WO2019026910A1 - 凍結保存用組成物、凍結保存物の製造方法、細胞製剤、細胞製剤の製造方法、凍結保存用キット - Google Patents
凍結保存用組成物、凍結保存物の製造方法、細胞製剤、細胞製剤の製造方法、凍結保存用キット Download PDFInfo
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0205—Chemical aspects
- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
- A01N1/0221—Freeze-process protecting agents, i.e. substances protecting cells from effects of the physical process, e.g. cryoprotectants, osmolarity regulators like oncotic agents
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0278—Physical preservation processes
- A01N1/0284—Temperature processes, i.e. using a designated change in temperature over time
Definitions
- the present invention relates to a composition for cryopreservation, a method for producing a cryopreserved substance, a cell preparation, a method for producing a cell preparation and a kit for cryopreservation.
- Patent Document 1 describes that scaffold-type cell preparations can be frozen by devising the composition and form of nanofibers and collagen sheets used as scaffolds.
- Patent Document 2 discloses a cell suspension-type cell therapeutic drug provided in a cryopreservation form in which autologous serum and DMSO are added to a cryopreservation solution.
- Japanese Patent Publication Japanese Patent Application Laid-Open No. 2015-198604
- Japanese Patent Publication Japanese Patent Publication "Japanese Patent Application Laid-Open No. 2009-107929”
- One aspect of the present invention has been made based on the above circumstances, and it is an object of the present invention to provide a cryopreservation composition which is less likely to produce dead cells and can be cryopreserved with good quality. Do.
- the composition for cryopreservation is a composition for cryopreservation for cryopreservation of cells, and contains a fatty acid.
- composition for cryopreservation is a composition for cryopreservation for cryopreservation of cells, wherein the cells form a cell mass having a three-dimensional structure. It may be for cryopreservation of the cell mass and comprises at least one component selected from the group consisting of fatty acids and fatty acid esters.
- the method for producing a cryopreserved material is a method for producing a cryopreserved product in which cells are cryopreserved, and is characterized by including the following steps (a) and (c) in this order: It is a manufacturing method of frozen preservation thing which is said.
- the method for producing a cryopreserved material is a method for producing a cryopreserved product in which cells are cryopreserved, wherein the cells form a cell mass having a three-dimensional structure.
- it is a method of producing a cryopreserved article characterized by including the following steps (a) and (c) in this order:
- a cell preparation according to an aspect of the present invention contains cells and a composition for cryopreservation containing a fatty acid, and is cryopreserved.
- a cell preparation according to one aspect of the present invention comprises a cell and a composition for cryopreservation comprising at least one component selected from the group consisting of fatty acids and fatty acid esters, and the cells have a three-dimensional structure.
- the cell mass may be formed and cryopreserved.
- the method for producing a cell preparation is a method for producing a cell preparation containing cells, and the cells may form a cell mass having a three-dimensional structure. It is a manufacturing method characterized by including the following processes (a) and (c) in this order.
- a method for producing a cell preparation is a method for producing a cell preparation containing cells, comprising the following steps (a) and (c) in this order: Manufacturing method.
- the kit for cryopreservation which concerns on 1 aspect of this invention is a kit for cryopreservation for cryopreservation of a cell, Comprising: A fatty acid is provided.
- kits for cryopreservation are kits for cryopreservation for cryopreservation of cells, which is a kit for cryopreservation of the cells having formed a cell mass having a three-dimensional structure. It is a kit for cryopreservation characterized by comprising at least one component selected from the group consisting of fatty acids and fatty acid esters.
- the generation of dead cells is less likely to occur after thawing, and it is possible to achieve high quality cryopreservation.
- FIG. 1 shows the results of the cryopreservation experiment. It is a figure which shows the result of the cryopreservation experiment of MSC as cell suspension. It is a figure which shows the result of the cryopreservation experiment of MSC as cell suspension. It is a figure which shows the result of the cryopreservation experiment of MSC as cell suspension. It is a figure which shows the result of the cryopreservation experiment of MSC as cell suspension.
- FIG. 6 shows the confirmation result of bone differentiation ability
- B is a figure which shows the confirmation result of fat differentiation ability.
- FIG. 7 shows the results of Example 3 (gMSC (registered trademark) 1). It is a graph which shows the result of the cryopreservation experiment of gMSC (registered trademark) 1. It is a graph which shows the result of the cryopreservation experiment of gMSC (registered trademark) 1.
- composition for cryopreservation is a composition for cryopreservation for cryopreservation of cells, and contains a fatty acid.
- the composition for cryopreservation according to one aspect of the present invention is a composition for cryopreservation for cryopreservation of cells, wherein the cells form a cell mass having a three-dimensional structure. It may be for cryopreservation of the cell mass, and may comprise at least one component selected from the group consisting of fatty acids and fatty acid esters.
- the composition for cryopreservation when simply referred to as “the composition for cryopreservation”, “the composition for cryopreservation for cryopreservation of cells” and “the composition for cryopreservation for cryopreservation of cells”, wherein the cells are It forms a cell mass that is a three-dimensional structure, and refers to any of the composition for cryopreservation for cryopreserving the cell mass.
- the composition for cryopreservation according to one aspect of the present invention it may be one aspect of the composition for cryopreservation for cryopreservation of cells suspended in a suspension, and has a three-dimensional structure. It may also be one aspect of a cryopreservation composition for cryopreserving cells forming a certain cell mass.
- mesenchymal stem cells are described here as an example, but cells to be cryopreserved are not limited to mesenchymal cells.
- the cells such as mesenchymal stem cells are well frozen with good recovery rate and viability, and cells obtained after cryopreservation and thawing The number was also found to be good.
- a composition for cryopreservation comprising at least one component selected from the group consisting of fatty acids and fatty acid esters, cells forming a cell mass having a three-dimensional structure have a good recovery rate and survival. It was found that the ratio was well frozen and that the recovery rate and survival rate after freezing and thawing were also good.
- the “recovery rate” is the ratio of the total number of cells (whether alive or dead) to the number of cells before cryopreservation and thawing.
- the total cell number refers to the number of cells recovered by cryopreservation and thawing without breaking, outflow, and the like.
- the "survival rate” is the ratio of the number of surviving cells to the number of all cells recovered after cryopreservation and thawing.
- the quality of cells after thawing was normal, and that the properties of the cells were not changed even by cryopreservation and thawing.
- the composition for cryopreservation according to one aspect of the present invention does not use a component that is not present in animal cells such as trihalos, polyethylene glycol, polylysine, etc., and there is a significant difference between lots at risk of biological contamination.
- the safety and the quality are also excellent because they can be suitably cryopreserved without using serum.
- freeze storage refers to freezing and storing cells, but preferably refers to freezing and storage in a very low temperature environment such as ⁇ 80 ° C. or less.
- Target cells to be frozen with the composition for cryopreservation include, for example, mesenchymal stem cells, CD34 cells, embryonic stem cells (ES cells), iPS (induced pluripotent stem cells) cells, chondrocytes, Examples include osteoblasts, fibroblasts, epidermal cells, epithelial cells, adipocytes, liver cells, pancreatic cells, muscle cells, nerve cells, neural stem cells, hematopoietic stem cells, and precursor cells thereof. Alternatively, the cells may be positive for a desired marker.
- the species from which the cell is derived is also not particularly limited, and examples of the species include microorganisms, non-human mammals, and humans.
- the cells may be cultured by a conventionally known method.
- cultivates the said cell is suitably selected according to the cell to culture.
- a culture vessel suitable for cell growth is also selected appropriately depending on the cells to be cultured.
- “cells” in another aspect of the present invention (a method for producing a cryopreserved product, a cell preparation, a method for producing a cell preparation, a kit for cryopreservation) described below are also based on the description herein.
- mesenchymal stem cells A mesenchymal stem cell (Mesenchymal stem cell, hereinafter also referred to as “MSC”), which is an example of a target to be frozen by the composition for cryopreservation according to one embodiment of the present invention, will be described.
- MSC mesenchymal stem cell
- Mesenchymal stem cells are considered to be one of the most promising cells in regenerative medicine due to their advantages such as the ability to differentiate into cells belonging to the mesenchymal system and also having an immunosuppressive action.
- mesenchymal stem cells refer to somatic stem cells that differentiate into tissues belonging to the mesenchymal system.
- mesenchymal stem cells those from which mesenchymal stem cells further having specific properties are isolated, those from which some kind of stimulation such as cytokine stimulation is applied to mesenchymal stem cells, and those into which genes are introduced are included.
- MUSE cells, MAPC cells, SP-1 cells and the like are also included.
- Mesenchymal stem cells have proliferation ability and differentiation ability to bone cells, chondrocytes, muscle cells, stromal cells, tendon cells, adipocytes and the like.
- mesenchymal stem cells are preferably human mesenchymal stem cells, but they may be mesenchymal stem cells derived from non-human animals such as rats and mice.
- Mesenchymal stem cells may be cultured by a conventionally known method, preferably serum-free culture or low-serum culture, and more preferably serum-free culture. If serum-free culture, culture components are known. In other words, since serum is derived from natural components, differences in components occur between lots, but such differences do not occur in serum-free media. Therefore, serum-free cultured mesenchymal stem cells are excellent in safety and quality. In addition, it is possible to minimize risks such as biological contamination and contamination of immunogenic substances, and to minimize substances that are not present in the body. In addition, since the biological material to be contained is clear, quality control is easy.
- mesenchymal stem cells cultured in a serum-free manner in a part of serum-free medium such as STK (registered trademark) medium are excellent in proliferation rate.
- the cells cultured in the serum-free culture as described above are subjected to freezing. Therefore, even after repeated passaging, the mesenchymal stem cells in such a good state are in a youthful state where aging does not occur, such as generation of pseudopods or change to a flat shape.
- mesenchymal stem cells can be cultured and used at an excellent proliferation rate even after cryopreservation and thawing.
- serum free culture is intended to be culture that does not use serum. For example, it is intended to culture using a serum free medium, which is a medium free of serum.
- low serum culture refers to culture using a medium containing a smaller amount of serum than general serum-containing medium (eg, medium containing 10% FBS), and general serum-containing medium It is intended that the culture period using a serum-containing medium is shorter than the culture.
- the basal medium for constructing the serum-free medium is not particularly limited as long as it is a medium for animal cells well known in the art, and preferable basal media include, for example, Ham's F12 medium, DMEM medium, RPMI-1640 medium , MCDB medium and the like. These basal media may be used alone or in combination of two or more.
- a basal medium for constituting a serum-free medium is preferably a medium in which MCDB and DMEM are mixed at a ratio of 1: 1.
- a serum-free medium obtained by adding FGF, PDGF, TGF- ⁇ , HGF, EGF, at least one phospholipid, and at least one fatty acid to the above-described basal medium may be used in the growth step.
- the content of FGF relative to the basal medium is preferably 0.1 to 100 ng / ml, more preferably 3 ng / ml, at a final concentration.
- the content of PDGF relative to the basal medium is preferably 0.5 to 100 ng / ml, more preferably 10 ng / ml, at a final concentration.
- the content of TGF- ⁇ in the basal medium is preferably 0.5 to 100 ng / ml, more preferably 10 ng / ml, at a final concentration.
- the content of HGF relative to the basal medium is preferably 0.1 to 50 ng / ml, more preferably 5 ng / ml, at a final concentration.
- the content of EGF relative to the basal medium is preferably 0.5 to 200 ng / ml, more preferably 20 ng / ml, at a final concentration.
- the total content of phospholipids to the basal medium is preferably 0.1 to 30 ⁇ g / ml at final concentration, more preferably 10 ⁇ g / ml.
- the total content of fatty acids relative to the basal medium is preferably 1/1000 to 1/10 of the basal medium, more preferably 1/100.
- Serum-free media may contain phospholipids.
- phospholipids include phosphatidic acid, lysophosphatidic acid, phosphatidyl inositol, phosphatidyl serine, phosphatidyl ethanolamine, phosphatidyl choline, phosphatidyl glycerol and the like.
- the phospholipid may be contained alone or in combination.
- the serum-free medium may contain a combination of phosphatidic acid and phosphatidyl choline, and these phospholipids may be of animal or plant origin. .
- Serum-free media may contain fatty acids.
- fatty acids include linoleic acid, oleic acid, linolenic acid, arachidonic acid, myristic acid, palmitic acid, palmitic acid, stearic acid and the like, and the culture medium additive according to the present embodiment contains only these fatty acids. It may be contained or in combination.
- the serum-free medium according to this embodiment may further contain cholesterol in addition to the above-mentioned fatty acids.
- FGF is intended a growth factor selected from the fibroblast growth factor (FGF) family, preferably FGF-2 (bFGF), although It may be selected from other FGF families such as -1.
- FGF fibroblast growth factor
- PDGF is intended a growth factor selected from the platelet derived growth factor (PDGF) family and is preferably PDGF-BB or PDGF-AB .
- TGF- ⁇ is a TGF- ⁇ 1 intended growth factor selected from the transforming growth factor- ⁇ (TGF- ⁇ ) family. Although preferred, it may be selected from other TGF- ⁇ families.
- HGF is intended a growth factor selected from the hepatocyte growth factor family and EGF is selected from the epidermal growth factor (EGF) family Growth factors are contemplated.
- EGF epidermal growth factor
- the serum-free medium is at least 2 selected from the group consisting of connective tissue growth factor (CTGF), connective tissue growth factor (VEGF) and ascorbic acid compounds. It may further contain one factor.
- CTGF connective tissue growth factor
- VEGF connective tissue growth factor
- ascorbic acid compounds may further contain one factor.
- Ascorbic acid compounds as used herein are contemplated as ascorbic acid (vitamin C) or ascorbic acid diphosphate, or compounds similar thereto.
- the above-mentioned growth factor contained in the serum-free medium may be natural or may be one produced by genetic recombination.
- the serum-free medium preferably contains a lipid antioxidant.
- the lipid antioxidant contained in the serum free medium may be DL- ⁇ -tocopherol acetate (Vitamin E).
- the serum free medium may also further contain a surfactant.
- the surfactant contained in the serum free medium may be Pluronic F-68 or Tween 80.
- the serum free medium may further contain insulin, transferrin and selenate.
- insulin may be an insulin-like growth factor and may be of natural cell origin or recombinantly produced.
- the culture medium additive according to the present invention may further contain dexamethasone or another glucocorticoid.
- mesenchymal stem cells isolated by conventional methods from animal tissues or cells such as humans are seeded in the above-described serum-free medium, and cultured until they grow to a desired number.
- culture conditions it is preferable to inoculate 1 to 2 ⁇ 10 4 mesenchymal stem cells to 1 ml of culture medium, culture temperature is 37 ° C. ⁇ 1 ° C., culture time is 48 to 96 hours, and 5% CO 2 It is preferable to be below. By culturing in this manner, it is possible to efficiently obtain a large amount of mesenchymal stem cells that maintain or improve the immunosuppressive ability.
- the culture container used for culture is not particularly limited as long as mesenchymal stem cells can be proliferated.
- a Falcon 75 cm 2 flask, a Sumitomo Bakelite 75 cm 2 flask, etc. can be suitably used.
- cell growth may be affected by the type of culture vessel used. Therefore, in order to proliferate mesenchymal stem cells more efficiently, a culture vessel suitable for proliferation is used for each mesenchymal stem cell to be proliferated in the proliferation step (hereinafter also referred to as "proliferation target cell"). It is preferable to carry out the proliferation step.
- a method of selecting a culture vessel suitable for the growth of the cells to be grown for example, a method of selecting an optimal culture vessel as the cells to be grown can be mentioned. Specifically, a plurality of types of culture vessels are prepared, cells to be grown are grown under the same culture conditions except for different types of culture vessels, and the number of cells two weeks after the start of culture is measured by a known method In addition, it can be judged that the culture vessels are suitable for the proliferation of the cells to be proliferated in descending order of cell number.
- the growth period of the cells to be grown in ascending order of time to reach 80 to 90% of confluent cell number It can be determined that it is a suitable culture vessel.
- the cell adhesion molecule is further contained.
- cell adhesion molecules include fibronectin, collagen, gelatin and the like. These cell adhesion molecules may be used alone or in combination of two or more.
- the content of cell adhesion molecules to the serum-free medium is preferably 1 to 50 ⁇ g / ml, more preferably 5 ⁇ g / ml, at a final concentration.
- the adhesion efficiency of the proliferation target cells to the culture vessel can be improved by adding the final concentration of fibronectin to the serum-free medium to 5 ⁇ g / ml. .
- mesenchymal stem cells may be passaged at least once. Since mesenchymal stem cells proliferate in a anchorage-dependent manner, culture conditions can be increased by passaging mesenchymal stem cells in the middle of the proliferation step when mesenchymal stem cells are locally biased and proliferated, etc. It can be improved.
- the passaging method for mesenchymal stem cells is not particularly limited, and passaging can be performed using a conventionally known passaging method for mesenchymal stem cells. Since the state of mesenchymal stem cells after passaging is good, in the above-mentioned proliferation step, when passaging, the above mesenchymal stem cells using a cell exfoliating agent which does not contain components derived from mammals and microorganisms It is preferable to peel off. Examples of the above-mentioned "cell release agent not containing components derived from mammals and microorganisms" include TrypLE Select CTS (Thermo Fisher Scientific Inc.), ACCUTASE (Innovative Cell Technologies, Inc.), and the like.
- the processed product of the cells to be frozen by the composition for cryopreservation may be in any form, for example, a suspension of cells, one cultured in a sheet form, A cell mass of a three-dimensional structure etc. are mentioned. Among them, mesenchymal stem cells forming a three-dimensional cell mass are more preferable, and mesenchymal stem cells forming a scaffold-free cell mass are more preferable.
- the cell mass is preferably composed only of mesenchymal stem cells, but may contain polysaccharides such as collagen and hyaluronic acid.
- collagen or polysaccharide such as hyaluronic acid is contained, it is preferably 0.1 to 50% (v / v) of the cell mass.
- the cell mass may or may not be subjected to treatment (equilibration) to make the cell mass penetrate the cryopreservation solution which is one aspect of the composition for cryopreservation of the present invention before it is frozen.
- treatment equilibration
- the cell mass of a three-dimensional structure can also be frozen suitably.
- a cell suspension is administered to the affected area, it is reported that even mesenchymal stem cells that are said to have immunoprivileged migration from the affected area are considered to be administered by the host immune cells. There are reports that it does not stay in the affected area.
- a cell mass of a three-dimensional structure it is possible to prevent cells from migrating and dropping out from the affected area, and exert a therapeutic effect for a long time.
- the cell preparation in a form processed into a three-dimensional structure is extremely difficult to cryopreserve the cell suspension type of cell preparation.
- the size and thickness of a cell mass to be frozen increase as in a three-dimensional structure, it becomes difficult to uniformly freeze the inside from the viewpoint of heat conduction.
- the cell mass of three-dimensional structure one obtained by processing the cell mass into a three-dimensional structure by a conventionally known method may be used.
- a “three-dimensional structure” when referred to a cell mass, a matrix is three-dimensionally oriented, and a cell in which cells are three-dimensionally arrayed and retains intercellular binding and orientation refers to an object that extends in three dimensions including
- the shape of the three-dimensional structure may be determined, for example, according to the purpose of treatment.
- the area, thickness, and strength may be set as appropriate depending on the part to be transplanted, and those skilled in the art can set the size as appropriate.
- This size can be set according to the environment to be transplanted.
- Cell clusters of small size have the merit of being able to be injected into a body cavity with an injection needle.
- there is an advantage that it is easy to administer a sufficient number of cells for example, because it is easy to handle, for example, it is easy to grip with pincent at the time of surgery.
- a certain size is, for example, 1 cm 2 or more, preferably 2 cm 2 or more, more preferably 3 cm 2 or more. More preferably, it is 4 cm 2 or more, 5 cm 2 or more, 6 cm 2 or more, 7 cm 2 or more, 8 cm 2 or more, 9 cm 2 or more, 10 cm 2 or more, 15 cm 2 or more Or 20 cm 2 or more, and may be, for example, 40 cm 2 or less, 30 cm 2 or less, 20 cm 2 or less, but is not limited thereto, and the area is 1 cm 2 or less or 40 cm 2 or more depending on the application It can be.
- the size is preferably 2 mm 3 or more, more preferably 40 mm 3 or more, and may be, for example, 40 cm 3 or less, or 20 cm 3 or less, but is not limited thereto. It may be 2 mm 3 or less.
- the sufficient thickness in the implantable artificial tissue may vary depending on the portion intended for transplantation, but one skilled in the art can appropriately set the thickness.
- This thickness can be set according to the environment to be implanted. May exceed 5 mm.
- the above, more preferably 3 mm or more, further preferably 5 mm or more is intended.
- it when applied to bones, cartilages, ligaments, tendons, etc., it may be, for example, 1 mm or more, preferably 2 mm or more, more preferably 3 mm or more, still more preferably 5 mm or more, like the heart. Further, in any case, it may be 1 mm or less, 10 mm or less, or 5 mm or less.
- the number of cells constituting the cell mass may be appropriately selected, and may be, for example, 50 to 200 masses, or 1,000,000 to 100,000,000 mass.
- the mass may be a small mass or a large mass.
- the migration of cells from the affected area can be more effectively prevented, and the therapeutic effect is exhibited for a longer time.
- the composition for cryopreservation which concerns on 1 aspect of this invention is used, even if it is a cell mass of such a size, a dead cell is less likely to be generated after thawing, and the property of a cell is also after cryopreservation and thawing. Allows cryopreservation without change.
- the cell mass of mesenchymal stem cells is preferably transplanted in an undifferentiated state where differentiation is not induced.
- it is finely cut Is preferred. It is because the differentiation-inducing medium more easily penetrates into the inside of the tissue when the cell mass is finely cut. It does not specifically limit as a method used in order to cut
- the size of the cell mass is more preferably about 10 mg to 20 mg.
- the cell mass to be frozen with the composition for cryopreservation according to one aspect of the present invention is a scaffold-free three-dimensional structure.
- natural products such as collagen may be used as scaffolds.
- Such natural products differ in ingredients from lot to lot.
- the components are known by being a scaffold-free three-dimensional structure, it is excellent in safety and quality stability.
- natural products as described above are at risk of biological contamination and inclusion of immunogenic substances. Such a risk can be reduced by being a scaffold-free three-dimensional structure.
- methods of cryopreserving even three-dimensional cell masses are being developed.
- these methods devise the composition and form of nanofibers or collagen sheets used as scaffolds. Therefore, it can not be used in principle for "scaffold free" cell preparations.
- such scaffold-free three-dimensional cell mass is also less likely to produce dead cells after thawing, and is cryopreserved without changing cell properties by freezing and thawing. it can.
- a method of obtaining a scaffold-free three-dimensional cell aggregate including mesenchymal stem cells as an example of a freezing target in the present invention for example, a method using a conventionally known low adhesion plate, micropattern surface plate, etc. And, a hanging drop method can be adopted. Moreover, you may produce using the method as described in Japanese Patent 4522994. In addition, commercially available products may be used, and for example, gMSC (registered trademark) 1 (manufactured by Two Cell Co., Ltd.) can be suitably used.
- the composition for cryopreservation according to one aspect of the present invention contains a fatty acid.
- the cryopreservation composition can freeze cells with high viability, and does not cause changes in cell properties after thawing.
- fatty acids examples include linoleic acid, oleic acid, linolenic acid, arachidonic acid, myristic acid, palmitic acid, palmitic acid and stearic acid.
- fatty acid it is preferable that as the fatty acid, at least one of linoleic acid and linolenic acid is contained in the composition for cryopreservation.
- the composition for cryopreservation can freeze mesenchymal stem cells with higher viability.
- short chain fatty acid, medium chain fatty acid, long chain fatty acid may be used.
- highly unsaturated fatty acids may be used. These may be individual or multiple types may be mixed. Among them, a mixture of a plurality of fatty acids is preferable, and a mixture in which the type and the amount of the contained fatty acids are specified is more preferable.
- Chemically defined lipid concentrate manufactured by Thermo Fisher Scientific Inc., product number 11905-031
- CD lipid registered trademark
- the composition of the stock solution of CD lipid is as follows.
- the fatty acids exemplified above may be used alone or in combination of two or more, but it is more preferable to use a mixture of two or more.
- the survival rate is better, mesenchymal stem cells can be frozen, and the change in cell properties after thawing does not occur.
- the content of the fatty acid in the composition for cryopreservation according to one aspect of the present invention is not particularly limited, and for example, the final concentration is 0.01 ⁇ g / ml to 500 ⁇ g / ml with respect to the total amount of the composition for cryopreservation Is preferred. For example, 1/1000 to 1/10 (v / v) is more preferable with respect to the total amount of the composition for cryopreservation.
- CD lipid registered trademark
- 1/1000 to 1/10 (v / v) PA: 0.5 to 100 ⁇ g / ml, PC: 0) relative to the total amount of the composition for cryopreservation .5 to 100 ⁇ g / ml
- the fatty acid content is preferably higher in the above range. That is, it is more preferable that fatty acid be contained abundantly in the composition for cryopreservation, and it is more preferable to be rich in various fatty acids. Thereby, the survival rate is better, mesenchymal stem cells can be frozen, and there is no change in cell properties after thawing.
- the composition for cryopreservation according to one aspect of the present invention contains a fatty acid ester.
- the composition for cryopreservation can freeze cells with high viability.
- fatty acid esters examples include phospholipids and neutral fats. Among them, it is preferable that a phospholipid is contained in the composition for cryopreservation.
- Phospholipids include, for example, phosphatidic acid (hereinafter, the sodium salt of phosphatidic acid is referred to as PA, and when simply referred to as “phosphatidic acid", salts thereof are also included), lysophosphatidic acid, Phosphatidyl inositol, phosphatidyl serine, phosphatidyl ethanolamine, phosphatidyl choline (hereinafter referred to as PC), and phosphatidyl glycerol may be mentioned.
- phosphatidic acid hereinafter, the sodium salt of phosphatidic acid is referred to as PA, and when simply referred to as “phosphatidic acid”, salts thereof are also included
- lysophosphatidic acid Phosphatidyl inositol
- phosphatidyl serine phosphatidyl serine
- phosphatidyl ethanolamine phosphatidyl choline
- PC phosphatidyl gly
- the final concentration in the cryopreservation composition at the time of use is 0.01 ⁇ g / ml to 500 ⁇ g / ml with respect to the total amount of the cryopreservation composition Is preferred.
- composition for cryopreservation may further contain a substance that aids in the water solubility (emulsification) of a fatty acid or fatty acid ester, such as a surfactant.
- a surfactant may be Pluronic F-68 or Tween 80.
- the composition for cryopreservation which further contains both a fatty acid or at least any one of a fatty acid ester and a surfactant.
- a composition for cryopreservation containing phosphatidic acid and Pluronic F-68 is preferable.
- the components of the composition for cryopreservation according to one aspect of the present invention described above may contain a lyoprotectant, which suppresses the growth of ice crystals in cells during the freezing and thawing process.
- a lyoprotectant dimethyl sulfoxide
- the amount is more preferably 0.5% to 50% (v / v) with respect to the total amount of the composition for cryopreservation.
- composition for cryopreservation may contain components other than fatty acids, and examples thereof include a basal medium, a thickener, a pH adjuster, a lyoprotectant and the like.
- composition for cryopreservation more preferably contains insulin, albumin, transferrin.
- Insulin, albumin, transferrin can enhance the action of fatty acids.
- the concentration when these are added is preferably such that the final concentration in the cryopreservation solution at the time of use is 0.5 ⁇ g / ml to 500 ⁇ g / ml.
- composition for cryopreservation which concerns on 1 aspect of this invention should just mix each component mentioned above, such as a fatty acid, suitably.
- the cryopreservation composition according to one aspect of the present invention can be provided in various forms. For example, it may be a liquid, or a solid such as a powder or a tablet. When provided as a solid, the user may appropriately dissolve in a solvent and use it as a cryopreservation solution, but the composition for cryopreservation according to one embodiment of the present invention may be used together with an instruction specifying a suitable solvent. It may be provided.
- the cryopreservation method is a method for producing a cryopreserved product in which cells are cryopreserved, and includes the following steps (a) and (c) in this order.
- a cryopreservation method is a method for producing a cryopreserved article in which cells are cryopreserved, wherein the cells form a cell mass having a three-dimensional structure, The steps (a) and (c) are included in this order.
- cryopreserved refers to a cryopreserved substance.
- the cryopreservation solution used in the method of producing a cryopreserved product according to one aspect of the present invention contains at least one component selected from the group consisting of fatty acids and fatty acid esters.
- the cryopreservation solution may use a liquid form of the composition for cryopreservation in the above-mentioned one aspect of the present invention, and is a solid form among the composition for cryopreservation in one aspect of the present invention As for things, a solution obtained by dissolving the solid in a solvent may be used.
- step (a) cells to be frozen are immersed in a cryopreservation solution containing fatty acid.
- a method of immersing for example, it is preferable to put a cryopreservation solution in a container that can be cryopreserved and put cells in the cryopreservation solution. It is because freezing of the below-mentioned (c) process can be performed as it is.
- the ratio of the amount of the cryopreservation solution to the cells to be frozen upon immersion is not particularly limited as long as it can be cryopreserved, for example, about 1: 1 to 1: 1000 (50 cells / cell volume). (approximately 100 ml / ml). With such an amount, cells can be sufficiently impregnated with the cryopreservation solution. Moreover, when using the cell mass of the above-mentioned three-dimensional structure, a cryopreservation solution can fully permeate a cell mass.
- the amount of the cryopreservation solution to the cells is reduced from the state in which the cells are immersed in the cryopreservation solution (b). It is more preferable to freeze the said cell after the said (b) process in the (c) process mentioned later including a process.
- the thawing time after storage can be made short, which can further improve the cell recovery rate by cryopreservation, the total cell number, and the survival rate.
- the cryopreservation solution may be removed from the cryopreservation solution containing mesenchymal stem cells, or the mesenchymal stem cells may be removed.
- the mesenchymal stem cells it is more preferable that the mesenchymal stem cells be not immersed in the cryopreservation solution. As a result, the thawing time can be further shortened, and the cell recovery rate by cryopreservation, the total cell number, and the survival rate can be further improved.
- cryopreservation solution is stored in a container in which the mesenchymal stem cells are stored. May remain.
- the cryopreservation solution may be aspirated from a container stored with mesenchymal stem cells immersed in the cryopreservation solution, or mesenchymal stem cells may be removed.
- a conventionally known suction device such as a pipette may be used.
- step (c) mesenchymal stem cells are frozen.
- the freezing temperature may be appropriately set to a temperature at which the mesenchymal stem cells to be frozen are frozen, and examples thereof include ⁇ 80 ° C. or less, or ⁇ 196 ° C. or less.
- freezing at ⁇ 80 ° C. or less for example, conventionally known examples may be used, and in the case of freezing at ⁇ 196 ° C. or less, liquid nitrogen may be used.
- the container used for cryopreservation should just be able to withstand freezing temperature. For example, it is preferable to be able to withstand -80 ° C, and more preferable to be able to withstand -196 ° C.
- a container made of a synthetic resin such as polyethylene, polypropylene or polyethylene terephthalate is more preferable.
- the container may be, for example, a commercially available cryovial (freeze vessel).
- the method of recovering cells after cryopreservation is not particularly limited, and for example, those cryopreserved may be thawed. As a method of thawing, it is sufficient to melt at a temperature at which cells are not damaged, and known methods are used. Examples of the method include general methods such as water bath melting method, heat block method, room temperature melting method and the like. The melting method by a water bath and the heat block method are preferable, and in particular, the melting method by a water bath is more preferable from the viewpoint of melting temperature, melting time and the like.
- the melting temperature is preferably 10 ° C. or more and 45 ° C. or less, more preferably 20 ° C. or more and 40 ° C. or less, and more preferably 35 ° C. or more and 40 ° C. or less.
- it may be allowed to stand at room temperature (25.degree. C.), but as shown in the examples, it is more preferable to melt using a 35-38.degree. C. water bath. This is because the cell recovery rate and survival rate are higher.
- the cell preparation according to one aspect of the present invention comprises a cell, a composition for cryopreservation containing a fatty acid, and is cryopreserved. Further, a cell preparation according to one aspect of the present invention comprises a cell, and a composition for cryopreservation containing at least one component selected from the group consisting of fatty acids and fatty acid esters, and the cells have a three-dimensional structure. It forms a cell mass that is a structure, and is cryopreserved. In addition, about the cell preparation which concerns on 1 aspect of this invention, having demonstrated about the composition for cryopreservation which concerns on 1 aspect of this invention is applied correspondingly.
- the "cell preparation” is a therapeutic agent prepared by formulating cells, which is used for regenerative medicine as a material for regenerative medicine, and is formulated without changing the function of cells in the original state. It includes not only ones, but also those in which cells having improved functions such as differentiation ability and immunosuppression ability are formulated by culturing and proliferating under specific conditions.
- the cell preparation according to one aspect of the present invention is suitably obtained by the method for producing a cell preparation according to one aspect of the present invention described later.
- the process (b) is performed in the method for producing a cell preparation according to one aspect of the present invention, the components of the composition for cryopreservation permeate into the cells.
- the method for producing a cell preparation according to one aspect of the present invention is a method for producing a cell preparation containing cells, and comprises the following steps (a) and (c) in this order.
- a method for producing a cell preparation comprising mesenchymal stem cells comprising the steps of (a) and (c) below in this order: (A) immersing the cells in a cryopreservation solution containing at least one component selected from the group consisting of fatty acids and fatty acid esters; (C) freezing the cells.
- the method for producing a cell preparation according to one aspect of the present invention may be one aspect of the method for producing a cryopreserved substance of the present invention.
- kits for frozen cells are a kit for cryopreservation for cryopreservation of cells, which comprises fatty acid.
- the kit for frozen cells according to one aspect of the present invention is a kit for cryopreservation for cryopreservation of cells, which is a kit for cryopreservation of the cells in which cell masses having a three-dimensional structure are formed. And at least one component selected from the group consisting of fatty acids and fatty acid esters.
- kits for frozen cells when simply referred to as a “kit for frozen cells”, “a kit for cryopreservation for cryopreserving mesenchymal stem cells” and “a kit for cryopreservation for cryopreservation of cells”, said cells are 3 It is forming a cell mass which is a dimensional structure, and refers to any of "a kit for cryopreservation for cryopreserving the cell mass".
- kit for frozen cells when referring to “kit for frozen cells according to one aspect of the present invention”, it may be one aspect of a kit for cryopreservation for cryopreservation of cells, and cells forming a cell mass having a three-dimensional structure It may also be one aspect of a cryopreservation kit for cryopreservation.
- the composition for cryopreservation and cell preparation according to one aspect of the present invention described above can be suitably obtained, and to the above-mentioned one aspect of the present invention
- the method for producing such a cryopreserved product and the method for producing a cell preparation can be suitably carried out.
- the kit for cryopreservation which concerns on 1 aspect of this invention having demonstrated about the cryopreservation composition which concerns on 1 aspect of this invention is applied mutatis mutandis, and a different point is mainly demonstrated here.
- the configuration of the kit for frozen cells according to one aspect of the present invention is not particularly limited as long as it comprises at least one component of fatty acid in some aspects, fatty acid in some aspects, and fatty acid esters in other aspects.
- An appliance may be included.
- components other than fatty acids and fatty acid esters of the composition for cryopreservation according to one aspect of the present invention described above may be provided.
- reagents, buffers, etc. for stably holding mesenchymal stem cells may be provided, and a serum-free medium for serum-free culture of mesenchymal stem cells may be provided.
- Reagents and devices may be provided to obtain scaffold-free cell clusters processed into three-dimensional structures from stem cells in a system.
- the kit for frozen cells according to one aspect of the present invention may be prepared by mixing a plurality of different reagents in an appropriate volume and / or form, or may be provided by separate containers.
- kit for frozen cells may include an instruction describing a procedure for obtaining the composition for cryopreservation and the like. It may be written or printed on paper or other medium, or may be attached to an electronic medium such as a magnetic tape, a readable disc such as a computer, or a CD-ROM.
- the cells are more preferably mesenchymal stem cells.
- the cells have a three-dimensional structure and form scaffold-free cell clusters, and the cell clusters are cryopreserved. It is more preferable that
- the cells form a scaffold-free cell mass.
- composition for cryopreservation according to one aspect of the present invention, it is more preferable that the cells are cultured without serum.
- the component is a fatty acid ester, and it is more preferable to further include a surfactant.
- the fatty acid is more preferably at least one of linoleic acid and linolenic acid.
- the fatty acid ester is more preferably a phospholipid.
- the phospholipid is more preferably phosphatidic acid.
- the fatty acid ester is preferably phosphatidic acid
- the surfactant is more preferably Pluronic F-68.
- composition for cryopreservation according to one aspect of the present invention is more preferably for cryopreservation at -80 ° C. or lower.
- the cryopreservation solution for the cells is placed from the state in which the cells are immersed in the cryopreservation solution. It is more preferable to freeze the said cell after the said (b) process in the said (c) process including the process of reducing a quantity.
- the cells are not immersed in the cryopreservation solution.
- the cryopreservation of the cells is performed from the state where the cells are immersed in the cryopreservation solution (b) after the step (a). It is more preferable to freeze the said cell after the said (b) process in the said (c) process including the process of reducing the amount of liquids.
- the cells are not immersed in the cryopreservation solution.
- Example 1 Active ingredient identification test for cryopreservation of cell suspension: Cryopreservation experiment for examining the influence on cell viability> In order to identify the active ingredient for cryopreservation of the cell suspension, the composition of the cell cryopreservation solution was shaken to evaluate the cryopreservation effect on MSC in the form of test cell suspension. The following protocol was followed in this example unless otherwise noted.
- Cell culture The primary cells of human synovial membrane-derived MSCs are immersed in a culture solution (STK (registered trademark) 1) in a culture vessel according to the following procedures (1) to (4) or shredding the tissues and The cells were obtained by outgrowth from a piece.
- STK registered trademark
- the primary cells obtained by the above method are passaged, and STK (registered trademark) 2 (serum free medium for MSC growth culture, marketed by DS Pharma Biomedical Co., Ltd .; same in the following description) by the manufacturer. It was made to grow by repeating subculture according to the instruction.
- Cell freezing MSCs grown by repeating subculture were further subjected to planar culture in STK (registered trademark) 2 medium and washed once with PBS in a subconfluent state.
- the cells were detached and single-celled with TrypLE Select CTS (Thermo Fisher Scientific Inc.), collected in a tube for centrifugation, and diluted with a washing medium (DMEM, Sigma).
- TrypLE Select CTS TrypLE Select CTS (Thermo Fisher Scientific Inc.)
- the cell suspension was divided into "1,000,000 cells per tube for centrifugation" and pelleted again in the same manner as above.
- each cryopreservation solution used under each condition was dispensed into each centrifuge tube and suspended.
- the composition of each cryopreservation solution is as described in Table 1, Table 2 and Table 3 below.
- cryovials used cryogenic vial (2 ml, WHEATON).
- the frozen vial was transferred to a -80 ° C freezer (Panasonic Healthcare Co., Ltd.) and cryopreserved.
- Cell count 1. The thawed cells were washed once with washing medium (DMEM, Sigma) and then suspended again in washing medium. 2. 10 ⁇ L each of the cell suspension and trypan blue solution were mixed in a microtube, and the number of cells was counted using a One-Cell Counter (registered trademark) to calculate the viability.
- cryopreservation experiments of MSC in the form of cell suspension were carried out using each of the following three cryopreservation solutions.
- cryopreservation solution "a mixture of MCDB and DMEM in a ratio of 1: 1" was used as the basal medium.
- cryopreservation fluid i
- cryopreservation fluid ii
- cryopreservation fluid ii
- FIG. 1 (a) is a view for briefly explaining the component comparison of each cryopreservation solution
- FIG. 1 (b) is a view showing the result of the cryopreservation experiment.
- Live indicates “the number of viable cells per frozen vial (vial)”
- viability indicates the ratio of "the number of viable cells per frozen vial” to "the total number of cells per frozen vial”.
- “before storage” is the result when the number of cells is measured without performing the cryopreservation, and the other is the result of performing the cryopreservation and thawing.
- cryopreservation solution (ii) when stored in the cryopreservation solution (ii) and the cryopreservation solution (iii) as compared with the cryopreservation solution (i), the number of MSC cells after cryopreservation / thawing is Significantly more (P ⁇ 0.001) and cell viability was also high. In addition, no difference in cell number and cell viability after cryopreservation and thawing was observed between the MSC stored in the cryopreservation solution (ii) and the MSC stored in the cryopreservation solution (iii). In cryopreserving MSCs, it has been shown that in addition to being able to be cryopreserved well according to one aspect of the present invention, cytokines are not essential.
- FIG. 2 shows the results of a cryopreservation experiment of MSCs.
- total cell number indicates “total cell number after freezing storage / thawing per frozen vial”
- living cells indicates “freeze storage / thawing per frozen vial”
- viability indicates the ratio of "the number of viable cells per frozen vial” to "the total number of cells per frozen vial”.
- basic medium described in each table etc. shown after this embodiment intends what mixed MCDB which is a basal medium, and DMEM by the ratio of 1: 1.
- STK2 (cytokine free) and STK (registered trademark) 2 are each (of parts other than DMSO) of the cryopreservation solutions (ii) and (iii) of the above [Result 1]. See FIG. 1 (a)).
- STK2 (cytokine free) may be abbreviated as STK2 (-) or the like.
- the composition contained in STK2 (cytokine free) is the same composition as the STK (registered trademark) 2 medium except that it does not contain a cytokine.
- FIG. 3 shows the results of a cryopreservation experiment of MSCs.
- total cell number indicates “total cell number after freezing storage / thawing per frozen vial”
- living cells indicates “freeze storage / thawing per frozen vial”
- the number of viable cells after is shown.
- N 3, all the results are shown as “mean value ⁇ standard deviation” (mean ⁇ SD).
- “Viability” means “total cell number per frozen vial”. The ratio of "the number of viable cells per frozen vial” is shown.
- FIG. 4 shows the results of a cryopreservation experiment of MSCs.
- total cell number indicates “total cell number after freezing storage / thawing per frozen vial”
- living cells indicates “freeze storage / thawing per frozen vial”
- the number of viable cells after is shown.
- N 3, all the results are shown as “mean value ⁇ standard deviation” (mean ⁇ SD).)
- “Viability” means “total cell number per frozen vial”. The ratio of "the number of viable cells per frozen vial” is shown.
- Example 2 Cryopreservation experiment using a cell mass of mesenchymal stem cells having scaffold-free three-dimensional structure>
- the cryopreservation solution described in [Result 1] of Example 1 (ii) And cryopreservation solution (iii) were used to conduct a cryopreservation experiment of gMSC (registered trademark) 1.
- the primary cells obtained by the above-mentioned method were passaged and expanded by repeating subculture with STK (registered trademark) 2 according to the manufacturer's instruction.
- the cell exfoliant TrypLE Select CTS After stripping and collecting with (Thermo Fisher Scientific Inc.) and suspending with washing medium (DMEM, Sigma), it was transferred to a tube for centrifugation and centrifuged at 1500 rpm for 5 minutes at room temperature. The separated single cells were again suspended in the washing medium, and the cell number was counted by trypan blue staining.
- PBS Phosphate buffered saline, calcium and magnesium free, PBS (-), Inc., Cell Science Research Institute, Inc.
- DMEM washing medium
- the tissue is mechanically detached from the culture dish, and a plurality of mesenchymal stem cells are collected and brought into a state of aggregation, thereby forming a scaffold-free three-dimensional structure.
- a cell mass of gMSC (registered trademark) 1 is obtained.
- the freeze preservation method of gMSC (registered trademark) 1
- Materials (1) Freeze preservation solution (in-house): As the cryopreservation solution, the cryopreservation solutions (ii) and (iii) described in [Result 1] of Example 1 were used respectively. These compositions are as described above (see FIG. 1 (a), etc.).
- Cryopreservation container WHEATON cryogenic vial, model number: W985868
- Storage solution consumption 1.0 mL / g MSC (registered trademark) 1 / cryovial 2.
- Preparation of gMSC (registered trademark) 1 and preparation of cryopreserved gMSC (registered trademark) 1 are performed according to the description of the above-mentioned “Preparation of gMSC (registered trademark) 1”. More preferably, it is desirable to perform an operation (equilibration) to allow the cryopreservation solution to penetrate the cell mass as described below.
- the equilibration may be performed "before processing the culture into gMSC (registered trademark) 1 shape" (see (2) below), or “after processing into gMSC (registered trademark) 1 shape” (described below Although (4) may be performed, in this embodiment, an example in which both are performed will be described. In particular, in order to explain the equilibration step "before processing into gMSC (registered trademark) 1 shape" in this example, the steps of preparation of gMSC (registered trademark) 1 detailed above are also briefly described. .
- MSCs with high density culture were physically exfoliated from the culture vessel using P200 pipetman (registered trademark) with a tip attached, and collected. It was processed into a three-dimensional shape gMSC (registered trademark) 1 by being put into a state.
- gMSC registered trademark 1 was transferred to a cryopreservation container in which 1 mL of a cryopreservation solution was previously placed, and was allowed to stand in a refrigerator (4 ° C.) for 10 minutes.
- the melting method of gMSC (registered trademark) 1
- Frozen gMSC.RTM. 1 (stored in a frozen vial) was removed from the -80.degree. C. freezer. 2. The frozen vial was warmed in a 37 ° C. water bath for 2.5 minutes. 3. After visually confirming that the globular gMSC 1 was completely thawed, the frozen vial was removed from the water bath.
- Method of measuring cell number of thawed gMSC (registered trademark) 1
- Materials (1) Collagenase-A (Animal Origin Free). Worthington Biochemical Corporation, Model Number: LS004154 (2) 0.4% trypan blue solution. Thermo Fisher Scientific Inc. Model number: 15250 (3) A collagenase solution of 560 U (unit) / mL prepared in DMEM was filtered with a 0.45 mm filter (Millipore model number: SLHV 033 RS). 2. Method (1) gMSC (registered trademark) 1 was added to 1 mL of a collagenase solution in a 15 mL centrifuge tube.
- FIG. 5 is a diagram showing the results of a cryopreservation experiment using gMSC (registered trademark) 1.
- “Live” indicates "the number of living cells per" gMSC (registered trademark) 1 "
- Total indicates “indicates the total number of cells per one" gMSC (registered trademark) 1 ""Is shown.
- cryopreservation solution As described above, as the cryopreservation solution, the cryopreservation solutions (ii) and (iii) described in column [Result 1] of Example 1 were respectively used. Although these compositions are as above-mentioned (refer FIG. 1 (a) etc.), CD lipid (trademark), PA, and PC were contained by the above-mentioned composition in each case.
- cryopreservation solution of one embodiment of the present invention can well cryopreserve cell clusters of mesenchymal stem cells having scaffold-free three-dimensional structure.
- cryopreservation solution of one embodiment of the present invention can well cryopreserve cell clusters of mesenchymal stem cells having scaffold-free three-dimensional structure.
- significant differences in total cell number and cell viability are obtained depending on the presence or absence of cytokines. It turned out that it was not recognized.
- the bone differentiation ability was evaluated by the following method in any of gMSC (registered trademark) 1 before cryopreservation and gMSC (registered trademark) 1 after cryopreservation and thawing. That is, gMSC (registered trademark) 1 is brought into a single cell state by collagenase treatment, washed, cultured in STK (registered trademark) 2 respectively, and when confluent, the medium is exchanged and the medium used is for bone differentiation The medium was switched to culture medium (STK (registered trademark) 3 (manufactured by DS Pharma Biomedical Co., Ltd.)) and cultured. The subsequent medium change was performed approximately once every three days. After 21 days of culture, gMSC (registered trademark) 1 after culture was stained with alizarin red S (Nacalai Tesque: 01303-52) to confirm whether or not bone differentiation was induced.
- the bone differentiation ability was evaluated by the following method in any of gMSC (registered trademark) 1 before cryopreservation and gMSC (registered trademark) 1 after cryopreservation and thawing. That is, the cells are put into a single cell state by collagenase treatment, washed, cultured in 6 well plates using STK (registered trademark) 2 respectively, and when confluent, the medium is exchanged and the medium used is a medium for fat differentiation.
- gMSC registered trademark 1 before cryopreservation
- gMSC registered trademark 1 after cryopreservation and thawing. That is, the cells are put into a single cell state by collagenase treatment, washed, cultured in 6 well plates using STK (registered trademark) 2 respectively, and when confluent, the medium is exchanged and the medium used is a medium for fat differentiation.
- FIG. 6 is a diagram showing the results of a confirmation experiment of bone / fat differentiation ability of cells after cryopreservation and thawing, and FIG. 6 (a) shows the confirmation result of bone differentiation ability, and FIG. 6 (b) Is a figure which shows the confirmation result of fat differentiation ability. As shown in FIG.
- cryopreservation / thawing in the cell mass of mesenchymal stem cells having a scaffold-free three-dimensional structure, even when frozen using any of the cryopreservation solutions (ii) and (iii), cryopreservation / thawing It has been confirmed that they have good bone differentiation ability and fat differentiation ability also later.
- Cartilage differentiation is induced in the following manner in any of gMSC (registered trademark) 1 before cryopreservation and gMSC (registered trademark) 1 after cryopreservation and thawing. Specifically, after gMSC (registered trademark) 1 is divided into four equal parts (approximately 10 mg to 20 mg in wet weight), the cells are washed once with a basal medium for cartilage differentiation, and then transferred one by one to a 15 mL conical tube.
- the medium was replaced with the same differentiation-inducing medium every 2 to 3 days sulfated glycosaminoglycan (GAG)
- GAG sulfated glycosaminoglycan
- the amount of sulfated glycosaminoglycan (GAG) of gMSC (registered trademark) 1 after culture was quantified using a kit for quantification (manufactured by Biocolor)
- the amount of GAG was normalized according to the DNA content of cells .
- FIG. 7 is a diagram showing the results of a confirmation experiment of the cartilage differentiation ability of cells after cryopreservation and thawing, and FIG. 7 (a) shows a result of photographing a sample for which a confirmation experiment of the cartilage differentiation ability was performed. 7 (b) shows the result of sulfated glycosaminoglycan (GAG) assay. As shown in FIG.
- cryopreservation / thawing is also performed on the cell mass of mesenchymal stem cells having a scaffold-free three-dimensional structure. It has been confirmed that it has good cartilage differentiation ability also later.
- the cell pellet was suspended and mixed in 5 mL of DMEM.
- PBS PBS
- HSA human albumin
- HSA human albumin
- FIG. 8 shows the results of experiments to confirm the expression of cell surface antigen markers after cryopreservation and thawing.
- FIG. 8 when cryopreserved and thawed using either of the cryopreservation solutions (ii) and (iii), also in the cell mass of mesenchymal stem cells having a scaffold-free three-dimensional structure, changes in the expression profile of cell surface antigens markers before and after cryopreservation after the decomposition of the cell mass were not.
- Example 3 Cryopreservation experiment using a cell mass of mesenchymal stem cells having a scaffold-free three-dimensional structure and the influence of the liquid volume of the cryopreservation solution at the time of thawing>
- the effect of the volume of the cryopreservation solution at the time of freezing (thawing) is evaluated The experiment to do was done as follows.
- Step 7b in Table 5 is one form of the above-mentioned "b (step)".
- FIG. 9 is a diagram showing the results of this example.
- Live indicates “the number of living cells per“ gMSC (registered trademark) 1 ”
- “ total ” indicates the total number of cells per“ “gMSC (registered trademark) 1” "Is shown.
- “before storage” is the result when the number of cells is measured without performing the cryopreservation of gMSC (registered trademark) 1, and in the other cases, it is the result after freezing and thawing.
- mesenchymal stem cells having scaffold-free three-dimensional structure could be cryopreserved with good viability. Furthermore, the total cell number and the survival rate were significantly higher in the method (7b) in which the cryopreservation solution was removed before cryopreservation, as compared to the method (7a) in which the cryopreservation solution was not removed. Thus, it was shown that the "liquid-freezing" in which the "(b) step" is performed is capable of cryopreservation with higher total cell number and survival rate.
- Example 4 Test for identifying active ingredient for cryopreservation of mesenchymal stem cells, in particular, cell mass of mesenchymal stem cells having scaffold-free three-dimensional structure: cryopreservation experiment for examining influence on cell viability>
- each of the cell cryopreservation solutions having different compositions is used for freezing using gMSC (registered trademark) 1 The preservation effect was evaluated.
- viability indicates the ratio of "the number of viable cells per one" gMSC (registered trademark) 1 "to the” the total number of cells per one "gMSC (registered trademark) 1". Also, “before storage” is the result when the number of cells is measured without performing the cryopreservation of gMSC (registered trademark) 1, and in the other cases, it is the result after freezing and thawing.
- the number of cells after thawing of gMSC (registered trademark) 1 cryopreserved in (1) was significantly higher than that in the control group (6-2) (P ⁇ 0.05).
- the cryopreservation solution (6-3) in which CD lipid (registered trademark) (which is a mixture of fatty acids) is added to the above (6-2)
- the number of cells after thawing is in the control group (6-2). It was significantly more than that.
- (6-2) will be linoleic acid (50.0 ⁇ g / mL), PA (10.0 ⁇ g / mL), PA (50.0 ⁇ g / mL), linolenic acid (10.0 ⁇ g / mL)
- gMSC registered trademark
- the cryopreservation of the cell mass of mesenchymal stem cells having a scaffold-free three-dimensional structure includes a plurality of components of the group consisting of fatty acids and fatty acid esters as in (6-1) or (6-3)
- the addition of a solution of linoleic acid, PA and linolenic acid was shown to be effective as well as the cryopreservation solution to which the above composition was added.
- FIG. 11 is a graph showing the results of a cryopreservation experiment of gMSC (registered trademark) 1 using a cryopreservation solution containing linoleic acid, linolenic acid, and PA, respectively.
- FIG. 12 is a graph showing the results of a cryopreservation experiment of gMSC (registered trademark) 1.
- live indicates “the number of living cells per" gMSC (registered trademark) 1 "
- viability indicates the ratio of "the number of viable cells per one" gMSC (registered trademark) 1 "to the” the total number of cells per one "gMSC (registered trademark) 1".
- before storage is the result when the number of cells is measured without performing the cryopreservation of gMSC (registered trademark) 1, and in the other cases, it is the result after freezing and thawing.
- the number (total cell number) was significantly higher (P ⁇ 0.05, P ⁇ 0.01, respectively).
- the cryopreservation solution containing linoleic acid solution has the cryopreservation effect of cryopreserving mesenchymal stem cells having scaffold-free three-dimensional structure with good survival rate, and it is of methyl- ⁇ -cyclodextrin Compared to the effect, the freeze preservation effect of linoleic acid is high, and it is supported that the main effect of the effect is in linoleic acid.
- FIG. 13 is a graph showing the results of a cryopreservation experiment of gMSC (registered trademark) 1.
- live indicates “the number of living cells per" gMSC (registered trademark) 1 "
- viability indicates the ratio of "the number of viable cells per one" gMSC (registered trademark) 1 "to the” the total number of cells per one "gMSC (registered trademark) 1".
- before storage is the result when the number of cells is measured without performing the cryopreservation of gMSC (registered trademark) 1, and in the other cases, it is the result after freezing and thawing.
- cryopreservation solution containing linoleic acid solution and Pluronic F-68 has a cryopreservation effect of cryopreserving mesenchymal stem cells having scaffold-free three-dimensional structure with good survival rate. It was found that the addition of both causes a synergistic effect and exerts a higher cryopreservation effect.
- FIG. 14 is a graph showing the results of a cryopreservation experiment of gMSC (registered trademark) 1.
- “live” indicates "the number of living cells per" gMSC (registered trademark) 1 "
- total indicates the total number of cells per one" gMSC (registered trademark) 1 "
- viability indicates the ratio of "the number of viable cells per one" gMSC (registered trademark) 1 "to the” the total number of cells per one "gMSC (registered trademark) 1". Also, “before storage” is the result when the number of cells is measured without performing the cryopreservation of gMSC (registered trademark) 1, and in the other cases, it is the result after freezing and thawing.
- cryopreservation solution containing PA solution has a cryopreservation effect of cryopreserving mesenchymal stem cells having scaffold-free three-dimensional structure with good survival rate, and dissolution that assists PA dissolution It can be seen that the addition of the adjuvant (Tween 80) alone has no effect on cryopreservation. Therefore, it was confirmed that the active ingredient was PA, and it was shown that PA has a cryopreservation effect.
- FIG. 15 is a graph showing the results of a cryopreservation experiment of gMSC (registered trademark) 1.
- gMSC registered trademark
- gMSC registered trademark
- PA and Pluronic F-68 were used in combination, rather than the above (11-4) and (11-5) to which each was added alone. It was also found that the number of cells was 1 and it was shown that the effect was further enhanced (P ⁇ 0.01).
- cryopreservation solution containing PA and Pluronic F-68 has the cryopreservation effect of cryopreserving mesenchymal stem cells having scaffold-free three-dimensional structure with good survival rate, and both of them It was found that the addition produces a synergistic effect, and a higher storage and preservation effect is exhibited.
- a safer and more valuable transplantation therapeutic material using mesenchymal stem cells can be provided, so that it can be suitably used for regenerative medicine such as transplantation therapy using mesenchymal stem cells. .
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Abstract
Description
(c)前記細胞を凍結する工程。
本発明の一態様に係る凍結保存用組成物は、細胞を凍結保存するための凍結保存用組成物であって、脂肪酸を含む。また、本発明の一態様に係る凍結保存用組成物は、細胞を凍結保存するための凍結保存用組成物であって、前記細胞は、3次元構造である細胞塊を形成しているものであってよく、当該細胞塊を凍結保存するためのものであり、脂肪酸及び脂肪酸エステルからなる群より選ばれる少なくとも1種の成分を含む。以下、単に「凍結保存用組成物」という場合、「細胞を凍結保存するための凍結保存用組成物」と、「細胞を凍結保存するための凍結保存用組成物であって、前記細胞は、3次元構造である細胞塊を形成しているものであり、当該細胞塊を凍結保存するための凍結保存用組成物」と、のいずれをも指すものとする。例えば、「本発明の一態様に係る凍結保存用組成物」という場合、懸濁液状に懸濁された細胞を凍結保存するための凍結保存用組成物の一態様でもあり得、3次元構造である細胞塊を形成している細胞を凍結保存するための凍結保存用組成物の一態様でもあり得る。なお、後述する通り、ここでは間葉系幹細胞を一例に説明しているが、凍結保存対象となる細胞は、間葉系細胞に限定されない。
本発明の一態様に係る凍結保存用組成物で凍結する対象の細胞は、例えば、間葉系幹細胞、CD34細胞、胚性幹細胞(ES細胞)、iPS(induced pluripotent stem cells)細胞、軟骨細胞、骨芽細胞、線維芽細胞、表皮細胞、上皮細胞、脂肪細胞、肝細胞、膵細胞、筋細胞、神経細胞、神経幹細胞、造血幹細胞又はこれらの前駆細胞等が挙げられる。また、所望のマーカーに対して陽性を示す細胞であってもよい。細胞が由来する生物種も特に限定されず、生物種は、例えば微生物、非ヒト哺乳類またはヒト等が挙げられる。
本発明の一態様に係る凍結保存用組成物で凍結する対象の一例である間葉系幹細胞(Mesenchymal stem cell、以下「MSC」ともいう。)について説明する。間葉系幹細胞は間葉系に属する細胞への分化能を有し、免疫抑制作用も併せ持つこと等といった利点により、再生医療において最も有望な細胞の一つと考えられている。
まず、本発明の一態様に係る凍結保存用組成物で凍結する対象となる細胞を無血清培養するために用いる無血清培地の一例について説明する。無血清培地を構成するための基礎培地は、当該分野において周知の動物細胞用培地であれば特に限定されず、好ましい基礎培地としては、例えば、Ham’s F12培地、DMEM培地、RPMI-1640培地、MCDB培地などが挙げられる。これらの基礎培地は、単独で使用されても、複数を混合して使用されてもよい。一実施形態において、無血清培地を構成するための基礎培地は、MCDBとDMEMとを1:1の比率で混合した培地が好ましい。
本発明の一態様に係る凍結保存用組成物で凍結する対象の細胞の加工物は、どのような形態のものであってもよく、例えば、細胞の懸濁液、シート状に培養したもの、3次元構造の細胞塊等が挙げられる。中でも3次元構造の細胞塊を形成している間葉系幹細胞がより好ましく、スキャフォールドフリーの細胞塊を形成している間葉系幹細胞がさらに好ましい。
本発明の一態様に係る凍結保存用組成物によれば、3次元構造の細胞塊も好適に凍結することができる。細胞懸濁液を患部に投与する場合、投与される細胞が免疫特権を持つと言われる間葉系幹細胞ですら患部から遊走するという報告、及び、ホストの免疫細胞により攻撃される等の理由で患部に留まらないという報告がある。しかし、3次元構造の細胞塊を用いれば、細胞が患部から遊走、脱落することを防ぐことができ、長時間治療効果を発揮させる。
間葉系幹細胞の細胞塊は分化誘導を行わない未分化な状態で移植するのが好ましい。細胞塊を移植した後の軟骨分化能をin vitroで評価する試験する場合には、凍結保存しない状態の細胞塊であっても、凍結保存・解凍後の細胞塊であっても、細かく切断することが好ましい。細胞塊を細かく切断した方が、分化誘導培地が組織内部までより浸透し易いためである。細胞塊を切断するために用いられる手法としては、特に限定されず、例えば、滅菌済みメスあるいはハサミ等が用いられる。また、切断する際、細胞塊の大きさは、概ね10mg~20mgにすることがより好ましい。
本発明の一態様に係る凍結保存用組成物で凍結する対象の細胞塊は、スキャフォールドフリーの3次元構造体であることがより好ましい。本明細書において「スキャフォールドフリー(足場フリー、基盤材料なし;scaffold-free)」とは、人工組織を生産するときに従来使用されている材料(基盤材料=スキャフォールド)を実質的に含まないことをいう。
本発明の一態様に係る凍結保存用組成物は脂肪酸を含む。脂肪酸を含むことによって、凍結保存用組成物は、細胞を生存率よく凍結することができ、解凍後の細胞の性質の変化も生じない。
Myristic Acid 10.00μg/ml, Oleic Acid 10.00μg/ml, Palmitoleic Acid 10.00μg/ml, Palmitic Acid 10.00μg/ml, Stearic Acid 10.00μg/ml, Pluronic F-68 100mg/ml, Tween 80 2.2mg/ml。
本発明の一態様に係る凍結保存用組成物は、脂肪酸エステルを含む。脂肪酸エステルを含むことによって、凍結保存用組成物は、細胞を生存率よく凍結することができる。
本発明の一態様に係る凍結保存用組成物は、界面活性剤のような、脂肪酸あるいは脂肪酸エステルの水溶(乳化)を補助する物質をさらに含有していてもよい。界面活性剤としては、Pluronic F-68又はTween 80であり得る。
上述した本発明の一態様に係る凍結保存用組成物の成分には、凍結・解凍過程で細胞内に氷結晶が成長することを抑制する、凍結保護剤が含まれていてもよい。凍結保護剤としてはDMSO(ジメチルスルホキシド)等が挙げられる。本発明の一態様に係る凍結保存用組成物が凍結保護剤を含む場合、その量は、凍結保存用組成物の全量に対して0.5%~50%(v/v)がより好ましい。
本発明の一態様に係る凍結保存用組成物は脂肪酸以外の成分を含んでもよく、例えば、基礎培地、増粘剤、pH調整剤、凍結保護剤等が挙げられる。
本発明の一態様に係る凍結保存用組成物は、脂肪酸等の上述した各成分を適宜混合すればよい。
本発明の一態様に係る凍結保存用組成物は様々な形態で提供され得る。例えば、液体であってもよく、粉末、錠剤等の固体等であってもよい。固体で提供する場合は、使用者が適宜溶媒で溶解して凍結保存液にして使用すればよいが、本発明の一態様に係る凍結保存用組成物は、好適な溶媒を指定する説明書と共に提供されてもよい。
本発明の一態様に係る凍結保存方法は、細胞を凍結保存した凍結保存物の製造方法であって、以下の(a)、(c)の工程をこの順に含む。
(c)前記細胞を凍結する工程。
(c)前記細胞を凍結する工程。
本発明の一態様に係る凍結保存物の製造方法で用いる凍結保存液は脂肪酸及び脂肪酸エステルからなる群より少なくとも1種の成分を含む。凍結保存液は、前述の本発明の一態様における凍結保存用組成物のうち液体の形態であるものを用いてもよく、本発明の一態様における凍結保存用組成物のうち固体の形態であるものについては、溶媒に当該固体を溶解して得られる溶液を用いてもよい。
(a)工程では、凍結対象の細胞を、脂肪酸を含む凍結保存液に浸漬する。浸漬する方法としては、例えば、凍結保存可能な容器に凍結保存液を入れ、当該凍結保存液中に細胞を入れることが好ましい。そのまま後述の(c)工程の凍結を行なうことができるからである。
本発明の一態様に係る凍結保存物の製造方法では、(a)工程の後に、(b)前記凍結保存液に前記細胞が浸漬された状態から、前記細胞に対する前記凍結保存液の量を減らす工程を含み、後述する(c)工程では、前記(b)工程後の前記細胞を凍結することがより好ましい。保存後の融解時間を短時間にすることができ、これにより、凍結保存による細胞の回収率、全細胞数、生存率をより向上させることができる。
(c)工程では、間葉系幹細胞を凍結する。(b)工程を行なった場合には、(b)工程によって浸漬されていない状態とした間葉系幹細胞を凍結する。凍結温度は凍結対象の間葉系幹細胞が凍結する温度に適宜設定すればよいが、例えば-80℃以下、又は-196℃以下が挙げられる。-80℃以下で凍結する場合は、例えば、従来公知の例等を用いればよく、-196℃以下で凍結する場合は、液体窒素を用いればよい。
凍結保存に用いる容器は、凍結温度に耐え得るものであればよい。例えば、-80℃に耐え得るものであることが好ましく、-196℃に耐え得るものであることがより好ましい。具体的には、ポリエチレン、ポリプロピレン又はポリエチレンテレフタレート等の合成樹脂製の容器がより好ましい。該容器は、例えば、市販のクライオバイアル(凍結ベッセル)を使えば良い。
凍結保存後に細胞を回収する方法は特に限定されず、例えば、凍結保存されたものを融解すればよい。融解させる方法としては、細胞が損傷しない温度で融解させればよく、公知の方法が用いられる。該方法としては例えば、ウォーターバスによる融解法、ヒートブロック法、室温融解法等の一般的な方法が挙げられる。ウォーターバスによる融解法、ヒートブロック法が好ましく、中でも融解温度および融解時間等の観点から、ウォーターバスによる融解法がさらに好ましい。
本発明の一態様に係る細胞製剤は、細胞を含み、脂肪酸を含む凍結保存用組成物と、を含み、凍結保存されている。また、本発明の一態様に係る細胞製剤は、細胞を含み、脂肪酸及び脂肪酸エステルからなる群より選ばれる少なくとも1種の成分を含む凍結保存用組成物と、を含み、前記細胞は、3次元構造である細胞塊を形成しているものであり、凍結保存されている。なお、本発明の一態様に係る細胞製剤については、本発明の一態様に係る凍結保存用組成物について説明したことが準用される。
本発明の一態様に係る細胞製剤の製造方法は、細胞を含む細胞製剤の製造方法であって、以下の(a)、(c)の工程をこの順に含む。
(c)前記細胞を凍結する工程。
(a)前記細胞を、脂肪酸及び脂肪酸エステルからなる群より選ばれる少なくとも1種の成分を含む凍結保存液に浸漬する工程、
(c)前記細胞を凍結する工程。
本発明の一態様に係る凍結細胞用キットは、細胞を凍結保存するための凍結保存用キットであって、脂肪酸を備える。また、本発明の一態様に係る凍結細胞用キットは、細胞を凍結保存するための凍結保存用キットであって、3次元構造である細胞塊を形成した前記細胞を凍結保存するためのキットであり、脂肪酸及び脂肪酸エステルからなる群より選ばれる少なくとも1種の成分を備える。以下、単に「凍結細胞用キット」という場合、「間葉系幹細胞を凍結保存するための凍結保存用キット」と、「細胞を凍結保存するための凍結保存用キットであり、前記細胞は、3次元構造である細胞塊を形成しているものであり、当該細胞塊を凍結保存するための凍結保存用キット」と、のいずれをも指すものとする。例えば、「本発明の一態様に係る凍結細胞用キット」という場合、細胞を凍結保存するための凍結保存用キットの一態様でもあり得、3次元構造である細胞塊を形成している細胞を凍結保存するための凍結保存用キットの一態様でもあり得る。
以上のように、本発明の一態様に係る凍結保存用組成物では、細胞は、間葉系幹細胞であることがより好ましい。
細胞懸濁液の凍結保存に対する有効成分を特定するために、細胞凍結保存液の組成を振って、試験細胞懸濁液状のMSCに対する凍結保存効果を評価した。本実施例では特に断りの無い限り、以下のプロトコールに従った。
「細胞培養」
ヒト滑膜由来MSCの初代細胞は、以下の(1)から(4)の手順、もしくは、組織を細断して培養容器中で培養液(STK(登録商標)1)中に浸漬し、組織片から細胞をOutgrowthさせることで得た。
継代培養を繰り返すことで増殖されたMSCを、STK(登録商標)2培地中でさらに平面培養し、サブコンフルエントになった状態で、PBSで1回洗浄した。
1.-80℃フリーザー中で1週間凍結保存したMSC(凍結バイアル中に保存)を取り出した。
2.前記の凍結バイアルを37℃湯浴(ウォーターバス)にて2.5分間温めた。
3.目視で氷の塊が完全に融解したことを確認し、前記の凍結バイアルをウォーターバスから取り出した。
1.融解した細胞を洗浄培地(DMEM, Sigma)で1回洗浄した後、再び洗浄培地にて懸濁した。
2.マイクロチューブ内で細胞懸濁液とトリパンブルー溶液を10μLずつ混合し、ワンセルカウンター(登録商標)を用いて細胞数をカウントし、生存率を算出した。
まず、次の3つの凍結保存液それぞれを用いて、細胞懸濁液状のMSCの凍結保存実験を行なった。以下の凍結保存液において、基礎培地は、「MCDBとDMEMとを1:1の比率で混合したもの」を用いた。以降、以下の(i)から(iii)の凍結保存液を、必要に応じ、それぞれ凍結保存液(i)、凍結保存液(ii)、凍結保存液(iii)等と略記することがある。
基礎培地
+Albumin(1.25mg/ml,Millipore,CellPrime rAlbumin:AF-S)+10%DMSO(Wako 031-24051)
(ii)STK2(サイトカインフリー)を用いた保存液:
基礎培地
+Albumin(1.25 mg/ml, Millipore, CellPrime rAlbumin AF-S)
+ Insulin(10 μg/ml, Wako, 090-06481)
+ Transferrin(5.5 μg/ml, Wako, 201-18081)
+ Ascorbic acid 2-phosphate(VC)(50μg/ml, SIGMA, A8960)
+ Dexamethasone (Dex)(10-8M, SIGMA(Fluka), 31375)
+ Na2SeO3(6.7 ng/ml, Wako, 194-10842)
+CD lipid(登録商標)(原液の1/100, Thermo Fisher Scientific Inc., 11905-031)
+Phosphatidic acid sodium salt (PA)(10μg/ml, Sigma, P9511)
+Phosphatidylcholine(PC)(10 μg/ml, Sigma P3556)
+L-Glutathione (reduced) (2μg/ml, メルクミリポア, 104090)
+Lithium chloride(LiCl)(1mM, メルクミリポア, 105679)
(iii)STK(登録商標)2を用いた保存液:
STK(登録商標)2+10%DMSO(Wako 031-24051)
結果を図1に示す。図1(a)は各凍結保存液の成分比較を簡単に説明するための図であり、図1(b)は凍結保存実験の結果を示す図である。この棒グラフのそれぞれの項目において、「Live」は「凍結バイアル(vial)一本あたりの生細胞数」を示し、「Total」は「凍結バイアル一本あたりの全細胞数」を示している(n=3、結果は全て、「平均値±標準偏差」(mean±SD)で示されている。)。また、「生存率」とは、「凍結バイアル一本あたりの全細胞数」に対する「凍結バイアル一本あたりの生細胞数」の比を示している。また、「保存前」とは、凍結保存を行わずに細胞数を測定した場合の結果であり、それ以外は凍結保存・解凍を行った結果である。
表1の凍結保存液それぞれを用いて細胞懸濁液状のMSCの凍結保存実験を行なった結果を図2に示す。図2はMSCの凍結保存実験の結果を示す図である。この棒グラフのそれぞれの項目において、「全細胞数」は「凍結バイアル一本あたりの凍結保存・解凍後の全細胞数」を示し、「生細胞」は「凍結バイアル一本あたりの凍結保存・解凍後の生細胞数」を示している(n=3、結果は全て、「平均値±標準偏差」(mean±SD)で示されている。)。また、「生存率」とは、「凍結バイアル一本あたりの全細胞数」に対する「凍結バイアル一本あたりの生細胞数」の比を示している。
表2に記載の凍結保存液それぞれを用いて細胞懸濁液状のMSCの凍結保存実験を行なった結果を図3に示す。図3はMSCの凍結保存実験の結果を示す図である。この棒グラフのそれぞれの項目において、「全細胞数」は「凍結バイアル一本あたりの凍結保存・解凍後の全細胞数」を示し、「生細胞」は「凍結バイアル一本あたりの凍結保存・解凍後の生細胞数」を示している。(n=3、結果は全て、「平均値±標準偏差」(mean±SD)で示されている。)また、「生存率」とは、「凍結バイアル一本あたりの全細胞数」に対する「凍結バイアル一本あたりの生細胞数」の比を示している。
表3に記載のそれぞれの凍結保存液を用いて細胞懸濁液状のMSCの凍結保存実験を行なった結果を図4に示す。図4はMSCの凍結保存実験の結果を示す図である。この棒グラフのそれぞれの項目において、「全細胞数」は「凍結バイアル一本あたりの凍結保存・解凍後の全細胞数」を示し、「生細胞」は「凍結バイアル一本あたりの凍結保存・解凍後の生細胞数」を示している。(n=3、結果は全て、「平均値±標準偏差」(mean±SD)で示されている。)また、「生存率」とは、「凍結バイアル一本あたりの全細胞数」に対する「凍結バイアル一本あたりの生細胞数」の比を示している。
本発明の一形態における凍結保存液のスキャフォールドフリーの3次元構造を有する間葉系幹細胞の細胞塊に対する効果を評価する為、実施例1の〔結果1〕に記載の凍結保存液(ii)及び凍結保存液(iii)を用いて、gMSC(登録商標)1の凍結保存実験を行なった。
「gMSC(登録商標)1の作製」
ヒト滑膜由来MSCの初代細胞は、以下の(1)から(4)の手順、もしくは、組織を細断して培養容器中で培養液(STK1)中に浸漬し、組織片から細胞をOutgrowthさせることで得た。
(4)メーカーの指示に従い、STK(登録商標)1中で初代培養を行った。
1.材料
(1)凍結保存液(自社製):
凍結保存液は、実施例1の〔結果1〕に記載の(ii)及び(iii)の凍結保存液をそれぞれ用いた。これらの組成は前述の通り(図1(a)、等参照)である。
(3)保存液使用量:1.0mL/gMSC(登録商標)1/cryovial
2.gMSC(登録商標)1の作製及び、凍結保存
gMSC(登録商標)1の作製は、前述の「gMSC(登録商標)1の作製」欄の記載に準拠して行う。より好適には、以下に記載するように凍結保存液を細胞塊へ浸透させるための動作(平衡化)を行うのが望ましい。
1.-80℃フリーザーから凍結したgMSC(登録商標)1(凍結バイアル中に収容されている)を取り出した。
2.前記の凍結バイアルを37℃湯浴(ウォーターバス)にて2.5分間温めた。
3.目視で氷塊状のgMSC(登録商標)1が完全に融解されたことを確認し、前記の凍結バイアルをウォーターバス中から取り出した。
1.材料
(1)Collagenase-A (Animal Origin Free)。Worthington Biochemical Corporation、型番:LS004154
(2)0.4%トリパンブルー溶液。Thermo Fisher Scientific Inc. 型番:15250
(3)DMEMにて調製した560U(unit)/mLのコラゲナーゼ溶液を0.45mmフィルター(ミリポア 型番:SLHV033RS)にて、フィルトレーションした。
2.方法
(1)gMSC(登録商標)1を15mL遠沈管のコラゲナーゼ溶液1mLへ入れた。
上記の実験の結果を図5に示す。即ち、図5はgMSC(登録商標)1を用いた凍結保存実験の結果を示す図である。この棒グラフのそれぞれの項目において、「Live」は「『gMSC(登録商標)1』一個当たりの生細胞数」を示し、「Total」は「『gMSC(登録商標)1』一個当たりの全細胞数」を示している。(n=3、結果は全て、「平均値±標準偏差」(mean±SD)で示されている。)。また、「生存率」とは、「『gMSC(登録商標)1』一個当たりの全細胞数」に対する「『gMSC(登録商標)1』一個当たりの生細胞数」の比を示している。また、「保存前」とは、gMSC(登録商標)1の凍結保存を行わずに細胞数を測定した場合の結果であり、それ以外は凍結保存・解凍後の結果である。
次に、凍結保存前のgMSC(登録商標)1、及び凍結保存液(ii)、(iii)を用いて凍結保存・解凍した後のgMSC(登録商標)1を用いて、骨・脂肪分化能の評価を行った。
凍結保存前のgMSC(登録商標)1、凍結保存・解凍後のgMSC(登録商標)1のいずれにおいても、以下の方法で骨分化能を評価した。即ち、gMSC(登録商標)1をコラゲナーゼ処理によりシングルセル状態にし、洗浄した後に、それぞれSTK(登録商標)2で培養し、コンフルエントになった時点で、培地交換を行い、用いる培地を骨分化用培地(STK(登録商標)3(株式会社DSファーマバイオメディカル製))に切り替えて培養した。この後の培地交換は、概ね3日に1回行った。培養21日後に、培養後のgMSC(登録商標)1を、アリザリンレッドS(ナカライテスク:01303-52)で染色し、骨分化誘導されているか否かを確認した。
凍結保存前のgMSC(登録商標)1、凍結保存・解凍後のgMSC(登録商標)1のいずれにおいても、以下の方法で骨分化能を評価した。即ち、コラゲナーゼ処理によりシングルセル状態にし、洗浄した後にそれぞれSTK(登録商標)2を用いて6well-plate中で培養し、コンフルエントになった時点で、培地交換を行い、用いる培地を脂肪分化用培地(DMEM(sigma:D5796)、FBS(Hyclone)、ペニシリン-ストレプトマイシン(Sigma:P0781)、インスリン(Wako:093-06471)、デキサメタゾン(Sigma:D1756)、インドメタシン(Wako:097-02471)、3-イソブチル-1-メチルキサンチン(Calbiochem:410957))に切り替えて3日間培養した。その後、脂肪分化誘導培地と脂肪分化維持培地(MEM(sigma:D5796)、FBS(Hyclone)、ペニシリン-ストレプトマイシン(Sigma:P0781)、インスリン(Wako:093-06471))とを3日おきに交互に交換し分化培養を続けた。培養21日後にオイルレッドO(WAKO:154-02072)で染色し、脂肪分化誘導されているか否かを確認した。
以上の実験を3株のgMSC(登録商標)1について行なった。結果を図6に示す。図6は、凍結保存・解凍後の細胞の骨・脂肪分化能の確認実験の結果を示す図であり、図6の(a)は骨分化能の確認結果を示し、図6の(b)は脂肪分化能の確認結果を示す図である。図6に示すとおり、スキャフォールドフリーの3次元構造を有する間葉系幹細胞の細胞塊においても、凍結保存液(ii)、(iii)の何れを用いて凍結した場合にも、凍結保存・解凍後にも良好な骨分化能及び脂肪分化能を有することが確認できた。
次に、凍結保存前のgMSC(登録商標)1、及び凍結保存液(ii)、(iii)を用いて凍結保存・解凍した後のgMSC(登録商標)1を用いて、軟骨分化能の評価を行なった。なお、以下に記載のように、軟骨分化誘導を行う前に、凍結保存前のgMSC(登録商標)1、凍結保存・解凍後のgMSC(登録商標)1のいずれにおいても、これを滅菌済みメスあるいはハサミで4等分(湿重量で概ね10mg~20mg)した。
凍結保存前のgMSC(登録商標)1、凍結保存・解凍後のgMSC(登録商標)1のいずれにおいても、以下の方法で軟骨分化誘導を行う。即ち、gMSC(登録商標)1を4等分(湿重量で概ね10mg~20mg)した後、軟骨分化用基礎培地で1回洗浄後、1個ずつを15mLコニカルチューブに移し、軟骨分化誘導培地(高グルコースα-MEM中に、10ng/mlのTGF-β3、100nMのDexamethasone、50μg/mlのL-Ascorbic acid 2‐phosphate、100μg/mlのSodium pyruvate、ITS-プラス(6.25μg/mlのTransferrin、6.25μg/mlのInsulin、6.25ng/mlのselenate、5.35μg/mlのlinoleic acid、1.25mg/mlのウシ血清アルブミン(BSA)を含む。)を前記のコニカルチューブに分注(1.0~1.8ml/本)し、5%炭酸ガス存在下にて37℃で28日間培養した。なお、2~3日おきに同一の分化誘導培地と交換した。硫酸化グリコサミノグリカン(glycosaminoglycan:GAG)定量用キット(Biocolor社製)を用いて培養後のgMSC(登録商標)1の硫酸化グリコサミノグリカン(glycosaminoglycan:GAG)の量を定量した。なおGAGの量は細胞のDNA含量によってノーマライズした。
以上の手順を3株のgMSC(登録商標)1について行なった。結果を図7に示す。図7は凍結保存・解凍後の細胞の軟骨分化能の確認実験の結果を示す図であり、図7の(a)は軟骨分化能の確認実験を行なったサンプルを撮影した結果を示し、図7の(b)は硫酸化グリコサミノグリカン(glycosaminoglycan:GAG)assayの結果を示す図である。図7に示すとおり、凍結保存液(ii)、(iii)の何れを用いて凍結した場合にも、スキャフォールドフリーの3次元構造を有する間葉系幹細胞の細胞塊においても、凍結保存・解凍後にも良好な軟骨分化能を有することが確認できた。
次に、凍結保存前のgMSC(登録商標)1、及び凍結保存液(ii)、(iii)を用いて凍結保存・解凍した後のgMSC(登録商標)1を用いて、細胞表面抗原マーカーの発現確認を行った。Fluorescence Activated Cell Sorting(FACS解析)によって細胞表面抗原マーカーの発現の確認を評価した。FACS解析に用いたフローサイトメータは、BD社製FACSVERSE(登録商標)を用いた。
(1)上述の方法で凍結保存・解凍後に融解して得られた細胞懸濁液の細胞数と細胞濃度を確認して、洗浄工程後に500万個の細胞を分取した。
(1)各抗体を薬用冷蔵ショーケース(4℃)より取り出して、前記の各チューブに添加した。各抗体の添加量は規定濃度(/1,000,000cells)より算出した。
以上の解析を3株のgMSC(登録商標)1について行なった。結果を図8に示す。図8は凍結保存・解凍後の細胞表面抗原マーカーの発現確認実験の結果を示す図である。図8に示すように、凍結保存液(ii)、(iii)の何れを用いて凍結保存・解凍した場合にも、スキャフォールドフリーの3次元構造を有する間葉系幹細胞の細胞塊においても、細胞塊の分解後の凍結保存前後の細胞表面抗原マーカーの発現プロファイルに変化はなかった。
本発明の一態様の方法で凍結保存されたスキャフォールドフリーの3次元構造を有する間葉系幹細胞の細胞塊の凍結保存実験において、凍結(融解)時の凍結保存液の液量の影響を評価する為の実験を以下のように行った。
本発明の一態様の方法で凍結保存された細胞の凍結保存効果に対する有効成分をさらに詳細に特定する為に、組成の異なる細胞凍結保存液をそれぞれ用いてgMSC(登録商標)1を用いて凍結保存効果を評価した。
表6の凍結保存液を用いて実施例2と同様の条件で凍結保存したgMSC(登録商標)1の解凍後の細胞数(生細胞数と全細胞数)を評価した。その結果を図10に示す。この棒グラフのそれぞれの項目において、「live」は「『gMSC(登録商標)1』一個当たりの生細胞数」を示し、「total」は「『gMSC(登録商標)1』一個当たりの全細胞数」を示している(n=3、結果は全て、「平均値±標準偏差」(mean±SD)で示されている。)。また、「生存率」とは、「『gMSC(登録商標)1』一個当たりの全細胞数」に対する「『gMSC(登録商標)1』一個当たりの生細胞数」の比を示している。また、「保存前」とは、gMSC(登録商標)1の凍結保存を行わずに細胞数を測定した場合の結果であり、それ以外は凍結・解凍後の結果である。
表7の凍結保存液を用いて、実施例2と同様の条件で凍結保存したgMSC(登録商標)1の解凍後の細胞数(生細胞数と全細胞数)を評価した。その結果を図11に示す。図11はリノール酸、リノレン酸、及びPAがそれぞれ含まれている凍結保存液を用いた、gMSC(登録商標)1の凍結保存実験の結果を示すグラフである。この棒グラフのそれぞれの項目において、「live」は「『gMSC(登録商標)1』一個当たりの生細胞数」を示し「total」は「『gMSC(登録商標)1』一個当たりの全細胞数」を示している(n=3、結果は全て、「平均値±標準偏差」(mean±SD)で示されている。)。また、「生存率」とは、「『gMSC(登録商標)1』一個当たりの全細胞数」に対する「『gMSC(登録商標)1』一個当たりの生細胞数」の比を示している。また、「保存前」とは、gMSC(登録商標)1の凍結保存を行わずに細胞数を測定した場合の結果であり、それ以外は凍結・解凍後の結果である。
表8の組成の凍結保存液を用いて、実験2と同様の条件で凍結保存したgMSC(登録商標)1の解凍後の細胞数を評価した。その結果を図12に示す。
表9の組成の凍結保存液を用いて、実施例2と同様の条件で凍結保存したgMSC(登録商標)1の解凍後の細胞数(生細胞数と全細胞数)を評価した。その結果を図13に示す。
表10の組成の凍結保存液を用いて、表記の条件で凍結保存したgMSC(登録商標)1の解凍後の細胞数(生細胞数と全細胞数)を評価した。その結果を図14に示す。図14は、gMSC(登録商標)1の凍結保存実験の結果を示すグラフである。この棒グラフのそれぞれの項目において、「live」は「『gMSC(登録商標)1』一個当たりの生細胞数」を示し、「total」は「『gMSC(登録商標)1』一個当たりの全細胞数」を示している(n=3、結果は全て、「平均値±標準偏差」(mean±SD)で示されている。)。また、「生存率」とは、「『gMSC(登録商標)1』一個当たりの全細胞数」に対する「『gMSC(登録商標)1』一個当たりの生細胞数」の比を示している。また、「保存前」とは、gMSC(登録商標)1の凍結保存を行わずに細胞数を測定した場合の結果であり、それ以外は凍結・解凍後の結果である。
表11の組成の凍結保存液で実施例2に記載した条件で凍結保存したgMSC(登録商標)1の解凍後の細胞数(生細胞数と全細胞数)を評価した。その結果を図15に示す。図15は、gMSC(登録商標)1の凍結保存実験の結果を示すグラフである。この棒グラフのそれぞれの項目において、「live」は「『gMSC(登録商標)1』一個当たりの生細胞数」を示し、「total」は「『gMSC(登録商標)1』一個当たりの全細胞数」を示している(n=3、結果は全て、「平均値±標準偏差」(mean±SD)で示されている。)。また、「生存率」とは、「『gMSC(登録商標)1』一個当たりの全細胞数」に対する「『gMSC(登録商標)1』一個当たりの生細胞数」の比を示している。また、「保存前」とは、gMSC(登録商標)1の凍結保存を行わずに細胞数を測定した場合の結果であり、それ以外は凍結・解凍後の結果である。
Claims (26)
- 細胞を凍結保存するための凍結保存用組成物であって、
脂肪酸を含むことを特徴とする凍結保存用組成物。 - 前記細胞は、間葉系幹細胞であることを特徴とする、請求項1に記載の凍結保存用組成物。
- 前記間葉系幹細胞は、3次元構造であり、スキャフォールドフリーの細胞塊を形成しているものであり、当該細胞塊を凍結保存するためのものであることを特徴とする請求項2に記載の凍結保存用組成物。
- 細胞を凍結保存するための凍結保存用組成物であって、
前記細胞は、3次元構造である細胞塊を形成しているものであり、当該細胞塊を凍結保存するためのものであり、
脂肪酸及び脂肪酸エステルからなる群より選ばれる少なくとも1種の成分を含むことを特徴とする凍結保存用組成物。 - 前記細胞は、間葉系幹細胞であることを特徴とする、請求項4に記載の凍結保存用組成物。
- 前記間葉系幹細胞は、スキャフォールドフリーの細胞塊を形成しているものである、請求項5に記載の凍結保存用組成物。
- 前記間葉系幹細胞は無血清培養されたものであることを特徴とする請求項2、3及び5のいずれか1項に記載の凍結保存用組成物。
- 前記成分は脂肪酸エステルであり、界面活性剤をさらに含む、請求項4に記載の凍結保存用組成物。
- 前記脂肪酸はリノール酸及びリノレン酸のうち少なくとも1種である、請求項1~8のいずれか1項に記載の凍結保存用組成物。
- 前記脂肪酸エステルはリン脂質である、請求項8に記載の凍結保存用組成物。
- 前記リン脂質はフォスファチジン酸である、請求項10に記載の凍結保存用組成物。
- 前記脂肪酸エステルはフォスファチジン酸であり、前記界面活性剤はPluronic F-68である、請求項8に記載の凍結保存用組成物。
- -80℃以下で凍結保存するためのものであることを特徴とする請求項1~12のいずれか1項に記載の凍結保存用組成物。
- 細胞を凍結保存した凍結保存物の製造方法であって、以下の(a)、(c)の工程をこの順に含むことを特徴とする凍結保存物の製造方法、
(a)前記細胞を、脂肪酸を含む凍結保存液に浸漬する工程、
(c)前記細胞を凍結する工程。 - 細胞を凍結保存した凍結保存物の製造方法であって、前記細胞は、3次元構造である細胞塊を形成しているものであり、以下の(a)、(c)の工程をこの順に含むことを特徴とする凍結保存物の製造方法、
(a)前記細胞を、脂肪酸及び脂肪酸エステルからなる群より選ばれる少なくとも1種の成分を含む凍結保存液に浸漬する工程、
(c)前記細胞を凍結する工程。 - 前記(a)工程の後に、(b)前記凍結保存液に前記細胞が浸漬された状態から、前記細胞に対する前記凍結保存液の量を減らす工程を含み、
前記(c)工程では、前記(b)工程後の前記細胞を凍結する、請求項14又は15に記載の凍結保存物の製造方法。 - 前記(b)工程では、前記細胞が、前記凍結保存液に浸漬されていない状態にする、請求項14~16のいずれか1項に記載の凍結保存物の製造方法。
- 前記(c)工程において、-80℃以下で凍結することを特徴とする請求項14~16のいずれか1項に記載の凍結保存物の製造方法。
- 細胞と、
脂肪酸を含む凍結保存用組成物と、を含み、
凍結保存されていることを特徴とする細胞製剤。 - 細胞と、
脂肪酸及び脂肪酸エステルからなる群より選ばれる少なくとも1種の成分を含む凍結保存用組成物と、を含み、
前記細胞は、3次元構造である細胞塊を形成しているものであり、
凍結保存されていることを特徴とする細胞製剤。 - 細胞を含む細胞製剤の製造方法であって、前記細胞は、3次元構造である細胞塊を形成しているものであり、以下の(a)、(c)の工程をこの順に含むことを特徴とする細胞製剤の製造方法、
(a)前記細胞を、脂肪酸を含む凍結保存液に浸漬する工程、
(c)前記細胞を凍結する工程。 - 細胞を含む細胞製剤の製造方法であって、以下の(a)、(c)の工程をこの順に含むことを特徴とする細胞製剤の製造方法、
(a)前記細胞を、脂肪酸及び脂肪酸エステルからなる群より選ばれる少なくとも1種の成分を含む凍結保存液に浸漬する工程、
(c)前記細胞を凍結する工程。 - 前記(a)工程の後に、(b)前記凍結保存液に前記細胞が浸漬された状態から、前記細胞に対する前記凍結保存液の量を減らす工程を含み、
前記(c)工程では、前記(b)工程後の前記細胞を凍結する、請求項22に記載の細胞製剤の製造方法。 - 前記(b)工程では、前記細胞が、前記凍結保存液に浸漬されていない状態にする、請求項23に記載の細胞製剤の製造方法。
- 細胞を凍結保存するための凍結保存用キットであって、
脂肪酸を備えることを特徴とする凍結保存用キット。 - 細胞を凍結保存するための凍結保存用キットであって、3次元構造である細胞塊を形成した前記細胞を凍結保存するためのキットであり、
脂肪酸及び脂肪酸エステルからなる群より選ばれる少なくとも1種の成分を備えることを特徴とする凍結保存用キット。
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