Preparation of adipose tissue-derived cell populations

Abstract

To provide effective means for the efficient preparation of stromal vascular cells.SOLUTION: The present invention provides a method for preparing stromal vascular cells from adipose tissue, which includes treating the adipose tissue with an enzyme solution containing collagenase and neutral protease, where neutral protease activity is 1 U or more per 10,000 U of collagenase activity, preferably an enzyme solution that does not contain clostripain or thermolysin, and then recovering the cells.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method for preparing a cell population derived from adipose tissue, and an enzyme preparation and the like used in said method. [Background technology]

[0002] Adipose tissue contains mesenchymal stem cells, various stromal cells, and their precursor cells, making it a promising source of cells for regenerative medicine. It is also easier to extract than blood or bone marrow, making it highly useful. Many research groups and institutions are currently attempting to use adipose tissue-derived mesenchymal stem cells and stromal vascular fraction (SVF) in regenerative medicine.

[0003] Vascular endothelial cells (ECs) / endothelial progenitor cells (EPCs) are useful as transplantation materials in regenerative medicine. Furthermore, their combined use is expected to enhance the therapeutic effects of transplantation therapy, making them useful as therapeutic tools in regenerative medicine. Enzymes are typically used to prepare SVF containing vascular endothelial cells / endothelial progenitor cells from adipose tissue. For example, enzyme preparations containing collagenase and protease (crude collagenase preparations, e.g., collagenase contaminated with clostripain, neutral protease, etc.) are used to disperse adipose tissue, i.e., enzymatically hydrolyze adipose tissue. Collagenase and thermolysin may also be used in combination, and such enzyme preparations (containing collagenase and thermolysin) for enzymatic hydrolysis are commercially available (e.g., Liberase from Roche and Celase from Cytori Therapeutics). [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Japanese Patent Application Publication No. 2019-88279 Summary of the Invention [Problem to be solved by the invention]

[0005] Thermolysin, which is used to prepare SVF, is known to cause significant damage to cells, affecting the activity of cells in SVF. Although efficient preparation methods have been proposed (see, for example, Patent Document 1), it remains difficult to easily and efficiently prepare highly purified vascular endothelial cells / vascular endothelial progenitor cells (for convenience of explanation, the term "vascular endothelial cells" will be used hereinafter to encompass vascular endothelial cells and vascular endothelial progenitor cells) from adipose tissue. In light of future developments in regenerative medicine, it is desirable to more efficiently prepare SVF containing adipose tissue-derived vascular endothelial cells. Therefore, an objective of the present invention is to provide an effective means for efficiently preparing SVF (in other words, improving the yield). Another objective is to increase the number of vascular endothelial cells in SVF and easily and efficiently prepare highly purified vascular endothelial cells. [Means for solving the problem]

[0006] In light of the above problems, the inventors of the present invention have conducted research and have concluded that the conditions for enzymatic hydrolysis of adipose tissue are most important for the efficient preparation of SVF, and have conducted detailed studies focusing on the enzymes used. As a result, it has been found that the combined use of collagenase and neutral protease is particularly effective, and that the ratio of the two enzymes is important, thereby successfully improving the yield of SVF and increasing the number of vascular endothelial cells in SVF. Based on these results, the following inventions are provided. [1] A method for preparing stromal vascular cells from adipose tissue, comprising the following step (1): (1) A step of treating adipose tissue with an enzyme solution containing collagenase and neutral protease, in which the neutral protease activity is 1 U or more per 10,000 U of collagenase activity, and then recovering cells. [2] The preparation method described in [1], wherein the neutral protease activity of the enzyme solution is 2 U or more per 10,000 U of collagenase activity. [3] The preparation method described in [1], wherein the neutral protease activity of the enzyme solution is 2.5 U or more per 10,000 U of collagenase activity. [4] The preparation method described in [1], wherein the activity ratio of collagenase to neutral protease in the enzyme solution is 34,000:9 to 34,000:45. [5] The preparation method described in any one of [1] to [4], wherein the collagenase content of the enzyme solution is 500 U or more per 1 g of adipose tissue. [6] A preparation method described in any one of [1] to [4], wherein the neutral protease content of the enzyme solution is 0.05 U or more per 1 g of adipose tissue. [7] A preparation method described in any one of [1] to [6], wherein the collagenase is derived from Clostridium histolyticum. [8] The preparation method according to any one of [1] to [7], wherein the neutral protease is derived from Clostridium histolyticum. [9] The preparation method according to any one of [1] to [8], wherein the enzyme solution further contains clostripain and / or thermolysin.

[10] The preparation method according to any one of [1] to [8], wherein the enzyme solution does not contain clostripain or thermolysin.

[11] The preparation method according to any one of [1] to

[10] , wherein the adipose tissue is human adipose tissue.

[12] A method for preparing a cell population containing adipose tissue-derived vascular endothelial cells and vascular endothelial progenitor cells, comprising the following step (2): (2) A step of enriching and / or expanding vascular endothelial cells and vascular endothelial progenitor cells in the stromal vascular cell population obtained by the preparation method according to any one of [1] to

[11] .

[13] An enzyme preparation for disintegrating adipose tissue, which contains collagenase and neutral protease, and in which the neutral protease activity is 1 U or more per 10,000 U of collagenase activity.

[14] The enzyme preparation according to

[13] , wherein the neutral protease activity is 2 U or more per 10,000 U of collagenase activity.

[15] The enzyme preparation according to

[13] , wherein the neutral protease activity is 2.5 U or more per 10,000 U of collagenase activity.

[16] The enzyme preparation according to

[13] , wherein the activity ratio of collagenase to neutral protease in the enzyme solution is 34,000:9 to 34,000:45.

[17] The enzyme preparation according to any one of

[13] to

[16] , wherein the collagenase is derived from Clostridium histolyticum.

[18] The enzyme preparation according to any one of

[13] to

[17] , wherein the neutral protease is derived from Clostridium histolyticum.

[19] The enzyme preparation according to any one of

[13] to

[18] , further comprising clostripain and / or thermolysin.

[20] The enzyme preparation according to any one of

[13] to

[18] , which does not contain clostripain or thermolysin. DETAILED DESCRIPTION OF THE INVENTION

[0007] 1. Method for preparing stromal vascular fraction (SVF) A first aspect of the present invention relates to a method for preparing stromal vascular cells (SVF) from adipose tissue (hereinafter referred to as "SVF preparation method"). The SVF preparation method of the present invention involves the following step (1): (1) A step of treating adipose tissue with an enzyme solution containing collagenase and neutral protease, in which the neutral protease activity is 1 U or more per 10,000 U of collagenase activity, and then recovering cells.

[0008] Adipose tissue can be collected by means of excision, suction, or the like from humans, non-human mammals (including pet animals, livestock, and laboratory animals; specifically, for example, mice, rats, guinea pigs, hamsters, monkeys, cows, pigs, goats, sheep, dogs, and cats), birds (chickens, quails, and the like), and the like. Examples of adipose tissue include subcutaneous fat, visceral fat, intramuscular fat, and intermuscular fat. Adipose tissue can also be obtained by suction by inserting a cannula into the subcutaneous adipose tissue of the abdomen, thigh, buttocks, or the entire body. The amount of adipose tissue obtained is, for example, 1 g to 1000 g, preferably 1 g to 500 g, more preferably 1 g to 100 g, even more preferably 2 g to 50 g, or even more preferably 2 g to 40 g. Subcutaneous fat is particularly preferred because it can be collected very easily, for example, under local anesthesia, and places less strain on the donor during collection. Usually, one type of adipose tissue is used, but two or more types of adipose tissue can also be used in combination. Alternatively, adipose tissues collected in multiple batches (which do not have to be the same type of adipose tissue) may be mixed and used in the subsequent procedures.

[0009] The collected adipose tissue is subjected to the following enzymatic treatment after removal of blood components adhering thereto (for example, by washing the adipose tissue in an appropriate buffer or culture medium to remove blood components) and / or fragmentation, if necessary. When aspirated lipoproteins are used, it is preferable to leave the aspirated lipoproteins to stand and separate the adipose and aqueous layers. Alternatively, the adipose and aqueous layers can be separated by treating the aspirated lipoproteins with a centrifuge. The adipose and aqueous layers can be isolated by recovering and removing the aqueous layer after separation. The obtained adipose tissue may be washed with, for example, physiological saline, before being subjected to enzymatic treatment. Before being subjected to enzymatic treatment, it is preferable to warm the adipose tissue at room temperature or in a water bath at about 37°C for about 5 to 15 minutes.

[0010] Adipose tissue is subjected to enzyme treatment (enzyme reaction). In the present invention, collagenase and neutral protease are used in combination in the enzyme treatment, and the neutral protease content in the enzyme solution (activity ratio to collagenase) is increased to improve the yield of SVF. Specifically, an enzyme solution containing collagenase and neutral protease is prepared, with the neutral protease activity being 1 U or more per 10,000 U of collagenase activity, and the adipose tissue is treated with the enzyme solution. The enzyme solution used in the present invention can be prepared, for example, by dissolving or diluting an enzyme agent prepared so that the collagenase and neutral protease each have the desired activity.

[0011] Preferably, to increase the yield of SVF, an enzyme solution with a higher neutral protease activity relative to collagenase activity (hereinafter, for convenience of explanation, referred to as "activity ratio") is used. Specifically, in a preferred embodiment, an enzyme solution with a neutral protease activity of 2 U or more (e.g., in the range of 2 U to 50 U) per 10,000 U of collagenase activity is used, a more preferred embodiment with a neutral protease activity of 2.5 U or more (e.g., in the range of 2.5 U to 50 U) per 10,000 U of collagenase activity is used, an even more preferred embodiment with a neutral protease activity of 3 U or more (e.g., in the range of 3 U to 50 U) per 10,000 U of collagenase activity is used, and an even more preferred embodiment with a neutral protease activity of 5 U or more (e.g., in the range of 5 U to 50 U) per 10,000 U of collagenase activity is used. A particularly preferred example of the activity ratio is 34,000: 9 to 34,000: 45. The activities of collagenase and neutral protease are calculated by the measurement method shown in the Examples section below.

[0012] To treat adipose tissue with an enzyme solution, for example, the enzyme solution is added to the adipose tissue or the adipose tissue is immersed in the enzyme solution, creating conditions that allow the enzyme in the enzyme solution to contact (act on) the adipose tissue. Under these conditions, the treatment is carried out under conditions that allow the enzyme in the enzyme solution to react, i.e., the enzymatic reaction. The conditions for the enzymatic reaction are not particularly limited as long as the collagenase and neutral protease are active and cells are separated from the adipose tissue. Examples of the conditions include a pH of 5 to 10, preferably 6 to 9, a temperature of 25°C to 50°C, preferably 30°C to 45°C, and more preferably 35°C to 40°C (e.g., 37°C), and a reaction time of 10 minutes to 3 hours, preferably 15 minutes to 1 hour (e.g., 20 minutes, 30 minutes, 40 minutes, or 50 minutes). To efficiently proceed with the enzymatic reaction, it is recommended to shake the reaction vessel (reciprocating or rotating, etc.).

[0013] The enzyme solution used in the present invention has a higher neutral protease content than when a crude collagenase preparation (e.g., Wako's "Collagenase") is used. Although collagenase also plays an important role in dispersing adipose tissue in the present invention, the high neutral protease content affects the yield of SVF and the number of vascular endothelial cells (vascular endothelial cells / vascular endothelial progenitor cells) in the SVF. According to the present invention, the yield of SVF is improved. Furthermore, the number of vascular endothelial cells in the SVF typically increases. Therefore, the present invention is extremely effective as a means for efficiently obtaining vascular endothelial cells derived from adipose tissue (a method for preparing vascular endothelial cells is described in the second aspect below).

[0014] The amounts of collagenase and neutral protease in the enzyme solution are not particularly limited as long as cells can be separated from the adipose tissue; however, the enzyme solution should contain, in terms of activity per 1 g of adipose tissue, for example, 500 U or more (e.g., 500 to 30,000 U) of collagenase and 0.05 U or more (e.g., 0.05 to 20 U) of neutral protease, preferably 1,000 to 20,000 U of collagenase and 0.1 to 15 U of neutral protease, and more preferably 3,000 to 10,000 U of collagenase and 0.15 to 10 U of neutral protease (however, the activity ratio between collagenase and neutral protease in the enzyme solution should be as described above).

[0015] The origin of collagenase and neutral protease is not particularly limited, as long as they are useful for separating cells from adipose tissue. For example, collagenase derived from Clostridium histolyticum and neutral protease derived from Clostridium histolyticum can be used. Collagenase and neutral protease derived from these microorganisms can be prepared by isolation and purification from the culture medium or cells of the microorganisms (producing strains) that produce them. A host microorganism into which a collagenase gene (or a modified version of said gene) obtained from a collagenase-producing strain has been introduced can also be used as a collagenase-producing strain. The same applies to neutral proteases. Collagenase and neutral protease can be isolated and purified using various chromatography techniques (e.g., ion exchange chromatography, hydrophobic chromatography, affinity chromatography), salting out, etc. For preparation of neutral protease, please refer to the literature "Dendo M, et al. Synergistic effect of neutral protease and clostripain on pancreatic islet isolation. Transplantation. In press, 2015." Purified collagenases are provided by Amano Enzyme (e.g., Collagenase "Amano" GMP), Worthington (e.g., Collagenase Purified), Vitacyte (e.g., Collagenase HA, Collagenase MA, rCollagenase HI), Roche (e.g., Collagenase A), and the collagenase used in the present invention is easily available.

[0016] Preferably, Clostridium histolyticum-derived collagenase and Clostridium histolyticum-derived neutral protease are used. Clostridium histolyticum-derived collagenase has broad substrate specificity, acting on almost all types of collagen, making it particularly useful for dissociating adipose tissue. Similarly, Clostridium histolyticum-derived neutral protease has specificity for the FAGFYA substrate and is characterized by low cytotoxicity, making it particularly useful for dissociating adipose tissue.

[0017] Ca is added to the enzyme solution to stabilize and activate collagenase. 2+ It is preferable that CaCl2 is present in the enzyme solution. Therefore, it is advisable to add, for example, CaCl2 to the enzyme solution. When CaCl2 is added, the concentration in the enzyme solution is, for example, 1 mM to 10 mM, preferably 2 mM to 5 mM, and more preferably 2 mM to 4 mM.

[0018] In a preferred embodiment of the present invention, the enzyme solution does not contain clostripain or thermolysin. That is, the enzymes for decomposing adipose tissue essentially contain only collagenase and neutral protease. This embodiment is advantageous in that the composition of the enzyme solution is simple and the enzyme solution is easy to prepare. Furthermore, the absence of clostripain in the enzyme solution is preferable, particularly in that it increases the number of vascular endothelial cells in the SVF.

[0019] Alternatively, clostripain or thermolysin, or both, may be added to the enzyme solution to utilize the effects of these enzymes to further improve the adipose tissue dispersion efficiency and SVF yield. In this case, the clostripain content is, for example, greater than 0 U to 2,000 U, preferably 1 U to 1,500 U, and more preferably 10 U to 1,100 U, relative to 10,000 U of collagenase activity. An excessively high clostripain content will affect the SVF yield. On the other hand, the thermolysin content is, for example, greater than 0 U to 10,000 U, preferably 1 U to 7,000 U, and more preferably 10 U to 5,000 U, relative to 10,000 U of collagenase activity. An excessively high thermolysin content will affect the SVF yield. The origin of clostripain and thermolysin is not particularly limited; for example, clostripain derived from Clostridium histolyticum, thermolysin derived from Bacillus thermoproteolyticus, or Geobacillus stearothermophilus can be used. Clostripain and thermolysin derived from these microorganisms can be prepared by isolation and purification from the culture medium or cells of the microorganisms that produce them (producing strains), or by genetic engineering techniques, as in the case of collagenase. The isolation and purification methods are also similar to those for collagenase. For the preparation of clostripain, please refer to the aforementioned publication, "Dendo M, et al. Synergistic effect of neutral protease and clostripain on pancreatic islet isolation. Transplantation. In press, 2015."

[0020] Furthermore, enzymes other than the above enzymes (collagenase, neutral protease, clostripain, thermolysin) may be included in the enzyme solution as long as they do not affect the action of the above enzymes (collagenase, neutral protease, clostripain, thermolysin) and the resulting effect (dispersion of adipose tissue).

[0021] The cell population obtained by enzyme treatment includes multipotent stem cells, vascular endothelial cells, stromal cells, blood cells, etc. Typically, the sediment (cell pellet) obtained by centrifugation is collected as SVF. Centrifugation conditions vary depending on the type and amount of cells, but are, for example, 1 to 20 minutes at 500 to 1000 G. Prior to centrifugation, it is preferable to perform a filter treatment (a cell strainer or the like) to remove tissues that have not been digested with the enzyme. Furthermore, the cells obtained by centrifugation may be subjected to a filter treatment or the like to remove unnecessary components. Furthermore, a hemolysis treatment may be performed before or after centrifugation. In this specification, a cell population refers to a population containing a large number of one or more types of cells.

[0022] The types and ratios of cell populations that make up SVF depend on the origin and type of adipose tissue used, the conditions of enzyme treatment, etc., but the SVF fraction usually consists of adipose tissue-derived cells (CD45 negative) and peripheral blood-derived cells (CD45 positive). The adipose tissue-derived cells (CD45 negative) are a CD34-positive and CD31-positive cell population (CD45 - CD34 + CD31 + ) and a CD34-positive, CD31-negative cell population (CD45 - CD34 + CD31 - ) ASCs (adipose tissue-derived stromal cells / adipose tissue-derived stem cells).

[0023] 2. Method for preparing vascular endothelial cells In a second aspect, the present invention provides a method for preparing a cell population containing adipose tissue-derived vascular endothelial cells (i.e., vascular endothelial cells and vascular endothelial progenitor cells) from SVF obtained by the SVF preparation method of the present invention (hereinafter referred to as the "vascular endothelial cell preparation method"). The vascular endothelial cell preparation method of the present invention utilizes SVF obtained by the SVF preparation method of the present invention to obtain a vascular endothelial cell population with a high abundance (purity) of vascular endothelial cells, i.e., a highly purified vascular endothelial cell population. In a highly purified vascular endothelial cell population, the proportion of vascular endothelial cells in the cell population, i.e., "(vascular endothelial cells / total cell number in the cell population) × 100 (%)," is, for example, 50% or more, preferably 60% or more, more preferably 70% or more, even more preferably 80% or more, even more preferably 90% or more, and even more preferably 95% or more. The number of vascular endothelial cells in SVF is usually about 1 to several %. Therefore, the method for preparing vascular endothelial cells of the present invention significantly increases the purity of vascular endothelial cells, and a cell population containing vascular endothelial cells at a much higher concentration (higher purity) than SVF can be obtained. Vascular endothelial cells can be identified as CD45-negative, CD31-positive cells. Markers such as CD144 and CD146 can also be used to identify vascular endothelial cells.

[0024] Vascular endothelial cells can be used to treat ischemic diseases of any organ through angiogenesis, and are also necessary for organ (tissue) regeneration in vitro or in vivo, along with organ-constituting cells, organ-specific progenitor cells, stem cells (including fetal stem cells and iPS cells), and cells derived from stem cells, in regenerative medicine for organs (organs, tissues). The cell population obtained by the method for preparing vascular endothelial cells of the present invention is useful as a valuable cellular medicine that can be used to treat a wide range of diseases.

[0025] In the method for preparing vascular endothelial cells of the present invention, a highly purified cell population containing vascular endothelial cells is obtained by selectively recovering and proliferating vascular endothelial cells in SVF. Typically, the following step (2) is carried out. (2) A step of concentrating and / or expanding vascular endothelial cells and vascular endothelial progenitor cells from the stromal vascular cell population obtained by the SVF preparation method of the present invention.

[0026] Although vascular endothelial cells in SVF are typically enriched or expanded en masse, it is also possible to concentrate or expand vascular endothelial cells or vascular endothelial progenitor cells. Various methods can be used for enrichment or expansion, including known methods as well as methods to be developed in the future. A typical enrichment procedure is selection (selection and recovery) using a cell marker. For example, cell markers such as CD45 and CD31, which are useful for selecting vascular endothelial cells, can be used. The enrichment procedure can also be performed multiple times. For example, cells enriched with a specific marker can be cultured and expanded, and then further enriched with another marker. This series of procedures can be repeated, in which case the markers used in each procedure can be the same or different. Furthermore, cell markers can be used alone or in combination. For example, the cell markers CD45 and CD31 can be used in the first selection to select and recover CD45-negative, CD31-positive cells, followed by culturing and expanding the recovered cells, followed by selection and recovery of CD31-positive cells using the cell marker CD31.

[0027] Cell selection and recovery using cell markers can be performed using techniques such as magnetic cell sorting (MACS) and FACS. MACS involves immobilizing antibodies against marker proteins onto magnetic beads, and then using a strong magnet to separate target cells from the inner wall of a cylindrical container (column) or simply within a tube. Common magnetic bead reagents, such as MACS (Miltenyi Biotec) and IMag (BD Japan), can be used. FACS allows the use of a flow cytometer with a cell sorter function to separate specific cells that emit a specified fluorescence. Examples of such instruments include the FACSAria II (BD Japan), JSAN (Bay Biosciences), and MoFlo XDP (Beckman Coulter).

[0028] Vascular endothelial cells can be grown by conventional methods, i.e., by using a medium suitable for culturing vascular endothelial cells and incubating under appropriate conditions (e.g., 37°C in a CO2 incubator). The culture period is not particularly limited, but may be, for example, 1 to 21 days. Cells may be passaged during the culture. The number of passages is not particularly limited. The medium may be changed, for example, every 1 to 2 days. Examples of culture media that can be used include EGM-2 (Lonza), αMEM, Dulbecco's Modified Eagle's Medium (DMEM), Dulbecco's Modified Eagle's Medium / Ham's F-12 Mixed Medium (DMEM / F12), and RPMI 1640. Examples of preferred culture media include EGM-2 medium (Lonza) and EGM-2MV (Lonza). The medium may contain various additives commonly used in cell culture, such as serum, various vitamins, various antibiotics, various hormones, and various growth factors.

[0029] Step (2) may utilize two methods (referred to as "method 1" and "method 2") disclosed in Japanese Patent Application Laid-Open No. 2019-88279. In the first method, CD31-positive cells are selected from SVF to obtain a cell population containing CD31-positive cells (this may involve selecting CD45-negative and CD31-positive cells or only CD31-positive cells), and the cell population is then cultured for 1 hour to 7 days. CD31-positive cells are then selected from the cell population obtained by the culture to obtain a cell population containing CD31-positive cells. On the other hand, in the second method, CD45-negative and CD31-positive cells are selected from SVF to obtain a cell population containing CD45-negative and CD31-positive cells, and the cell population is then cultured for 2 to 6 days (or 3 to 6 days), and CD31-positive cells are then selected from the cell population obtained by the culture to obtain a cell population containing CD31-positive cells. The SVF may be cultured for 1 hour to 5 days (or 1 to 4 days) and then subjected to Method 1 or Method 2. For details of Method 1 and Method 2, see JP 2019-88279 A.

[0030] 3. Enzyme preparations Another aspect of the present invention relates to an enzyme preparation for dispersing adipose tissue. The enzyme preparation of the present invention is typically used in the above-mentioned SVF preparation method and vascular endothelial cell preparation method of the present invention. That is, it is used to prepare an enzyme solution for use in treating adipose tissue. Therefore, it is characterized by containing collagenase and neutral protease, and having a neutral protease activity of 1 U or more per 10,000 U of collagenase activity. The neutral protease activity is preferably 2 U or more per 10,000 U of collagenase activity (e.g., in the range of 2 U to 50 U), more preferably 2.5 U or more per 10,000 U of collagenase activity (e.g., in the range of 2.5 U to 50 U), even more preferably 3 U or more per 10,000 U of collagenase activity (e.g., in the range of 3 U to 50 U), and even more preferably 5 U or more per 10,000 U of collagenase activity (e.g., in the range of 5 U to 50 U). A particularly preferred activity ratio is 34,000:9 to 34,000:45. The contents of collagenase and neutral protease are not particularly limited, but may be, for example, 1,000 U / g to 6,000,000 U / g (per 1 g of the enzyme preparation) of collagenase and 0.1 U / g to 9,000 U / g (per 1 g of the enzyme preparation) of neutral protease (provided that the ratio of collagenase to neutral protease in the enzyme preparation is as described above).

[0031] The collagenase and neutral protease are not particularly limited, and for example, collagenase derived from Clostridium histolyticum and neutral protease derived from Clostridium histolyticum are used.

[0032] In a preferred embodiment, the enzyme preparation does not contain clostripain or thermolysin. That is, the enzymes used to disperse adipose tissue are essentially collagenase and neutral protease. Alternatively, the enzyme preparation may contain clostripain, thermolysin, or both, to further improve the efficiency of dispersing adipose tissue and the yield of SVF by utilizing the effects of these enzymes. In this case, the clostripain content is, for example, greater than 0 U / g to 100,000 U / g (per gram of enzyme preparation), preferably 10 U / g to 50,000 U / g (per gram of enzyme preparation). The thermolysin content is, for example, greater than 0 U / g to 5,000,000 U / g (per gram of enzyme preparation), preferably 10 U / g to 1,000,000 U / g (per gram of enzyme preparation).

[0033] Furthermore, enzymes other than the above enzymes may be included in the enzyme preparation as long as they do not affect the action of the above enzymes (collagenase, neutral protease, clostripain, thermolysin) and the resulting effect (dispersion of adipose tissue).

[0034] In addition to the active ingredients (i.e., each enzyme useful for dispersing adipose tissue), the enzyme preparation may contain excipients, buffers, suspending agents, stabilizers, preservatives, surfactants, physiological saline, etc. Examples of excipients that can be used include lactose, sorbitol, D-mannitol, maltodextrin, trehalose, and sucrose. Examples of buffers that can be used include Good's buffer (e.g., HEPES), phosphates, citrates, and acetates. Examples of stabilizers that can be used include propylene glycol, ascorbic acid, sodium chloride, and calcium chloride. Examples of preservatives that can be used include phenol, benzalkonium chloride, benzyl alcohol, chlorobutanol, and methylparaben. Examples of preservatives that can be used include benzalkonium chloride, parahydroxybenzoic acid, and chlorobutanol. Examples of surfactants that can be used include poloxamer.

[0035] The enzyme preparation may be in the form of a liquid or a solid (including a powder). In the latter case, the enzyme preparation is typically prepared by powdering an enzyme solution containing the necessary ingredients (each enzyme as an active ingredient and other ingredients added as needed) by freeze-drying, vacuum drying, spray-drying, or the like. [Example]

[0036] 1.Activity measurement / activity definition (1) Collagenase activity measurement method and activity definition Collagenase activity was measured using the Molecular Probes EnzChek Gelatinase / Collagenase Assay Kit (Invitrogen). One mL of water was added to one DQ gelatin vial (Molecular Probes) to make a 1 mg / mL solution. This solution was diluted 40-fold with 0.05 mol / L Tris buffer (pH 7.6) containing NaCl·CaCl2 to make a 25 μg / mL substrate solution. Enzymes with known enzymatic activity were diluted with 0.05 mol / L Tris buffer (pH 7.6) containing NaCl·CaCl2 to prepare 0.1–0.4 U / mL solutions. The sample was diluted with 0.05 mol / L Tris buffer (pH 7.6) containing NaCl·CaCl2 to make the sample solution. To 100 μL of each of these solutions, 100 μL of substrate solution was added. The mixture was shaken and fluorescence measurements (excitation wavelength 485 nm, emission wavelength 528 nm) were performed every minute at room temperature for 1 hour. A calibration curve is created with fluorescence on the vertical axis and enzyme concentration (0, 0.1, 0.2, 0.3, 0.4 U / mL) on the horizontal axis. Enzyme activity is calculated using the following formula, where 1 unit (1U) is the amount of enzyme required to liberate 1 μmol of L-leucine from collagen in 5 hours at 37°C and pH 7.5. Collagenase potency (U / g, mL) = {(AT-AB)-b} / a × n AB: Fluorescence intensity of the reaction solution at the start of the reaction AT: Fluorescence level of the reaction solution after 1 hour a: Slope of the calibration curve obtained using the calibration curve stock solution b: y-intercept of the calibration curve obtained using the calibration stock solution n: dilution factor

[0037] (2) Measurement method and definition of clostripain activity Add 3 mL of substrate solution (0.76 mmol / L N-Benzoyl-L-arginine ethyl ester hydrochloride, 0.4 mmol / L calcium chloride, 0.1 mol / L monopotassium phosphate / dipotassium phosphate buffer (pH 7.6)) to a quartz cell and leave at 25°C for 5 minutes. After this, add 0.05 mL of appropriately diluted enzyme solution (0.0025 mol / L MOPS buffer (pH 7.4), 0.001 mol / L calcium chloride) and immediately shake. While maintaining this solution at 25°C, measure the absorbance (AT) at a wavelength of 255 nm every 10 seconds for 5 minutes. Under these conditions, the amount of enzyme that produces 1 μmol of N-Benzoyl-L-arginine per minute is defined as 1 unit (1 U), and calculate the enzyme activity using the following formula: Protein digestion capacity (U / g) = (AT / min-AB / min) / 0.81 × 3.05 / 0.05 × n AT / min: Change in absorbance of reaction solution per minute AB / min: Change in absorbance of blank solution per minute 0.81: millimolar absorption coefficient of N-Benzoyl-L-arginine at 255 nm 3.05: Volume of reaction mixture (mL) 0.05: Volume of sample added during enzyme reaction (mL) n: dilution factor

[0038] (3) Measurement method and definition of neutral protease (NP) activity Add 2.88 mL of substrate solution (0.4 mmol / L N-[3-(2-Furyl)acryloyl]-Gly-Phe-Tyr-amide, 10% dimethyl sulfoxide, 0.1 mol / L Tris buffer (pH 7.5)) to a quartz cell and leave at 37 ± 0.5°C for 5 minutes. Then, add 0.12 mL of appropriately diluted enzyme solution and mix. While maintaining the temperature at 37°C, measure the absorbance (AT) at 344 nm every 10 seconds for 100 seconds. Under these conditions, the amount of enzyme that produces 1 μmol of N-[3-(2-Furyl)acryloyl]-Gly per minute is defined as 1 unit (1 U), and calculate the enzyme activity using the following formula: Protein digestion capacity (U / g) = -{(AT80-AB80)-(AT20-AB20)} / 0.524 × 3000 / 120 × n AT20: absorbance of the reaction solution at 20 seconds AT80: absorbance of the reaction solution at 80 seconds AB20: Absorbance of blank solution at 20 seconds AB80: Absorbance of blank solution at 80 seconds 0.524: millimolar extinction coefficient of FAGFYA at 344 nm 3000: Volume of reaction mixture (μL) 120: Amount of sample added during enzyme reaction (μL) n: dilution factor

[0039] (4) Thermolysin activity measurement method and activity definition Add 1 mL of enzyme solution to 5 mL of casein solution preheated to 37°C using substrate solution (0.6% W / V 2-amino-2-hydroxymethyl-1,3-propanediol, 0.7% W (dry weight) / V milk casein, pH 7.0), mix, and after 30 minutes at 37°C, add 5 mL of 0.11 mol / L trichloroacetic acid test solution to stop the reaction. After leaving the mixture at 37°C for 30 minutes, mix well and filter through an 11 cm Whatman No. 42 filter paper. Measure the absorbance of the filtrate at 275 nm. Under these conditions, the amount of enzyme that liberates a substance equivalent to the absorbance of 1.5 μg of L-tyrosine per minute is defined as 1 protein digestion unit (1 U), and calculate the enzyme activity using the following formula: Protein digestion capacity (U / g) = (A30-A0) / As × 11 / 30 × n A30: Absorbance of enzyme reaction solution A0: Absorbance of blank solution As: Amount of tyrosine (μg) when the absorbance difference obtained from the tyrosine calibration curve is 1 11: Final volume of reaction solution (mL) 30: Reaction time (min) n: Dilution factor per 1g of enzyme

[0040] 2. Preparation of SVF nucleated cells Divide the adipose tissue into 50 mL centrifuge tubes, approximately 10 mL each, and measure the volume and mass. Mix the adipose tissue with approximately equal volumes of HBSS buffer (pH 6.7-7.8) containing the enzyme of interest. Wrap the tube cap with parafilm and shake at 120 rpm at 37°C for 20 minutes to allow the reaction (disperse the adipose tissue). Then, add HBSS buffer pre-chilled to 4°C in a volume twice the amount of adipose tissue to terminate the enzyme reaction. Centrifuge at 800 g for 10 minutes, and remove the tissue debris and supernatant with a pipette, leaving only 2-3 mL. Add an appropriate amount of HBSS buffer pre-chilled to 4°C, and pass the cell suspension through a cell strainer (mesh size: 1st mesh: 100 μm, 2nd mesh: 40 μm). Centrifuge at 800 g for 10 minutes, remove the supernatant, and suspend in 2 mL of DMEM / F12 medium (Wako) to obtain the SVF nucleated cell suspension.

[0041] 3. Measurement of SVF nucleated cell count and vascular endothelial cell (EC and EPC) count (1) Method for measuring the number of SVF nucleated cells The number of SVF nucleated cells in the SVF nucleated cell suspension was measured using Luna-stem (Logos Biosystems).

[0042] (2) Method for measuring the number of vascular endothelial cells in SVF The number of vascular endothelial cells in SVF was measured by the FACS (Fluorescence activated cell sorting) method (see Patent Document 1).

[0043] 4. Examination of the combination of clostripain (CP) and neutral protease (NP) The number of SVF nucleated cells was compared when collagenase (CL) was used in combination with CP and / or NP during adipose tissue dispersion. The following test groups were set up using different enzymes, and SVF nucleated cell suspensions were prepared using the method described above. The number of SVF nucleated cells in the SVF nucleated cell suspension was measured, and evaluated as a ratio, assuming the number of SVF nucleated cells obtained when 0.2% (w / v) Wako CL (product name: collagenase) was used. Test group 1: A combination of 34,000 U of purified CL derived from Clostridium histolyticum, 3,600 U of purified CP derived from Clostridium histolyticum, and 9 U of purified NP derived from Clostridium histolyticum Test group 2: A combination of 34,000 U of purified CL derived from Clostridium histolyticum and 3,600 U of purified CP derived from Clostridium histolyticum Test group 3: 34,000 U of purified CL derived from Clostridium histolyticum and 9 U of purified NP derived from Clostridium histolyticum Test group 4: 34,000 U of purified CL derived from Clostridium histolyticum

[0044] The experimental results are shown in Table 1. Compared to using CL alone (Test Group 4), the combined use of CP and NP increased the yield of SVF nucleated cells (Test Groups 1 to 3). In particular, the combined use of CL and CP significantly increased the SVF yield (Test Group 3). The combined use of CL and CP (Test Group 3) did not result in a significant increase in SVF yield, but it was found that the combined use of NP (Test Group 1) significantly increased the SVF yield.

[0045] [Table 1]

[0046] 4. Consideration of the dosage of clostripain (CP) We investigated the relationship between the amount of CP used (in combination with collagenase (CL)) during adipose tissue dispersion and the number of SVF nucleated cells. The following test groups were established, each differing in the type and amount of enzyme used, and SVF nucleated cell suspensions were prepared using the method described above. The number of SVF nucleated cells in the SVF nucleated cell suspension was measured, and evaluated as a ratio, assuming the number of SVF nucleated cells obtained using 0.2% (w / v) Wako CL (product name: collagenase) as 1. Test group 1: Wako CL 0.0067% (w / v) Test group 2: 34,000 U of purified CL derived from Clostridium histolyticum and 360 U of purified CP derived from Clostridium histolyticum Test group 3: A combination of 34,000 U of purified CL derived from Clostridium histolyticum and 3,600 U of purified CP derived from Clostridium histolyticum Test group 4: A combination of 34,000 U of purified CL derived from Clostridium histolyticum and 18,000 U of purified CP derived from Clostridium histolyticum

[0047] The experimental results are shown in Table 2. When the amount of CL used was reduced from 0.2% to 0.067% (Test Group 1), the yield of SVF nucleated cells decreased by approximately 10%. On the other hand, when CP was used alone in combination with CL, the SVF yield did not increase even with an increased amount of CP (Test Groups 2 to 4). In other words, it was found that the amount of CP does not directly affect the number of SVF nucleated cells.

[0048] [Table 2]

[0049] 5. Examination of the amount of neutral protease (NP) used We investigated the relationship between the amount of NP used (in combination with collagenase (CL)) during adipose tissue dispersion and the number of SVF nucleated cells. The following test groups were established, each differing in the type and amount of enzyme used, and SVF nucleated cell suspensions were prepared using the method described above. The number of SVF nucleated cells in the SVF nucleated cell suspension was measured, and evaluated as a ratio, assuming the number of SVF nucleated cells obtained when 0.2% (w / v) Wako CL (product name: collagenase) was used. Test group 1: Wako CL 0.0067% (w / v) Test group 2: 34,000 U of purified CL derived from Clostridium histolyticum and 0.9 U of purified NP derived from Clostridium histolyticum Test group 3: 34,000 U of purified CL derived from Clostridium histolyticum and 9 U of purified NP derived from Clostridium histolyticum Test group 4: 34,000 U of purified CL derived from Clostridium histolyticum and 45 U of purified NP derived from Clostridium histolyticum

[0050] The experimental results are shown in Table 3. Even when NP was used alone in combination with CL, increasing the amount of NP increased the SVF yield. Furthermore, when the NP activity was increased to 9 or higher, the SVF yield was significantly improved compared to when Wako CL was used. Thus, it became clear that the combined use of CL and purified NP is effective for efficient SVF preparation, and that increasing the amount of purified NP used improves the SVF yield.

[0051] [Table 3]

[0052] 6. Examination of the effects and dosage of thermolysin (TP) We investigated the relationship between the amount of TP used (in combination with collagenase (CL)) during adipose tissue dispersion and the number of SVF nucleated cells. The following test groups were created using different amounts of enzyme, and SVF nucleated cell suspensions were prepared using the method described above. The number of SVF nucleated cells in the SVF nucleated cell suspension was measured and evaluated as a ratio, assuming the number of SVF nucleated cells obtained using 0.2% (w / v) Wako CL (product name: collagenase) as 1. Test group 1: 6,9000 U of purified CL derived from Clostridium histolyticum and 6,000 U of Thermolysin "Amano" GMP TP Test group 2: 6,9000 U of purified CL derived from Clostridium histolyticum and 9,000 U of Thermolysin "Amano" GMP TP Test group 3: 6,9000 U of purified CL derived from Clostridium histolyticum and 12,000 U of Thermolysin "Amano" GMP TP

[0053] The experimental results are shown in Table 4. Increasing the amount of TP reduced the SVF yield. Thus, although TP can be used to prepare SVF, using an amount greater than necessary resulted in a reduction in the SVF yield.

[0054] [Table 4]

[0055] 7. Comparison of vascular endothelial cell numbers in SVF We investigated whether not using CP during adipose tissue dispersion would change the number of vascular endothelial cells in SVF. After preparing an SVF nucleated cell suspension using the method described above, the number of vascular endothelial cells in the SVF was measured and evaluated as a ratio, assuming the number of vascular endothelial cells in SVF when 0.2% (w / v) Wako CL (product name: collagenase) was used.

[0056] The experimental results are shown in Table 5. When adipose tissue was dispersed without CP (treatment with purified CL and purified NP), the number of vascular endothelial cells in the extracted SVF increased. In other words, a larger number of vascular endothelial cells could be recovered. Thus, it became clear that not using CP (not including CP in the enzyme solution) and using purified CL and purified NP in combination is an effective means of efficiently preparing or recovering vascular endothelial cells from adipose tissue.

[0057] [Table 5] [Industrial Applicability]

[0058] According to the present invention, it becomes possible to efficiently prepare SVF that is useful in regenerative medicine, research, etc. Therefore, the present invention is expected to be used and utilized particularly in the field of regenerative medicine.

[0059] The present invention is not limited to the above-described embodiments and examples. Various modifications within the spirit and scope of the appended claims that are easily conceivable to those skilled in the art are also included in the present invention. The contents of the publications cited in this specification, such as papers, published patent applications, and patent publications, are incorporated herein by reference to the same extent as if they were expressly set forth in their entirety. This application is based on Patent Application No. 2019-219192 filed in Japan on December 4, 2019, the contents of which are incorporated herein by reference in their entirety.

Claims

1. A method for preparing stromal vascular cells from adipose tissue, comprising the following step (1): (1) A step of treating adipose tissue with an enzyme solution containing collagenase and neutral protease, in which the neutral protease activity is 1 U or more per 10,000 U of collagenase activity, and then recovering cells.

2. 2. The method according to claim 1, wherein the neutral protease activity of the enzyme solution is 2 U or more per 10,000 U of collagenase activity.

3. 2. The method according to claim 1, wherein the neutral protease activity of the enzyme solution is 2.5 U or more per 10,000 U of collagenase activity.

4. 2. The method according to claim 1, wherein the activity ratio of collagenase to neutral protease in the enzyme solution is 34,000:9 to 34,000:

45.

5. The method according to any one of claims 1 to 4, wherein the collagenase content of the enzyme solution is 500 U or more per gram of adipose tissue.

6. The method according to any one of claims 1 to 4, wherein the neutral protease content of the enzyme solution is 0.05 U or more per 1 g of adipose tissue.

7. The method according to any one of claims 1 to 6, wherein the collagenase is derived from Clostridium histolyticum.

8. The method according to any one of claims 1 to 7, wherein the neutral protease is derived from Clostridium histolyticum.

9. The method according to any one of claims 1 to 8, wherein the enzyme solution further comprises clostripain and / or thermolysin.

10. The method according to any one of claims 1 to 8, wherein the enzyme solution does not contain clostripain or thermolysin.

11. The method according to any one of claims 1 to 10, wherein the adipose tissue is human adipose tissue.

12. A method for preparing a cell population containing adipose tissue-derived vascular endothelial cells and vascular endothelial progenitor cells, comprising the following step (2): (2) A step of enriching and / or expanding vascular endothelial cells and vascular endothelial progenitor cells in the stromal vascular cell population obtained by the preparation method according to any one of claims 1 to 11.

13. An enzyme preparation for disintegrating adipose tissue that contains collagenase and neutral protease, with neutral protease activity of 1 U or more per 10,000 U of collagenase activity.

14. 14. The enzyme preparation according to claim 13, wherein the neutral protease activity is 2 U or more per 10,000 U of collagenase activity.

15. 14. The enzyme preparation according to claim 13, wherein the neutral protease activity is 2.5 U or more per 10,000 U of collagenase activity.

16. The enzyme preparation according to claim 13, wherein the activity ratio of collagenase to neutral protease in the enzyme solution is 34,000:9 to 34,000:

45.

17. The enzyme preparation according to any one of claims 13 to 16, wherein the collagenase is derived from Clostridium histolyticum.

18. The enzyme preparation according to any one of claims 13 to 17, wherein the neutral protease is derived from Clostridium histolyticum.

19. The enzyme preparation according to any one of claims 13 to 18, further comprising clostripain and / or thermolysin.

20. The enzyme preparation according to any one of claims 13 to 18, which does not contain clostripain or thermolysin.

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