Pharmaceutical composition for soft tissue regeneration containing a cell population including mesenchymal stem cells

Abstract

To provide a method for improving a survival rate of adipose tissue by using a mesenchymal stem cell derived from other materials than the adipose tissue, and a pharmaceutical composition for regeneration of soft tissue that is primarily composed of a mesenchymal stem cell derived from other materials than the adipose tissue.SOLUTION: A pharmaceutical composition for regeneration of soft tissue contains a cell population containing a mesenchymal stem cell derived from other materials than adipose tissue, as an active ingredient. There is also provided a method for improving a survival rate of adipose tissue by mixing a mesenchymal stem cell, not derived from the adipose tissue, with the adipose tissue and transplanting the mixture into an organism.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a pharmaceutical composition for soft tissue regeneration containing a cell population including mesenchymal stem cells, and a method for improving the survival rate of adipose tissue by mixing a cell population including mesenchymal stem cells with adipose tissue and transplanting the mixture into a living body. [Background technology]

[0002] Fat injection is a method of regenerating soft tissue by collecting one's own fat tissue and injecting it into a depressed lesion, and is one of the options for treating facial depressions caused by factors such as collagen diseases such as lupus erythematosus, congenital diseases such as Parry-Romberg syndrome, surgical operations aimed at treating diseases such as cancer, and trauma from traffic accidents (Non-Patent Document 1). In this procedure, a thin cannula is used to collect fat tissue and a syringe is used to inject it, making it less invasive and less likely to leave scars. Another major advantage is that autologous tissue is transplanted, so there are no problems such as sequelae associated with foreign body transplants. For this reason, fat injection is also used for cosmetic improvement purposes such as breast augmentation.

[0003] Although fat injection has the above-mentioned advantages, the problem of reduced survival rate of adipose tissue due to necrosis of the adipose tissue after collection has been raised (Non-Patent Document 2). In recent years, a method (Cell-assisted lipotransfer: CAL method) has been devised to improve the survival rate of adipose tissue by mixing mesenchymal stem cells derived from autologous adipose tissue during fat transplantation (Patent Document 1).

[0004] Mesenchymal stem cells are somatic stem cells that were first discovered as stem cells present in bone marrow, and are capable of differentiating into mesenchymal cells such as osteoblasts, adipocytes, and chondrocytes. Mesenchymal stem cells are known to exist not only in bone marrow, but also in tissues such as fat, dental pulp, and fetal appendages (placenta, umbilical cord, fetal membrane, and amniotic fluid), and adipose mesenchymal stem cells in particular are used in the CAL method.

[0005] Adipose mesenchymal stem cells have the ability to differentiate into adipocytes, and are induced to differentiate into adipocytes by co-culturing with mature adipocytes (Non-Patent Document 3). Adipose mesenchymal stem cells also have the ability to differentiate into vascular endothelial cells, and furthermore, since they secrete large amounts of VEGF (Vascular endothelial growth factor) in a hypoxic environment, they can contribute to angiogenesis (Non-Patent Documents 4 and 5). Due to these cellular characteristics, it is believed that adipose mesenchymal stem cells improve the engraftment rate of adipose tissue when mixed with adipose tissue.

[0006] The amniotic membrane is a translucent membrane located closest to the fetus in the placenta of a pregnant woman, and has the role of enveloping the fetus and retaining amniotic fluid. It has been reported that cells belonging to the mesenchymal system are present in the amniotic membrane (Non-Patent Document 6). The amniotic membrane can be collected non-invasively as medical waste at the time of childbirth, and no additional invasion is required for cell collection. Mesenchymal cells present in the amniotic membrane are considered to be mesenchymal stem cells because they have cellular characteristics similar to mesenchymal stem cells derived from bone marrow or fat, but it has also been reported that they have almost no ability to differentiate into fat cells or vascular endothelial cells (Non-Patent Document 7), and therefore their cellular characteristics are thought to be different from those of bone marrow mesenchymal stem cells and fat mesenchymal stem cells. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] Special table 2007-507202 [Non-patent literature]

[0008] [Non-Patent Document 1] Francesco M Egro. et al, Clin Plast Surg. 2020 Jan;47(1):1-6. [Non-Patent Document 2] Coleman SR, Clin Plast Surg. 2001 Jan;28(1):111-9. [Non-Patent Document 3] Considine RV, et al. Am J Physiol. 1996 May;270(5 Pt 1):E895-9. [Non-Patent Document 4] Cao Y, et al. Biochem Biophys Res Commun. 2005 Jul 1;332(2):370-9. [Non-Patent Document 5] Rehman J, et al. Circulation. 2004 Mar 16;109(10):1292-8. [Non-Patent Document 6] Casey ML, et al. Biol Reprod. 1996 Dec;55(6):1253-60. [Non-Patent Document 7] Chen L, et al. Stem Cell Res. 2019 Oct;40:101537. Summary of the Invention [Problem to be solved by the invention]

[0009] Up until now, stem cells used in the CAL method have been limited to adipose mesenchymal stem cells because they must have the ability to differentiate into adipocytes and vascular endothelial cells and high angiogenic ability. However, in the case of adipose mesenchymal stem cells, it is difficult to collect adipose tissue from thin individuals and separate the cells, so the number of patients to whom the CAL method can be applied is limited. In such a situation, the objective of the present invention is to provide a method for improving the engraftment rate of adipose tissue using mesenchymal stem cells derived from a tissue other than adipose tissue, and a pharmaceutical composition for soft tissue regeneration containing mesenchymal stem cells derived from a tissue other than adipose tissue as the main component. [Means for solving the problem]

[0010] As a result of intensive research to solve the above-mentioned problems, the inventors discovered that the engraftment rate of adipose tissue can be improved by mixing adipose tissue with a cell population containing mesenchymal stem cells derived from a source other than adipose tissue and transplanting the mixture into a living body, thereby completing the present invention.

[0011] That is, according to the present specification, the following invention is provided. (1) A pharmaceutical composition for soft tissue regeneration, comprising as an active ingredient a cell population containing mesenchymal stem cells derived from tissue other than adipose tissue. (2) The pharmaceutical composition for soft tissue regeneration according to (1), wherein the mesenchymal stem cells are derived from amniotic tissue. (3) The pharmaceutical composition for soft tissue regeneration described in (1) or (2), wherein the mesenchymal stem cells are obtained from a living organism other than the subject to which the composition is to be administered. (4) The pharmaceutical composition for soft tissue regeneration according to (1) or (2), wherein the mesenchymal stem cells are obtained from a living organism of the same species as the subject to which the composition is administered. (5) A pharmaceutical composition for soft tissue regeneration described in any one of (1) to (4), wherein the subject to be administered is a human. (6) The pharmaceutical composition for soft tissue regeneration described in any one of (1) to (5), wherein the mesenchymal stem cells are derived from a human. (7) The pharmaceutical composition for soft tissue regeneration described in any one of (1) to (6), wherein the mesenchymal stem cells have no or low ability to differentiate into adipocytes. (8) The pharmaceutical composition for soft tissue regeneration described in any one of (1) to (7), wherein the mesenchymal stem cells have an inhibitory effect on fibrosis. (9) The pharmaceutical composition for soft tissue regeneration described in any one of (1) to (8), wherein the mesenchymal stem cells are mesenchymal stem cells that have been passaged twice or more. (10) A pharmaceutical composition for soft tissue regeneration according to any one of (1) to (9), wherein the surface markers of the mesenchymal stem cells are positive for CD44, CD73, CD90, and CD105, and negative for CD45. (11) A pharmaceutical composition for soft tissue regeneration according to any one of (1) to (10), which is mixed with adipose tissue and administered to soft tissue. (12) A pharmaceutical composition for soft tissue regeneration described in any one of (1) to (11), which is mixed with adipose tissue and mesenchymal stem cells derived from adipose tissue and administered to soft tissue. (13) The pharmaceutical composition for soft tissue regeneration according to any one of (1) to (12), wherein the content of dimethyl sulfoxide is 5 to 10% by mass, the content of hydroxyethyl starch is 4 to 10% by mass, and the content of human albumin is 5% by mass or less. (14) A pharmaceutical composition for soft tissue regeneration according to any one of (1) to (13), which has been cryopreserved prior to administration. (15) The pharmaceutical composition for soft tissue regeneration according to (14), which can be frozen and stored for one month or longer before administration. (16) The pharmaceutical composition for soft tissue regeneration according to (14), which can be frozen for 6 months or longer before administration. (17) The pharmaceutical composition for soft tissue regeneration according to (14), which can be frozen and stored for one year or more before administration. (18) Adipose tissue for transplantation, which contains a mixture of cell populations including mesenchymal stem cells derived from a tissue other than adipose tissue and has high engraftment ability in the body. (19) A method for improving the engraftment rate of adipose tissue, comprising the steps of mixing a cell population containing mesenchymal stem cells derived from a tissue other than adipose tissue with adipose tissue and transplanting the mixture into a subject. (20) A method for regenerating soft tissue in a subject, comprising the steps of mixing a cell population containing mesenchymal stem cells derived from a source other than adipose tissue with adipose tissue, and transplanting the mixture into the subject. Effect of the Invention

[0012] According to the present invention, it is possible to obtain a pharmaceutical composition for soft tissue regeneration that can improve the survival rate of adipose tissue when mixed with adipose tissue and transplanted into a living body. This makes it possible to perform soft tissue regeneration by the CAL method even in thin people from whom adipose tissue is difficult to obtain, thereby greatly expanding the scope of application of the CAL method.

[0013] The isolation of transplant cells from biological tissues must be performed aseptically in a cell processing center (CPC) with high indoor cleanliness, but the work of isolating transplant cells aseptically from biological tissues in a CPC is very complicated, and it is difficult to isolate cells in parallel with the collection and transplantation of adipose tissue. Since the pharmaceutical composition for soft tissue regeneration obtained by the present invention can be stored for a long period of time by cryopreservation, it can be used in the CAL method without the need to collect new tissue or isolate cells in parallel with the collection and transplantation of adipose tissue, and the steps of the CAL method can be simplified. This significantly reduces the requirements for the implementation of the CAL method, making it easier to implement the CAL method. [Brief description of the drawings]

[0014] [Figure 1] These are macroscopic photographs of adipose tissue over time after adipose tissue and amniotic mesenchymal stem cells (AMSCs) were mixed and subcutaneously transplanted into a mouse. [Diagram 2] This is a graph showing the volume of adipose tissue over time after adipose tissue and amniotic mesenchymal stem cells were mixed and subcutaneously transplanted into mice. Data are shown as mean ± standard error (SE). Measurements were performed on n=7 for each group. "#" in the figure indicates that a significant difference was observed when comparing groups (p<0.05). [Diagram 3] This is a graph showing the weight of adipose tissue over time after adipose tissue and amniotic mesenchymal stem cells were mixed and subcutaneously transplanted into mice. Data are shown as mean ± standard error (SE). Measurements were performed on n=7 for each group. "#" in the figure indicates that a significant difference was observed when comparing groups (p<0.05). [Figure 4] This is an Elastica-Masson (EM) stained image of an adipose tissue specimen 16 weeks after adipose tissue and amniotic mesenchymal stem cells were mixed and subcutaneously transplanted into a mouse. [Diagram 5]This is a graph showing the EM stained area of ​​adipose tissue specimens over time after adipose tissue and amniotic mesenchymal stem cells were mixed and subcutaneously transplanted into mice. Data are shown as mean ± standard error (SE). Measurements were performed on n=7 for each group. "#" in the figure indicates that a significant difference was observed when comparing groups (p<0.05). [Figure 6] This is a graph showing the expression levels of fibrosis-related genes in adipose tissue 4 weeks after adipose tissue and amniotic mesenchymal stem cells were mixed and subcutaneously transplanted into mice. Data are shown as mean ± standard error (SE). Measurements were performed on n=6-7 subjects for each group. "#" in the figure indicates that a significant difference was observed when comparing groups (p<0.05). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] The following describes the embodiments of the present invention in detail. The following description is intended to facilitate understanding of the present invention, and the scope of the present invention is not limited to the embodiments described below. Other embodiments in which a person skilled in the art appropriately replaces the configuration of the embodiments described below are also included in the scope of the present invention.

[0016] [1] Explanation of terms In this specification, "fetal appendages" refers to the fetal membrane, placenta, umbilical cord, and amniotic fluid. Furthermore, the "fetal membrane" is a gestational sac containing fetal amniotic fluid, and is composed of the amnion, chorion, and decidua from the inside out. Of these, the amnion originates from the fetus. The "amnion" refers to a transparent thin film with few blood vessels that is the innermost layer of the fetal membrane. The inner layer of the amnion (also called the epithelial cell layer) is covered with a single layer of epithelial cells with secretory function and secretes amniotic fluid, and the outer layer of the amnion (also called the extracellular matrix layer, equivalent to the interstitium) contains mesenchymal stem cells.

[0017] In the present specification, "mesenchymal stem cells (MSCs)" are used without distinction from "mesenchymal stromal cells". In the present specification, "mesenchymal stem cells" may be written as "MSCs". Mesenchymal stem cells adhere to plastic containers and grow, and have the ability to differentiate into one or more types of cells belonging to the mesenchymal system (e.g., osteoblasts, adipocytes, chondrocytes). In addition, the surface antigen expression pattern for identifying mesenchymal stem cells is positive for CD44, CD73, CD90, and CD105, and negative for CD45.

[0018] The expression of a surface antigen on a cell can be detected by any detection method known in the art. Methods for detecting an expression marker include, but are not limited to, flow cytometry or cell staining. In flow cytometry using a fluorescently labeled antibody, when a cell is detected that emits stronger fluorescence compared to a negative control (isotype control), the cell is judged to be "positive" for the marker. The fluorescently labeled antibody can be any antibody known in the art, and includes, but is not limited to, antibodies labeled with fluorescein isothiocyanate (FITC), phycoerythrin (PE), allophycocyanin (APC), etc. In cell staining, when a colored or fluorescent cell is observed under a microscope, the cell is judged to be "positive" for the marker. Cell staining may be immune cell staining using an antibody, or non-immune cell staining using no antibody.

[0019] As used herein, the term "amniotic mesenchymal stem cells" refers to mesenchymal stem cells derived from the amniotic membrane, and is used interchangeably with "amniotic mesenchymal stromal cells."

[0020] In the present specification, the "cell population containing mesenchymal cells" is not particularly limited as long as it is a cell population containing at least mesenchymal cells, and may be a population containing other cells. The mesenchymal stem cells may be only those isolated from one tissue, or may be a mixture of those isolated from multiple tissues. The form of the "cell population containing mesenchymal cells" is not particularly limited, and examples thereof include cell pellets, cell aggregates, cell sheets, cell suspensions, cell suspensions, frozen products thereof, and the like.

[0021] In this specification, the term "soft tissue" refers to connective tissue that plays a role in connecting the tissues that make up the body, and is a general term that includes adipose tissue, muscle tissue, peripheral nerve tissue, blood vessels, and the like.

[0022] In this specification, the "pharmaceutical composition for soft tissue regeneration" contains components for regenerating and repairing damaged and repaired soft tissue.

[0023] [2] Pharmaceutical composition for soft tissue regeneration The pharmaceutical composition for soft tissue regeneration of the present invention is characterized by containing, as an active ingredient, a cell population containing mesenchymal stem cells derived from tissue other than adipose tissue.

[0024] The subject of administration of the pharmaceutical composition for soft tissue regeneration of the present invention is typically a human, but may be another animal. Examples of other animals include mammals such as dogs, cats, cows, horses, pigs, sheep, monkeys, and ferrets, and birds such as chickens. The preferred subject of administration is a human. Hereinafter, the pharmaceutical composition for soft tissue regeneration of the present invention will be described in an embodiment in which the subject of administration is a human, but it is not intended that the subject of administration in the present invention is limited to a human.

[0025] Examples of mesenchymal stem cells derived from tissues other than adipose tissue include amniotic mesenchymal stem cells, placental mesenchymal stem cells, umbilical cord mesenchymal stem cells, bone marrow mesenchymal stem cells, and dental pulp mesenchymal stem cells. Among them, amniotic mesenchymal stem cells can be obtained in a short period of time because a large number of cells can be isolated from the source tissue and they are highly proliferative. Therefore, amniotic mesenchymal stem cells can be stably produced at low cost and with a small number of passages, and are high quality cells. Therefore, it is preferable to use amniotic mesenchymal stem cells for industrial use. As the mesenchymal stem cells, those obtained from a living organism other than the subject to be administered, that is, allogeneic mesenchymal stem cells, can be used. By using allogeneic mesenchymal stem cells, it becomes possible to perform soft tissue regeneration by the CAL method even when it is difficult to collect tissue containing autologous mesenchymal stem cells.

[0026] The mesenchymal stem cells may be derived from any animal, for example, from mammals such as rodents, such as mice, rats, and hamsters; primates, such as humans, gorillas, and chimpanzees; and livestock or pet animals, such as dogs, cats, rabbits, cows, horses, sheep, and goats, but are preferably derived from the same species of animal as the subject of administration. When the subject of administration is a human, it is preferable to use mesenchymal stem cells derived from humans.

[0027] The mesenchymal stem cells can be those that have no or low differentiation ability into adipocytes. The adipose mesenchymal stem cells used in the conventional CAL method have the ability to differentiate into adipocytes and vascular endothelial cells, and improve the engraftment rate of adipose tissue by inducing angiogenesis. However, even mesenchymal stem cells that have no or low differentiation ability have the effect of improving the engraftment rate of adipose tissue, and therefore the mechanism of action is considered to be essentially different from that of the conventional CAL method. In this specification, mesenchymal stem cells that have no or low differentiation ability into adipocytes refer to mesenchymal stem cells that have no or low differentiation ability into adipocytes when cells cultured for 21 days or more in any known adipocyte differentiation induction medium (for example, commercially available media such as StemMACS (trademark) AdipoDiff Media, MesenCult (trademark) Adipogenic Differentiation Kit, StemPro (trademark) Adipogenesis Differentiation Kit, etc. can be used) are stained with Oil Red O, and the percentage of stained cells in the cell group under the microscope field of view is not 20% or more (most preferably 0%). More preferably, when mesenchymal stem cells that have no or low ability to differentiate into adipocytes are cultured using two or three of the adipocyte differentiation induction media of StemMACS™ AdipoDiff Media, MesenCult™ Adipogenic Differentiation Kit, and StemPro™ Adipogenesis Differentiation Kit, in either case, when cells cultured for 21 days or more are stained with Oil Red O, the proportion of stained cells in the cell group under a microscope does not reach 20% or more.

[0028] The mesenchymal stem cells preferably have an inhibitory effect on fibrosis, which can inhibit fibrosis of adipose tissue and improve the engraftment rate of adipose tissue.

[0029] Hereinafter, a cell population containing amniotic mesenchymal stem cells will be described as an example. The method for producing amniotic mesenchymal stem cells may include a step of enzymatically treating the amniotic membrane or fetal appendages containing the amniotic membrane and / or a step of attaching the amniotic membrane to a culture vessel to obtain a cell population containing mesenchymal stem cells. The obtained cell population may contain other miscellaneous cells as long as it contains mesenchymal stem cells derived from the amniotic membrane. When obtaining cells from tissue, blood cells may be mixed in from blood adhering to the tissue.

[0030] The amniotic membrane is composed of an epithelial cell layer and an extracellular matrix layer, the latter of which contains amniotic mesenchymal stem cells. Like other epithelial cells, amniotic epithelial cells characteristically express epithelial cadherin (E-cadherin: CD324) and epithelial adhesion factor (EpCAM: CD326), whereas amniotic mesenchymal stem cells do not express these epithelial-specific surface antigen markers and can be easily distinguished by flow cytometry. The above-mentioned cell obtaining step may include a step of obtaining the amniotic membrane by Caesarean section.

[0031] In the method for producing amniotic mesenchymal stem cells of the present invention, the cells are preferably obtained by treating the amniotic membrane with at least collagenase.

[0032] The enzyme treatment of the amniotic membrane is preferably a treatment with an enzyme (or a combination thereof) that can release mesenchymal stem cells contained in the extracellular matrix layer of the amniotic membrane and does not degrade the epithelial cell layer. Such an enzyme is not particularly limited, but may be, for example, collagenase and / or metal proteinase. The metal proteinase may be, but is not particularly limited to, thermolysin and / or dispase, which are metal proteinases that cleave the N-terminal side of nonpolar amino acids.

[0033] The collagenase activity concentration is preferably 50 PU / ml or more, more preferably 100 PU / ml or more, even more preferably 200 PU / ml or more, even more preferably 300 PU / ml or more, even more preferably 400 PU / ml or more. The collagenase activity concentration is not particularly limited, but is, for example, 1000 PU / ml or less, 900 PU / ml or less, 800 PU / ml or less, 700 PU / ml or less, 600 PU / ml or less, or 500 PU / ml or less. Here, PU (Protease Unit) is defined as the amount of enzyme that decomposes 1 μg of FITC-collagen in 1 minute at pH 7.5 and 30° C.

[0034] The activity concentration of the metal proteinase (e.g., thermolysin and / or dispase) is preferably 50 PU / ml or more, more preferably 100 PU / ml or more, even more preferably 200 PU / ml or more, even more preferably 300 PU / ml or more, even more preferably 400 PU / ml or more. The activity concentration of the metal proteinase is preferably 1000 PU / ml or less, more preferably 900 PU / ml or less, even more preferably 800 PU / ml or less, even more preferably 700 PU / ml or less, even more preferably 600 PU / ml or less, even more preferably 500 PU / ml or less. Here, in an embodiment in which dispase is used as the metal proteinase, PU (Protease Unit) is defined as the amount of enzyme that releases amino acids equivalent to 1 μg of tyrosine from lactate casein per minute at pH 7.5 and 30° C. Within the above enzyme concentration range, mesenchymal stem cells contained in the extracellular matrix layer can be efficiently released while preventing contamination of epithelial cells contained in the epithelial cell layer of the fetal appendage. A preferred combination of collagenase and / or metalloproteinase concentrations can be determined by microscopic observation of the fetal appendages after enzyme treatment or by flow cytometry of the obtained cells.

[0035] From the viewpoint of efficiently recovering living cells, it is preferable to treat the fetal appendages simultaneously and in a lump with a combination of collagenase and metal proteinase. In this case, the metal proteinase may be, but is not limited to, thermolysin and / or dispase. By treating the fetal appendages only once with an enzyme solution containing collagenase and metal proteinase, mesenchymal stem cells can be easily obtained. In addition, by treating them simultaneously and in a lump, the risk of contamination by bacteria, viruses, etc. can be reduced.

[0036] The enzyme treatment of the amniotic membrane is preferably performed by immersing the amniotic membrane, which has been washed with a washing solution such as physiological saline or Hank's balanced salt solution, in the enzyme solution and stirring the solution with a stirring means. As the stirring means, from the viewpoint of efficiently releasing the mesenchymal stem cells contained in the extracellular matrix layer of the fetal appendage, for example, a stirrer or a shaker can be used, but is not limited thereto. The stirring speed is not particularly limited, but when a stirrer or a shaker is used, for example, 5 rpm or more, 10 rpm or more, 20 rpm or more, 30 rpm or more, 40 rpm or more, or 50 rpm or more. In addition, the stirring speed is not particularly limited, but when a stirrer or a shaker is used, for example, 100 rpm or less, 90 rpm or less, 80 rpm or less, 70 rpm or less, or 60 rpm or less. The enzyme treatment time is not particularly limited, but for example, 10 minutes or more, 20 minutes or more, 30 minutes or more, 40 minutes or more, 50 minutes or more, 60 minutes or more, 70 minutes or more, 80 minutes or more, or 90 minutes or more. The enzyme treatment time is not particularly limited, but may be, for example, 6 hours or less, 5 hours or less, 4 hours or less, 3 hours or less, 2 hours or less, 110 minutes or less, or 100 minutes or less. The enzyme treatment temperature is not particularly limited, but may be, for example, 15°C or more, 16°C or more, 17°C or more, 18°C ​​or more, 19°C or more, 20°C or more, 21°C or more, 22°C or more, 23°C or more, 24°C or more, 25°C or more, 26°C or more, 27°C or more, 28°C or more, 29°C or more, 30°C or more, 31°C or more, 32°C or more, 33°C or more, 34°C or more, 35°C or more, or 36°C or more. The enzyme treatment temperature is not particularly limited, but may be, for example, 40°C or less, 39°C or less, 38°C or less, or 37°C or less.

[0037] In the production method of the present invention, if desired, the liberated mesenchymal stem cells can be separated and / or recovered from the enzyme solution containing the liberated mesenchymal stem cells by known methods such as a filter, centrifugation, a hollow fiber separation membrane, or a cell sorter. Preferably, the enzyme solution containing the liberated mesenchymal stem cells is filtered through a filter. In an embodiment in which the enzyme solution is filtered through a filter, only the liberated cells pass through the filter, and the epithelial cell layer that has not been decomposed cannot pass through the filter and remains on the filter, so that not only can the liberated mesenchymal stem cells be easily separated and / or recovered, but also the risk of contamination by bacteria, viruses, etc. can be reduced. The filter is not particularly limited, but for example, a mesh filter can be mentioned. The pore size (mesh size) of the mesh filter is not particularly limited, but is, for example, 40 μm or more, 50 μm or more, 60 μm or more, 70 μm or more, 80 μm or more, or 90 μm or more. The pore size of the mesh filter is not particularly limited, but may be, for example, 200 μm or less, 190 μm or less, 180 μm or less, 170 μm or less, 160 μm or less, 150 μm or less, 140 μm or less, 130 μm or less, 120 μm or less, 110 μm or less, or 100 μm or less. The filtration speed is not particularly limited, but by setting the pore size of the mesh filter in the above range, the enzyme solution containing mesenchymal stem cells can be filtered by gravity, thereby preventing a decrease in cell viability.

[0038] Nylon is preferably used as the material for the mesh filter. Tubes containing 40 μm, 70 μm, 95 μm, or 100 μm nylon mesh filters, such as Falcon cell strainers, which are widely used for research, can be used. Medical mesh cloths (nylon and polyester) used in hemodialysis can also be used. Furthermore, arterial filters (polyester mesh filters, pore size: 40 μm to 120 μm) used during extracorporeal circulation can also be used. Other materials, such as stainless steel mesh filters, can also be used.

[0039] When passing mesenchymal stem cells through a filter, natural (free) fall is preferable. Forced passage through a filter, such as by suction using a pump, is also possible, but it is preferable to use as little pressure as possible to avoid damaging the cells.

[0040] The filtered mesenchymal stem cells can be collected by centrifugation after diluting the filtrate with twice or more of a medium or a balanced salt buffer, such as, but not limited to, Dulbecco's phosphate buffer (DPBS), Earle's balanced salt solution (EBSS), Hank's balanced salt solution (HBSS), and phosphate buffered saline (PBS).

[0041] The method may include a step of culturing the amniotic mesenchymal stem cells obtained by the above steps to proliferate them. The seeding density of the cell population in the culture step is, for example, 500 to 10,000 cells / cm. 2 The density at which the cell population is seeded is more preferably 500 cells / cm. 2 More than 1,000 cells / cm 2 More than 2,000 cells / cm 2 More than 3,000 cells / cm 2 More than 4,000 cells / cm 2 More than 5,000 cells / cm 2 The density at which the cell population is seeded is more preferably 10,000 cells / cm. 2 Below, 9,000 cells / cm 2 Below 8,000 cells / cm 2 Below, 7,000 cells / cm 2 The following is the result.

[0042] The above-mentioned culturing step may include a subculture step, or may include a step of repeating the culture multiple times under different culture conditions.

[0043] The culture period for one culture cycle can be, for example, 4 to 10 days, and more specifically, can be 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, or 10 days.

[0044] The medium used for the above culture can be prepared by using any liquid medium for animal cell culture as a basal medium and appropriately adding other components (serum, serum replacement reagent, growth factor, etc.) as necessary. In an embodiment in which a growth factor is added to the basal medium, the medium may be prepared by further adding a reagent (heparin, etc.) for stabilizing the growth factor in the medium in addition to the growth factor, or the growth factor may be stabilized in advance with a gel, polysaccharide, etc., and then the stabilized growth factor may be added to the basal medium.

[0045] Examples of basal media that can be used include, but are not limited to, BME medium, BGJb medium, CMRL1066 medium, Glasgow MEM medium, Improved MEM Zinc Option medium, IMDM medium (Iscove's Modified Dulbecco's Medium), Medium 199 medium, Eagle MEM medium, αMEM (Alpha Modification of Minimum Essential Medium Eagle) medium, DMEM medium (Dulbecco's Modified Eagle's Medium), Ham's F10 medium, Ham's F12 medium, RPMI 1640 medium, Fischer's medium, and mixtures thereof (e.g., DMEM / F12 medium (Dulbecco's Modified Eagle's Medium / Nutrient Mixture F-12 Ham)).

[0046] Examples of other components include albumin, serum, serum replacement reagents, and growth factors. In the case of albumin, the concentration is preferably more than 0.05% and not more than 5%. In the case of serum, the concentration is preferably 5% or more.

[0047] The medium used for the above culture may be a commercially available serum-free medium, such as, but not limited to, STK1 or STK2 (DS Pharma Biomedical), EXPREP MSC Medium (Biomimetics Sympathies), Corning stemgro human mesenchymal stem cell medium (Corning).

[0048] The culture of a cell population containing mesenchymal stem cells can be carried out, for example, by the following steps. First, the cell suspension is centrifuged, the supernatant is removed, and the obtained cell pellet is suspended in a medium. Next, the cells are seeded in a plastic culture vessel and incubated at 3% to 5% CO. 2 The cells are cultured at a concentration of 100 mM KOH at 37° C. in a medium to a confluence rate of 95% or less. Examples of the medium include, but are not limited to, αMEM, M199, or media based on these. The cells obtained by the above-mentioned culture are cells that have been cultured once.

[0049] The cells cultured once can be further passaged and cultured, for example, as follows. First, the cells cultured once are treated with ethylenediaminetetraacetic acid (EDTA) and then with trypsin to detach them from the plastic culture vessel. Next, the obtained cell suspension is centrifuged, the supernatant is removed, and the obtained cell pellet is suspended in a medium. Finally, the cells are seeded in a plastic culture vessel and incubated at 3% to 5% CO. 2The cells are cultured at a concentration of 1000 mM, at 37°C, using a medium to a confluence rate of 95% or less. Examples of the medium include, but are not limited to, αMEM, M199, or media based on these. The cells obtained by the above-mentioned passage and culture are cells that have been passaged once. By performing the same passage and culture, cells that have been passaged N times can be obtained (N represents an integer of 1 or more). From the viewpoint of mass production of cells, the lower limit of the number of passages N is, for example, 2 times or more, preferably 3 times or more, more preferably 4 times or more, more preferably 5 times or more, more preferably 6 times or more, more preferably 7 times or more, more preferably 8 times or more, more preferably 9 times or more, more preferably 10 times or more, more preferably 11 times or more, more preferably 12 times or more, more preferably 13 times or more, more preferably 14 times or more, more preferably 15 times or more, more preferably 16 times or more, more preferably 17 times or more, more preferably 18 times or more, more preferably 19 times or more, more preferably 20 times or more, and more preferably 25 times or more. Moreover, from the viewpoint of suppressing cellular aging, the upper limit of the number of passages N is preferably, for example, 50 or less, 45 or less, 40 or less, 35 or less, or 30 or less.

[0050] In the above-mentioned culture process, the amniotic mesenchymal stem cells can be cultured without proliferation arrest for preferably 40 days or later, more preferably 45 days or later, 50 days or later, 55 days or later, 60 days or later, 65 days or later, 70 days or later, 75 days or later, 80 days or later, 85 days or later, 90 days or later, 95 days or later, 100 days or later, 105 days or later, or 110 days or later after the start of ex vivo culture.

[0051] In the above-mentioned culture process, the amniotic mesenchymal stem cells can be cultured until the number of doublings after initiation of ex vivo culture is preferably 10 or more, more preferably 15 or more, 20 or more, 25 or more, 30 or more, 35 or more, 40 or more, 45 or more, or 50 or more.

[0052] The doubling number is the number of times a cell divides during a certain culture period, and is expressed as [log 10 (Number of cells at the end of culture)-log 10 (cell number at the start of culture)] / log 10 (2) The doubling time is calculated using the formula below. When subcultures are performed, the doubling times for each subculture are calculated using the formula above, and then accumulated to calculate the total doubling times.

[0053] Furthermore, the method for producing amniotic mesenchymal stem cells can include a step of cryopreserving the cell population containing the mesenchymal stem cells. In an embodiment including a step of cryopreserving the cell population, the cell population may be thawed, and then separated, collected, and / or cultured as necessary. Furthermore, the cell population may be used as is after thawing.

[0054] The means for cryopreserving the cell population containing mesenchymal stem cells is not particularly limited, but examples include a programmed freezer, a deep freezer, immersion in liquid nitrogen, etc. The temperature during freezing is preferably -30°C or lower, -40°C or lower, -50°C or lower, -60°C or lower, -70°C or lower, -80°C or lower, -90°C or lower, -100°C or lower, -110°C or lower, -120°C or lower, -130°C or lower, -140°C or lower, -150°C or lower, -160°C or lower, -170°C or lower, -180°C or lower, -190°C or lower, or -196°C or lower (liquid nitrogen temperature). The freezing rate is preferably, for example, 1° C. / min, 2° C. / min, 3° C. / min, 4° C. / min, 5° C. / min, 6° C. / min, 7° C. / min, 8° C. / min, 9° C. / min, 10° C. / min, 11° C. / min, 12° C. / min, 13° C. / min, 14° C. / min, or 15° C. / min. When a programmable freezer is used as the freezing means, the temperature can be lowered to a temperature between −50° C. and −30° C. (e.g., −40° C.) at a freezing rate of 1° C. / min to 2° C. / min, and further lowered to a temperature between −100° C. and −80° C. (e.g., −90° C.) at a freezing rate of 9° C. / min to 11° C. / min (e.g., 10° C. / min).

[0055] When freezing by the above freezing means, the cell population may be frozen in any storage container, such as, but not limited to, cryotubes, cryovials, freezing bags, infusion bags, etc.

[0056] From the viewpoint of increasing the survival rate of cells, the cryopreservation solution preferably contains albumin at a predetermined concentration of more than 0% by mass. The preferred concentration of albumin is, for example, 0.5% by mass or more, 1% by mass or more, 2% by mass or more, 3% by mass or more, 4% by mass or more, 5% by mass or more, 6% by mass or more, 7% by mass or more, or 8% by mass or more. The preferred concentration of albumin is, for example, 40% by mass or less, 35% by mass or less, 30% by mass or less, 25% by mass or less, 20% by mass or less, 15% by mass or less, 10% by mass or less, or 9% by mass or less. Examples of albumin include, but are not limited to, bovine serum albumin, mouse albumin, and human albumin.

[0057] The pharmaceutical composition for soft tissue regeneration of the present invention can be used as a therapeutic agent for depression lesions due to trauma caused by sports or traffic accidents on fat, muscle, ligaments, etc., deep lupus erythematosus, Parry-Romberg syndrome, scleroderma, facial degenerative disease (facial lipoatrophy) caused by lipoatrophy associated with HIV infection, etc. The pharmaceutical composition for soft tissue regeneration of the present invention can also be used for cosmetic improvement purposes such as breast augmentation and removal of facial wrinkles and sagging caused by aging. By mixing the pharmaceutical composition for soft tissue regeneration of the present invention with adipose tissue and administering it to the treatment site or cosmetic improvement site in an amount that can be measured, fibrosis of adipose tissue can be suppressed and the survival rate of adipose tissue can be improved.

[0058] According to the present invention, there is provided a pharmaceutical composition for soft tissue regeneration according to the present invention, which is used for the treatment of depressed lesions of fat, muscle, ligaments, etc. due to injuries caused by sports or traffic accidents, lupus erythematosus profundus, Parry-Romberg syndrome, scleroderma, facial degenerative diseases caused by fat transplantation associated with HIV infection (facial lipoatrophy), and the like.

[0059] According to the present invention, there is provided a pharmaceutical composition for soft tissue regeneration according to the present invention, which is used for cosmetic improvement such as breast augmentation and removal of facial wrinkles and sagging caused by aging.

[0060] According to the present invention, there are provided a method for transplanting cells into a patient or subject, a method for treating a disease in a patient or subject, and a method for improving the cosmetic appearance of a patient or subject, which comprise the step of administering to the patient or subject an effective amount of a pharmaceutical composition for soft tissue regeneration according to the present invention.

[0061] According to the present invention, there is provided use of the pharmaceutical composition for soft tissue regeneration according to the present invention for soft tissue regeneration treatment for the purpose of treating the aforementioned depressed lesion or improving cosmetic appearance.

[0062] According to the present invention, there is provided use of the pharmaceutical composition for soft tissue regeneration according to the present invention for treatment required for repairing soft tissue, promoting soft tissue survival, and inhibiting soft tissue fibrosis by administration to a patient or subject.

[0063] The pharmaceutical composition for soft tissue regeneration of the present invention is preferably mixed with adipose tissue and administered to soft tissue, and may be mixed with adipose tissue and mesenchymal stem cells derived from adipose tissue and administered to soft tissue.

[0064] The dosage of the pharmaceutical composition for soft tissue regeneration of the present invention is the amount of cells that, when administered to a patient or subject, can provide an effect on disease treatment or cosmetic improvement compared to a patient or subject not administered the composition. The specific dosage can be appropriately determined depending on the dosage form, administration method, purpose of use, age and symptoms of the patient or subject, and the amount of adipose tissue to be engrafted. The dosage is not particularly limited, but for example, 10 5 More than 10 pieces 6 More than 10 7 The dosage is not particularly limited, but may be, for example, 10 10 Less than or equal to 10 9 10 or less 8 1 or less.

[0065] The method of administration of the pharmaceutical composition for soft tissue regeneration of the present invention is not particularly limited, but examples thereof include direct injection into a local area or direct transplantation into a local area.

[0066] The pharmaceutical composition for soft tissue regeneration of the present invention can also be used for the treatment of other diseases as an injectable preparation, or a transplant preparation of a cell mass or sheet-like structure, or a gel preparation mixed with any gel.

[0067] The pharmaceutical composition for soft tissue regeneration of the present invention may be used immediately after culture, but is preferably stored in a frozen state until immediately before use. The period during which frozen storage is possible before administration is preferably one month or more, more preferably six months or more, and even more preferably one year or more. The pharmaceutical composition for soft tissue regeneration of the present invention may contain any component used in human treatment. Examples of such components include, but are not limited to, salts, polysaccharides (e.g., HES, dextran, etc.), proteins (e.g., albumin, etc.), DMSO, medium components (e.g., components contained in RPMI1640 medium, etc.). For example, it is preferable that the pharmaceutical composition contains dimethyl sulfoxide, hydroxyethyl starch, and human albumin, and it is more preferable that the pharmaceutical composition contains 5 to 10% by mass of dimethyl sulfoxide, 4 to 10% by mass of hydroxyethyl starch, and 5% by mass or less of human albumin.

[0068] The pharmaceutical composition of the present invention may also be a cell population containing mesenchymal stem cells diluted with an infusion preparation used as a pharma- ceutical acceptable medium. The "infusion preparation (pharma-ceutical acceptable medium)" in this specification is not particularly limited as long as it is a solution used in human treatment, and examples thereof include physiological saline, 5% glucose solution, Ringer's solution, lactate Ringer's solution, acetate Ringer's solution, initiation solution (solution No. 1), dehydration replacement solution (solution No. 2), maintenance infusion solution (solution No. 3), postoperative recovery solution (solution No. 4), etc.

[0069] [3] Adipose tissue for transplantation and method for improving the survival rate of adipose tissue The transplantable adipose tissue of the present invention is characterized by being mixed with a cell population including mesenchymal stem cells derived from a source other than adipose tissue and having high engraftment ability in a living body. More specifically, the transplantable adipose tissue is produced by mixing adipose tissue with the pharmaceutical composition for soft tissue regeneration of the present invention.

[0070] The present specification provides a method for improving the survival rate of adipose tissue. The method for improving the survival rate of adipose tissue in the present specification is characterized by comprising a step of mixing a cell population containing mesenchymal stem cells derived from a non-adipose tissue with adipose tissue and transplanting the mixture into a subject. More specifically, the method comprises a step of transplanting the transplantable adipose tissue of the present invention into a subject.

[0071] The adipose tissue as the source of the transplant adipose tissue may be derived from the same species or different species as the subject to be transplanted, but is preferably derived from the same species. When the subject is a human, it is preferably derived from a human. Of the allogeneic origin, it may be derived from an autologous or heterologous system, but is preferably derived from an autologous system.

[0072] The detailed conditions for the method of harvesting adipose tissue, the method of processing the adipose tissue before mixing with the pharmaceutical composition for soft tissue regeneration of the present invention, and the method of transplanting transplantable adipose tissue may be those described in, for example, Patent Document 1.

[0073] Details of the soft tissue regeneration composition to be mixed, the mixing ratio of adipose tissue and soft tissue regeneration composition, etc. are as described in the section [2] Soft tissue regeneration composition, unless otherwise stated.

[0074] [4] Methods for regenerating soft tissue The present specification provides a method for regenerating soft tissue of a subject. The method for regenerating soft tissue of a subject in the present specification is characterized by comprising a step of mixing a cell population containing mesenchymal cells derived from a source other than adipose tissue with adipose tissue and transplanting the mixture into a subject. Specifically, the method for regenerating soft tissue of a subject comprises a step of transplanting the transplantable adipose tissue of the present invention into a subject.

[0075] Detailed conditions for the method of preparing adipose tissue, a cell population containing mesenchymal cells derived from a tissue other than adipose tissue, and a method of preparing adipose tissue for transplantation, used in the method of regenerating soft tissue, are as described in the sections [2] Composition for soft tissue regeneration and [3] Method for improving the survival rate of adipose tissue and transplantable adipose tissue, unless otherwise stated. EXAMPLES

[0076] The present invention will be specifically described in the following examples, but the present invention is not limited to these examples.

[0077] <Example 1: Preparation of amniotic mesenchymal stem cells> (1) Collection of amniotic membrane Fetal appendages, the amniotic membrane and placenta, were collected aseptically from pregnant women undergoing elective cesarean section who had given informed consent. The obtained amniotic membrane and placenta were placed in a sterile tray containing physiological saline, and the amniotic membrane was manually peeled off from the stump of the amniotic membrane. The amniotic membrane was washed with Hanks' balanced salt solution (Ca·Mg-free) to remove any attached blood and blood clots.

[0078] (2) Enzyme treatment of amniotic membrane and collection of mesenchymal stem cells The amniotic membrane containing the epithelial cell layer and the mesenchymal cell layer was immersed in Hanks' balanced salt solution (containing Ca·Mg) containing 240 PU / mL collagenase and 200 PU / mL dispase I, and the amniotic membrane was enzymatically treated by shaking at 50 rpm for 90 minutes at 37°C. The solution after the enzyme treatment was filtered through a nylon mesh with 95 μm openings to remove undigested material from the amniotic membrane, and a cell suspension containing mesenchymal cells was collected.

[0079] (3) Cultivation of mesenchymal stem cells The cell population containing mesenchymal stem cells obtained in the above "(2) Enzyme treatment of amniotic membrane and collection of mesenchymal stem cells" was seeded in a culture vessel CellSTACK (registered trademark) (manufactured by Corning Incorporated). The seeding density was 5,000 cells / cm. 2After cell seeding, the cells were cultured in Alpha Modification of Minimum Essential Medium Eagle (αMEM) containing 10% fetal bovine serum (FBS) until the cells became subconfluent. After the culture, 15 mL of 0.5% trypsin-ethylenediaminetetraacetic acid was added per stack of CellSTACK (registered trademark), and the cells were incubated at 37°C for 10 minutes to completely detach and recover the cell population. The cell population obtained here is the 0th passage cell population. Then, 1 / 5 of the cell population was seeded on CellSTACK (registered trademark) of the same scale as the previous culture, and subcultured in αMEM containing 10% FBS. The above process was repeated 2-4 times to prepare the 3rd-5th passage cell population.

[0080] (4) Cryopreservation of mesenchymal stem cells The cell population at passages 3-5 obtained in the above-mentioned "(3) Culture of mesenchymal stem cells" was suspended in Cell Banker and cryopreserved to produce amniotic mesenchymal stem cells.

[0081] <Example 2: Study to improve the engraftment rate of adipose tissue using amniotic mesenchymal stem cells> (1) Subcutaneous transplantation of adipose tissue and amniotic mesenchymal stem cells into mice The adipose tissue was collected from the excess fat generated during human fat transplantation. Subcutaneous fat was aspirated from the abdomen, thighs, buttocks, etc. of the patient using the Coleman method, and after centrifugation at 700 x g for 3 minutes, only the middle fat layer was collected.

[0082] After thawing the amniotic mesenchymal stem cells at 37℃, they were seeded on a 150 mm culture dish and cultured in αMEM supplemented with 10% FBS, 100 U / mL penicillin and 100 μg / mL streptomycin. When the cells became subconfluent, they were passaged using 0.5% trypsin-ethylenediaminetetraacetic acid, and the cells of the 1st to 3rd passages were collected and cultured as 5.0 × 10 amniotic mesenchymal stem cells. 5 pieces, 5.0×106 The pieces were suspended in 100μL of phosphate buffered saline and placed in a 1mL syringe. 0.5g of human fat was placed in the same syringe and mixed by manually moving the inner cylinder. The prepared amniotic mesenchymal stem cell-mixed fat was injected subcutaneously into the left side of the back of a CB-17 / IcrHsd-Prkdcscid mouse, and 0.5g of fat not mixed with amniotic mesenchymal stem cells was injected in the right side of the back as a control.

[0083] (2) Macroscopic observation of the grafted fat over time Evaluations were performed 2, 4, 8, 12, and 16 weeks after transplantation. The transplanted fat was extracted after subcutaneous dissection from a midline skin incision on the back. The extracted transplanted fat was covered with a thin capsule and was relatively easy to peel off from the surrounding tissue. Capillaries were visible within the capsule in some of the transplanted fat, but there was no clear difference in the degree of capillaries due to the addition of amniotic mesenchymal stem cells. Macroscopic findings are shown in Figure 1.

[0084] (3) Measurement of the remaining volume of grafted fat over time The volume of the transplanted fat was measured by water displacement using a mouse foot volume measuring device. The volume of the transplanted fat decreased over time. 5 Individual addition group, 5.0×10 6 In both groups, the volume of the remaining grafted fat tended to be larger than in the control group, with a 5.0×10 5 12 weeks in the group with 5.0×10 6 At 4, 8, and 12 weeks, the amniotic mesenchymal stem cell group had a significantly larger volume than the control group (p < 0.05). The measurement data are shown in Figure 2.

[0085] (4) Measurement of the remaining weight of transplanted fat over time The mass of the transplanted fat was measured using an electronic balance. The mass of the transplanted fat decreased over time. 5 Individual addition group, 5.0×10 6In both groups, the mass of the remaining transplanted fat tended to be larger than in the control group, and in both groups, the mass was significantly larger in the amniotic mesenchymal stem cell group at 4 weeks and 12 weeks (p < 0.05). The measurement data are shown in Figure 3.

[0086] (5) Measurement of the area of ​​fibrosis in the transplanted fat over time The collected fat mass was divided in half, immersed in 4% paraformaldehyde-phosphate buffer, fixed, embedded in paraffin, and stained with Elastica-Masson (EM). The stained image of the tissue slide is shown in Figure 4.

[0087] The average positive area of ​​20 randomly selected fields (400x magnification) was measured on the prepared tissue slide. The area occupied by collagen fibers was 5.0 × 10 amniotic mesenchymal stem cells. 5 Individual addition group, 5.0×10 6 In both groups, fibrosis tended to be suppressed by the addition of amniotic mesenchymal stem cells. Quantification showed that fibrosis tended to increase from 8 weeks after transplantation, but fibrosis was significantly suppressed in the amniotic mesenchymal stem cell addition group compared to the control group. The measurement data are shown in Figure 5.

[0088] (6) Evaluation of the expression levels of fibrosis-related genes in transplanted fat The collected fat mass was divided in half, crushed, and total RNA was extracted using the spin column method. DNase treatment was performed to prevent contamination of genomic DNA. cDNA was created from total RNA by reverse transcription reaction, and the amount of PCR amplified product was measured using the intercalation method and analyzed using the ΔΔCT method. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an endogenous control.

[0089] Gene expression of α-SMA and TGF-β at 4 weeks after transplantation was significantly higher in amniotic mesenchymal stem cells (5.0×10 5 Individual addition group, 5.0×10 6 The expression of collagen I gene was also significantly suppressed in both amniotic mesenchymal stem cell (5.0×10) and ES cell (5.0×10) groups. 6The results were significantly suppressed in the group treated with 1,000 mg of glycerol. The evaluation data are shown in Figure 6.

[0090] Example 3: Preparation of pharmaceutical composition A portion of the amniotic mesenchymal stem cells obtained in the above "Example 1: Production of amniotic mesenchymal stem cells" is used to prepare a pharmaceutical composition. Amniotic mesenchymal stem cells 1.0 x 10 7 A pharmaceutical composition (cell preparation) consisting of 1 mL of physiological saline containing 5 v / v% dimethyl sulfoxide (DMSO), 6 w / v% hydroxyethyl starch (HES), and 46 w / v% human serum albumin is prepared. The pharmaceutical composition is sealed in a freezing bag and stored in a frozen state. When used, the pharmaceutical composition can be thawed and provided to the patient or subject.

Claims

1. A method for treating amniotic fibrosis comprising administering to the patient a therapeutically effective amount of a cell population comprising mesenchymal stem cells derived from amniotic tissue as an active ingredient, It is mixed with adipose tissue and administered to soft tissue, A pharmaceutical composition for soft tissue regeneration.

2. The pharmaceutical composition for soft tissue regeneration according to claim 1 , wherein the mesenchymal stem cells are obtained from a living body other than the subject to which the composition is administered.

3. The pharmaceutical composition for soft tissue regeneration according to claim 1 , wherein the mesenchymal stem cells are obtained from a living body of the same species as the subject to which the composition is administered.

4. The pharmaceutical composition for soft tissue regeneration according to any one of claims 1 to 3, wherein the subject to be administered is a human.

5. The pharmaceutical composition for soft tissue regeneration according to claim 1 , wherein the mesenchymal stem cells are derived from humans.

6. The pharmaceutical composition for soft tissue regeneration according to any one of claims 1 to 5, wherein the mesenchymal stem cells have no or low ability to differentiate into adipocytes.

7. The pharmaceutical composition for soft tissue regeneration according to claim 1 , wherein the mesenchymal stem cells have an inhibitory effect on fibrosis.

8. The pharmaceutical composition for soft tissue regeneration according to claim 1 , wherein the mesenchymal stem cells are mesenchymal stem cells that have been passaged twice or more.

9. The pharmaceutical composition for soft tissue regeneration according to any one of claims 1 to 8, wherein the mesenchymal stem cell surface markers are positive for CD44, CD73, CD90, and CD105, and negative for CD45.

10. The pharmaceutical composition for soft tissue regeneration according to any one of claims 1 to 9, which is mixed with adipose tissue and mesenchymal stem cells derived from adipose tissue and administered to soft tissue.

11. The pharmaceutical composition for soft tissue regeneration according to any one of claims 1 to 10, wherein the dimethyl sulfoxide content is 5 to 10% by mass, the hydroxyethyl starch content is 4 to 10% by mass, and the human albumin content is 5% by mass or less.

12. The pharmaceutical composition for soft tissue regeneration according to any one of claims 1 to 11, which is frozen and stored prior to administration.

13. The pharmaceutical composition for soft tissue regeneration according to claim 12, which can be frozen for one month or more before administration.

14. The pharmaceutical composition for soft tissue regeneration according to claim 12, which can be frozen for a period of 6 months or more before administration.

15. The pharmaceutical composition for soft tissue regeneration according to claim 12, which can be frozen for a period of one year or more before administration.

16. Adipose tissue for transplantation, which contains a mixture of cells including mesenchymal stem cells derived from amniotic tissue and has high ability to engraft in the body.

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