Composition for isolation and cryopreservation of adipose tissue-derived stem cells

Abstract

The present invention relates to a composition for cell isolation and cryopreservation of adipose tissue-derived stem cells, the composition comprising N-acetylcysteine and human serum albumin. A solution for cell isolation containing albumin and / or N-acetylcysteine according to the present invention increased the cell yield when stromal vascular fraction (SVF) was isolated from adipose tissue. The isolated adipose stem cells exhibited the differentiation potential into adipocytes, chondrocytes, and osteocytes. In addition, a cryopreservation agent comprising albumin and / or N-acetylcysteine increased the cell recovery rate upon thawing after freezing. Therefore, the composition for cell isolation and cryopreservation according to the present invention can be used for isolating and preserving stromal vascular fractions.

Description

Composition for isolating and cryopreserving adipose tissue-derived stem cells

[0001] The present invention relates to a composition for isolating and cryopreserving adipose tissue-derived stem cells.

[0002] Recently, adipose tissue has been attracting attention as a source of undifferentiated adult stem cells (A. Miranville et al., Circulation 110:349, 2004). Adult stem cells within adipose tissue can differentiate into adipocytes, osteoblasts, and chondroblasts, and are known to be particularly capable of cross-differentiation into various tissues (PA Zuk et al., Tissue Eng., 7:211, 2001). Furthermore, adipose tissue-derived stem cells have been reported to promote the regeneration of cardiac muscle and nerve cells.

[0003] Adipose tissue is considered a promising source of stem cells because it has the advantage of allowing stem cells to be extracted in relatively large quantities from patients compared to other tissues, and the use of autologous adipose tissue can eliminate immune-related side effects.

[0004] However, conventional methods for isolating adipose tissue-derived stem cells present various challenges in clinical applications, including cell yield, cell activity, and cell quality. Furthermore, it is difficult to preserve isolated stem cells while maintaining their activity. Therefore, there is a pressing need for improved methods for isolating and preserving isolated adipose tissue-derived stem cells.

[0005] Accordingly, the inventors of the present invention studied a method for improving the yield of adipose tissue-derived stem cells compared to conventional methods, and confirmed that when cells are isolated using a composition comprising N-acetylcysteine ​​and albumin, the yield of stem cells obtained from adipose tissue is improved. Furthermore, the activity of stem cells isolated using the composition of the present invention was maintained, thereby completing the present invention.

[0006] To achieve the above purpose, one aspect of the present invention provides a cell separation composition and a cell separation kit comprising N-acetylcysteine ​​or a pharmaceutically acceptable salt thereof represented by the following chemical formula I:

[0007] <Chemical Formula I>

[0008] .

[0009] Another aspect of the present invention provides a method for isolating cells derived from adipose tissue, comprising the steps of: i) mixing adipose tissue separated from an individual with the cell separation composition and washing it; and ii) treating the washed adipose tissue with a tissue dissociation enzyme to isolate cells derived from the adipose tissue.

[0010] Another aspect of the present invention provides a composition for cell cryopreservation comprising N-acetylcysteine ​​or a pharmaceutically acceptable salt thereof represented by the following chemical formula I:

[0011] <Chemical Formula I>

[0012] .

[0013] Another aspect of the present invention provides a method for cryopreservation of cells, comprising the steps of: i) mixing cells and the cryopreservation composition; and ii) cryopreserving the mixture.

[0014] The cell separation solution comprising human serum albumin and / or N-acetylcysteine ​​according to the present invention improved the cell yield when isolating the stromal vascular fraction (SVF) from adipose tissue. The isolated adipose stem cells exhibited differentiation potential into adipocytes, chondrocytes, and osteocytes. In addition, the cryopreservation agent comprising albumin and / or N-acetylcysteine ​​improved the yield of SVF upon thawing after freezing. Therefore, the cell separation solution and cryopreservation solution according to the present invention can be used for SVF separation and cryopreservation.

[0015] Figure 1 is a drawing showing the result of separating layers by centrifuging fat tissue obtained through a liposuction method.

[0016] Figure 2 is a drawing showing the result of washing the fat layer of Figure 1 using the cell separation composition of the present invention.

[0017] Figure 3 is a drawing showing the results of treating with collagenase after removing the top oil layer and the bottom tumescent layer in Figure 2.

[0018] Figure 4 is a drawing showing the results of performing a blood cell hemolysis (RBC lysis) process to remove blood cells present in the pellet of Figure 2.

[0019] Figure 5 is a graph showing the results of counting the number of cells and analyzing the viability of the stromal vascular fraction (SVF) separated from adipose tissue using a cell separation solution containing human serum albumin (HSA) and / or N-acetylcysteine ​​(NAC). HS: Hartmann solution, HSA: human serum albumin (1%), NAC: N-acetylcysteine ​​(200 ug / mL).

[0020] Figure 6 is a graph showing the results of counting the number of cells and analyzing the viability of SVF after freezing and thawing using a cryopreservation solution containing human serum albumin and / or N-acetylcysteine. HS: Hartmann's solution, HSA: human serum albumin (10%), NAC: N-acetylcysteine ​​(24.5 mM (approximately 4 mg / mL)).

[0021] Figure 7 is a drawing showing the results of confirming the differentiation ability into adipocytes, chondrocytes, and bone cells for adipose stem cells separated according to the present invention.

[0022] Composition for cell separation

[0023] One aspect of the present invention provides a cell separation composition comprising N-acetylcysteine ​​or a pharmaceutically acceptable salt thereof. The composition may further comprise a buffer. Furthermore, the composition may further comprise albumin.

[0024] As used herein, the term "N-acetylcysteine ​​(NAC)" refers to (2R)-2-acetylamino-3-sulfanylpropanoic acid, a thiol-containing amino acid derivative in which an acetyl group (CH3CO-) is attached to the nitrogen atom of cysteine, and is represented by the following chemical formula I:

[0025] <Chemical Formula I>

[0026] .

[0027] The above N-acetylcysteine ​​has an antioxidant effect as a ROS (reactive oxygen species) scavenger.

[0028] In addition, the N-acetylcysteine ​​may be in the form of a solvate thereof or a pharmaceutically acceptable salt thereof. The solvate refers to a compound solvated in an organic or inorganic solvent. The solvate may be, for example, a hydrate. The pharmaceutically acceptable salt refers to an inorganic acid salt, an organic acid salt, or an addition salt of a metal salt of the compound. The pharmaceutically acceptable salt may be a salt that does not cause serious irritation to an organism to which the compound is administered and does not impair the biological activity and physical properties of the compound. The inorganic acid salt may be a hydrochloride salt, a bromate salt, a phosphate salt, a sulfate salt, or a disulfate salt. The organic acid salt may be formate, acetate, acetate, propionate, lactate, oxalate, tartrate, malate, maleate, citrate, fumarate, besylate, camsylate, edicyl, trichloroacetic acid, trifluoroacetate, benzoate, gluconate, methanesulfonate, glycolate, succinate, 4-toluenesulfonate, galacturonate, embonic acid, glutamate, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, or aspartate. The metal salt may be calcium salt, sodium salt, magnesium salt, strontium salt, or potassium salt. N-acetylcysteine ​​can be chemically synthesized or commercially purchased.

[0029] In the present invention, the composition may contain N-acetylcysteine ​​or a pharmaceutically acceptable salt thereof in an amount of about 0.01 mg / mL to about 10 mg / mL based on the total volume of the composition. Specifically, the concentration of N-acetylcysteine ​​or a pharmaceutically acceptable salt thereof in the composition may be about 0.01 mg / mL to about 10 mg / mL, about 0.05 mg / mL to about 8 mg / mL, about 0.1 mg / mL to about 6 mg / mL, or about 0.2 mg / mL to about 5 mg / mL. Preferably, the concentration of N-acetylcysteine ​​in the composition may be about 0.2 mg / mL to about 5 mg / mL. In one embodiment of the present invention, the concentration of N-acetylcysteine ​​may be 0.2 mg / mL.

[0030] In the present invention, the yield of cells can be improved by adding NAC or a pharmaceutically acceptable salt thereof to the composition.

[0031] The term "albumin" used in this specification is the major protein most abundant in plasma (approximately 50-60%), and is involved in plasma osmotic pressure, intracellular transport of nutrients and ions, immunity, and energy metabolism.

[0032] In the present invention, the albumin may be, but is not limited to, human serum albumin, bovine serum albumin, conalbumin, ovalbumin, lactalbumin, etc. The albumin may include, but is not limited to, sodium stearoyl lactalbumin, lithospermum root extract serum albumin succinate, succinoyl serum albumin, etc. In one embodiment of the present invention, the albumin may be human serum albumin (HSA).

[0033] The above "human serum albumin" is serum albumin found in human blood and is the most abundant protein present in human plasma. In the present invention, it may be isolated from human serum or may be recombinant human serum albumin. The amino acid sequence and polynucleotide sequence for albumin can be obtained from known databases such as GenBank of the National Institutes of Health (NCBI) of the United States (Genbank: ABS29264.1), and specifically, it may include the amino acid sequence of SEQ ID NO: 1. In addition, as long as it has the same activity as the protein or the gene location encoding the protein on the chromosome is the same, it may be composed of a sequence in which one or more amino acids of the protein are added, deleted, or substituted. Specifically, the albumin may comprise or consist of an amino acid sequence having about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identity, or about 100% identity, to the amino acid sequence of SEQ ID NO: 1.

[0034] Additionally, the albumin may include a fragment of albumin or a variant thereof.

[0035] As used herein, the term "albumin fragment" refers to a fragment in which a portion of the N-terminus and / or C-terminus of wild-type albumin is deleted (truncated). Specifically, the albumin fragment may be in a form that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids consecutively from the N-terminus and / or C-terminus of a protein having the amino acid sequence of SEQ ID NO: 1.

[0036] Additionally, the albumin fragment may be composed of a sequence in which one or more amino acids of the fragment are added, deleted, or substituted, as long as the albumin fragment has the same activity as the albumin fragment or the gene encoding the albumin on the chromosome is at the same location.

[0037] As used herein, the term "mutant" refers to a form in which some amino acids have been substituted, deleted, and / or inserted compared to the wild type. In the present invention, the "albumin mutant" refers to a form in which some amino acids of the above-described albumin or a fragment thereof have been substituted. The albumin mutant may have a sequence different from that of the wild type albumin or a fragment thereof.

[0038] In the present invention, the composition may contain albumin in an amount of about 0.1% (v / v) to about 20% (v / v) based on the total volume of the composition. Specifically, it may contain about 0.1% (v / v) to about 20% (v / v), about 0.5% (v / v) to about 15% (v / v), or about 1% (v / v) to about 10% (v / v). Preferably, the composition may be characterized in that it contains albumin in an amount of about 1% (v / v) to about 10% (v / v). In one embodiment of the present invention, the albumin may be contained in an amount of 1% (v / v) based on the total volume of the composition.

[0039] In the present invention, the albumin may be replaced with or combined with other proteins that perform similar roles.

[0040] In the present invention, the above "buffer" refers to a liquid composition that is a solution that has properties similar to human blood or body fluid and does not cause damage to cells, and is preferably included in the cell separation composition of the present invention in the largest volume ratio to serve as a base.

[0041] The above buffer may include at least one selected from the group consisting of calcium chloride, potassium chloride, sodium chloride, and sodium lactate.

[0042] The calcium chloride may be included in an amount of about 0.1 to about 1.0 parts by weight based on 1.0 parts by weight of potassium chloride. Specifically, it may be included in an amount of about 0.1 to about 1.0 parts by weight, about 0.2 to about 0.9 parts by weight, about 0.3 to about 0.8 parts by weight, about 0.4 to about 0.7 parts by weight, or about 0.5 to about 0.6 parts by weight.

[0043] The sodium chloride may be included in an amount of about 5.0 to about 40 parts by weight based on 1.0 parts by weight of potassium chloride. Specifically, it may be included in an amount of about 5.0 to about 40 parts by weight, about 10 to about 30 parts by weight, or about 15 to about 20 parts by weight.

[0044] The sodium lactate may be included in an amount of about 1.0 to about 30 parts by weight based on 1.0 parts by weight of potassium chloride. Specifically, it may be included in an amount of about 1.0 to about 30 parts by weight, about 5.0 to about 20 parts by weight, or about 10 to about 15 parts by weight.

[0045] In the present invention, a specific example of the components and contents of the buffer may include about 1.0 parts by weight of potassium chloride, about 0.5 to about 0.8 parts by weight of calcium chloride, about 15 to about 25 parts by weight of sodium chloride, and about 15 to about 25 parts by weight of sodium lactate based on 1.0 parts by weight of potassium chloride.

[0046] Additionally, the buffer may contain additional salts, sugars, lipids, proteins, etc., in addition to the substances disclosed above, as long as they do not negatively affect the activity and viability of the cells.

[0047] In the present invention, the buffer may be characterized by having an appropriate pH and osmolality to prevent damage to cells, and preferably, may be characterized by having a pH and osmolality similar to human plasma or body fluid.

[0048] Specifically, the pH of the buffer may be from about 4.0 to about 8.0. More specifically, the pH of the buffer may be from about 4.0 to about 8.0, from about 6.0 to about 8.0, or from about 7.0 to about 8.0. Preferably, the pH may be about 7.4.

[0049] Specifically, the osmolality of the buffer may be about 200 mOsm / L to about 400 mOsm / L. More specifically, it may be about 200 mOsm / L to about 400 mOsm / L, about 250 mOsm / L to about 350 mOsm / L, or about 280 mOsm / L to about 310 mOsm / L. Preferably, it may be about 273 mOsm / L.

[0050] In the present invention, the buffer may be a commercially available solution or a modified composition thereof. For example, Plasma solution A, Plasma-lyte (e.g., Plasma-lyte A, Plasma-lyte 148, Plasma-lyte 56, etc.), Hartmann's solution, Ringer's solution, about 0.45% saline, Sterofundin, about 0.9% Normal Saline, or a solution having a similar composition to these may be used.

[0051] In one embodiment of the present invention, the buffer may be a Hartmann solution.

[0052] The cell separation composition according to the present invention may be for separating cells.

[0053] At this time, the cells can be isolated from adipose tissue. Specifically, the cells can be adipose tissue-derived stem cells.

[0054] As used herein, the term "stem cell" refers to a relatively undifferentiated cell that, under suitable conditions, has the ability to differentiate into specific tissue cells. The stem cell can self-renew and produce daughter cells that maintain their undifferentiated characteristics, while simultaneously producing daughter cells that differentiate into specific cell types. The stem cell may be an embryonic stem cell or an adult stem cell.

[0055] The term "adult stem cell" used herein refers to a stem cell discovered after the developmental process, as distinct from an embryonic stem cell obtained in the early stage of development of a fertilized egg. It refers to cells with various forms of regenerative ability even after the developmental process is completed in each part of the body, and these exhibit various degrees of multi-potentiality. Therefore, it does not refer to a single type of stem cell, but rather a general collection of stem cells found in bone marrow, umbilical cord blood, skin, adipose tissue, nerve tissue, liver, pancreatobiliary tract, etc. In the present invention, the adult stem cell may be an adipose-derived stem cell (ADSC).

[0056] The above "adipose tissue" refers to connective tissue primarily composed of adipocytes. From the adipose tissue, a stromal vascular fraction (SVF) containing adipocytes, adipocyte precursor cells, fibroblasts, vascular endothelial cells, various immune cells such as adipose tissue macrophages, stem cells, and endothelial precursor cells can be isolated.

[0057] The adipose tissue may be obtained from a living human or animal, preferably, but not limited to, adipose tissue from the lower abdomen, calf, thigh, upper arm, breast, or knee joint.

[0058] The above "stromal vascular fraction (SVF)" may include adipocytes, stem cells (e.g., mesenchymal stem cells), endothelial progenitor cells, T cells, B cells, mast cells, adipose tissue macrophages, other stromal cells, etc.

[0059] As used herein, the term "adipose tissue-derived stem cell" refers to a stem cell extracted from adipose tissue among various types of adult stem cells, including bone, muscle, fat, and umbilical cord blood. The adipose tissue-derived stem cell can be obtained from the stromal vascular fraction (SVF) of adipose tissue. Adipose tissue-derived stem cells (ADSCs) with multipotency can differentiate into most mesenchymal cells, including adipocytes, osteoblasts, chondrocytes, and myofibroblasts.

[0060] Method for isolating adipose tissue-derived stem cells

[0061] Another aspect of the present invention provides a method for isolating adipose tissue-derived cells comprising the following steps:

[0062] i) a step of washing the adipose tissue separated from the subject by mixing it with the cell separation composition; and

[0063] ii) A step of treating the washed adipose tissue with a tissue dissociation enzyme to obtain a fraction containing cells.

[0064] Additionally, the method may further include the following steps:

[0065] iii) a step of removing blood cells by adding a blood cell hemolysis solution to the fraction containing the obtained adipose tissue-derived cells; and / or

[0066] iv) A step of filtering the cell fraction from which the blood cells have been removed.

[0067] The above adipose tissue can be separated from the body according to a method known in the art.

[0068] Specifically, the method for isolating adipose tissue-derived cells according to the present invention may include the following steps. In this case, the cells may include adipose tissue-derived stem cells. The adipose tissue-derived stem cells are the same as those described above.

[0069] First, it may include a step of mixing the adipose tissue separated from the subject with the cell separation composition and washing it.

[0070] In the present invention, the adipose tissue may be separated from an individual.

[0071] The adipose tissue can be isolated from the body using methods known in the art. For example, the adipose tissue can be isolated from the subject using suction-assisted liposuction (lipoplasty), ultrasound-assisted liposuction, excisional lipectomy, or a combination thereof. The tissue extraction may be performed in a sterile or aseptic manner.

[0072] The above entity may be a human, mammal or bird, preferably a human.

[0073] If the above-mentioned adipose tissue is an aspirate obtained through liposuction, a step of separating the adipose tissue from the aspirate may be performed. The separation step may be performed through centrifugation.

[0074] Specifically, the centrifugation may be performed at about 100 g to about 500 g, about 200 g to about 400 g, or about 250 g to about 300 g. In one embodiment of the present invention, the centrifugation may be performed at about 300 g.

[0075] Specifically, the centrifugation may be performed for about 1 minute to about 10 minutes, about 2 minutes to 10 minutes, about 3 minutes to 10 minutes, about 4 minutes to 10 minutes, or about 5 minutes to 10 minutes. In one embodiment of the present invention, the centrifugation may be performed for about 5 minutes.

[0076] From the fraction separated by centrifugation as described above, the uppermost oil layer and the lowermost red blood cell layer (RBC layer) can be removed, and the middle layer (fat layer) containing adipose tissue can be obtained.

[0077] Additionally, the above separation process may be repeated more than once. Specifically, it may be repeated once, twice, or three times. In one embodiment of the present invention, the process may be performed three times.

[0078] In one embodiment of the present invention, the fat tissue separation process may include a process of obtaining a middle layer (fat layer) by centrifugation at 300 g for 5 minutes, and may include repeating the process three times.

[0079] The adipose tissue separated as described above can be washed by mixing it with the cell separation composition according to the present invention.

[0080] Specifically, in the present invention, the adipose tissue and the cell separation composition according to the present invention can be washed by mixing at a ratio of about 1:0.5 to about 1:30. Specifically, the washing can be performed by mixing at a ratio of about 1:0.5 to about 1:30, about 1:1 to about 1:25, about 1:2 to about 1:20, or about 1:5 to about 1:10, but the present invention is not limited thereto as long as an amount capable of sufficiently washing the adipose tissue is used.

[0081] Washing in the above step may be performed by centrifugation, but is not limited thereto. After mixing the adipose tissue and the cell separation composition of the present invention through the washing, centrifugation is performed to remove the uppermost oil layer and the lowermost tumescent layer, thereby obtaining the middle layer containing the adipose tissue (Fig. 2). The centrifugation is the same as described above.

[0082] Additionally, the above washing process may be repeated more than once. Specifically, it may be repeated once, twice, or three times. In one embodiment of the present invention, the washing process may be performed three times.

[0083] In one embodiment of the present invention, the washing may include mixing a composition for separating adipose tissue and cells and centrifuging at 300 g for 5 minutes to obtain a middle layer (fat layer), repeating the process three times.

[0084] Second, a step of treating the washed adipose tissue with a tissue dissociation enzyme to separate cells may be included.

[0085] In the present invention, the adipose tissue obtained as described above can be separated into fat and cells through tissue dissociation. At this time, an enzyme may be used for tissue dissociation. In the present invention, the enzyme for tissue dissociation may be collagenase or dispase.

[0086] The above "collagenase" refers to an enzyme that cleaves peptide bonds in collagen. Here, collagen is a major component of the extracellular matrix. In the present invention, the collagenase may be any one selected from the group consisting of collagenase I, II, III, IV, V, and combinations thereof. Specifically, it may be collagenase type I or II, but is not limited thereto. The treatment concentration of the collagenase may be about 0.1 units / mL to about 0.5 units / mL.

[0087] The above "dispase" is a proteolytic enzyme that cleaves fibronectin, collagen IV, and to a lesser extent collagen I. The dispase treatment concentration may be about 1.0 unit / mL to about 2.5 unit / mL.

[0088] In the present invention, collagenase, dispase, or a mixture thereof can be used as the tissue dissociation enzyme.

[0089] The above-mentioned tissue dissociation enzyme is not limited thereto, but can be treated on washed adipose tissue in a stirrer for 0.5 to 6 hours. Considering optimization of enzyme activity, the temperature in the stirrer during enzyme treatment is preferably maintained at about 25°C to about 37°C, and the stirring speed can be about 10 rpm to 300 rpm.

[0090] In one embodiment of the present invention, the tissue dissociation enzyme can be treated by stirring at about 37°C for 1.5 hours at a speed of about 30 rpm.

[0091] The above step may additionally include a centrifugation step. In other words, the adipose tissue treated with the enzyme as described above may be centrifuged to isolate a fraction (cell pellet, FIG. 3) containing adipose tissue-derived cells. The fraction containing the adipose tissue-derived cells may be a stromal vascular fraction (SVF). Furthermore, the above step may additionally include a washing step. The washing step is the same as described above.

[0092] Third, a step of removing blood cells by adding a blood cell hemolysis solution to the obtained cell fraction may be additionally included.

[0093] As described above, a hemolytic solution can be added to the separated cells to remove the blood cells. The hemolytic solution may include, but is not limited to, ammonium chloride, potassium bicarbonate, and EDTA. The composition of the hemolytic solution is known in the art and can be prepared using commercially available products (e.g., RBC lysis buffer (Biolegend, 420301)).

[0094] The fraction containing the above-described separated adipose tissue-derived cells can be reacted with a hemolytic solution for about 1 to about 10 minutes. In one embodiment, the fraction containing the above-described adipose tissue-derived stem cells can be treated with a hemolytic solution for about 10 minutes.

[0095] At this time, the above solution can be treated and reacted at room temperature. Specifically, the reaction can be performed at about 10°C to about 27°C. More specifically, the reaction can be performed at about 10°C to about 22°C or about 23°C to about 27°C.

[0096] The above step may additionally include centrifugation and washing steps. The centrifugation and washing steps are the same as described above.

[0097] Fourth, the step of filtering the cell fraction from which the blood cells have been removed may be additionally included.

[0098] The above step may be performed using a mesh filter. In this case, the filtering may be performed using a mesh filter of about 50 um to about 150 um, but is not limited thereto. Preferably, it may be performed using a mesh filter of about 70 um.

[0099] In one embodiment of the present invention, it was confirmed that the method for isolating adipose tissue-derived stem cells according to the present invention increased the yield of adipose tissue-derived stem cells by about 3 to about 5 times compared to a conventional method (Fig. 5).

[0100] Cell separation kit

[0101] Another aspect of the present invention provides a cell separation kit comprising N-acetylcysteine ​​or a pharmaceutically acceptable salt thereof represented by the following chemical formula I:

[0102] <Chemical Formula I>

[0103] .

[0104] The above kit may further comprise a buffer and may further comprise albumin. Each of the N-acetylcysteine, buffer and albumin may be included separately or in a mixed form.

[0105] Additionally, the kit may further comprise a tissue dissociation enzyme and / or a blood cell hemolysis solution.

[0106] If the above kit includes a tissue dissociation enzyme and / or a blood cell hemolysis solution, each may be included separately in a separate container.

[0107] The above N-acetylcysteine ​​or a pharmaceutically acceptable salt thereof, buffer, albumin, tissue dissociation enzyme and blood cell hemolysis solution are the same as described above.

[0108] Composition for cryopreservation

[0109] Another aspect of the present invention provides a composition for cell cryopreservation comprising N-acetylcysteine ​​or a pharmaceutically acceptable salt thereof represented by the following chemical formula I:

[0110] <Chemical Formula I>

[0111] .

[0112] The above cell cryopreservation composition may further comprise DMSO.

[0113] As used herein, the term "cryopreservation" means maintaining cells stable over long periods of time through freezing.

[0114] The term "cryopreservation composition" as used herein, also called a cryopreservative or cryoprotectant, refers to a substance added for the purpose of alleviating various stresses and damages when preserving cells in a frozen state.

[0115] The above cryopreservation composition may comprise N-acetylcysteine ​​or a pharmaceutically acceptable salt thereof, and may further comprise DMSO. Furthermore, it may further comprise albumin and / or a buffer. The N-acetylcysteine ​​or a pharmaceutically acceptable salt thereof, albumin, and buffer are the same as those described above.

[0116] In one embodiment of the present invention, the concentration of N-acetylcysteine ​​or a pharmaceutically acceptable salt thereof included in the cell cryopreservation composition may be about 4 mg / mL (about 24.5 mM).

[0117] The term "dimethyl sulfoxide (DMSO)" used in this specification is used as a cell membrane permeable cryopreservative, which permeates the cell membrane to replace intracellular water and inhibits the formation of ice crystals during freezing.

[0118] The content of DMSO included in the cell cryopreservation composition of the present invention may be about 0.001% (v / v) to about 10% (v / v) based on the total volume of the composition. Specifically, it may be about 0.001% (v / v) to about 10% (v / v), about 0.01% (v / v) to about 9% (v / v), about 0.1% (v / v) to about 8% (v / v), about 1% (v / v) to about 7% (v / v), or about 3% (v / v) to about 6% (v / v). In one embodiment of the present invention, the content of albumin included in the cell cryopreservation composition may be about 5% (v / v) based on the total volume of the composition.

[0119] In one embodiment of the present invention, the buffer may be a Hartmann solution.

[0120] The cells that can be preserved by the cryopreservation composition of the present invention may be animal cells or insect cells, but any cell that can be stably cryopreserved by the composition of the present invention may be used. The cells may be cells derived from humans, monkeys, pigs, horses, cows, sheep, dogs, cats, mice, rabbits, insects, etc. Specifically, the cells may be immune cells, tumor cells, umbilical cord blood cells, stem cells, epithelial cells, primary cells, etc. derived from the animals. Specifically, the cells may be cells derived from adipose tissue, and more specifically, adipose tissue-derived stem cells. The adipose tissue-derived stem cells are the same as described above.

[0121] Cell cryopreservation methods

[0122] Another aspect of the present invention provides a method for cell cryopreservation, comprising the steps of: i) mixing cells with the cell cryopreservation composition; and ii) cryopreserving the mixture. The cells and the cell cryopreservation composition are the same as described above.

[0123] In step ii) above, freezing can be performed through a conventional method.

[0124] For example, the freezing may be performed by methods such as vitrification, slow freezing, etc., but is not limited thereto. In freezing, the cryopreservation composition of the present invention can prevent intracellular ice crystal formation, perform the function of protecting cells, and significantly increase the yield / survival rate of cells after thawing.

[0125] The above "vitrification freezing" refers to a method of freezing cells by controlling the temperature at a constant rate per minute using equipment such as CRF (controlled rate freezing), and when the temperature reaches -80℃ or lower, the frozen cells are stored in a nitrogen tank.

[0126] The above "gentle freezing" method involves placing a vial containing a cryopreservation composition containing cells in a cryogenic container box containing isopropyl alcohol or an alcohol-free cell freezing container, and freezing the cells by steadily lowering the temperature in an ultra-low temperature freezer below -80°C for 12 to 24 hours. The frozen cells are then stored in a liquid nitrogen tank.

[0127] In one embodiment of the present invention, the freezing may be a gentle freezing.

[0128] Another aspect of the present invention provides a cell cryopreservation kit comprising a cell cryopreservation composition comprising N-acetylcysteine ​​or a pharmaceutically acceptable salt thereof represented by the following chemical formula I:

[0129] <Chemical Formula I>

[0130] .

[0131] The above cell cryopreservation kit may further comprise DMSO, albumin, and a buffer. These components may be provided separately or mixed.

[0132] The above cells, cell cryopreservation composition, DMSO, albumin and buffer are the same as described above.

[0133] The present invention will be described in more detail below through examples. However, these examples are intended to exemplify one or more specific examples, and the scope of the present invention is not limited to these examples.

[0134] I. Isolation of adipose tissue-derived stem cells

[0135] Example 1. Preparation of solution for cell separation

[0136] The raw materials used in the present invention are shown in Table 1 below.

[0137] Raw material name Abbreviation Manufacturer Cat. No. Use Storage condition (℃) Hartmann Solution Hartmann Solution (H / S) Daehan Pharmaceutical Co., Ltd. Specialized drug separation solution Manufacturing 1 - 30 Nordmark NB6 Collagenase Nordmark N0002779 Collagenase solution Manufacturing 2 - 8 Acetylcysteine ​​(Muteran injection) NAC Hanwha Pharmaceutical Co., Ltd. Specialized drug separation solution Manufacturing 1 - 30 Albumin 20% 100 mL / 1 vial (refrigerated) HSA Green Cross Co., Ltd. Specialized drug separation solution Manufacturing 2 - 8 Acridine Orange / Propidium Iodide Stain AO / PI Stain Logos Biosystems F23001 Cell viability confirmation 2 - 8 10X RBC lysis buffer RBC lysis buffer Biolegend 420301 RBC removal 2 - 8DMSODMSOMylan67457-178-50Cell freezing20-30Water for injectionDWDaehan Pharmaceutical Industry Specialty Pharmaceutical RBC lysis buffer 1x for dilution1-30

[0138] Example 1.1. Preparation of separation solution

[0139] Hartmann's solution (H / S) and human serum albumin (HSA) were warmed in a 37°C water bath in a cell culture room, and then mixed with N-acetylcysteine ​​(NAC, Muteran injection) in a biological safety cabinet (BSC). At this time, HSA and NAC were mixed to prepare a final concentration of 1% (v / v) and 200 ug / mL, respectively. In addition, a separation solution containing HSA or NAC alone was also prepared. A specific example of the separation solution according to the present invention is shown in Table 2.

[0140] Solution composition for separationNAC+HSANACHSANAC (mL)0.320.32-HSA (mL)8-8H / S (mL)151.68159.68152Total (mL)160160160

[0141] Example 1.2. Preparation of collagenase solution

[0142] Collagenase (Nordmark, N0002779) was measured to a concentration of 2.85 mg / mL and added to Hartmann's solution (H / S) containing 1% HSA, followed by mixing using a vortex. A specific example of the collagenase solution of the present invention is shown in Table 3. The mixture was centrifuged (300 g, 5 minutes) to remove bubbles and prepared. The collagenase solution prepared as above was prepared and used immediately before use as a 2-fold concentrate.

[0143] Collagenase Solution (2X)Collagenase (mg)71.4142.9214.3285.7HSA (mL)1.252.503.755.00H / S (mL)23.7547.5071.2595.00Total (mL)25.050.075.0100.0

[0144] Example 2. Isolation of stromal vascular fraction from adipose tissue

[0145] Example 2.1. Adipose tissue separation and washing process

[0146] To isolate the stromal vascular fraction (SVF) from adipose tissue, first, only the yellow fat layer of the adipose tissue obtained through liposuction was collected and transferred to 50 mL tubes, followed by centrifugation (300 g, 5 min) (Fig. 1). After centrifugation, the free oil (oil layer) in the upper layer was removed, and only the middle fat layer (fat layer) was collected and transferred to 50 mL tubes.

[0147] The separation solution prepared by the method of Example 1.1 was added to the tube so that the final volume was 50 mL, and centrifuged at 300 g for 5 minutes. After centrifugation, the oil at the top and the tumescent solution at the bottom were removed. The same process as above was performed three times in total, and the washed adipose tissue aspirate was collected in one tube (Fig. 2).

[0148] Example 2.2. Collagenase treatment process

[0149] Lipoaspirate collected in the same manner as in Example 2.1 and collagenase solution (2X) prepared in the same manner as in Example 1.2 were mixed in a 1:1 ratio, gently inverted, and reacted for 1.5 hours at a speed of 30 rpm using a rotator in a 37°C incubator.

[0150] The above reaction solution was centrifuged at 300 g for 10 minutes, and the supernatant was removed to obtain a cell pellet (Fig. 3).

[0151] Example 2.3. Blood cell lysis process

[0152] 1X RBC lysis solution (10 mL) was added to the cell pellet obtained by the method of Example 2.2 above, the pellet was resuspended, and the reaction was performed at room temperature for 10 minutes. At this time, the 1X RBC lysis solution (RBC solution) was prepared by mixing pre-warmed DW (9 mL) and 10X RBC lysis solution (1 mL). The cell suspension after the reaction was centrifuged at 300 g for 5 minutes, and the supernatant was removed to obtain a cell pellet.

[0153] The cell pellet obtained as described above was resuspended by adding the separation solution (5 mL) prepared by the method of Example 1.1, and then 15 mL of the separation solution was added, mixed by inverting, and washed. The suspension was centrifuged (300 g, 5 minutes) to obtain a cell pellet (Fig. 4).

[0154] Example 2.4. Filtration process

[0155] The cell pellet obtained by the method of Example 2.3 was resuspended by adding a separation solution (5 mL), and then filtered using a 70 μm cell strainer (SPL, 93070). The separation solution (10 mL) was added to the filtered suspension as described above, and the supernatant was removed by centrifugation at 300 g for 5 minutes. The cell pellet was resuspended in the separation solution (5 mL), 15 mL of the separation solution was added, centrifuged (300 g, 5 minutes), and the supernatant was removed, and washed (Fig. 4).

[0156] The cell pellet obtained by the above method was resuspended in 1 mL of separation solution, and the cell number was counted to calculate the cell yield.

[0157] Example 3. Confirmation of cell yield

[0158] The cells resuspended in the above Example 2.4 were counted using a LuNA-FX7 cell counter. Cell counting was performed twice.

[0159] As a result, as shown in Fig. 5, it was confirmed that the yield (number of cells / fat (mL)) and viability of adipose tissue-derived SVF were improved when cells were separated using a separation solution containing HSA and NAC (thiol antioxidant) compared to when Hartmann's solution was used alone. In particular, the yield was improved by about 2.41 times and about 3.56 times when a separation solution containing only HSA or NAC was used, respectively, whereas the yield was improved by about 5 times when a separation solution containing both HSA and NAC was used.

[0160] Example 4. Confirmation of differentiation culture and multidifferentiation characteristics of adipose stem cells.

[0161] Adipose stem cells isolated from aspirated fat were cultured in an incubator at 37℃, 5% CO, and the culture medium (α-MEM, Sol Biopharm, CAT# ALP-001-500) was replaced every 2-3 days. This experiment used passage 5 cells to confirm the multipotency, one of the characteristics of adipose stem cells. After inducing differentiation of adipose stem cells into adipocytes, chondrocytes, and osteocytes, respectively, differentiation into each cell type was confirmed through Oil Red O staining, Alizarin Red S staining, and Alcian blue staining.

[0162] Example 4.1. Confirmation of adipocyte differentiation potential

[0163] The adipocyte differentiation potential was confirmed by the following method.

[0164] Specifically, 1×10 in a 12-well plate 4 cells / cm 2 After inoculation, when the cells reached 80-90% confluence, the cell culture medium was replaced with differentiation medium, StemPro™ Adipogenesis Differentiation kit (Thermofisher scientific, CAT# A1007001). During additional culture for 2-3 weeks, differentiation into adipocytes was observed using an optical microscope.

[0165] At 14 and 21 days after the start of differentiation, adipocyte differentiation was confirmed through Oil-red O staining.

[0166] Specifically, after removing the cell medium, the cells were washed with PBS (T&I CAT# BPB-9104) and fixed with 4% paraformaldehyde solution (T&I, CAT# BPP9004) at room temperature for 30 minutes. Then, the cells were washed with DW, treated with 60% isopropanol (DUKSAN, CAT# 856), and reacted for 5 minutes. After that, the cells were treated with Oil-red O staining solution (sigma, CAT# O1391) and reacted for 10-15 minutes at room temperature. Afterwards, the cells were washed with DW (Biosesang, CAT# WR2018) and the stained cells were observed under an optical microscope.

[0167] As a result, as shown in Fig. 7, it was confirmed that the separated adipose stem cells differentiated into adipocytes.

[0168] Example 4.2. Confirmation of chondrocyte differentiation potential

[0169] 1×10 in a 24-well plate 4 cells / cm 2 After inoculation, when the cells reached 80-90% confluence, the cell culture medium was replaced with differentiation medium, StemPro™ Chondrogenesis Differentiation kit (Thermofisher scientific, CAT# A1007101). During culturing for 2-3 weeks, chondrocyte differentiation was confirmed using an optical microscope.

[0170] After 14 and 21 days of differentiation induction, chondrocyte differentiation was confirmed through Alcian blue staining.

[0171] Specifically, cells were fixed in the same manner as in Example 4.12, and then treated with Alcian Blue staining solution (Sigma, CAT# TMS010) and incubated at room temperature for 45 minutes. Thereafter, the cells were washed with PBS and the stained cells were observed under an optical microscope.

[0172] As a result, as shown in Fig. 7, it was confirmed that the separated adipose stem cells differentiated into chondrocytes.

[0173] Example 4.3. Confirmation of osteocyte differentiation potential

[0174] 1×10 in a 24-well plate 4 cells / cm 2 After inoculation, when the cells reached 80-90% confluence, the cell culture medium was replaced with differentiation medium, StemPro™ Osteogenesis Differentiation kit (Thermofisher scientific, CAT# A1007201). During culturing for 2-3 weeks, differentiation into osteocytes was confirmed using an optical microscope.

[0175] At 14 and 21 days after the initiation of differentiation, osteocyte differentiation was confirmed through Alizarin Red S staining. Afterwards, the cells were washed with DW for 3 minutes, and the stained cells were observed under an optical microscope.

[0176] As a result, as shown in Fig. 7, it was confirmed that the separated adipose stem cells differentiated into bone cells.

[0177] II. Freezing and thawing of adipose tissue-derived stem cells

[0178] Example 5.1. Cryopreservation

[0179] Example 5.1. Preparation of cryopreservation solution

[0180] In one specific example of the present invention, a cell cryopreservation solution was prepared by mixing Hartmann's solution (H / S), HSA (human serum albumin), DMSO, and Hartmann's solution in the ratios shown in Table 4 below. At this time, HSA and NAC were prepared by mixing them to have a final concentration of 10% and 24.5 mM (approximately 4 mg / mL), respectively, and DMSO was prepared to have a final concentration of 5%. In addition, a cryopreservation solution containing HSA or NAC alone was also prepared.

[0181] Cryopreservative compositionNAC+HSANACHSANAC (mL)0.040.04-HSA (mL)0.5-0.5DMSO (mL)0.050.050.05H / S (mL)0.410.910.45Total (mL)111

[0182] Example 5.2. Cryopreservation process

[0183] After centrifuging (300 g, 5 minutes) the cells counted by the method of Example 3 above, 1X10 7 The cell pellet was resuspended by adding cryopreservation solution to a concentration of 1 cell / mL / vial. The cell suspension was dispensed into cryovials (NUNC, 368632), placed in cryocontainers (Corning, CLS432001), and frozen overnight in an ultra-low temperature freezer at -80°C. The following day, the frozen cells were transferred to a nitrogen tank and stored.

[0184] Example 6. Thawing frozen cells

[0185] Hartmann's solution (H / S) (washing solution, 10 mL) containing HSA at a 5% concentration, warmed in a BSC, was dispensed into a 50 mL tube, and the cells were thawed (3 minutes) at 37°C and mixed dropwise into the prepared wash solution. The mixture was centrifuged (300 g, 5 minutes), the supernatant was removed, and the cell pellet was resuspended in 1 mL of wash solution and the cells were counted. The cell counting was performed in the same manner as in Example 3.

[0186] As a result, as shown in Fig. 6, when using a cryopreservation solution containing HSA or HSA and NAC, a recovery rate of 80% or more was observed in all cases. In particular, when using a cryopreservation solution containing HSA and NAC, a high recovery rate of approximately 90% was observed.

Claims

1. A composition for cell separation comprising N-acetylcysteine represented by the following chemical formula I or a pharmaceutically acceptable salt thereof: <Chemical Formula I> .

2. In paragraph 1, The above composition is a cell separation composition further comprising a buffer.

3. In paragraph 2, A composition for cell separation, wherein the composition further comprises albumin.

4. In paragraph 1, A composition for cell separation, characterized in that the concentration of the N-acetylcysteine or a pharmaceutically acceptable salt thereof is 0.01 mg / mL to 10 mg / mL.

5. In paragraph 3, A composition for cell separation, characterized in that the concentration of the albumin is 0.1% (v / v) to 20% (v / v).

6. In paragraph 2, A composition for cell separation, wherein the buffer comprises at least one selected from the group consisting of calcium chloride, potassium chloride, sodium chloride, and sodium lactate.

7. In paragraph 2, A composition for cell separation, characterized in that the pH of the buffer is 4.0 to 8.

0.

8. In paragraph 2, A composition for cell separation, characterized in that the osmolarity of the buffer is 200 mOsm / L to 400 mOsm / L.

9. In paragraph 1, A composition for cell separation, wherein the cells are derived from adipose tissue.

10. In paragraph 9, A composition for cell separation, wherein the above cells are adipose tissue-derived stem cells.

11. A method for isolating cells derived from adipose tissue, comprising the following steps: i) a step of washing the adipose tissue separated from the subject by mixing it with the cell separation composition of claim 1; and ii) A step of treating the washed adipose tissue with a tissue dissociation enzyme to obtain a fraction containing cells.

12. In paragraph 11, A method for isolating cells derived from adipose tissue, wherein step ii) of the above method further includes a washing step.

13. In paragraph 11, A method for isolating cells derived from adipose tissue, wherein the tissue dissociation enzyme of step ii) is selected from the group consisting of collagenase, dispase, and a combination thereof.

14. In paragraph 13, A method for isolating cells derived from adipose tissue, wherein the collagenase is treated at a concentration of 0.1 unit / mL to 0.5 unit / mL.

15. In paragraph 13, A method for isolating cells derived from adipose tissue, wherein the above dispase is treated at a concentration of 1.0 unit / mL to 2.5 unit / mL.

16. In paragraph 11, The above method further comprises the following steps: iii) a step of removing blood cells by adding a blood cell hemolysis solution to the fraction containing adipose tissue-derived cells of clause 11; and / or iv) A step of filtering the cell fraction from which the blood cells have been removed.

17. In paragraph 16, A method for isolating cells derived from adipose tissue, wherein steps iii) and iv) of the above method further include a washing step.

18. In paragraph 12 or paragraph 17, A method for isolating cells derived from adipose tissue, wherein the washing is performed with the cell separation composition of claim 1.

19. In Article 16, A method for isolating cells derived from adipose tissue, wherein the above filtration is performed through a filter.

20. In paragraph 19, A method for isolating cells derived from adipose tissue, wherein the filter has a size of 50 um to 150 um.

21. In paragraph 11, A method for isolating adipose tissue-derived cells, wherein the adipose tissue-derived cells include adipose tissue-derived stem cells.

22. A kit for cell separation comprising N-acetylcysteine represented by the following chemical formula I or a pharmaceutically acceptable salt thereof: <Chemical Formula I> .

23. In paragraph 22, A kit for cell separation, wherein the above kit further comprises a buffer.

24. In paragraph 22, The above kit is a cell separation kit further comprising albumin.

25. A composition for cell cryopreservation comprising N-acetylcysteine represented by the following chemical formula I or a pharmaceutically acceptable salt thereof: <Chemical Formula I> .

26. In paragraph 25, A composition for cell cryopreservation, wherein the composition for cell cryopreservation further comprises DMSO.

27. In paragraph 26, A composition for cell cryopreservation, wherein the composition for cell cryopreservation further comprises albumin.

28. In paragraph 25, A composition for cell cryopreservation, wherein the composition for cell cryopreservation further comprises a buffer.

29. In paragraph 25, A composition for cell cryopreservation, characterized in that the concentration of the N-acetylcysteine or a pharmaceutically acceptable salt thereof is 0.01 mg / mL to 10 mg / mL.

30. In paragraph 26, A composition for cell cryopreservation, characterized in that the concentration of the DMSO is 0.001% (v / v) to 10% (v / v).

31. In paragraph 27, A composition for cell cryopreservation, characterized in that the concentration of the albumin is 0.1% (v / v) to 20% (v / v).

32. In paragraph 28, A composition for cell cryopreservation, wherein the buffer comprises at least one selected from the group consisting of calcium chloride, potassium chloride, sodium chloride, and sodium lactate.

33. In paragraph 28, A composition for cell cryopreservation, characterized in that the pH of the buffer is 4.0 to 8.

0.

34. In paragraph 28, A composition for cell cryopreservation, characterized in that the osmolarity of the buffer is 200 mOsm / L to 400 mOsm / L.

35. In paragraph 25, A composition for cryopreservation of cells, wherein the cells are derived from adipose tissue.

36. In paragraph 35, A composition for cryopreservation of cells, wherein the cells are adipose tissue-derived stem cells. 37.i) a step of mixing cells with the cell cryopreservation composition of clause 25; and ii) A method for cryopreserving cells, comprising the step of cryopreserving the mixture.

38. In paragraph 37, A method for cryopreserving cells, wherein the cells are derived from adipose tissue.

39. In paragraph 38, A method for cryopreserving cells, wherein the cells are adipose tissue-derived stem cells.

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