포(islet cell), 암 치료용으로 투여되는 수지상 세포(dendritic cell), 자연살해세포(NK cell), T 세포(특히 세포독성 T 세포, cytotoxic T lymphocyte), CAR-T 세포, CAR-NK 세포 등일 수 있의며, 생물 의약품 생산에 이용되는 CHO 세포, HEK 세포, MCF-7 세포, MDCK 세포, Vero 세포 등일 수 있다. In addition to stem cells, animal cells that can be used in the composition for cryopreservation of the present invention are islet cells administered intravenously to patients with type I diabetes, dendritic cells administered for cancer treatment, and natural killer cells. (NK cell), T cell (particularly cytotoxic T cell, cytotoxic T lymphocyte), CAR-T cell, CAR-NK cell, etc. CHO cell, HEK cell, MCF-7 cell used in biopharmaceutical production , MDCK cells, Vero cells, and the like.
또 동물세포는 생체로부터 분리된 것이거나 시험관내(In vitro)에서 계대배양된 것일 수 있다.In addition, animal cells may be isolated from living organisms or subcultured in vitro.
또 동물세포는 세포가 서로 응집하지 않은 상태의 단일 세포 상태인 것이 바람직할 수 있다. 단일 세포 상태의 포유동물 세포는 인비트로에서 배양한 포유동물 세포를 트립신/EDTA 등으로 효소처리한 후, 피펫팅, 탭핑 등의 당업계에서 주지된 방법에 의해 세포를 현탁함으로써 얻어질 수 있다. 단일 세포 상태의 세포 비율은 세포를 PBS에 분산하고, 이를 현미경으로 관찰하여, 무작위로 선택된 일정 수의 세포에 대해 응집 유무를 조사함으로써 결정할 수 있다.In addition, it may be preferable that the animal cells are in a single cell state in which the cells do not aggregate with each other. Mammalian cells in a single cell state can be obtained by enzymatically treating mammalian cells cultured in vitro with trypsin/EDTA, and then suspending the cells by a method known in the art, such as pipetting or tapping. The cell ratio in a single cell state can be determined by dispersing the cells in PBS, observing them under a microscope, and examining the presence or absence of aggregation of a certain number of randomly selected cells.
본 발명은 다른 측면에 있어서, 세포 동결 보존 방법을 제공한다.In another aspect, the present invention provides a cell cryopreservation method.
본 발명의 세포 동결 보존 방법은 펙틴과 알라닌을 포함하는 보존액을 제조하는 단계, 상기 보존액에 세포를 현탁하는 단계, 세포가 현탁된 보존액을 동결하고 보존하는 단계를 포함한다.The cell cryopreservation method of the present invention includes preparing a preservative solution containing pectin and alanine, suspending cells in the preservative solution, and freezing and preserving the preservative solution in which the cells are suspended.
본 발명의 방법에서 펙틴은 전술한 바와 같이 고메톡시 펙틴이나 저메톡시 메틴일 수 있으며, 알라닌은 엘-알라닌(L-alanine)일 수 있다.In the method of the present invention, pectin may be high methoxy pectin or low methoxy methine as described above, and alanine may be L-alanine.
또 본 발명의 방법에서, 상기 보존액은 전술한 바와 같이 세포 배양용 배지, 완충액, 등장액일 수 있다.In addition, in the method of the present invention, the preservation solution may be a cell culture medium, buffer, or isotonic solution as described above.
또 본 발명의 방법에서, 상기 보존액에는 소 등의 동물 유래 또는 인간 유래의 혈청 또는 혈청 유래 성분(예컨대 알부민)이 포함되지 않을 수 있다.In addition, in the method of the present invention, the preservation solution may not contain animal-derived or human-derived serum or serum-derived components (eg, albumin).
또 본 발명의 방법에서, 상기 세포는 줄기세포, 췌도세포, 수지상세포, 자연살해세포, T 세포, CAR-T 세포, CAR-NK 세포, CHO 세포, HEK 세포, MCF-7 세포, MDCK 세포, Vero 세포 등일 수 있다. In addition, in the method of the present invention, the cells are stem cells, pancreatic islet cells, dendritic cells, natural killer cells, T cells, CAR-T cells, CAR-NK cells, CHO cells, HEK cells, MCF-7 cells, MDCK cells, Vero cells or the like.
또 본 발명의 방법에서, 세포 현탁은 보존액 1ml에 대하여 1 × 103 세포 내지 약 1 × 109 세포 범위 특히 1 × 105 내지 1 × 108 세포 범위로 첨가되어 이루어질 수 있다. In addition, in the method of the present invention, the cell suspension is in the range of 1 × 10 3 cells to about 1 × 10 9 cells, particularly 1 × 10 5 to 1 × 10 8 cells, based on 1 ml of the preservation solution. It can be made by adding to the range.
또 본 발명의 방법에서, 동결은 당업계의 공지된 방법 예컨대 완만 동결법, 급속 동결법(유리화 동결법)을 통하여 수행될 수 있다. 완만 동결법은 일반적으로 세포가 현탁된 보존액을 저온 프리저나 초저온 프리저(통상적으로 -20℃ ~ -150℃ 범위)에서 12시간 내지 24시간 동안 온도를 일정하게 천천히 떨어뜨려 동결시키는 방법이다. 급속 동결법은 일반적으로 세포가 현탁된 보존액을 CRF(controlled rate freezing)와 같은 장비를 이용하여 통상적으로 -150℃ ~ -196℃ 온도 범위의 액체 질소를 이용하여 급속 동결하는 방법이다. 세포를 동결 보존하고, 그 후 융해했을 때의 세포 생존율은 세포의 종류에 따라 상이한 경우가 있다. 따라서 동결 보존 대상 세포에 따라 융해 후의 세포 생존율이 높아지도록 적절한 동결 보존 방법을 채택하는 것이 필요할 수 있다. In addition, in the method of the present invention, freezing may be performed through a method known in the art, such as a slow freezing method or a rapid freezing method (vitrification freezing method). Slow freezing is generally a method of freezing a preservative solution in which cells are suspended in a low-temperature freezer or an ultra-low-temperature freezer (usually in the range of -20 ° C to -150 ° C) by slowly dropping the temperature at a constant level for 12 to 24 hours. The rapid freezing method is generally a method of rapidly freezing a preservative solution in which cells are suspended using equipment such as CRF (controlled rate freezing), usually using liquid nitrogen in a temperature range of -150 ° C to -196 ° C. The cell viability when cells are cryopreserved and then thawed may differ depending on the cell type. Therefore, it may be necessary to adopt an appropriate cryopreservation method to increase the cell viability after thawing depending on the cells to be cryopreserved.
본 발명의 방법에서, 상기 보존은 액체 질소 탱크 또는 초저온 냉장고(deep freezer)에서 수행되는 것일 수 있다. 상기 보관 온도는 -100℃ 내지 약 -300℃, 특히 -150℃ 내지 약 -220℃, 특히 바람직하게는 -150℃ ~ -196℃ 온도 범위일 수 있다. 보존 기간은 상기 보존은 연구용, 의약품 개발용 등 세포의 사용 용도에 따라 1일 또는 3일 이상 특히 1개월, 특히 바람직하게는 3개월이나 6개월 또는 1년 이상 장기간일 수 있다. In the method of the present invention, the preservation may be performed in a liquid nitrogen tank or a deep freezer. The storage temperature may be -100 ° C to about -300 ° C, particularly -150 ° C to about -220 ° C, particularly preferably -150 ° C to -196 ° C. The preservation period may be 1 day or 3 days or more, particularly 1 month, particularly preferably 3 months, 6 months or 1 year or more depending on the use of the cells, such as for research or drug development.
본 발명에 따르면, 펙틴과 알라닌을 이용한 세포 동결 보존용 조성물과 이를 이용한 세포 동결 보존 방법을 제공할 수 있다. 본 발명의 세포 동결 보존용 조성물과 이를 이용한 세포 동결 보존 방법은 기존 동결 보존제인 DMSO와 FBS를 사용한 경우와 유사하게 해동 후 세포 생존율이 높고 세포 특성이 유지되는 효과를 가지며 또한 소 등의 동물 유래 또는 인간 유래의 혈청 또는 혈청 유래 성분을 사용하지 않고도 우수한 동결 보존 효과를 가진다. According to the present invention, a cell cryopreservation composition using pectin and alanine and a cell cryopreservation method using the same can be provided. The cell cryopreservation composition of the present invention and the cell cryopreservation method using the same have an effect of maintaining high cell viability after thawing and maintaining cell characteristics, similar to the case of using DMSO and FBS, which are conventional cryopreservatives, and also derived from animals such as cattle or It has an excellent cryopreservation effect without using human-derived serum or serum-derived components.
도 1은 본 발명의 동결 보존제의 펙틴(Pectin)과 알라닌 (L-alanine)에 의한 지방유래 줄기세포(ADSC; Adipose-derived stem cell)에서의 독성을 확인한 결과이다.1 is a result of confirming the toxicity of the cryopreservative of the present invention in adipose-derived stem cells (ADSC) by pectin and alanine.
도 2는 본 발명의 동결 보존제의 동결 보존 효과를 확인한 것으로, 상기 동결 보존제를 이용하여 3일간 지방유래 줄기세포를 동결보존한 뒤 해동하여 생존율을 측정하여 동결 보존 효과를 확인한 결과이다.FIG. 2 confirms the cryopreservation effect of the cryopreservative of the present invention. Adipose-derived stem cells are cryopreserved for 3 days using the cryopreservative and then thawed to measure the viability to confirm the cryopreservation effect.
도 3은 해동 후 세포를 배양접시에 배양하여 3일간 배양한 세포를 현미경으로 상, 하부 전체 4x의 배율로 관찰한 사진이다.Figure 3 is a photograph of the cells cultured in a culture dish after thawing and observing the cells cultured for 3 days under a microscope at a magnification of 4x over the entire upper and lower parts.
도 4는 본 발명의 동결 보존제를 이용하여 동결 보존한 뒤 해동 한 줄기세포의 전분화능 표지 유전자의 발현 정도를 확인한 결과이다.4 is a result of confirming the expression level of pluripotency marker genes in stem cells thawed after cryopreservation using the cryopreservative of the present invention.
도 5는 본 발명의 동결 보존제를 이용하여 동결 보존한 뒤 해동한 세포의 지방유래 줄기세포 특이적 표지 유전자의 발현 정도를 확인한 결과이다.5 is a result of confirming the expression level of adipose-derived stem cell-specific marker genes in cells cryopreserved using the cryopreservative of the present invention and then thawed.
이하 본 발명을 실시예를 참조하여 설명한다. 그러나 본 발명의 범위가 이러한 실시예에 한정되는 것은 아니다.Hereinafter, the present invention will be described with reference to examples. However, the scope of the present invention is not limited to these examples.
<실시예 1> 지방유래 줄기세포의 분리 및 배양<Example 1> Isolation and culture of adipose-derived stem cells
본 실시예에서 사용한 인간 지방조직은 고마바이오텍(주)(Seoul, Korea)에서 구입하였으며, 지방조직에서 줄기세포를 분리하는 방법은 Zuk 등의 방법(Mol Biol Cell. 2002 Dec; 13(12): 4279-4295)을 참고 및 변형하여 사용하였다. 지방조직에서 줄기세포를 분리한 후, 페니실린(100 U/ml), 스트렙토마이신 (100 ug/ml) 및 10% 열비활성 혈청을 첨가한 DMEM(Dulbecco's Modified Eagle's Medium)에서 95% 공기, 5% CO2와 37℃ 조건으로 배양하였다. 배양 접시에 부착되어 세포가 자라면 0.25% 트립신/10mM EDTA를 이용하여 세포를 모으고, 1:3의 비율로 나눠 10% DMEM에서 유지하였다.The human adipose tissue used in this example was purchased from Goma Biotech Co., Ltd. (Seoul, Korea), and the method for isolating stem cells from adipose tissue was described by Zuk et al. (Mol Biol Cell. 2002 Dec; 13(12): 4279-4295) was used for reference and modification. After isolating stem cells from adipose tissue, penicillin (100 U/ml), streptomycin (100 ug/ml) and 10% heat-inactivated serum were added to DMEM (Dulbecco's Modified Eagle's Medium) in 95% air and 5% CO. 2 and cultured at 37 ℃ conditions. When the cells adhered to the culture dish and grew, the cells were collected using 0.25% trypsin/10 mM EDTA, divided at a ratio of 1:3, and maintained in 10% DMEM.
<실시예 2> 펙틴 및 알라닌의 지방유래 줄기세포에서의 독성<Example 2> Toxicity of pectin and alanine in adipose-derived stem cells
실험에서 사용된 펙틴(Pectin)(194.14g/mol) 및 알라닌 (L-alanine)은 시그마-알드리치(Sigma-aldrich) 사에서 구입하여 사용하였으며, 지방유래 줄기세포는 상기 실시예 1에서 분리한 세포를 이용하였다.Pectin (194.14 g/mol) and alanine (L-alanine) used in the experiment were purchased from Sigma-Aldrich and used, and adipose-derived stem cells were the cells isolated in Example 1 above. was used.
실험에서 사용된 시약인 CCK-8은 (주)동인바이오텍(Seoul, Korea)에서 구입하여 사용하였다. 지방유래 줄기세포를 96-well plates에 1 × 104 cells/well의 농도로 100 ul씩 처리한 다음 24시간 배양하였다. 세포가 바닥에 부착되면 용매인 DPBS(Dulbecco's phosphate-buffered saline)와 최대 100 μM까지 펙틴 및 알라닌을 최대 48시간동안 처리하였다. 이후 10 ul의 CCK-8 시약과 지방유래 줄기세포 배양 배지 100 ul의 조건으로 3시간동안 배양시킨 후 540 nm에서 스펙트로포토미터(spectrophotometer)로 흡광도를 측정하였다.The reagent used in the experiment, CCK-8, was purchased from Dongin Biotech Co., Ltd. (Seoul, Korea). Adipose-derived stem cells were treated in 96-well plates at a concentration of 1 × 10 4 cells/well by 100 ul, and then cultured for 24 hours. When the cells adhered to the bottom, they were treated with the solvent DPBS (Dulbecco's phosphate-buffered saline), pectin and alanine up to 100 μM for up to 48 hours. After culturing for 3 hours under conditions of 10 ul of CCK-8 reagent and 100 ul of adipose-derived stem cell culture medium, absorbance was measured at 540 nm with a spectrophotometer.
그 결과, 도 1의 A와 같이 펙틴 및 도 1의 B와 같이 알라닌은 최대 100 uM까지 독성을 보이지 않고 안전히 사용할 수 있는 것을 알 수 있었다.As a result, it was found that pectin as shown in A of FIG. 1 and alanine as shown in B of FIG. 1 can be safely used without showing toxicity up to 100 uM.
<실시예 3> 펙틴 및 알라닌을 포함하는 동결 보존용 조성물의 제조<Example 3> Preparation of composition for cryopreservation containing pectin and alanine
본 발명자들은 지방유래 줄기세포를 보존하는 새로운 조성물을 제조하기 위하여, 펙틴 및 알라닌을 유효성분으로 사용하였다. The present inventors used pectin and alanine as active ingredients in order to prepare a new composition for preserving adipose-derived stem cells.
먼저, 100 μM의 펙틴 용액을 제조하기 위하여, 먼저 펙틴 분말 1 g에 DPBS 51.5 mL을 혼합하고 80 ℃에서 1시간 가온하여 완전히 녹여 100 mM 펙틴 용액을 제조한 후 최종 농도가 100 μM에 이르도록 DPBS와 혼합하였다.First, in order to prepare a 100 μM pectin solution, first, 1 g of pectin powder was mixed with 51.5 mL of DPBS and heated at 80 ° C. for 1 hour to completely dissolve to prepare a 100 mM pectin solution, and then DPBS to a final concentration of 100 μM mixed with.
또, 300 μM의 펙틴 용액을 제조하기 위하여, 먼저 알라닌 분말 250 mg에 DPBS 28.06 mL을 혼합 및 Voltexing 하여 완전히 녹여 100 mM의 알라닌 용액을 제조한 후, 최종 농도가 300 μM에 이르도록 DPBS와 혼합하였다. 상기 제조 방법으로 제조한 발명 동결보존제의 조성은 하기 표 1과 같으며, 편의상 FNCP(Frombio New Cryoprotective)로 명명하여 표시하였다. In addition, in order to prepare a 300 μM pectin solution, first, 250 mg of alanine powder was mixed with 28.06 mL of DPBS by vortexing to completely dissolve to prepare a 100 mM alanine solution, and then mixed with DPBS to a final concentration of 300 μM. . The composition of the inventive cryoprotective prepared by the above manufacturing method is shown in Table 1 below, and was named FNCP (Frombio New Cryoprotective) for convenience.
동결 보존용 조성물의 조성Composition of composition for cryopreservation
FNCPFNCP
용액 농도solution concentration 용량 (1mL)Capacity (1mL) 최종 농도 final concentration
PectinPectin 100 μM100 µM 100 μL100 µL 10 μM10 µM
L-alanineL-alanine 300 μM300 µM 300 μL300 µL 30 μM30 µM
DPBSDPBS -- 700 μL700 µL --
<실시예 4> 발명 동결 보존용 조성물의 동결보존 효과 확인<Example 4> Confirmation of the cryopreservation effect of the composition for cryopreservation of the invention
본 실시예에서는 상기 실시예 3에서 제조된 동결 보존용 조성물의 지방유래 줄기세포에서의 동결보존 효과를 확인하였다.In this example, the cryopreservation effect of the composition for cryopreservation prepared in Example 3 on adipose-derived stem cells was confirmed.
75T Flask에 배양한 지방유래 줄기세포를 0.25% 트립신/10mM EDTA로 모은 다음, 1 × 106 cells/mL의 세포를 상기 표 1의 조성대로 제조한 동결 보존용 조성물에 현탁하여 Freezing Container를 이용, 동결시켰다. 또, 10 w/v% DMSO 및 90 w/v% FBS를 사용한 군을 Control로 사용하였다. 그 다음, 3일간 LN2 탱크에 보관하고 해동한 다음, 트리판 블루 익스클루젼 어세이(Trypan blue exclusion assay)를 이용하여 생존 세포를 계수하였다.Adipose-derived stem cells cultured in a 75T flask were collected with 0.25% trypsin/10 mM EDTA, and then the cells at 1 × 10 6 cells/mL were suspended in a composition for cryopreservation prepared according to the composition of Table 1 above, using a Freezing Container, Frozen. In addition, a group using 10 w/v% DMSO and 90 w/v% FBS was used as a control. Then, after 3 days of storage in LN2 tank and thawing, viable cells were counted using Trypan blue exclusion assay.
그 결과, 도 2와 같이 발명 동결보존용 조성물(FNCP)을 사용할 경우 대조군과 유사하게 높은 세포 생존율을 확인하여, 본 발명 동결보존제의 동결 보존 효과를 확인하였다.As a result, when using the composition for cryopreservation (FNCP) of the present invention as shown in FIG. 2, a high cell viability was confirmed similarly to the control group, confirming the cryopreservation effect of the cryopreservative of the present invention.
또한, 배양 접시에 접종하여 3일간 배양하였을 때, 도 3과 같이 우태아혈청 및 디메틸설폭사이드 (Dimethyl sulfoxide)를 포함하는 기존 동결보존제 대비 동결 보존 후에도 생존한 지방유래 줄기세포가 동등한 수준의 밀도로 부착, 배양되는 것을 확인하였다. 형태적으로도 대조군과 유사하여 해동 후에도 세포의 부착능력에 영향을 주지 않고 본 발명 동결보존용 조성물을 이용하여 무 이종성 (Xeno-free) 및 무 동물성 (Animal-free) 상태로 안정적인 보관이 가능함을 확인하였다.In addition, when inoculated in a culture dish and cultured for 3 days, as shown in FIG. 3, compared to conventional cryopreservatives containing fetal bovine serum and dimethyl sulfoxide, adipose-derived stem cells that survived after cryopreservation were at the same density. Attachment and culture were confirmed. It is morphologically similar to the control group, so it does not affect the adhesion ability of cells even after thawing, and stable storage is possible in a Xeno-free and animal-free state using the composition for cryopreservation of the present invention. Confirmed.
<실시예 5> 해동 후 줄기세포 전분화능 표지 유전자 발현량 확인<Example 5> Determination of stem cell pluripotency marker gene expression level after thawing
상기 실시예 4에 따라 동결 보존하여 해동 후 3일간 배양한 다음 줄기세포 전분화능 표지 유전자의 발현량을 조사하였다. 동결 보존하지 않은 세포를 음성대조군 (Negative Control; NC)로, 10 w/v% DMSO 및 90 w/v% FBS로 동결 보존한 세포를 양성대조군 (Positive Control; PC)로 사용하였다.It was cryopreserved according to Example 4, thawed, cultured for 3 days, and then the expression level of the stem cell pluripotency marker gene was examined. Cells that were not cryopreserved were used as a negative control (NC), and cells cryopreserved with 10 w/v% DMSO and 90 w/v% FBS were used as a positive control (PC).
동결 보존 후 해동한 세포를 100mm 세포 배양용 접시에 접종하여 3일간 배양 후 부착 된 세포를 Trizol Reagent(Invitrogen)를 이용하여 Total RNA를 분리하였다. 다음 SuperscriptII reverse transcriptase (Invitrogen)와 올리고 dT를 이용하여 1 ㎍ total RNA로부터 cDNA를 합성하였다. 합성된 cDNA 및 아래 표 2의 줄기세포 전분화능 표지 유전자의 프라이머를 이용하여 SYBR green method 방법으로 실시간 PCR(Real-time PCR)을 수행하였다. SYBR Green I는 이중가닥 DNA에 결합하여 형광을 나타내는 시약 (interchelator)이다. 인터킬레이트(Interchelator)는 PCR 반응으로 합성된 이중 가닥 DNA에 결합하여 형광을 발하며 이 형광강도를 검출하여 증폭산물의 생성량을 측정할 수 있었다.Cells thawed after cryopreservation were inoculated into a 100 mm cell culture dish, and after incubation for 3 days, total RNA was isolated from the attached cells using Trizol Reagent (Invitrogen). Next, cDNA was synthesized from 1 μg total RNA using SuperscriptII reverse transcriptase (Invitrogen) and oligo dT. Real-time PCR was performed by the SYBR green method using the synthesized cDNA and the primers of the stem cell pluripotency marker genes in Table 2 below. SYBR Green I is an interchelator that exhibits fluorescence by binding to double-stranded DNA. The interchelator binds to the double-stranded DNA synthesized by the PCR reaction and emits fluorescence, and the amount of amplification product can be measured by detecting the fluorescence intensity.
줄기세포의 전분화능 표지 유전자로 알려진 Sox2, Oct4, c-myc, Klf4, Nanog의 발현량을 실시간 PCR을 이용하여 확인하였다. 이때, 데이터는 각 조건당 세 번의 독립적인 실험결과를 바탕으로 평균±표준 편차로 표기하였다. 각 인자의 프라이머 서열은 하기 표 2와 같다.The expression levels of Sox2, Oct4, c-myc, Klf4, and Nanog, known as pluripotency marker genes of stem cells, were confirmed using real-time PCR. At this time, the data were expressed as mean ± standard deviation based on the results of three independent experiments for each condition. The primer sequence of each factor is shown in Table 2 below.
줄기세포 전분화능 표지 유전자의 프라이머 서열Primer sequences of stem cell pluripotency marker genes
Sox2-ForwardSox2-Forward GCTACAGCATGATGCAGGACCAGCTACAGCATGATGCAGGACCA
Sox2-ReverseSox2-Reverse TCTGCGAGCTGGTCATGGAGTTTCTGCGAGCTGGTCATGGAGTT
Oct4-ForwardOct4-Forward CCTGAAGCAGAAGAGGATCACCCCTGAAGCAGAAGAGGATCACC
Oct4-ReverseOct4-Reverse AAAGCGGCAGATGGTCGTTTGGAAAGCGGCAGATGGTCGTTTGG
c-myc-Forwardc-myc-Forward CCTGGTGCTCCATGAGGAGACCCTGGTGCTCCATGAGGAGAC
c-myc-Reversec-myc-Reverse CAGACTCTGACCTTTTGCCAGGCAGACTCTGACCTTTTGCCAGG
Klf4-ForwardKlf4-Forward CATCTCAAGGCACACCTGCGAACATCTCAAGGCACACCTGCGAA
Klf4-ReverseKlf4-Reverse TCGGTCGCATTTTTGGCACTGGTCGGTCGCATTTTTGGCACTGG
Nanog-ForwardNanog-Forward CTCCAACATCCTGAACCTCAGCCTCCAACATCCTGAACCTCAGC
Nanog-ReverseNanog-Reverse CGTCACACCATTGCTATTCTTCGCGTCACACCATTGCTATTCTTCG
그 결과, 도 4와 같이 본 발명 동결보존용 조성물을 사용하여 세포의 동결 보존한 경우에도 줄기세포 전분화능 표지 유전자의 발현량은 음성대조군 및 양성대조군과 상이하지 않았으며, 따라서 본 발명 동결보존용 조성물을 사용할 경우 지방유래 줄기세포의 전분화능 표지 유전자의 발현에 대한 영향 없이 보존 가능함을 확인할 수 있었다.As a result, as shown in FIG. 4, even when cells were cryopreserved using the composition for cryopreservation of the present invention, the expression level of the stem cell pluripotency marker gene was not different from that of the negative control group and the positive control group. It was confirmed that the composition can be preserved without affecting the expression of the pluripotency marker gene of adipose-derived stem cells.
<실시예 6> 해동 후 줄기세포의 특이적 표지 유전자 발현 확인<Example 6> Confirmation of specific marker gene expression of stem cells after thawing
상기 실시예 5에서 제조한 cDNA를 사용하여 동일한 방법으로 지방유래 줄기세포의 특이적 표지 유전자의 발현량을 조사하였다.Using the cDNA prepared in Example 5, the expression level of the specific marker gene of adipose-derived stem cells was investigated in the same manner.
지방유래 줄기세포의 특이적 표지 유전자로 알려진 CD73. CD90, CD105의 발현량을 실시간 PCR을 이용하여 확인하였으며, 각 인자의 프라이머 서열은 하기 표 3과 같다.CD73 known as a specific marker gene for adipose-derived stem cells. The expression levels of CD90 and CD105 were confirmed using real-time PCR, and the primer sequences of each factor are shown in Table 3 below.
지방유래 줄기세포의 특이적 표지 유전자의 프라이머 서열Primer sequences of specific marker genes for adipose-derived stem cells
CD73-ForwardCD73-Forward AAGTGTCGAGTGCCCAGTTAAAGTGTCGAGTGCCCCAGTTA
CD73-ReverseCD73-Reverse TGATCCGACCTTCAACTGCTTGATCCGACCTTCAACTGCT
CD90-ForwardCD90-Forward AGTACGAGTTCAGCCTGACCAGTACGAGTTCAGCCTGACC
CD90-ReverseCD90-Reverse TCTGAGCACTGTGACGTTCTTCTGAGCACTGTGACGTTCT
CD105-ForwardCD105-Forward TCCATTGTGACCTTCAGCCTTCCATTGTGACCTTCAGCCT
CD105-ReverseCD105-Reverse CTTGGATGCCTGGAGAGTCACTTGGATGCCTGGAGAGTCA
그 결과, 도 5와 같이 본 발명 동결보존용 조성물을 사용하여 세포를 동결 보존하여도 지방유래 줄기세포 특이적 표지 유전자의 발현량이 음성대조군 및 양성대조군과 상이하지 않았으며, 따라서 본 발명 동결보존용 조성물은 지방유래 줄기세포의 고유한 특성을 보존하면서 안정적으로 동결 보존이 가능함을 확인하였다.As a result, as shown in FIG. 5, even when the cells were cryopreserved using the cryopreservation composition of the present invention, the expression level of the adipose-derived stem cell-specific marker gene was not different from that of the negative control group and the positive control group. It was confirmed that the composition can be stably cryopreserved while preserving the unique characteristics of adipose-derived stem cells.
통계학적 유의성을 검증하고자 각 실험별 음성대조군 결과와의 통계 분석을 Independent T-Test를 통해 분석하였으며, 이를 p-value로 환산하여 나타내었다. 이상의 모든 통계처리는 Excel 프로그램을 이용하여 분석하였다. p-value가 0.05보다 낮은 경우를 통계학적으로 유의하게 분리하고 결과에 별표(*)로 표시하였다.In order to verify the statistical significance, the statistical analysis with the negative control results for each experiment was analyzed through an independent T-Test, and this was converted into a p-value and shown. All statistical processing above was analyzed using the Excel program. Cases with a p-value lower than 0.05 were statistically significant and marked with an asterisk (*).

Claims (12)

  1. 펙틴과 알라닌을 포함하는 세포 동결 보존용 조성물.A composition for cryopreservation of cells containing pectin and alanine.
  2. 제1항에 있어서,According to claim 1,
    펙틴은 분자량이 100 내지 300 범위의 펙틴이고,Pectin is pectin with a molecular weight ranging from 100 to 300,
    상기 알라닌은 엘-알라닌(L-alanine)인 것을 특징으로 하는 조성물.The alanine is a composition characterized in that L-alanine (L-alanine).
  3. 제1항에 있어서,According to claim 1,
    상기 펙틴은 5 내지 15μM의 범위의 농도로 상기 조성물에 포함되고,The pectin is included in the composition at a concentration ranging from 5 to 15 μM,
    상기 알라닌은 알라닌 20 내지 40μM의 범위의 농도로 상기 조성물에 포함되는 것을 특징으로 하는 조성물.Wherein the alanine is included in the composition at a concentration ranging from 20 to 40 μM alanine.
  4. 제1항에 있어서,According to claim 1,
    상기 조성물은 보존액을 포함하고,The composition includes a preservative solution,
    상기 보존액은 세포 배양 배지이거나, 완충액이거나 등장액인 것을 특징으로 하는 조성물.The composition, characterized in that the preservation solution is a cell culture medium, buffer or isotonic solution.
  5. 제1항에 있어서, According to claim 1,
    동물이나 인간 유래의 혈청이나 혈청 유래 성분을 포함하지 않는 것을 특징으로 하는 조성물. A composition characterized in that it does not contain animal or human-derived serum or serum-derived components.
  6. 제1항에 있어서,According to claim 1,
    상기 세포는 포유동물세포이고,The cell is a mammalian cell,
    상기 포유동물세포는 줄기세포, 췌도세포(islet cell), 수지상세포(dendritic cell), 자연살해세포(NK cell), T 세포, CAR-T 세포, CAR-NK 세포, CHO 세포, HEK 세포, MCF-7 세포, MDCK 세포 또는 Vero 세포인 것을 특징으로 하는 조성물.The mammalian cells include stem cells, islet cells, dendritic cells, natural killer cells (NK cells), T cells, CAR-T cells, CAR-NK cells, CHO cells, HEK cells, MCF -7 cells, MDCK cells or Vero cells.
  7. 제1항에 있어서,According to claim 1,
    상기 세포는 지방 유래 줄기세포인 것을 특징으로 하는 조성물.The cell is a composition, characterized in that the adipose-derived stem cells.
  8. 펙틴과 알라닌을 포함하는 보존액을 제조하는 단계, 상기 보존액에 세포를 현탁하는 단계, 세포가 현탁된 보존액을 동결하고 보존하는 단계를 포함하는 세포 동결 보존 방법.A cell cryopreservation method comprising preparing a preservative solution containing pectin and alanine, suspending cells in the preservative solution, and freezing and preserving the preservative solution in which the cells are suspended.
  9. 제8항에 있어서,According to claim 8,
    상기 펙틴은 분자량이 100 내지 300 범위의 펙틴이고,The pectin is a pectin having a molecular weight in the range of 100 to 300,
    상기 알라닌은 엘-알라닌(L-alanine)이고,The alanine is L-alanine,
    상기 펙틴은 5 내지 15μM의 범위의 농도로 상기 보존액에 포함되고,The pectin is contained in the preservation solution at a concentration ranging from 5 to 15 μM,
    상기 알라닌은 알라닌 20 내지 40μM의 범위의 농도로 상기 보존액에 포함되며,The alanine is contained in the preservation solution at a concentration in the range of 20 to 40 μM alanine,
    상기 보존액은 세포 배양 배지이거나, 완충액이거나 등장액인 것을 특징으로 하는 방법.The method according to claim 1 , wherein the preservation solution is a cell culture medium, a buffer solution, or an isotonic solution.
  10. 제8항에 있어서,According to claim 8,
    상기 보존액은 동물이나 인간 유래의 혈청이나 혈청 유래 성분을 포함하지 않는 것을 특징으로 하는 방법. The method characterized in that the preservation solution does not contain animal or human-derived serum or serum-derived components.
  11. 제8항에 있어서,According to claim 8,
    상기 세포는 포유동물세포이고,The cell is a mammalian cell,
    상기 포유동물세포는 줄기세포, 췌도세포(islet cell), 수지상세포(dendritic cell), 자연살해세포(NK cell), T 세포, CAR-T 세포, CAR-NK 세포, CHO 세포, HEK 세포, MCF-7 세포, MDCK 세포 또는 Vero 세포인 것을 특징으로 하는 방법.The mammalian cells include stem cells, islet cells, dendritic cells, natural killer cells (NK cells), T cells, CAR-T cells, CAR-NK cells, CHO cells, HEK cells, MCF -7 cells, MDCK cells or Vero cells.
  12. 제8항에 있어서,According to claim 8,
    상기 세포는 지방 유래 줄기세포인 것을 특징으로 하는 방법.The method characterized in that the cells are adipose-derived stem cells.
PCT/KR2021/015995 2021-11-05 2021-11-05 Cell cryopreservation composition using pectin and alanine and cell cryopreservation method using same WO2023080281A1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
PCT/KR2021/015995 WO2023080281A1 (en) 2021-11-05 2021-11-05 Cell cryopreservation composition using pectin and alanine and cell cryopreservation method using same

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/KR2021/015995 WO2023080281A1 (en) 2021-11-05 2021-11-05 Cell cryopreservation composition using pectin and alanine and cell cryopreservation method using same

Publications (1)

Publication Number Publication Date
WO2023080281A1 true WO2023080281A1 (en) 2023-05-11

Family

ID=86241646

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/KR2021/015995 WO2023080281A1 (en) 2021-11-05 2021-11-05 Cell cryopreservation composition using pectin and alanine and cell cryopreservation method using same

Country Status (1)

Country Link
WO (1) WO2023080281A1 (en)

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020031527A1 (en) * 1998-11-16 2002-03-14 Introgen Therapeutics, Inc. Formulation of adenovirus for gene therapy
JP2005270006A (en) * 2004-03-25 2005-10-06 Kyudo Kk Agent for preserving sperm by freezing
KR20160033129A (en) * 2013-07-02 2016-03-25 오스트리아노바 싱가포르 피티이 리미티드 A method of freeze-drying encapsulated cells, freeze-dried encapsulated cells, compositions containing freeze-dried encapsulated cells and uses of such cells and compositions
US20180020658A1 (en) * 2016-07-22 2018-01-25 Tissue Testing Technologies Llc Enhancement of cell cryopreservation with glycolipids
US20190357525A1 (en) * 2016-12-23 2019-11-28 Cellular Biomedicine Group (Shanghai) Ltd. Cell freezing medium for clinical use
KR102391629B1 (en) * 2021-11-04 2022-04-28 주식회사 프롬바이오 Composition for cryopreservation of cells using pectin and alanine and the cryopreservation using the same

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020031527A1 (en) * 1998-11-16 2002-03-14 Introgen Therapeutics, Inc. Formulation of adenovirus for gene therapy
JP2005270006A (en) * 2004-03-25 2005-10-06 Kyudo Kk Agent for preserving sperm by freezing
KR20160033129A (en) * 2013-07-02 2016-03-25 오스트리아노바 싱가포르 피티이 리미티드 A method of freeze-drying encapsulated cells, freeze-dried encapsulated cells, compositions containing freeze-dried encapsulated cells and uses of such cells and compositions
US20180020658A1 (en) * 2016-07-22 2018-01-25 Tissue Testing Technologies Llc Enhancement of cell cryopreservation with glycolipids
US20190357525A1 (en) * 2016-12-23 2019-11-28 Cellular Biomedicine Group (Shanghai) Ltd. Cell freezing medium for clinical use
KR102391629B1 (en) * 2021-11-04 2022-04-28 주식회사 프롬바이오 Composition for cryopreservation of cells using pectin and alanine and the cryopreservation using the same

Similar Documents

Publication Publication Date Title
CN113423268B (en) Cryopreservation of stem cells
US11821006B2 (en) Method of isolating mesenchymal stem cells from the amniotic membrane of the umbilical cord, a mesenchymal stem cell population isolated from the amniotic membrane of the umbilical cord and a cell culture medium for isolating mesenchymal stem cells from the amniotic membrane of the umbilical cord
KR102391629B1 (en) Composition for cryopreservation of cells using pectin and alanine and the cryopreservation using the same
AU2017377309B9 (en) Mammalian cell cryopreservation liquid
WO2012091206A1 (en) A freezing medium composition for cryopreserving amniotic fluid-derived stem cells and a method for cryopreserving the same
KR101321144B1 (en) Vitrification medium for stem cells and vitrification method for stem cells using the same
US20210102171A1 (en) Mesenchymal stem cell storing or transport formulation and methods of making and using the same
US11998569B2 (en) Method of transporting mesenchymal stem cells by means of a transporting solution and a method of administering stem cells to wounds
CN114557337B (en) Protein-free non-program freezing solution of umbilical cord mesenchymal stem cells and preparation method thereof
TWI732298B (en) Mammalian cell preservation solution containing acarbose or stachyose
WO2023080281A1 (en) Cell cryopreservation composition using pectin and alanine and cell cryopreservation method using same
WO2019103529A2 (en) Composition for cell freeze and preservation, and method for cell freeze and preservation using same
WO2019199230A1 (en) A method of transporting mesenchymal stem cells by means of a cell culture medium and a method of administering stem cells to wounds
KR20210134701A (en) Mammalian Cell Preservation Solution Containing Trehalose
WO2023113109A1 (en) Method for isolating adipose-derived stem cells using double wells having upper part/lower part structure
WO2021193606A1 (en) Mammalian cell preservative solution containing acarbose and dextran

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 21963381

Country of ref document: EP

Kind code of ref document: A1