JP7372736B2 - Ischemic disease therapeutic agent containing adipose tissue-derived stromal cells and method for producing the same - Google Patents

Description

The present invention relates to an ischemic disease therapeutic agent containing adipose tissue-derived stromal cells and a method for producing the same.

Ischemic diseases develop when the blood supply falls below the demand in tissues downstream of narrowed or occluded blood vessels due to thrombi, emboli, or various other factors. Depending on the site of the narrowed or occluded blood vessel, pathological conditions such as cerebral infarction, ischemic optic neuropathy, myocardial infarction, ischemic colitis, ischemic osteonecrosis, leg ulcer, or intermittent claudication occur. For cerebral infarction, myocardial infarction, etc., treatments are performed to send blood downstream of the occluded blood vessel, such as thrombolytic therapy, vasodilation using drugs or catheters, vascular diversion, and amputation of the affected area.

Administration of angiogenic factors such as granulocyte colony-stimulating factor (G-CSF) or hepatocyte growth factor (HGF) for the purpose of promoting angiogenesis for peripheral arterial disease caused by narrowing or occlusion of arteries in the extremities ( Patent Document 1 or Patent Document 2), gene therapy that expresses the factor, and treatment methods that transplant cells have been proposed. As cells used for the treatment of such peripheral artery diseases, hematopoietic stem cells in peripheral blood and interstitial cells present in adipose tissue (Non-Patent Document 1 or Patent Document 3) are used.

International Publication No. 2002/022163 International Publication No. 2000/007615 International Publication No. 2005/034843

Planat-Benard V. et al. , Circulation, 2004, 109, pp. 656-663 Francois M. et al. , Cytotherapy, 2012, 14: pp. 147-152 Moll G. et al. , Stem Cells, 2014, 32(9): pp. 2430-2442 Quimby J. M. et al. , Stem Cell Res. Ther. , 2013, 4:48

When adipose tissue-derived stromal cells are cultured immediately before administration, it is necessary to culture them at a medical institution. However, since it is necessary to prepare special culture equipment for such culture, the medical institutions that can administer the cells are limited, and as a result, its versatility is reduced.

Therefore, in order to increase convenience, it is desirable to administer cryopreserved adipose tissue-derived stromal cells for transplantation as is, but when using cryopreserved cells for treatment, the cells immediately after thawing should be It has been reported that the therapeutic effect is reduced and adverse events are increased compared to cells or cells that have undergone a culture process after thawing (Non-patent Documents 2 to 4).

Therefore, the effectiveness and safety of frozen and thawed adipose tissue-derived stromal cells in treating ischemic diseases requires actual administration and confirmation of the results. Furthermore, when freezing and thawing are repeated, it becomes more difficult to estimate the effect.

The present invention has been made in view of the above-mentioned problems, and the present invention has been made in view of the above-mentioned problems. It is an object of the present invention to provide a method for manufacturing a therapeutic agent for ischemic disease containing derived stromal cells, and a therapeutic agent for ischemic disease obtained by such a manufacturing method.

That is, the present invention relates to:
[1] A therapeutic agent for ischemic disease comprising adipose tissue-derived stromal cells that have been cryopreserved and thawed at least twice.
[2] The therapeutic agent for ischemic disease according to [1], which is used for treating peripheral artery disease.
[3] The therapeutic agent for ischemic disease according to [1] or [2], wherein the adipose tissue-derived stromal cells are allogeneic to the subject.
[4] The therapeutic agent for ischemic disease according to any one of [1] to [3], wherein the adipose tissue-derived stromal cells are human adipose tissue-derived stromal cells.
[5] The therapeutic agent for ischemic disease according to any one of [1] to [4], wherein performing the cryopreservation and thawing at least twice includes the following steps;
(1) Cryopreserving and thawing adipose tissue-derived stromal cells,
(2) a step of culturing the thawed cells; and (3) a step of cryopreserving and thawing the cultured cells.
[6] The ischemic disease therapeutic agent according to [5], which is mixed with an infusion preparation and administered within 6 hours after thawing in step (3).
[7] The ischemic disease therapeutic agent according to [6], wherein the mixing is performed by mixing a cryopreservation solution containing adipose tissue-derived stromal cells and an infusion preparation.
[8] The therapeutic agent for ischemic disease according to any one of [1] to [7], wherein the adipose tissue-derived stromal cells are adipose tissue-derived stromal cells of 4 passages or less.
[9] The therapeutic agent for ischemic disease according to any one of [1] to [8], wherein the cryopreservation is performed in a gas phase on liquid nitrogen.
[10] The therapeutic agent for ischemic disease according to any one of [7] to [9], wherein the cryopreservation solution is a solution containing DMSO (dimethyl sulfoxide).
[11] (i) A step of culturing stromal cells separated from the collected adipose tissue, (ii) A step of cryopreserving and thawing the adipose tissue-derived stromal cells obtained in step (i), and (iii) A method for producing an ischemic disease therapeutic agent containing adipose tissue-derived stromal cells, the method comprising the steps of cryopreserving and thawing thawed adipose tissue-derived stromal cells.
[12] The manufacturing method according to [11], wherein the ischemic disease is peripheral artery disease.
[13] The production method according to [11] or [12], wherein the adipose tissue-derived stromal cells are allogeneic to the subject.
[14] The production method according to any one of [11] to [13], wherein the adipose tissue-derived stromal cells are human adipose tissue-derived stromal cells.
[15] (iv) The manufacturing method according to any one of [11] to [14], further comprising a step of mixing the adipose tissue-derived stromal cells thawed in step (iii) with an infusion preparation.
[16] The manufacturing method according to [15], wherein the step (iv) is performed within 6 hours after melting in the step (iii).
[17] The cryopreservation is performed using a cryopreservation solution, and step (iv) is a step of mixing the cryopreservation solution containing the thawed adipose tissue-derived stromal cells and the infusion preparation. ] or the manufacturing method according to [16].
[18] The production method according to any one of [11] to [17], wherein the culture in step (i) is carried out for at least one passage.
[19] The manufacturing method according to any one of [11] to [18], wherein step (ii) further includes a step of culturing thawed adipose tissue-derived stromal cells.
[20] The manufacturing method according to any one of [11] to [19], wherein the cryopreservation is performed in a gas phase above liquid nitrogen.
[21] The production method according to any one of [17] to [20], wherein the cryopreservation solution is a solution containing DMSO.

According to the present invention, it is possible to treat ischemic diseases using cryopreserved and thawed adipose tissue-derived stromal cells. Therefore, the present invention provides a therapeutic agent for ischemic diseases comprising cryopreserved and thawed adipose tissue-derived stromal cells. Furthermore, the present invention provides a method for producing a therapeutic agent for ischemic disease containing cryopreserved adipose tissue-derived stromal cells.

FIG. 1 shows the ratio of blood flow in the left limb to the right limb in a mouse model of left limb ischemia. The graphs show the results of administering PBS alone (negative control), 3 x 10 ⁵ adipose tissue-derived stromal cells, and 1 x 10 ⁶ cells, respectively. FIG. 2A shows the ratio of blood flow in the left limb to the right limb in a mouse model of left limb ischemia. In the figure, control indicates a negative control in which Ringer's lactate alone was administered, and P3 and P2 indicate the results of administration of cells that were passaged for 3 and 2 passages, respectively. FIG. 2B shows the average score of the condition of the ischemic side of the left limb lower limb ischemia model mouse. FIG. 3A shows the ratio of blood flow in the right limb to the left limb in a right limb ischemia model rabbit. The results of administering only the vehicle (negative control), 1×10 ⁶ adipose tissue-derived stromal cells, and 5×10 ⁶ cells are shown. FIG. 3B shows the number of CD31-positive cells per 1 mm ² in the ischemic limb of the right limb ischemia model rabbit.

In the present invention, "adipose tissue" refers to a tissue containing adipocytes and interstitial cells including microvascular cells, and is, for example, a tissue obtained by surgically removing or aspirating subcutaneous fat of a mammal. be. Adipose tissue can be obtained from subcutaneous fat. It is preferable that the adipose tissue-derived stromal cells described below be obtained from an animal of the same species as the subject to whom the adipose tissue-derived stromal cells are administered, and in consideration of administration to humans, human subcutaneous fat is more preferable. Although the subcutaneous fat supplying individual may be alive or dead, the adipose tissue used in the present invention is preferably tissue collected from a living individual. When collecting liposuction from an individual, examples of liposuction include PAL (power assisted) liposuction, Erconia laser liposuction, or body jet liposuction. It is preferable not to use.

In the present invention, "stromal cells" refer to cells that have the ability to differentiate into mesenchymal cells (bone cells, cardiomyocytes, chondrocytes, tendon cells, adipocytes, etc.) and can proliferate while maintaining this ability. means. The term stromal cell used in the present invention means the same cell as mesenchymal stem cell, and there is no particular distinction between the two. Therefore, adipose tissue-derived stromal cells refer to stromal cells contained in adipose tissue, and may also be referred to as adipose tissue-derived mesenchymal stem cells.

In the present invention, adipose tissue-derived stromal cells refer to any cell population containing adipose tissue-derived stromal cells. The cell population is at least 20%, preferably 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 93%, 96%, 97%, 98%. % or 99% are adipose tissue-derived stromal cells.

The adipose tissue-derived stromal cells may be obtained, for example, by the manufacturing method described in US Pat. No. 6,777,231, and include, for example, the following steps (a) to (c). be able to:
(a) obtaining a cell suspension by enzymatic digestion of adipose tissue;
(b) sedimenting the cells and resuspending the cells in a suitable medium; and (c) culturing the cells on a solid surface and removing cells that do not show binding to the solid surface.

The adipose tissue used in step (a) is preferably washed. Washing may be performed using a physiologically compatible saline solution, such as phosphate buffered saline (PBS), by sedimentation with vigorous agitation. This is to remove impurities (also called debris, such as damaged tissue, blood, red blood cells, etc.) contained in the fat tissue from the tissue. Therefore, washing and sedimentation are generally repeated until the supernatant is thoroughly cleared of debris. The remaining cells exist as clumps of various sizes, so in order to dissociate them while minimizing damage to the cells themselves, the washed cell clumps are treated with enzymes that weaken or break intercellular bonds (e.g. Treatment with collagenase, dispase or trypsin is preferred. The amount of such enzymes and duration of treatment will vary depending on the conditions used, but are known in the art. Instead of or in conjunction with such enzymatic treatment, cell clumps can be broken down by other treatments such as mechanical agitation, ultrasonic energy, thermal energy, etc., but with minimal damage to the cells. In order to suppress this, it is preferable to use only enzyme treatment. When using an enzyme, it is desirable to deactivate the enzyme using a medium or the like after an appropriate period of time in order to minimize harmful effects on cells.

The cell suspension obtained by step (a) comprises a slurry or suspension of aggregated cells, as well as various contaminant cells, such as red blood cells, smooth muscle cells, endothelial cells, and fibroblasts. Therefore, the aggregated cells and these contaminant cells may be separated and removed subsequently, but since they can be removed by adhesion and washing in step (c) described later, such separation and removal are omitted. It's okay. Separation and removal of contaminant cells can be achieved by centrifugation that forcibly separates the cells into a supernatant and a precipitate. The resulting precipitate containing contaminant cells is suspended in a physiologically compatible solvent. The suspended cells may contain red blood cells, but red blood cells are excluded by selection based on adhesion to the solid surface, which will be described later, so a lysing step is not necessarily necessary. Methods well known in the art can be used to selectively lyse red blood cells, such as incubation in hypertonic or hypotonic media by lysis with ammonium chloride. After lysis, the lysate may be separated from the desired cells, eg, by filtration, centrifugation, or density fractionation.

In step (b), the suspended cells may be washed once or multiple times in succession, centrifuged, and resuspended in a medium to increase the purity of the stromal cells. Alternatively, cells may be separated based on cell surface marker profiles or based on cell size and granularity.

The medium used for resuspension is not particularly limited as long as it is a medium that can culture stromal cells, but such a medium may be a basal medium supplemented with serum and/or albumin, transferrin, fatty acids, insulin. , sodium selenite, cholesterol, collagen precursors, trace elements, 2-mercaptoethanol, 3'-thiolglycerol, etc. may be added. These media can be supplemented with substances such as lipids, amino acids, proteins, polysaccharides, vitamins, growth factors, low molecular weight compounds, antibiotics, antioxidants, pyruvate, buffers, and inorganic salts, as necessary. Also good. Examples of the basal medium include IMDM medium, Medium 199 medium, Eagle's Minimum Essential Medium (EMEM) medium, αMEM medium, Dulbecco's modified Eagle's Medium (DMEM) medium, and Ham's F12 medium. Medium, RPMI 1640 medium, Examples include Fischer's medium, MCDB201 medium, and a mixed medium thereof. Examples of serum include, but are not limited to, human serum, fetal bovine serum (FBS), bovine serum, calf serum, goat serum, horse serum, pig serum, sheep serum, rabbit serum, rat serum, and the like. When using serum, it may be added in an amount of 5v/v% to 15v/v%, preferably 10v/v%, to the basal medium. Examples of fatty acids include, but are not limited to, linoleic acid, oleic acid, linoleic acid, arachidonic acid, myristic acid, palmitoic acid, palmitic acid, and stearic acid. Examples of lipids include, but are not limited to, phosphatidylserine, phosphatidylethanolamine, phosphatidylcholine, and the like. Amino acids include, but are not limited to, for example, L-alanine, L-arginine, L-aspartic acid, L-asparagine, L-cysteine, L-cystine, L-glutamic acid, L-glutamine, L-glycine, and the like. Examples of proteins include, but are not limited to, ecotin, reduced glutathione, fibronectin, and β2-microglobulin. Examples of the polysaccharide include glycosaminoglycans, and among the glycosaminoglycans, hyaluronic acid, heparan sulfate, and the like are particularly exemplified, but the polysaccharide is not limited thereto. Growth factors include, for example, platelet-derived growth factor (PDGF), basic fibroblast growth factor (bFGF), transforming growth factor beta (TGF-β), hepatocyte growth factor (HGF), and epidermal growth factor (EGF). Examples include, but are not limited to, connective tissue growth factor (CTGF), vascular endothelial growth factor (VEGF), and the like. From the viewpoint of using the adipose-derived stromal cells obtained in the present invention for cell transplantation, it is preferable to use a medium that does not contain xeno-derived components such as serum (xeno-free). Such a medium is provided as a pre-prepared medium for mesenchymal stem cells (stromal cells) by, for example, PromoCell, Lonza, Biological Industries, Veritas, R&D Systems, Corning, and Rohto. has been done.

Subsequently, in step (c), the cells in the cell suspension obtained in step (b) are grown on a solid surface without differentiation to an appropriate cell density and using an appropriate cell culture medium as described above. Cultivate under culture conditions. In the present invention, "solid surface" refers to any material that allows attachment of the adipose tissue-derived stromal cells of the present invention. In certain embodiments, such material is a plastic material that has been treated to promote attachment of mammalian cells to its surface. Although the shape of the culture container having a solid surface is not particularly limited, petri dishes, flasks, etc. are preferably used. Cells are washed after incubation to remove unbound cells and cell debris.

In the present invention, cells that ultimately remain bound to a solid surface can be selected as a cell population of adipose tissue-derived stromal cells.

In order to confirm that the selected cells are adipose tissue-derived stromal cells of the present invention, surface antigens may be analyzed using conventional methods such as flow cytometry. Additionally, the ability to differentiate into each cell lineage may be tested, and such differentiation can be performed using conventional methods.

The adipose tissue-derived stromal cells of the present invention can be produced as described above, but may also be defined as cells having the following characteristics;
(a) Shows adhesiveness to plastic under culture conditions in standard medium;
(b) Positive for surface antigens CD105, CD73, and CD90, negative for CD45, CD34, CD11b, CD19, and HLA-DR, and (c) Can be differentiated into osteocytes, adipocytes, and chondrocytes under culture conditions. .

In order to use the adipose tissue-derived stromal cells of the present invention as a therapeutic agent for ischemic diseases, they may be cryopreserved and thawed repeatedly as appropriate, and preferably cryopreserved and thawed at least twice. Performing cryopreservation and thawing at least twice means performing the operation of cryopreserving adipose tissue-derived stromal cells and thawing them after an arbitrary period of time at least twice. In addition, "doing it at least twice" means, for example, doing it 2 times or more, 3 times or more, 4 times or more, 5 times or more, 10 times or less, 9 times or less, 8 times or less, 7 times or less, 6 times or less. can be mentioned. More preferably, cryopreservation and thawing are performed twice.

If non-compliance is confirmed in an evaluation test conducted during the process of preparing an ischemic disease therapeutic agent containing adipose tissue-derived stromal cells, the cells will need to be discarded. However, if time elapses before obtaining the results of the evaluation test, it is necessary to continue culturing the cells even during the test. Therefore, by performing cryopreservation before the evaluation test, it is possible to avoid the process of culturing cells that would eventually be discarded. By eliminating such wasteful steps, overall manufacturing costs can be reduced. Furthermore, by interrupting the work to produce a therapeutic agent for ischemic diseases, the manufacturing schedule of the therapeutic agent can be artificially controlled. Examples demonstrate that the adipose tissue-derived stromal cells of the present invention retain their excellent therapeutic effects as the ischemic disease therapeutic agent of the present invention even after being cryopreserved and thawed two or more times. It has been confirmed.

When performing cryopreservation and thawing at least twice, for example, (1) cryopreserving and thawing adipose tissue-derived stromal cells, (2) culturing the thawed cells, and (3) the cultured cells. A method including the steps of cryopreserving and thawing can be used. Note that the final melting step is preferably performed at a medical institution. When adipose tissue collected from a donor is obtained, (i) culturing stromal cells separated from the collected adipose tissue, (ii) cryopreservation and cryopreservation of the adipose tissue-derived stromal cells obtained in step (i). thawing, (iii) cryopreserving and thawing the thawed adipose tissue-derived stromal cells, and (iv) mixing the thawed adipose tissue-derived stromal cells in step (iii) with an infusion preparation. Preferably, after the step (ii), the method further includes a step of culturing the thawed adipose tissue-derived stromal cells. Step (iii) can be repeated if necessary. When repeating step (iii), it may further include a step of culturing thawed adipose tissue-derived stromal cells.

In the present invention, cryopreservation can be performed by suspending adipose tissue-derived stromal cells in a cryopreservation solution well known to those skilled in the art and cooling the suspension. Suspension can be performed by detaching the cells with a detachment agent such as trypsin, transferring them to a cryopreservation container, treating them as appropriate, and then adding a cryopreservation solution.

The cryopreservation solution may contain DMSO (dimethyl sulfoxide) as a cryoprotectant, but since DMSO has the property of inducing differentiation of interstitial cells in addition to its cytotoxicity, the DMSO content should be reduced. It is preferable to reduce it. Glycerol, propylene glycol or polysaccharides are exemplified as substitutes for DMSO. If DMSO is used, it contains a concentration of 5% to 20%, preferably a concentration of 5% to 10%, more preferably a concentration of 10%. In addition to this, additives described in WO2007/058308 may also be included. Examples of such cryopreservation solutions include cryopreservation solutions provided by BioVerde, Nippon Genetics, Reprocell, Zenoac, Cosmo Bio, Kojin Bio, and Thermo Fisher Scientific. A liquid may also be used.

When the above-mentioned suspended cells are cryopreserved, they can be stored at a temperature between -80°C and -100°C (for example, -80°C), using any freezer that can achieve this temperature. obtain. Although not particularly limited, in order to avoid sudden temperature changes, a programmable freezer may be used to appropriately control the cooling rate. The cooling rate may be appropriately selected depending on the components of the cryopreservation solution, and may be performed according to the instructions of the cryopreservation solution manufacturer.

The storage period is not particularly limited, as long as the cells cryopreserved under the above conditions retain the same properties as before freezing after thawing, but for example, 1 week or more, 2 weeks or more, 3 weeks or more, Examples include 4 weeks or more, 2 months or more, 3 months or more, 4 months or more, 5 months or more, 6 months or more, 1 year or more, or more. Since cell damage can be suppressed by storing at a lower temperature, it may be stored by transferring to a gas phase above liquid nitrogen (approximately -150°C or lower to -180°C or lower). When storing in the gas phase on liquid nitrogen, storage containers well known to those skilled in the art can be used. Although not particularly limited, for example, when storing for two weeks or more, it is preferable to store in a gas phase over liquid nitrogen.

The thawed adipose tissue-derived stromal cells may be cultured as appropriate before the next cryopreservation. The adipose tissue-derived stromal cells are cultured using the above-mentioned medium capable of culturing stromal cells, and is not particularly limited, at a culture temperature of about 30 to 40°C, preferably about 37°C, and in CO2 _- containing air. It may be carried out in an atmosphere of The CO ₂ concentration is about 2-5%, preferably about 5%. During culture, after reaching an appropriate confluency for the culture vessel (for example, cells occupy 50% to 80% of the culture vessel), cells are detached using a detachment agent such as trypsin. However, the cells may be seeded at an appropriate cell density in a separately prepared culture container and the culture may be continued. When seeding cells, typical cell densities include about 100 cells/cm ² to about 100,000 cells/cm ² , about 500 cells/cm ² to about 50,000 cells/cm ² , about 1,000 cells/cm 2 Examples include ~10,000 cells/cm ² and approximately 2,000 to 10,000 cells/cm ² . In certain embodiments, the cell density is between 2,000 and 10,000 cells/cm ² . It is preferable to adjust the period until appropriate confluency is reached from 3 days to 7 days. During culture, the medium may be replaced as necessary.

Thawing of cryopreserved cells can be performed by methods well known to those skilled in the art. For example, a method is exemplified in which the mixture is left standing or shaken in a constant temperature bath or hot water bath at 37°C. The thawed cells may be cultured before being cryopreserved again, or may be prepared as a therapeutic agent as described below. If the cells are to be cultured again after thawing, they can be cultured by seeding them at an appropriate cell density in a separately prepared culture container as described above.

Depending on the culture method, adipose tissue-derived stromal cells can be passaged several times to increase their number without losing their therapeutic effect on ischemic diseases. In order to ensure the number of cells necessary for the treatment of ischemic diseases, it is preferable to passage the cells. The adipose tissue-derived stromal cells used in the treatment of the present invention may be cells that have been passaged no more than 8 times, no more than 7 times, no more than 6 times, no more than 5 times, no more than 4 times, or no more than 3 times. preferable. More preferred are cells that have been passaged four times or less. In the present invention, the step of detaching and collecting cells after culture is counted as one passage. The cells after detachment are subjected to re-seeding or cryopreservation. One embodiment of obtaining such adipose tissue-derived stromal cells includes (1) culturing stromal cells separated from adipose tissue at least once with cell detachment and reseeding, (2) culturing the cells. Examples include a step of detaching and cryopreserving cells, (3) a step of thawing, (4) a step of culturing, (5) a step of detaching cells and cryopreserving them, and (6) a step of thawing. The step (4) may further include a step of culturing accompanied by cell detachment and seeding.

The adipose tissue-derived stromal cells prepared above can be used as a therapeutic agent for ischemic diseases. In the present invention, ischemic disease is a disease in which cell death progresses due to a decrease in blood supply in tissues located downstream of arteries that are narrowed or occluded due to thrombus, embolism, or various other factors. Examples include cerebral infarction, angina pectoris, myocardial infarction, peripheral artery disease, and the like. In the present invention, the treatment of ischemic disease means promoting improvement of blood flow in tissues with reduced blood supply, and the improvement of blood flow includes vascular regeneration, that is, new blood vessel construction (development of collateral blood circulation). including what can be achieved by promoting Therefore, it is preferable to apply the method to pathological conditions in which tissue cell proliferation is expected in ischemic sites, and peripheral artery disease is an example of such pathological conditions. In the present invention, peripheral artery disease refers to pathological conditions of poor blood circulation in the periphery caused by diabetes, Buerger's disease, thromboangiitis obliterans, arteriosclerosis obliterans, etc., all of which are associated with ischemic disease. Contains. That is, in the present invention, treatment of peripheral artery disease means promoting vascular regeneration, that is, new blood vessel construction, in tissues downstream of peripheral arteries that cause ischemia, particularly in the peripheral limbs.

In order to be used as a therapeutic agent for ischemic diseases, adipose tissue-derived stromal cells are preferably stromal cells isolated from human-derived adipose tissue, and from the viewpoint of using cryopreserved adipose tissue-derived stromal cells. It is desirable to collect adipose tissue from a subject other than the subject suffering from an ischemic disease. That is, the adipose tissue-derived stromal cells are preferably allogeneic to the subject.

According to the present invention, there is provided a therapeutic agent for ischemic disease containing the above-described adipose tissue-derived stromal cells or a kit for treating ischemic disease containing the adipose tissue-derived stromal cells. Further, according to the present invention, the above-described adipose tissue-derived stromal cells used for cell transplantation therapy are provided. Further, according to the present invention, there is provided a method for transplanting cells into a subject suffering from an ischemic disease, the method comprising the step of administering a therapeutically effective amount of the above adipose tissue-derived stromal cells to a subject, and a method for treating a disease in a subject. is provided. Note that the therapeutic agent for ischemic diseases in the present invention is not particularly limited in its form as long as it is a medicament containing the above-mentioned adipose tissue-derived stromal cells. That is, various forms such as those in which the adipose tissue-derived stromal cells described above are cryopreserved, and those in which adipose tissue-derived stromal cells are suspended in an infusion solution for administration or preservation. are included in the ischemic disease therapeutic agent of the present invention.

The cryopreserved adipose tissue-derived stromal cells obtained by the above method can be thawed and then mixed with an infusion preparation to produce a therapeutic agent for ischemic diseases containing adipose tissue-derived stromal cells. . For mixing, a cryopreservation solution in which thawed cells are suspended may be mixed with the infusion preparation, or cells may be separated from the solvent by centrifugation etc. and then only the cells mixed with the infusion preparation. good. In order to avoid the complexity of the procedure, it is preferable not to include the step of culturing after thawing, or to directly mix the cryopreservation solution in which the thawed cells are suspended with the infusion preparation. In the present invention, the adipose tissue-derived stromal cells are preferably mixed with the infusion preparation within 3 hours, 2 hours, or 1 hour after thawing, and more preferably within 3 hours after mixing with the infusion preparation. Preferably, it is administered to a subject within 2 hours or within 1 hour. That is, it is desirable to administer the adipose tissue-derived stromal cells to the subject within 6 hours, 5 hours, 4 hours, 3 hours, 2 hours, or 1 hour after thawing.

The "infusion preparation" in the present invention is not particularly limited as long as it is a solution used in human treatment, but examples include physiological saline, 5% glucose solution, Ringer's solution, lactated Ringer's solution, acetic Ringer's solution, No. 1 solution, Examples include liquid No. 2, liquid No. 3, liquid No. 4, and the like.

The ischemic disease therapeutic agent of the present invention also includes a kit that combines frozen adipose tissue-derived stromal cells and an infusion preparation.

The infusion preparation containing cells may further include a pharmaceutically acceptable carrier. In the present invention, carriers include suspending agents, solubilizers, stabilizers, isotonic agents, preservatives, adsorption inhibitors, surfactants, diluents, media, and pH adjusters for administering therapeutic agents. It refers to agents, soothing agents, buffering agents, sulfur-containing reducing agents, antioxidants, etc., and these can be appropriately added.

Examples of suspending agents include methylcellulose, polysorbate 80, hydroxyethylcellulose, gum arabic, powdered tragacanth, sodium carboxymethylcellulose, polyoxyethylene sorbitan monolaurate, and the like. Examples of solution adjuvants include polyoxyethylene hydrogenated castor oil, polysorbate 80, nicotinamide, polyoxyethylene sorbitan monolaurate, macrogol, and castor oil fatty acid ethyl ester. Examples of the stabilizer include dextran 40, methylcellulose, gelatin, sodium sulfite, sodium metasulfate, and the like. Examples of tonicity agents include D-mannitol and sorbitol. Examples of the preservative include methyl paraoxybenzoate, ethyl paraoxybenzoate, sorbic acid, phenol, cresol, and chlorocresol. Examples of the adsorption inhibitor include human serum albumin, lecithin, dextran, ethylene oxide propylene oxide copolymer, hydroxypropyl cellulose, methyl cellulose, hydrogenated castor oil, and polyethylene glycol. Examples of the sulfur-containing reducing agent include N-acetylcysteine, N-acetylhomocysteine, thiochitoic acid, thiodiglycol, thioethanolamine, thioglycerol, thiosorbitol, thioglycolic acid and its salts, sodium thiosulfate, glutathione, Examples include those having a sulfohydryl group such as thioalkanoic acids having 1 to 7 carbon atoms. Examples of antioxidants include erythorbic acid, dibutylhydroxytoluene, butylated hydroxyanisole, α-tocopherol, tocopherol acetate, L-ascorbic acid and its salts, L-ascorbic acid palmitate, L-ascorbic acid stearate, sodium hydrogen sulfite. , sodium sulfite, triamyl gallate, propyl gallate, sodium ethylenediaminetetraacetate (EDTA), sodium pyrophosphate, sodium metaphosphate, and other chelating agents. Furthermore, inorganic salts such as sodium chloride, potassium chloride, calcium chloride, sodium phosphate, potassium phosphate, and sodium bicarbonate, organic salts such as sodium citrate, potassium citrate, and sodium acetate, and sugars such as glucose are commonly used. Components added to may be added as appropriate.

Infusion formulations mixed with cells can be prepared for topical administration using organic materials such as biopolymers, inorganic materials such as hydroxyapatite, specifically collagen matrices, polylactic acid polymers or copolymers, polyethylene glycol polymers or copolymers and their chemical derivatives. May be mixed with

The method for administering the therapeutic agent for ischemic diseases of the present invention is preferably by direct injection into the ischemic site, and examples include, but are not limited to, intracerebral administration, intramuscular administration (intramyocardial administration, intraskeletal muscle administration, etc.). ), subcutaneous administration, etc. The administration technique is not particularly limited as long as it is capable of delivering cells to the ischemic site, and may be performed using, for example, a needle or catheter.

The dose of the therapeutic agent for ischemic disease of the present invention is the amount of cells that, when administered to a subject, can provide a therapeutic effect on the disease compared to a subject to whom it has not been administered. The specific dosage can be determined as appropriate depending on the age, body weight, extent of ischemia, symptoms, etc. of the subject. per administration of 1×10 ⁵ to 1×10 ⁹ cells/kg body weight, more preferably 1×10 ⁵ to 1×10 ⁸ cells/kg body weight, even more preferably 1×10 ⁵ to 1×10 ⁷ cells/kg body weight.

Regarding the number of administrations of the therapeutic agent for ischemic diseases of the present invention, the above-mentioned dose may be administered once or in multiple doses to the entire ischemic area. When administering multiple times, it is preferable to change the administration site as appropriate, and the administration site depends on the size of the ischemic tissue, but for example, 10 or more, 20 or more, 30 or more, 40 or more, Examples include administration to 50 or more locations, 60 or more locations, 70 or more locations, or more. The amount of injection at one point is preferably 1 mL or less, 0.9 mL or less, 0.8 mL or less, 0.7 mL or less, 0.6 mL or less, or 0.5 mL or less. Therefore, it is preferable to manufacture a therapeutic agent for ischemic diseases by adjusting the cell concentration as appropriate.

The therapeutic agent for ischemic diseases of the present invention may be used in combination with other therapeutic agents or as a single agent.

The present invention will be specifically explained in the following examples, but the present invention is not limited by the examples.

[Example 1] Preparation of the therapeutic agent for ischemic diseases of the present invention
Establishment of adipose tissue-derived stromal cells
After consent was obtained from the human donor, the subcutaneous adipose tissue obtained by liposuction was washed with physiological saline. To achieve destruction of the extracellular matrix and isolation of cells, collagenase (Roche) (solvent: physiological saline) was added and dispersed by shaking at 37° C. for 90 minutes. Subsequently, this suspension was centrifuged at 800 g for 5 minutes to obtain a precipitate of interstitial vascular cells.

Add a serum-free medium for interstitial cells (Rohto) to the above cell precipitate, centrifuge the cell suspension at 400g for 5 minutes, remove the supernatant, and refill the serum-free medium for interstitial cells (Rohto). The cells were suspended and seeded into flasks.

Cells were cultured for several days at 37 °C in 5% _CO2 . After several days, the culture was washed with PBS to remove residual blood cells and adipose tissue contained in the culture solution, and stromal cells adhering to the plastic container were obtained.

Trypsin was added to detach the cells, the cells were diluted, and then seeded in a flask, and a serum-free medium for interstitial cells (Rohto) was added and cultured.

Cryopreservation of adipose tissue-derived stromal cells The obtained adipose tissue-derived stromal cells were detached using trypsin, transferred to a centrifuge tube, and centrifuged at 400 g for 5 minutes to obtain cell precipitates. After removing the supernatant, an appropriate amount of cell cryopreservation solution (STEM-CELLBANKER (Zenoac)) was added and suspended. The cell suspension solution was dispensed into cryotubes, stored in a freezer at -80 degrees, and then transferred to a gas phase above liquid nitrogen for continued storage.

Thawing of adipose tissue-derived stromal cells : Thaw cryopreserved cryotubes in a 37°C constant temperature bath, transfer to centrifuge tubes, dilute with serum-free medium for stromal cells (Rohto), and centrifuge at 400 g for 5 minutes. Separation was performed to obtain a cell pellet. After removing the supernatant, the cells were suspended in a serum-free medium for stromal cells (Rohto) and seeded in flasks. After several days, trypsin was added to detach the cells, subculturing was continued, and the cells were cryopreserved. When administering to a diseased individual, the frozen cells were thawed and suspended in an appropriate suspension, as shown in Examples below, and used as the ischemic disease therapeutic agent of the present invention.

That is, in Examples 2 to 4 below, expansion with a total of 2 passages, 3 passages, or 4 passages after seeding the treated subcutaneous adipose tissue and before the second cryopreservation. Adipose tissue-derived stromal cells obtained by culturing were used.

[Example 2]
Preparation of administration cells
The adipose tissue-derived stromal cells, which were expanded and cultured for a total of 4 passages in Example 1 and preserved, were thawed using a water bath (37°C) and transferred to a centrifuge tube containing 10 mL of DMEM. The cells were centrifuged at 400g for 5 minutes to obtain a cell pellet. After removing the supernatant, the cells were suspended in a serum-free medium for stromal cells (Rohto), transferred to a flask, and cultured for 6 days. The medium was removed and StemPro Accutase (Thermo Fisher Scientific) was added and incubated for 5 minutes to detach the cells and transfer to a centrifuge tube. After centrifugation, remove the supernatant, add DMEM, count the cell concentration, remove the supernatant, add PBS to 1.67 x 10 ⁶ cells/mL and 5.56 x 10 ⁶ cells/mL. It was adjusted.

Preparation of mouse model and therapeutic effects BALB/c mice were anesthetized by inhalation of 2% isoflurane, the left inguinal region was incised, and the left femoral artery was exposed. The origin of the femoral artery was ligated, and then the portion just before the bifurcation of the popliteal artery and the saphenous artery was ligated. After ligation, the femoral artery was excised. After adding kanamycin solution to the surgical site, the incised inguinal region was sutured to create a lower limb ischemia model mouse. On the day after surgery, PBS alone (negative control), 3 x 10 ⁵ cells/180 μL or 1 x 10 ⁶ cells/180 μL was administered to 3 sites (gastrocnemius muscle, gracilis muscle, quadriceps femoris muscle) to induce ischemia in 3 model mice each. It was administered intramuscularly near the site.

Blood flow in the hind limbs was measured on the 7th and 14th days after surgery using a blood flow imaging device, moorFLPI (Moor Instrument). Blood flow was evaluated using the ratio of blood flow in the left limb to that in the right limb. As a result, it was confirmed that blood flow was improved in a dose-dependent manner (Figure 1).

[Example 3]
Preparation of administration cells
The adipose tissue-derived stromal cells that were expanded and preserved in Example 1 with a total of 2 or 3 passages were thawed using a water bath (37°C), transferred to a centrifuge tube, and then cultured for stromal cells. A serum-free medium (Rohto) was added to bring the total volume to 13 mL. Centrifugation was performed at 380 g for 5 minutes to obtain a cell pellet. After removing the supernatant, the cells were suspended in 12 mL of a serum-free medium for stromal cells (Rohto), transferred to four flasks of 3 mL each, and cultured for 4 days. The medium was removed and StemPro Accutase (Thermo Fisher Scientific) was added and incubated for 5 minutes to detach the cells and transfer to a centrifuge tube. After centrifugation, remove the supernatant, add serum-free medium for interstitial cells (Rohto), count the cell concentration, remove the supernatant, and add lactated Ringer's solution to 5.56 x 10 ⁶ cells/mL. Adjusted to.

Preparation of mouse model and therapeutic effects BALB/c mice were anesthetized by inhalation of 2% isoflurane, the left inguinal region was incised, and the left femoral artery was exposed. The origin of the femoral artery was ligated, and then the portion just before the bifurcation of the popliteal artery and the saphenous artery was ligated. After ligation, the femoral artery was excised. After adding kanamycin solution to the surgical site, the incised inguinal region was sutured to create a lower limb ischemia model mouse. On the day after surgery, lactated Ringer's solution alone (negative control), 1×10 ⁶ cells/180 μL, was administered intramuscularly to 3 locations (gastrocnemius muscle, gracilis muscle, and quadriceps muscle) near the ischemic site of each of 5 model mice. did.

Blood flow in the hind limbs was measured on the 7th and 14th days after surgery using a blood flow imaging device, moorFLPI. Blood flow was evaluated using the ratio of blood flow in the left limb to that in the right limb. As a result, improvement in blood flow was confirmed regardless of the passage number (Fig. 2A). Furthermore, the condition of the ischemic limb was observed on the 7th and 14th day after the surgery, and scored according to the following criteria.
0: Normal appearance 1: Change in nail color 2: Damage to toe 3: Early toe fall off 4: Early toe fall off beyond the toe or damage to the lower limb 5: Fall off beyond the ankle or expansion of lower limb damage as a result It was confirmed that the deterioration of scores was suppressed in the cell-administered group (FIG. 2B).

[Example 4]
Preparation of administration cells
The adipose tissue-derived stromal cells, which were expanded and cultured for a total of 4 passages in Example 1 and preserved, were thawed using a water bath (37°C), transferred to a centrifuge tube, and PBS was added to bring the total volume to 14 mL. did. The cells were centrifuged at 400g for 5 minutes to obtain a cell pellet. After removing all but a small amount of the supernatant, the cell density was counted and PBS was added to give a concentration of 8.33 x 10 ⁵ cells/mL or 4.17 x 10 ⁶ cells/mL to prepare an administration solution.

Preparation of model rabbit and therapeutic effect A JW rabbit was anesthetized by inhalation of 3% isoflurane, the abdomen was incised, and the common iliac artery was exposed from the abdominal aorta. The right common iliac artery was ligated at two locations, and the right common iliac artery between them was excised. After adding kanamycin solution to the surgical site, the incision was sutured to create a lower limb ischemia model rabbit. On the day after surgery, PBS alone (negative control), 1 x 10 ⁶ cells/1200 μL or 5 x 10 ⁶ cells/1200 μL was intramuscularly administered to 6 locations near the tensor fascia latae muscle on the ischemic side of 3 model rabbits each. did. After the cells were thawed, an administration solution was prepared and administered within 3 hours.

The blood flow in the hind limbs was measured on the 4th, 7th, and 14th day after the surgery using a blood flow imaging device moorFLPI. Blood flow was evaluated using the ratio of blood flow in the right limb to that in the left limb. As a result, on the 14th day, improvement in blood flow was confirmed at all doses (FIG. 3A). Furthermore, after measuring the blood flow on the 14th day, the patient was sacrificed by exsanguination, and the skeletal muscles (tensor fascia latae and adductor muscles) of the ischemic limb were collected. Immunostaining was performed using When the number of CD31-positive cells per 1 mm ² was calculated and angiogenesis was evaluated, the number of CD31-positive cells tended to increase in the adductor muscle at all doses. (Figure 3B).

Claims (18)

The following step comprises subcutaneous adipose tissue-derived stromal cells cultured in a serum-free medium for stromal cells and cryopreserved and thawed at least twice, and the cryopreservation and thawing are performed at least twice.
(1) Cryopreservation and thawing of subcutaneous adipose tissue-derived stromal cells,
(2) a step of culturing the thawed cells in a serum-free medium for stromal cells, and
(3) Step of cryopreserving and thawing the cultured cells
including;
An ischemic disease in which the subcutaneous adipose tissue-derived stromal cells are positive for CD105, CD73, and CD90, negative for CD45, CD34, CD11b, CD19, and HLA-DR, and are allogeneic to the subject. therapeutic agent. The therapeutic agent for ischemic disease according to claim 1, which is used for treating peripheral artery disease. The therapeutic agent for ischemic disease according to claim 1 or 2, wherein the subcutaneous adipose tissue-derived stromal cells are human subcutaneous adipose tissue-derived stromal cells. The ischemic disease therapeutic agent according to any one of claims 1 to 3 , which is mixed with an infusion preparation and administered within 6 hours after thawing in the step (3). The ischemic disease therapeutic agent according to claim 4 , wherein the mixing is performed by mixing a cryopreservation solution containing subcutaneous adipose tissue-derived stromal cells and an infusion preparation. The therapeutic agent for ischemic disease according to any one of claims 1 to 5 , wherein the subcutaneous adipose tissue-derived stromal cells are subcutaneous adipose tissue-derived stromal cells of 4 passages or less. The ischemic disease therapeutic agent according to any one of claims 1 to 6 , wherein the cryopreservation is performed in a gas phase on liquid nitrogen. The ischemic disease therapeutic agent according to any one of claims 5 to 7 , wherein the cryopreservation solution is a solution containing DMSO (dimethyl sulfoxide). (i) a step of culturing stromal cells separated from the collected subcutaneous adipose tissue in a serum-free medium for stromal cells;
(ii) a step of cryopreserving and thawing the subcutaneous adipose tissue-derived stromal cells obtained in step (i); and (iii) a step of cryopreserving and thawing the thawed subcutaneous adipose tissue-derived stromal cells;
, the subcutaneous adipose tissue-derived stromal cells are positive for CD105, CD73, and CD90, negative for CD45, CD34, CD11b, CD19, and HLA-DR, and are allogeneic to the subject; The method for producing the therapeutic agent for ischemic diseases according to claim 1 . The manufacturing method according to claim 9 , wherein the ischemic disease is peripheral artery disease. The manufacturing method according to claim 9 or 10 , wherein the subcutaneous adipose tissue-derived stromal cells are human subcutaneous adipose tissue-derived stromal cells. The manufacturing method according to any one of claims 9 to 11 , further comprising the step of (iv) mixing the subcutaneous adipose tissue-derived stromal cells thawed in step (iii) with an infusion preparation. The manufacturing method according to claim 12 , wherein the step (iv) is performed within 6 hours after melting in the step (iii). 13. The cryopreservation is performed using a cryopreservation solution, and step (iv) is a step of mixing the cryopreservation solution containing the thawed subcutaneous adipose tissue-derived stromal cells and an infusion preparation. 13. The manufacturing method according to 13 . The manufacturing method according to any one of claims 9 to 14 , wherein the culture in step (i) is carried out for at least one passage. The manufacturing method according to any one of claims 9 to 15 , further comprising the step of culturing thawed subcutaneous adipose tissue-derived stromal cells in step (ii). The manufacturing method according to any one of claims 9 to 16 , wherein the cryopreservation is performed in a gas phase above liquid nitrogen. The manufacturing method according to any one of claims 14 to 17 , wherein the cryopreservation solution is a solution containing DMSO.

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