Cell population containing vascular endothelial (progenitor) cells, method for producing same, and pharmaceutical composition

Abstract

To provide cell populations produced from adipose tissue and having a high content of vascular endothelial (progenitor) cells, and to provide methods for producing a cell population having a high content of endothelial cells from adipose tissue.SOLUTION: Provided is a method for producing a cell population comprising vascular endothelial (progenitor) cells comprising: (1) treating adipose tissue with an enzyme to obtain a cell population comprising at least vascular endothelial (progenitor) cells and adipose stem cells; (2) obtaining a cell population containing CD31 positive cells by selecting CD31 positive cells from the cell population obtained in the step (1); (3) culturing the cell population containing the CD31 positive cells obtained in the step (2) for 1 hour to 7.0 days; and (4) obtaining a cell population containing CD31 positive cells by selecting CD31 positive cells from the cell population obtained in the step (3).SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a cell population comprising vascular endothelial (progenitor) cells. The present invention further relates to a method for producing a cell population comprising vascular endothelial (progenitor) cells, and a pharmaceutical composition comprising said cell population. [Background technology]

[0002] Since it is expected that the therapeutic effect can be improved by using vascular endothelial (precursor) cells in combination with transplantation therapy, etc., human-derived vascular endothelial (precursor) cells are expected to be a therapeutic tool in regenerative medicine. A method for recovering human-derived vascular endothelial (precursor) cells from umbilical veins or large blood vessels is established by filling the vascular lumen with an enzyme solution for cell dispersion, recovering and seeding vascular endothelial (precursor) cells, which makes it possible to easily recover, purify and culture the vascular endothelial (precursor) cells. However, since it is difficult to collect blood vessels as a cell source for regenerative medicine, vascular endothelial (precursor) cells recovered from umbilical veins or large blood vessels are used only in basic research and not as a therapeutic tool.

[0003] On the other hand, subcutaneous adipose tissue, which is abundant and requires little sacrifice or invasion when harvesting, contains a large number of vascular endothelial (precursor) cells (approximately 1 million per gram of adipose tissue), but is difficult to isolate and purify. This is because vascular endothelial (precursor) cells are separated and collected as a mixture with other cells such as adipose stem cells (stromal vascular fraction, hereafter referred to as SVF (Stroma Vascular Fraction)), and during the subsequent culturing process, the vascular endothelial (precursor) cells are eliminated by the adipose stem cells.

[0004] Patent Document 1 describes a method for preparing endothelial cells from adipose tissue. The method described in Patent Document 1 includes the steps of washing adipose tissue obtained from a patient who has undergone liposuction treatment; recovering cells from the washed adipose tissue; enzymatically treating the cells with a purified collagenase preparation, which is devoid of pepsin, trypsin, and thermolysin; selecting the treated cells by contacting them with magnetic beads containing a first antibody specific to an antigen selected from a first group consisting of CD31, CD34, CD144, and CD146, or an antigen selected from a second group consisting of CD14, CD45, and F19; recovering cells bound to the magnetic beads if the antibody is specific to an antigen of the first group, and recovering cells not bound to the magnetic beads if the antibody is specific to an antigen of the second group. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Special Publication No. 2009-528841 Summary of the Invention [Problem to be solved by the invention]

[0006] As described above, the production of vascular endothelial (precursor) cells from adipose tissue has the problem that vascular endothelial (precursor) cells are eliminated due to contamination with other cells such as adipose stem cells. In the method described in Patent Document 1, cells are purified using positive selection with CD31 microbeads, but the ratio of endothelial (precursor) cells after concentration is 17.2% to 86.7% (see Table 1 in paragraph 0031 of Patent Document 1), and the purity of endothelial cells is not sufficient.

[0007] The present invention aims to provide a cell population having a high content of vascular endothelial (precursor) cells produced from adipose tissue, and a method for producing a cell population having a high content of vascular endothelial (precursor) cells from adipose tissue. Another aim of the present invention is to provide a pharmaceutical composition comprising the above-mentioned cell population. [Means for solving the problem]

[0008] The cell population dispersed and collected from the aspirated and crushed adipose tissue through enzyme treatment contains many other cells such as adipose stem cells and fibroblasts in addition to vascular endothelial (precursor) cells, and the percentage of vascular endothelial (precursor) cells is about 1% to several%. In order to isolate only vascular endothelial (precursor) cells from such a cell population, the present inventors focused on the expression of two types of surface antigens (CD45 and CD31) and performed flow cytometry using magnetic beads to obtain a cell population containing CD45-negative and CD31-positive cells. After that, they cultured the obtained cell population for a certain period of time and then attempted to collect CD31-positive cells by flow cytometry using magnetic beads. As a result, it was found that by using the above method, it is possible to obtain a cell population containing vascular endothelial (precursor) cells at a content rate of 92% or more. The present invention was completed based on these findings.

[0009] That is, according to the present invention, the following inventions are provided. <1> A cell population comprising vascular endothelial (progenitor) cells and adipose stem cells, wherein the proportion of CD45-negative and CD31-positive vascular endothelial (progenitor) cells in said cell population is 92% or more, and the proportion of adipose stem cells in said cell population is 8% or less.

[0010] <2> (1) treating adipose tissue with an enzyme to obtain a cell population containing at least vascular endothelial (precursor) cells and adipose stem cells; (2) obtaining a cell population containing CD31-positive cells by selecting CD31-positive cells from the cell population obtained in the step (1); (3) culturing the cell population containing CD31-positive cells obtained in the step (2) for 1 hour to 7.0 days; and (4) obtaining a cell population containing CD31-positive cells by selecting CD31-positive cells from the cell population obtained in the step (3); A method for producing a cell population containing vascular endothelial (progenitor) cells, comprising:

[0011] <3> The step (2) is a step of obtaining a cell population containing CD45-negative and CD31-positive cells by selecting CD45-negative and CD31-positive cells from the cell population obtained in the step (1), and the step (3) is a step of culturing the cell population containing CD45-negative and CD31-positive cells obtained in the step (2) for 2.0 to 6.0 days (or 3.0 to 6.0 days). <2> The method according to <4> Between the step (1) and the step (2), a step of culturing the cell population obtained in the step (1) for 1.0 hour to 5.0 days (also may be 1.0 to 4.0 days) is included. <2> The method according to <5> The cell population containing vascular endothelial (precursor) cells is a cell population containing vascular endothelial (precursor) cells and adipose stem cells, wherein the proportion of vascular endothelial (precursor) cells in the cell population is 92% or more, and the proportion of adipose stem cells in the cell population is 8% or less. <2> ~ <4> 13. The method according to claim 12, wherein <6> In the step (1), the enzyme is collagenase. <2> from <5> 13. The method according to claim 12, wherein <7> The collagenase concentration during the enzyme treatment in the step (1) is 0.02% to 0.5%. <6> The method according to <8> The enzyme treatment solution used in the enzyme treatment in the step (1) further contains DNase I. <6> or <7> The method according to <9> The enzyme treatment solution used in the enzyme treatment in the step (1) does not contain poloxamer or pronase. <6> from <8> 13. The method according to claim 12, wherein

[0012] <10> In the step (2), CD45-negative and CD31-positive cells are selected using magnetic beads labeled with an anti-CD45 antibody and magnetic beads labeled with an anti-CD31 antibody. <2> from <9> 13. The method according to claim 12, wherein <11> In the step (4), CD31-positive cells are selected using magnetic beads labeled with an anti-CD31 antibody. <2> from <10> 13. The method according to claim 12, wherein <12> The method further comprises the step of culturing a cell population containing CD31-positive cells selected in the step (4). <2> from <11> 13. The method according to claim 12, wherein <13> <1> or <2> from <12> A pharmaceutical composition comprising a cell population produced by any one of the methods described above.

[0013]

[14] For the preparation of a pharmaceutical composition: <1> or <2> from <12> Use of a cell population produced by any one of the methods described above.

[15] for use in the treatment of a disease, <1> or <2> from <12> A cell population produced by any one of the methods described above.

[16] <1> or <2> from <12> 2. A method for treating a disease, comprising administering to a patient in need of treatment a cell population produced by any one of the methods described above. Effect of the Invention

[0014] According to the present invention, a cell population having a high content of vascular endothelial (precursor) cells can be produced from subcutaneous adipose tissue. Since the cell population of the present invention has a high content of vascular endothelial (precursor) cells, the vascular endothelial (precursor) cells are not eliminated by other cells during culture, and the vascular endothelial (precursor) cells can be stably cultured in large quantities. [Brief description of the drawings]

[0015] [Figure 1] FIG. 1 shows the results of investigating the optimal collagenase concentration. [Diagram 2]FIG. 2 shows the results of investigating the optimal collagenase concentration. [Diagram 3] FIG. 3 shows the results of FACS analysis of the cell populations recovered when the enzyme composition was changed. [Figure 4] FIG. 4 shows the results of confirming the effect of purification by MACS using FACS. [Diagram 5] FIG. 5 shows the cells over time when the second MACS was performed on day 4 of culture and on day 7 of culture. [Figure 6] 6 shows cells when SVF after hemolysis treatment was seeded and cultured, and shows a comparative example showing that the first cell separation by MACS is necessary. [Figure 7] 7 shows the cells when a CD45-negative, CD31-positive cell population was seeded and cultured, showing a comparative example in which a first MACS cell separation was performed and a second MACS cell separation was also required. [Figure 8] FIG. 8 shows purified and cultured vascular endothelial (progenitor) cells. [Figure 9] FIG. 9 shows the results of cell characterization by network formation assay after cryopreservation. [Figure 10] FIG. 10 shows the results of cell characterization by cell fluorescent immunostaining after cryopreservation. [Figure 11] FIG. 11 shows the purification process of vascular endothelial precursor cells derived from human adipose tissue in Example 8. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] Hereinafter, an embodiment of the present invention will be described in detail. [1] A cell population containing vascular endothelial (progenitor) cells and adipose stem cells The present invention relates to a cell population comprising vascular endothelial (progenitor) cells and adipose stem cells, wherein the proportion of CD45-negative, CD31-positive vascular endothelial (progenitor) cells in the cell population is 92% or more, and the proportion of adipose stem cells in the cell population is 8% or less.

[0017] Vascular endothelial (progenitor) cells are cells that constitute the inner surface of blood vessels and are in contact with the lumen through which blood circulates. Vascular endothelial (progenitor) cells are a concept that encompasses vascular endothelial cells and vascular endothelial progenitor cells. The vascular endothelial (progenitor) cells of the present invention are vascular endothelial (progenitor) cells derived from adipose tissue and retain the ability to divide and proliferate. Vascular endothelial (progenitor) cells can be identified using CD45-negative and CD31-positive as indicators, and can also be identified as CD146-positive and CD144-positive.

[0018] Vascular endothelial (progenitor) cells can be used to treat ischemic diseases of any organ through angiogenesis, and are also necessary cells for regenerating organs in vitro or in vivo together with organ-constituting cells, organ-specific progenitor cells, stem cells (including fetal stem cells and iPS cells), and cells induced from stem cells in regenerative medicine for organs (tissues). The cell population containing vascular endothelial (progenitor) cells according to the present invention is useful as a valuable cell medicine that can be used to treat a wide range of diseases.

[0019] Adipose stem cells are multipotent cells derived from fat, and can differentiate into adipocytes, bone matrix cells, cartilage matrix cells, muscle fiber matrix cells, bone matrix cells, muscle cells, nerve cells, and the like.

[0020] A cell population means a population containing one or more types of many cells. A cell population is generally an impure cell population containing one predominant cell type and one or more minor cell types. In the present invention, vascular endothelial (progenitor) cells correspond to the predominant cell type, and adipose stem cells correspond to the minor cell type.

[0021] CD45 negative and CD31 positive means that the expression of CD45 is negative and the expression of CD31 is positive. CD45 negative and CD31 positive cells can be collected by magnetic cell sorting (MACS) or the like using CD45 antibody and CD31 antibody, as described below.

[0022] In the cell population of the present invention, the percentage of CD45-negative and CD31-positive vascular endothelial (progenitor) cells is 92% or more, preferably 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, and may be 100%. The percentage of CD45-negative and CD31-positive vascular endothelial (progenitor) cells can be measured by FACS (fluorescence activated cell sorting).

[0023] In the cell population of the present invention, the percentage of adipose stem cells is 8% or less, preferably 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2% or less, or 1% or less, and may be 0%. The percentage of adipose stem cells can be confirmed as cells that are CD31 negative, CD146 negative, and CD90 positive. The percentage of adipose stem cells can be further confirmed as CD45 negative, CD44 positive, CD29 positive, and CD13 positive. The above markers can be measured by FACS (fluorescence activated cell sorting).

[0024] [2] Method for producing cell populations containing vascular endothelial (progenitor) cells The present invention relates to (1) treating adipose tissue with an enzyme to obtain a cell population containing at least vascular endothelial (precursor) cells and adipose stem cells; (2) obtaining a cell population containing CD31-positive cells by selecting CD31-positive cells from the cell population obtained in the step (1); (3) culturing the cell population containing CD31-positive cells obtained in the step (2) for 1 hour to 7.0 days; and (4) obtaining a cell population containing CD31-positive cells by selecting CD31-positive cells from the cell population obtained in the step (3); The present invention relates to a method for producing a cell population containing vascular endothelial (progenitor) cells, comprising: In step (2), "selecting CD31-positive cells" may mean selecting CD45- / CD31+ (i.e., selecting CD45-negative and CD31-positive cells) or selecting CD31+ (i.e., selecting only CD31-positive cells). As an example of the present invention, (1) treating adipose tissue with an enzyme to obtain a cell population containing at least vascular endothelial (precursor) cells and adipose stem cells; (2) obtaining a cell population containing CD45-negative and CD31-positive cells by selecting CD45-negative and CD31-positive cells from the cell population obtained in the step (1); (3) culturing the cell population containing CD45-negative and CD31-positive cells obtained in the step (2) for 2.0 to 6.0 days (or 3.0 to 6.0 days); and (4) obtaining a cell population containing CD31-positive cells by selecting CD31-positive cells from the cell population obtained in the step (3); The present invention relates to a method for producing a cell population containing vascular endothelial (progenitor) cells, comprising: Furthermore, between the step (1) and the step (2), a step of culturing the cell population obtained in the step (1) for 1.0 hour to 5.0 days (or 1.0 to 4.0 days) may be included.

[0025] The method for producing a cell population containing vascular endothelial (precursor) cells according to the present invention relates to a method for isolating, purifying, and culturing vascular endothelial (precursor) cells, which are useful cells in regenerative medicine, from adipose tissue (subcutaneous adipose tissue).

[0026] In the present invention, adipose tissue, which is the source of isolation of vascular endothelial (precursor) cells, is a tissue found throughout the body and whose main component is adipocytes. It is mainly present subcutaneously and has the functions of storing energy, protecting the body against physical shocks from the outside world and temperature changes, and secreting hormones, cytokines, etc.

[0027] Adipose tissue can be obtained by surgical excision from, for example, humans, or non-human mammals or birds. Examples of non-human mammals include dogs, cats, cows, horses, pigs, goats, sheep, monkeys, ferrets, rabbits, mice, rats, gerbils, guinea pigs, and hamsters. Examples of birds include chickens. Local anesthesia may be used during surgical excision. Adipose tissue can also be obtained by suction by inserting a cannula into the subcutaneous adipose tissue of the abdomen, thighs, 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, 1 g to 100 g, 2 g to 50 g, or 2 g to 40 g, but is not limited thereto.

[0028] The obtained adipose tissue is preferably confirmed by visual inspection to be free of tumor lesions and contamination. The adipose tissue may be confirmed to be negative for HBV, HCV, HIV, HTLV-1, and TPHA / RPR. The adipose tissue may be confirmed to be less than 1:128 for mycoplasma (PA method) and less than 1:320 for herpes simplex (CF method).

[0029] When using aspirated fat tissue, it is preferable to leave the aspirated fat tissue to separate the fat layer and the aqueous layer. Alternatively, the aspirated fat tissue can be separated using a centrifuge to separate the fat layer and the aqueous layer. After the fat layer and the aqueous layer are separated, the aqueous layer can be collected and removed to isolate only the fat layer. The obtained adipose tissue may be washed with, for example, physiological saline, and then subjected to enzyme treatment.

[0030] Before being subjected to enzyme treatment, the adipose tissue is preferably warmed at room temperature or in a 37°C water bath for 5 to 15 minutes.

[0031] <Regarding process (1)> Step (1) in the present invention is a step of treating adipose tissue with an enzyme to obtain a cell population containing at least vascular endothelial (precursor) cells and adipose stem cells.

[0032] The enzyme treatment can be carried out by adding an appropriate amount of enzyme reaction solution to the adipose tissue in a tube, fixing the tube to a thermostatic shaker, and shaking. The temperature of the enzyme treatment is not particularly limited as long as the enzyme reaction proceeds, but is generally 25°C to 50°C, preferably 30°C to 45°C, for example, 37°C. The shaking may be reciprocating shaking or rotary shaking. In the case of reciprocating shaking, the reciprocating shaking speed is not particularly limited, but is generally 10 rpm to 300 rpm, preferably 50 rpm to 200 rpm, for example, 120 rpm. The reaction time is not particularly limited, but is generally 10 minutes to 3 hours, preferably 15 minutes to 1 hour, for example, 30 minutes.

[0033] As the enzyme, it is preferable to use at least collagenase. Collagenase is a recombinant protein produced by animal tissue cells, inflammatory cells, tumor cells, or bacteria such as Clostridium histolyticum, or artificially produced by genetic engineering technology, and is an enzyme that breaks down types I, II, and III collagen.

[0034] The collagenase concentration in the enzyme treatment solution is preferably 0.02% to 0.5%, more preferably 0.1% to 0.5%, even more preferably 0.1% to 0.4%, still more preferably 0.1% to 0.3%, and most preferably 0.2%.

[0035] It is also preferable that the enzyme treatment solution used for the enzyme treatment further contains, in addition to collagenase, DNase I. When DNase I is used, the concentration of DNase I in the enzyme treatment solution is preferably 100 to 10,000 U / mL, more preferably 200 to 5,000 U / mL, and even more preferably 500 to 2,000 U / mL.

[0036] The enzyme treatment solution used for the enzyme treatment preferably does not contain poloxamer or pronase.

[0037] The enzyme treatment solution used for the enzyme treatment preferably further contains CaCl2. The concentration of CaCl2 in the enzyme treatment solution is preferably 1 mM to 10 mM, more preferably 2 mM to 5 mM, and further preferably 2 mM to 4 mM, for example, 3 mM.

[0038] The enzyme treatment solution used in the enzyme treatment is preferably a buffer solution, more preferably HBSS (Hanks' Balanced Salt Solution).

[0039] A specific preferred composition of the enzyme treatment solution used in the enzyme treatment is 0.2% collagenase, HBSS, 3 mM CaCl2, and 1000 U / ml DNaseI.

[0040] <Regarding step (2)> Step (2) in the present invention is a step of obtaining a cell population containing CD31-positive cells by selecting CD31-positive cells from the cell population obtained in the above step (1). Step (2) in the present invention may be a step of obtaining a cell population containing CD45-negative and CD31-positive cells by selecting CD45-negative and CD31-positive cells from the cell population obtained in step (1) above.

[0041] The method for selecting CD31-positive cells or CD45-negative and CD31-positive cells is not particularly limited, and a method using an antibody to select and separate cells that express a marker protein or cells that do not express a marker protein may be used.

[0042] In the present invention, cells can be selected and separated using an antibody capable of specifically binding to CD31, and further, if desired, an antibody capable of specifically binding to CD45. The antibody is not particularly limited as long as it can specifically bind to the above-mentioned marker protein, and may be either a polyclonal antibody or a monoclonal antibody. Furthermore, the antibody may be a fragment as long as it can specifically bind to the above-mentioned marker protein. Examples of antibody fragments include Fab fragments, F(ab')2 fragments, and single chain antibodies (scFv).

[0043] Methods for selecting and separating cells that express a marker protein or cells that do not express a marker protein using an antibody include, for example, fluorescence activated cell sorting (FACS) and magnetic activated cell sorting (MACS), and among the above, MACS is preferred.

[0044] In MACS, an antibody against a marker protein is immobilized on magnetic beads, and a strong magnet is used to separate the target cells on the inner wall of a cylindrical container (column) or simply in a tube. A general magnetic bead reagent can be used for immobilization. Examples include MACS (Miltenyi Biotec), IMag (BD Japan), etc.

[0045] In the step (2) above, CD31-positive cells or CD45-negative and CD31-positive cells can be selected by MACS using magnetic beads labeled with anti-CD31 antibodies and, if desired, magnetic beads labeled with anti-CD45 antibodies.

[0046] In FACS, if a flow cytometer with a cell sorter function is used, it is possible to separate only specific cells that emit a specified fluorescence. Examples of such instruments include FACSAriaII (BD Japan), JSAN (Bay Biosciences), and MoFlo XDP (Beckman Coulter).

[0047] <Regarding step (3)> Step (3) in the present invention is a step of culturing the cell population containing CD31-positive cells obtained in the above step (2) for 1 hour to 7.0 days (or 3.0 to 6.0 days).

[0048] One of the features of the present invention is that the cell population obtained in step (2) is cultured for 1 hour to 7.0 days, particularly 3.0 to 6.0 days. The cell population obtained in step (2) is cultured for preferably 3.5 to 5.5 days, more preferably 3.5 to 5.0 days, and particularly preferably 4.0 to 4.5 days.

[0049] The culture conditions for culturing the cell population obtained in step (2) can be set in accordance with the conditions suitable for culturing normal animal cells (preferably vascular endothelial (precursor) cells).

[0050] The medium is not particularly limited as long as it is a medium capable of culturing vascular endothelial (progenitor) cells, and examples thereof include EGM-2 (Lonza), αMEM, Dulbecco's modified Eagle medium (DMEM), Dulbecco's modified Eagle medium / Ham's F-12 mixed medium (DMEM / F12), RPMI1640, etc. These culture solutions may generally contain various additives applicable to normal cell culture, such as serum, various vitamins, various antibiotics, various hormones, and various growth factors. As the medium, it is particularly preferable to use EGM-2 medium (Lonza), EGM-2MV (Lonza), etc.

[0051] The culture is preferably carried out in a culture vessel such as a flask at 5% CO2 and 37° C. The medium may be changed, for example, every two days.

[0052] <Regarding step (4)> Step (4) is a step of obtaining a cell population containing CD31-positive cells by selecting CD31-positive cells from the cell population obtained in step (3).

[0053] The method for selecting a cell population containing CD31-positive cells is not particularly limited, and a method for selecting and separating cells expressing the marker protein CD31 using an antibody may be used, as in the case of step (2).Specific examples of the method include FACS, MACS, and the like, as in the case of step (2), and among the above, MACS is preferred. In step (4), CD31-positive cells can be selected using magnetic beads labeled with an anti-CD31 antibody.

[0054] <After step (4)> The method of the present invention may further comprise a step of culturing the cell population containing the CD31-positive cells selected in the step (4) above.

[0055] The culture conditions for culturing the cell population obtained in step (4) can be set in accordance with the conditions suitable for culturing normal animal cells (preferably vascular endothelial (precursor) cells).

[0056] The medium is not particularly limited as long as it is a medium capable of culturing vascular endothelial (progenitor) cells, and examples thereof include EGM-2 (Lonza), αMEM, Dulbecco's modified Eagle medium (DMEM), Dulbecco's modified Eagle medium / Ham's F-12 mixed medium (DMEM / F12), RPMI1640, etc. These culture solutions may generally contain various additives applicable to normal cell culture, such as serum, various vitamins, various antibiotics, various hormones, and various growth factors. As the medium, it is particularly preferable to use EGM-2 medium (Lonza), EGM-2MV (Lonza), etc.

[0057] The culture is preferably carried out in a culture vessel such as a flask at 5% CO2 and 37° C. The medium may be changed, for example, every two days. The culture period is not particularly limited, but may be, for example, 1 to 14 days. After culturing for 1 to 6 days, the cells may be passaged and cultured again for, for example, 3 to 6 days. The number of passages and the number of cultures after step (4) are not particularly limited.

[0058] The seeding of the cell suspension is not particularly limited, but for example, 1x10 4 viable nucleated cells / cm 2 , medium volume is 1mL / 5cm 2 After P3, when subculture is stable, the cell density can be increased to about 2,500 viable nucleated cells / cm. 2 , medium volume is 1mL / 5cm 2 It can be said that:

[0059] [3] Pharmaceutical composition The present invention relates to a pharmaceutical composition comprising the cell population according to the present invention, or a cell population produced by the production method of the present invention (hereinafter, these are also collectively referred to as the cell population of the present invention).

[0060] The cell population of the present invention contains vascular endothelial (progenitor) cells, and can be used as a transplant material for the treatment or cosmetic purposes of damage, degeneration, disorder or dysfunction of biological tissues or organs.

[0061] The cell populations of the present invention can be used alone or in combination with stem cells and / or somatic cells to promote angiogenesis in a variety of pathological tissues where blood flow is compromised. When used in combination with stem cells, the cell population of the present invention can efficiently regenerate various organs and tissues and engraft them into the transplant recipient. The cell population of the present invention can be used in combination with adipose tissue for transplantation for the purposes of breast augmentation, breast reconstruction after breast cancer resection, and improvement of wrinkles and loss of firmness around the eyes and cheeks. The cell population of the present invention can be used in combination with osteoblasts for transplantation for the purpose of treating bone fractures, or for bone lengthening surgery to correct short stature, bone deformity, or leg length discrepancy. The cell population of the present invention can be used in combination with chondrocytes for transplantation for the purpose of treating diseases associated with degeneration, degeneration, deformation and other abnormalities of articular cartilage (e.g., osteoarthritis, rheumatoid arthritis, periarthritis of the shoulder, temporomandibular joint disorders, etc.). The cell population of the present invention can be used in combination with smooth muscle cells to perform transplantation for the purpose of treating diseases caused by damage or abnormalities in smooth muscle cells (e.g., urinary disorders (urinary leakage, frequent urination, urinary retention, etc.), leiomyoma, leiomyosarcoma, etc.).

[0062] The pharmaceutical composition of the present invention may be prepared by diluting the cell population of the present invention with an infusion preparation or culture medium used as a pharma- ceutical acceptable medium. Infusion preparations are not particularly limited, but 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 rehydration solution (solution No. 2), maintenance infusion solution (solution No. 3), and postoperative recovery solution (solution No. 4). The number of cells to be diluted is not particularly limited, but may be, for example, 1×10 3 ~1×10 7 It can be expressed as pieces / mL.

[0063] The pharmaceutical composition of the present invention may contain various additives for increasing storage stability, sterility, isotonicity, absorbency and / or viscosity, such as emulsifiers, dispersing agents, buffers, preservatives, wetting agents, antibacterial agents, antioxidants, chelating agents, thickening agents, gelling agents, pH adjusting agents, etc. Examples of thickening agents include, but are not limited to, HES, dextran, methylcellulose, xanthan gum, carboxymethylcellulose, hydroxypropylcellulose, etc.

[0064] The pH of the pharmaceutical composition of the present invention may be near neutral, for example, pH 6.5 or higher or pH 7.0 or higher, and may be pH 8.5 or lower or pH 8.0 or lower, but is not limited thereto.

[0065] The pharmaceutical composition of the present invention can be stored in a frozen state until immediately before use. When the pharmaceutical composition of the present invention is administered to a patient or subject, it can be rapidly thawed at 37° C. before use.

[0066] The dosage of the pharmaceutical composition of the present invention can be appropriately determined depending on the dosage form, administration method, purpose of use, and the age, weight, and symptoms of the patient or subject. The dosage of vascular endothelial (progenitor) cells per administration is not particularly limited, but may be, for example, 10 4 pcs / kg body weight or more, 10 5 Pieces / kg body weight or more or 10 6 The amount of vascular endothelial (progenitor) cells administered at one time is not particularly limited, but may be, for example, 10 9 pieces / kg weight or less, 10 8 10 pieces / kg body weight or less 7 per kg body weight or less.

[0067] The method of administration of the pharmaceutical composition of the present invention is not particularly limited, and may be, for example, subcutaneous injection, intralymph node injection, intravenous injection, intraarterial injection, intraperitoneal injection, intrathoracic injection, or direct injection into a local area, or direct implantation into a local area using a catheter or the like.

[0068] Furthermore, the pharmaceutical composition of the present invention is not limited to being administered as a cell suspension. (1) Attached to or mixed with other tissues such as fat or skin; (2) Attached to or mixed with other cells or scaffolds and administered; (3) As a cellular product created by attaching or mixing it with other cells or scaffolds and administering it; Also possible are dosage forms such as:

[0069] The subject of administration of the pharmaceutical composition of the present invention is typically a human, but may be another animal. Examples of other animals include mammals and birds. Examples of mammals include dogs, cats, cows, horses, pigs, goats, sheep, monkeys, ferrets, rabbits, mice, rats, gerbils, guinea pigs, and hamsters. Examples of birds include chickens.

[0070] The present invention will be specifically described in the following examples, but the present invention is not limited to these examples. EXAMPLES

[0071] <Reagents, equipment, etc.> HBSS without Ca++, without Mg++ (Gibco, #14175-095) Crudely purified collagenase from Clostridium histolyticum (Wako, #032-22364) Centrifuge tubes (500 mL, Corning, #431123; 250 mL, Corning #430776; 50 mL, Falcon, #352070,15 mL, FALCON #352096) Tabletop centrifuge (Kubota, main unit #S700T, support #RS-7504M, bucket #053-0100) Constant temperature shaker (Yamato, #100) electronic scale Cal Pipette 10 mL (Iwaki, #K-PIPET-LT10) Pipette sterilizer square type (Sansho, #92-0655-4) Silicone rubber dropper for 10 mL (As One, #6-356-04) Cell culture dish (Falcon, #353025, 150mm dish, growth area 156.36 cm2) Cell strainer φ100 μm, FALCON #352360 Cell strainer φ40 μm, FALCON # 352340 DNaseI crude purification (Worthington, #LS002138) Blood cell lysis kit (Miltenyi, #130-094-183) BSA fatty acid free, low endotoxin, lyophilized powder, BioReagent, suitable for cell culture, ≥96% (agarose gel electrophoresis) (Sigma, #A8806) EDTA-2Na (Dojindo, #N001) MS column (Miltenyi, #120-000-472) MACS separator (Miltenyi) CD45 Microbeads (Miltenyi, #130-045-801) CD31 Microbeads kit (human) (Miltenyi, #130-091-935) Culture sure CaCl2(wako, #037-24031, MW110.98) 0.22 μmφ syringe filter (Millipore, Millex-GV, #2LGV-33RS) Endothelial (progenitor) cell medium (EGM-2, Lonza #CC-3162) TrypLE express (Gibco, #12604-021) Dimethyl sulfoxide for molecular biology (DMSO) (Wako Pure Chemical Industries, #047-29353) Deactivation Fetal Bovine Serum (FBS) Cryopreservation unit (Thermo Fisher Scientific, #5100-0001) -80℃ deep freezer, vapor-phase liquid nitrogen cryogenic freezer Sonicator (Branson, #M2800J) Disposable pipettes (Corning, COSTER, 5 mL #4487, 10 mL #4488, 25 mL #4489, 50 mL #4490) Matrigel matrix phenol red-free (Corning, #356237) 96 well plate, SpectraPlate-96 TC (PerkinElmer, #6005650) 4% paraformaldehyde-phosphate buffer (Wako Pure Chemical Industries, #163-20145) Glycine (Wako Pure Chemical Industries, #077-00735) Triton-X100 (Wako Pure Chemical Industries, #591-12191) Human CD31 antibody (R&D, #BBA7) Normal mouse IgG antibody (Santa cruz, # sc-2025) Alexa-488 Goat anti-mouse IgG1 antibody (Invitrogen, #A21121) DAPI (Dojindo, #340-07971) 4-well chamber slide (Iwaki, # 5722-004) VECTA shield mounting medium (VECTOR Laboratories, #H-1000) Confocal microscope (OLYMPUS, FV1000) Cover glass (Matsunami, 24x50 mm, No.1, 0.12-0.17)

[0072] <Preparation of Reagents> (Preparation of 1M CaCl2 stock) Culture sure CaCl2 (wako, #037-24031, MW110.98) 11.1 g MiliQ water 100mL The above was mixed thoroughly and autoclaved (121°C, 20 minutes).

[0073] (Preparation of Collagenase basal 2x mix) [Table 1]

[0074] Collagenase was dissolved in HBSS to a concentration of 0.4%. To completely dissolve, it was dissolved on a rotator for 15 minutes. In a safety cabinet, 0.4% collagenase / HBSS was applied to a syringe filter and sterilized by filtration. Then, 1 M CaCl2 was added to prepare a collagenase basal 2x mix containing 6 mM CaCl2.

[0075] (Preparation of enzyme reaction solution) [Table 2] TIFF0007628689000003.tif189168

[0076] Preparation of enzyme reaction solution in Example 3 Regarding the enzyme reaction solution under condition B, enzyme reaction solutions were also prepared in which the collagenase concentration was changed to 0.0%, 0.02%, 0.1%, 0.2%, 0.4%, 0.8%, 1.0% or 2.0%.

[0077] The callus pipette was placed in a sterile tube and sterilized by dry heat (180°C, 2 hours).

[0078] (Preparation of 100 mM EDTA stock) 0.37 g of EDTA-2Na was dissolved in 10 mL of MiliQ water and sterilized by filtration using a syringe filter with a diameter of 0.22 μm.

[0079] (Preparation of MACS buffer) The solution was dissolved in 0.5% BSA / 2 mM EDTA / HBSS, sterilized through a syringe filter (0.22 μmφ), sonicated for 10 minutes to degas the solution, and stored at 4° C.

[0080] Example 1: Enzyme treatment <Dispensing of aspirated fat tissue> The conical tube was weighed. The aspirated fat tissue was left to stand to separate the fat layer and the aqueous layer. Once separated, a disposable pipette was inserted into the aqueous layer under positive pressure using a silicone rubber dropper, and the aqueous layer was gently removed. The tip of a 10 mL disposable pipette was broken off to make the opening wider. The aspirated fat tissue was stirred with the pipette to make the tissue as homogenous as possible, and then dispensed into conical tubes.

[0081] Conical tube dispense volume: 50 mL conical tube: Approximately 5 g of aspirated fat (small scale) 250 mL conical tube: Approximately 25 g of aspirated fat (medium scale) 500 mL conical tube: Approximately 50 g of aspirated fat (large scale)

[0082] The weight of the aspirated lipoaspirate was measured as follows. "Weight of aspirated fat" = "Weight after tissue dispensing" - "Weight of empty conical tube"

[0083] The dispensed aspirated fat tissue was pre-warmed in a 37°C water bath for 5 to 30 minutes or left at room temperature.

[0084] <Enzyme treatment> The enzyme reaction solution was prepared under the above conditions and either pre-warmed at 37°C for 10 minutes or left at room temperature. An equal amount of enzyme reaction solution (condition A, B, C, D, E or F) was added to the aspirated fat tissue. The conical tube was placed on its side in a thermostatic shaker and shaken at 37℃ at 120 rpm for 30 minutes to allow the enzyme reaction. The cap of the conical tube was wrapped with parafilm to prevent contamination. The tube was removed and centrifuged at 800G for 10 minutes. After centrifugation, the tissue-enzyme reaction solution was separated into an oil layer, a residual fat layer, an emulsion layer, a water layer, and a cell pellet from the top of the centrifuge tube. The oil layer, fat layer, and emulsion layer were removed from the tube. In the case of medium and large scale, they were removed with a cal pipette, and in the case of small scale, they were removed with a 50 mL disposable pipette. Care was taken not to break up the precipitated cell mass. The water layer was removed by aspirating with a Pasteur pipette. The cell pellet in the tube was gently suspended in 45 mL of HBSS (4°C) using a 50 mL disposable pipette for both large, medium and small scales to obtain a cell suspension.

[0085] A φ100um cell strainer was placed in a new 50 mL conical tube, and the milky white fibrous mass was transferred onto the cell strainer with a pipette together with the cell suspension (approximately 10 mL) to pass the cell suspension through. The fibrous mass trapped on the cell strainer was squeezed with the tip of a 5 mL disposable pipette to recover the cells adhering to the fibrous mass. The remaining cell suspension (approximately 35 mL) was passed through the cell strainer. The cells were thoroughly suspended (15 times) using a 50 mL disposable pipette (Corning ##4490 pipette tip with an inner diameter of 3.15 mm). (The cells were suspended by passing through a hole with an inner diameter of 3.15 mm 15 times).

[0086] A φ40 μm cell strainer was placed on a new 50 mL conical tube, and the cell suspension that had been passed through the 100 μm φ cell strainer was then passed through the φ40 μm cell strainer again.

[0087] Centrifugation was performed at 800G for 5 minutes at 4°C. The supernatant was aspirated and the mixture was made up to 45 mL with HBSS (4°C) and resuspended. Centrifugation was performed at 800G for 5 minutes at 4°C. The supernatant was aspirated and the mixture was resuspended in HBSS (4°C). The mixture was suspended in 0.5 mL per 1 g of fat.

[0088] The number of live nucleated cells was counted using Luna-stem. The number of live nucleated cells was determined by co-staining with Acridine Orange (AO) and Propidium Iodide (PI). This number of live nucleated cells was taken as the number of cells in the stromal vascular fraction (SVF).

[0089] Hemolysis was performed using the Miltenyi Red Blood Cell Lysis Solution Kit (all procedures were performed according to the kit's protocol). The supernatant was aspirated and resuspended in HBSS (4°C). The fat was suspended at 1 mL per 5 g. The number of live nucleated cells was counted using Luna-stem. This number of live nucleated cells was used as the number of cells in the SVF after hemolysis.

[0090] Regarding the SVF after hemolysis, the ratio of the cell population contained in the SVF after hemolysis was investigated by FACS (Example 2), the ratio of the cell population contained in the SVF after hemolysis was investigated by MACS (Example 3), and adipose tissue-derived vascular endothelial (precursor) cells (EC cells) were purified by MACS (Example 4).

[0091] Example 2: Investigation of the ratio of cell populations contained in SVF after hemolysis treatment by FACS The ratios of CD45, CD34, and CD31 were confirmed by FACS when enzyme treatment was performed using enzyme reaction solutions in which the collagenase concentration was changed to 0.0%, 0.02%, 0.1%, 0.2%, 0.4%, 0.8%, 1.0%, or 2.0% in the enzyme reaction solution under condition B.

[0092] (Preparation of FACS buffer) The solution was dissolved in 0.5% BSA / 2 mM EDTA / DPBS, sterilized through a syringe filter (0.22 μmφ), sonicated for 10 minutes to degas the solution, and stored at 4° C.

[0093] (Preparation of FcR block reaction solution) BD Fc block TM Reagent for Human (BD, #564220) was added to the FACS buffer at a concentration of 2.5 μg / mL to prepare an FcR block reaction solution. 4×10 5 After hemolysis, the SVF was suspended in 80 μL of FcR block reaction solution and incubated on ice for 10 minutes. Antibody or FACS buffer was added according to the experimental series below, mixed thoroughly, and incubated on ice for 10 minutes. After the antibody reaction, 0.5 mL of FACS buffer was added, and the samples were left to stand on ice and then subjected to FACS in the same order. Voltage and compensation settings were performed for series #01-05, and the setting conditions were fixed, and sample group series #06-13 was subjected to FACS to obtain data.

[0094] Experimental series [Table 3] TIFF0007628689000005.tif154165

[0095] APC-IgG1 antibody (BD, #550854) PE-IgG1 antibody (BD, #555749) FITC-IgG1 antibody (BD, #555748) APC-CD45 antibody (BD, #555485) PE-CD31 antibody (BD, #555446) FITC-CD34 antibody (BD, #564221) The results are shown in Figure 1. The relationship between collagenase concentration and cell number is shown in Figure 2.

[0096] Population verdict: CD45- / CD34+ / CD31+: A population containing many endothelial (progenitor) cells CD45- / CD34+ / CD31-: A population containing many adipose stem cells CD45+: A population containing many blood cells

[0097] In addition, the ratios of CD45, CD34, and CD31 were confirmed by FACS for each enzyme reaction solution (conditions A, B, C, D, E, or F). The FACS method was the same as described above in Example 2. The experimental sequence is shown below.

[0098] (Experiment series) [Table 4] TIFF0007628689000007.tif129160

[0099] The results are shown in Figure 3. It was found that the ratio of the cell population that could be recovered changed depending on the enzyme composition. Condition B had the highest ratio of vascular endothelial (progenitor) cells.

[0100] Example 3: Investigation of the ratio of cell populations contained in SVF after hemolysis treatment using MACS <Preparation of enzyme reaction solution in Example 3> Collagenase basal 2x mix was prepared according to Table 1 (Preparation of Collagenase basal 2x mix). Enzyme reaction solutions under the following conditions AD were prepared.

[0101] [Table 5] TIFF0007628689000009.tif113165

[0102] The results are shown in the table below. [Table 6]

[0103] As in the case of FACS, the ratio of vascular endothelial (progenitor) cells (MACS: ratio of CD45-negative and CD31-positive cells) was higher in condition B.

[0104] Example 4: Purification of adipose tissue-derived vascular endothelial (progenitor) cells (EC cells) by MACS Adipose tissue-derived vascular endothelial (precursor) cells (EC cells) were purified by MACS using SVF obtained under the following conditions, which were the enzyme treatment conditions described in Example 1. The time when SVF was collected was designated as passage number P0.

[0105] [Table 7]

[0106] The time when SVF is collected is designated as passage number P0. (1) Collection of CD45- / CD31+ cell populations by MACS The procedure was carried out according to the Miltenyi MACS protocol as follows. After counting the number of nucleated cells, the cells to be subjected to MACS were dispensed into a 15 mL conical tube. The target number of viable nucleated cells was 1x10^7 per column. Centrifugation (300G, 3 min, 4℃) was performed. The cell pellet was gently suspended in 80 μL of MACS buffer. 20 μL of CD45 microbeads were added and gently pipetted. Reaction with CD45 antibody was performed for 15 minutes at 4℃. 1 mL of MACS buffer was added and the liquid was mixed by tapping. Centrifugation (300G, 3 min, 4℃) was performed. The cell pellet was resuspended in 0.5 mL of MACS buffer and left to stand at 4℃. A Miltenyi MS column was placed in the separator and the column was washed once with 0.5 mL of MACS buffer. Immediately after column washing, the cell suspension treated with CD45 microbeads was added to the column. The flow-through fraction was collected as the CD45- cell population. The fraction captured by the magnetic column and eluted by flushing after the column left the separator was collected as a CD45+ cell population.

[0107] The CD45- cell population was centrifuged (300G, 3 min, 4°C). The cell pellet was gently resuspended in 60 μL of MACS buffer. 20 μL of Fc receptor (FcR) block reagent was added to the cell suspension and vortexed for 2-3 seconds. 20 μL of CD31 microbeads were added and gently pipetted. The CD31 antibody reaction was carried out for 15 min at 4°C. 1 mL of MACS buffer was added and the liquid was mixed by tapping. Centrifugation (300G, 3 min, 4°C) was carried out. The cell pellet was resuspended in 0.5 mL of MACS buffer and left to stand at 4°C. A new Miltenyi MS column was placed in the separator and the column was washed once with 0.5 mL of MACS buffer. Immediately after column washing, the cell suspension treated with CD31 microbeads was added to the column. 0.5 mL of MACS buffer was passed through the column three times. The flow-through was collected as a CD45- / CD31- cell population (rich in adipose stem cells). The fraction captured by the magnetic column and eluted by flushing after separation from the separator was collected as a CD45- / CD31+ cell population (rich in vascular endothelial (progenitor) cells). The CD45- / CD31+ cell population was centrifuged (300G, 3 minutes, 4℃).

[0108] Immediately after the MACS procedure, the following three types of cell populations were subjected to FACS using CD45 / CD31 / CD34, and the differences in the populations of each cell population, particularly the ratios of vascular endothelial (progenitor) cells and adipose stem cells, were confirmed. (Cell population) 1. Post-hemolysis (non-MACS) SVF 2. CD45- / CD31+ cell population 3. CD45- / CD31+ Cell Population The FACS method was performed according to the same protocol as in Example 2. The experimental sequence is shown below. (Experiment series)

[0109] [Table 8]

[0110] The effect of purification by MACS described above was confirmed by FACS, and the results are shown in FIG.

[0111] (2) Cell culture The CD45- / CD31+ cell population obtained above was resuspended in EGM-2 medium and counted. The number of nucleated cells and cell viability were counted using a Luna-stem. 4 The cells were suspended at 100 viable nucleated cells / 0.25 mL EGM-2 medium / cm2 and seeded. A portion of the cell suspension of the CD45- / CD31+ cell population was subjected to FACS under condition B in Table 9. Hereinafter, conditions AH correspond to the culture schedule conditions shown in Table 9. Culture (EGM-2 medium) (P0) was performed for 4 or 7 days at 37°C, 5% CO 2、 The medium was changed every 2 days. In addition, a portion of the cell suspension of the SVF after hemolysis was subjected to FACS under condition A. The cell culture schedule is shown in the table below.

[0112] [Table 9]

[0113] To perform the second MACS on the 4th and 7th days of culture, the following cell detachment and dispersion procedures were performed on the 4th and 7th days of culture. After washing the cells with HBSS (37°C), an appropriate amount of 1x TrypLE express (37°C) was added (3 mL TrypLE express / 25 cm 2 ) was added and cells were detached at 37℃, 5% CO2 for 5 minutes. The reaction was stopped with EGM-2 medium and cell pellets were obtained by centrifugation (300G, 5 minutes, 4℃). The number of nucleated cells was counted using Luna stem. A portion of the cell suspension on day 4 of culture was subjected to FACS under condition C, and a portion of the cell suspension on day 7 of culture was subjected to FACS under condition F.

[0114] (3) Collection of CD31+ cell populations by MACS The cell suspension was centrifuged (300G, 3 min, 4℃). The cell pellet was gently resuspended in 60μL of MACS buffer. 20μL of Fc receptor (FcR) block reagent was added to the cell suspension and vortexed for 2-3 seconds. 20μL of CD31 microbeads were added and gently pipetted. The CD31 antibody reaction was carried out for 15 min at 4℃. 1mL of MACS buffer was added and the liquid was mixed by tapping. Centrifugation (300G, 3 min, 4℃) was carried out. The cell pellet was resuspended in 0.5mL of MACS buffer and left to stand at 4℃. A Miltenyi MS column was placed in the separator and the column was washed once with 0.5mL of MACS buffer. Immediately after column washing, the cell suspension treated with CD31 microbeads was added to the column. 0.5mL of MACS buffer was passed through the column three times. The flow-through was collected as a CD31- cell population (rich in adipose stem cells). The fraction captured by the magnetic column and eluted by flushing after separation from the separator was collected as a CD31+ cell population (rich in vascular endothelial (progenitor) cells). The CD31+ cell population was centrifuged (300G, 3 minutes, 4°C). The cell suspension from day 4 of culture was subjected to MACS and a portion of the cell suspension from day 7 of culture was subjected to FACS under condition D and condition G, respectively.

[0115] (4)Cell culture 2500 viable nucleated cells / 0.2 mL EGM2 medium / cm 2 The cells were suspended and seeded in EGM-2 medium (P1) for 6 days at 37°C and 5% CO2. The medium was changed every 2 days. The cells were cultured until they were 80-90% confluent. Figure 5 shows microscopic images of a cell population cultured under condition D, where the cell suspension on day 4 of culture was subjected to MACS, as condition E, and a cell population cultured under condition G, where the cell suspension on day 7 of culture was subjected to MACS, as condition H. Under condition E, the purification of vascular endothelial (progenitor) cells was completed. On the other hand, under condition H, an increase in adipose stem cells was confirmed, and vascular endothelial (progenitor) cells tended to be eliminated. This shows that the purification of vascular endothelial (progenitor) cells was completed only under the culture schedule of condition E.

[0116] The results of FACS analysis under conditions AD and conditions F and G, and photographs of the cells under conditions E and H are shown in FIG.

[0117] Comparative Example 1: For comparison, FIG. 6 shows a photograph of cells in the case where the SVF cell population was recovered under enzyme treatment condition B in Example 4 above, and the first MACS was not performed.

[0118] Comparative Example 2: For comparison, in Example 4 above, after recovering the SVF cell population under enzyme treatment condition B, the CD45- / CD31+ cell population was recovered by the first MACS, and a photograph of the cells is shown in Figure 7 when the second MACS was not performed.

[0119] Example 5: Example 5 is a cell culture and passaging step for cell expansion and large scale acquisition of purified EC cells.

[0120] The cells of Example 4, Condition E, were detached and dispersed in the same manner as above. The cells were suspended and seeded at 2500 viable nucleated cells / 0.2 mL EGM2 medium / cm2. Culture (EGM-2 medium) (P2) was carried out for 5-6 days at 37°C and 5% CO2. The medium was changed every 2 days. Culture was carried out until 80-90% confluent.

[0121] The cells were detached and dispersed as described above. They were suspended and seeded at 2500 viable nucleated cells / 0.2 mL EGM2 medium / cm2. Culture (EGM-2 medium) (P3) was carried out for 5-6 days at 37°C and 5% CO2. Medium was changed every 2 days. Culture was carried out until 80-90% confluent.

[0122] The resulting photograph of cells under enzyme treatment condition B is shown in FIG. 8A.

[0123] (result) From the above results, it was revealed that it is preferable to recover the CD45- / CD31+ cell population by MACS immediately after recovering the SVF after the hemolysis treatment, and to perform the second MACS on day 4 of culture.

[0124] Example 6 The cell pellet obtained in Example 5 was suspended in a cell preservation solution (80% EGM-2, 10% inactivated FBS, 10% DMSO). It was placed in a cryopreservation unit and frozen in a -80°C deep freezer. It was then transferred to a gas-phase liquid nitrogen tank and cryopreserved. The cells preserved here were warmed in a 37°C water bath for 2 minutes immediately after being removed from the cryopreservation container, and the cells were suspended and seeded in pre-warmed EGM-2 medium at 2500 viable nucleated cells / 0.2 mL EGM2 medium / cm2. Culture (EGM-2 medium) (P4) was performed for 5-6 days at 37°C and 5% CO2. The medium was replaced every 2 days. The cells were cultured until 80-90% confluent. The cells were detached and dispersed in the same manner as in Examples 4 and 5. The cells were suspended and seeded at 2500 viable nucleated cells / 0.25 mL EGM2 medium / cm2. Culture (EGM-2 medium) (P5) was performed for 5-6 days at 37°C, 5% CO2. Medium was changed every 2 days. Culture was performed until 80-90% confluent. The above steps of freezing, preserving, and suspending the purified endothelial (progenitor) cells are shown in Figure 8B.

[0125] The cells were thawed, suspended and seeded at 2500 viable nucleated cells / 0.2 mL EGM2 medium / cm2. Culture (EGM-2 medium) (P4) was carried out for 5-6 days at 37°C and 5% CO2. Medium was replaced every 2 days. As a result, vascular endothelial (progenitor) cells that can be used for various purposes were obtained.

[0126] The results of cell characterization after cryopreservation are shown in Figures 9 and 10. It is known that one of the characteristic properties of vascular endothelial (progenitor) cells is that they form a tubular network structure when cultured on Matrigel. This is a method commonly used for cell characteristic analysis of umbilical vein endothelial (progenitor) cells, etc. Therefore, we confirmed whether adipose tissue-derived vascular endothelial (progenitor) cells (P5) form a network structure by a network formation assay. For comparison, we set umbilical vein endothelial (progenitor) cells (HUVEC), which form a network structure, and adipose stem cells, which have been reported to form a slightly weaker network structure. The adipose stem cells were collected as SVF and were obtained by subculture in DMEM / F12 / penicillin-streptomycin / 10% inactivated FBS medium.

[0127] The method for the network formation assay is shown below. Matrigel was melted overnight at 4°C. Matrigel was added to a 96-well plate cooled on ice at 50 mL / well. The plate was left to stand in an incubator at 37°C and 5% CO2 for 30 minutes or more to gel the Matrigel. The adipose-derived vascular endothelial (precursor) cells (P4), umbilical vein endothelial (precursor) cells (P4), and adipose stem cells (P3) obtained in Example 6 were dispersed in the same manner as in Examples 4 and 5. The adipose-derived vascular endothelial (precursor) cells and umbilical vein endothelial (precursor) cells were dispersed at 5x10 3 A cell suspension was prepared so that viable cells / 50 mL EGM-2 / well was added to the Matrigel as P5. Adipose stem cells (P2) were 5x10 3 A cell suspension was prepared so that the cells were viable cells / 50 mL DMEM / F12 / penicillin-streptomycin / 10% inactivated FBS medium / well, and added onto the Matrigel as P3. After culturing for 6 hours in an incubator at 37°C and 5% CO2, the cells were photographed with an inverted phase contrast microscope. Figure 9 shows a photograph of the cells.

[0128] (result) Adipose tissue-derived vascular endothelial (progenitor) cells (P5) formed a tubular network structure equivalent to that of umbilical vein vascular endothelial (progenitor) cells.

[0129] The procedure for cell fluorescent immunostaining is as follows. Preparation of PBS: 137 mM NaCl / 2.7 mM KCl / 10 mM Na2HPO4 / 1.8 mM KH2PO4 / MiliQ water. 10 mM glycine / PBS. 0.1% Triton X-100 / PBS. 3% BSA / PBS, 1% BSA / PBS, and 0.1% BSA / PBS were prepared. CD31 antibody as the sample group and Normal Mouse IgG antibody as the control group were diluted in 1% BSA / PBS at a concentration of 10 μg / mL to prepare the primary antibody reaction solution. Alexa-488 goat anti-mouse IgG1 antibody was diluted 1000 times with % BSA / PBS to prepare the secondary antibody reaction solution. DAPI was diluted 2000 times with PBS to prepare the nuclear staining reaction solution.

[0130] Example 7 As in Example 6, vascular endothelial (progenitor) cells (P4) and umbilical vein endothelial cells (P4) were dispersed, and then suspended and seeded in a 4-well chamber slide at 2500 viable nucleated cells / 0.2 mL EGM2 medium / cm2. Culture (EGM-2 medium) (P5) was performed for 4 days at 37°C and 5% CO2. The medium was replaced every 2 days. The medium was removed, and the cells were fixed with 4% paraformaldehyde-phosphate buffer solution 0.4 mL / well for 10 minutes at room temperature. The cells were washed three times with 10 mM glycine / PBS solution, 1 mL / well, for 5 minutes at room temperature. The 10 mM glycine / PBS solution was removed, and the cells were permeabilized with 0.1% Triton X-100 / PBS 0.4 mL / well for 5 minutes at room temperature. The cells were washed three times with PBS 1 mL / well for 5 minutes at room temperature. The PBS was removed, and the cells were blocked with 0.4 mL / well of 3% BSA / PBS for 10 minutes at room temperature. The 3% BSA / PBS was removed, and the cells were washed with 1 mL / well of PBS for 5 minutes at room temperature. 0.25 mL / well of the sample and control primary antibody reaction solution was added, and the cells were incubated at 4℃ overnight in a humid environment, protected from light. The cells were washed three times with 1 mL / well of 0.1% BSA / PBS for 5 minutes at room temperature. 0.25 mL / well of the secondary antibody reaction solution was added, and the cells were incubated for 20 minutes at room temperature in a humid environment, protected from light. The cells were washed three times with 1 mL / well of 0.1% BSA / PBS for 5 minutes at room temperature. 0.25 mL / well of the nuclear staining reaction solution was added, and the cells were incubated for 5 minutes at room temperature in a humid environment, protected from light. The cells were washed once with 1 mL / well of PBS for 5 minutes at room temperature. The cells were mounted with VECTA shield, and the mounting medium was solidified at room temperature for 24 hours. Images were taken with a confocal microscope. The imaging results are shown in FIG.

[0131] (result) Cell characteristic analysis by cell fluorescent immunostaining. The cell morphology of vascular endothelial (progenitor) cells (P5) that had been repeatedly subcultured was confirmed by CD31, a vascular endothelial (progenitor) cell marker, and nuclear staining.

[0132] Example 8: Culture method for purifying endothelial progenitor cells derived from human adipose tissue (new method) 25–200 g of aspirated and crushed subcutaneous adipose tissue was collected. Stromal vascular cells (SVF, a mixture containing endothelial progenitor cells as well as other cells such as adipose stem cells) were prepared from adipose tissue by enzyme treatment (enzyme composition 1: 0.2% collagenase + 1000 U / mL DNase1 + 3 mM CaCl2 + HBSS) and centrifugation. At this time, SVF was prepared at a concentration of 2–10 × 10 per 1 g of fat. 5 Obtain nucleated cells. The cells were seeded in a plastic container for culture (culture flask, etc.). The seeding density was 5x10 4 Nucleated cells / cm 2 (5×10 3 ~5×10 5 Nucleated cells / cm 2 ) and the medium was 0.2 mL / cm 2 (0.02-20 mL / cm 2 ) vascular endothelial cell culture medium (e.g., EGM-2 or EGM2MV, Lonza) was used. The SVF was cultured in a vascular endothelial cell culture medium for 2 days (1.0 hour to 5 days is sufficient).

[0133] The cultured SVF was dispersed from the culture vessel using a cell dispersion liquid to obtain a cell suspension of the SVF cell population. CD31-positive cells were selected from the SVF cell population by MACS to obtain a cell population containing CD31-positive cells (CD31-positive cell population). At this time, the purity can be further increased by repeating the MACS operation twice to select using CD31 microbeads (recovering CD31+ / CD31+ cells).

[0134] The CD31 positive cell population was seeded in a plastic container for culture (culture flask, etc.). The seeding density was 5 × 10 4 Nucleated cells / cm 2 (5×10 3 ~5×10 5 Nucleated cells / cm 2 ) and the medium was 0.2 mL / cm 2 (0.02-20 mL / cm 2A vascular endothelial cell culture medium (for example, EGM-2 or EGM2MV, Lonza) was used. The CD31 positive cell population was cultured in the vascular endothelial cell culture medium for 3 days (any time between 1 hour and 7 days is acceptable).

[0135] The cultured CD31 positive cell population was dispersed from the culture vessel using a cell dispersion liquid to obtain a cell suspension of the CD31 positive cell population. CD31 positive cells were further selected by MACS to obtain a cell population containing CD31 positive cells with a high efficiency (high purity CD31 positive cell population). The purity can be further increased by repeating the MACS operation twice to select using CD31 microbeads. At this time, a cell population with a ratio of CD45 negative, CD31 positive, and CD146 positive cells of 90% or more by flow cytometry analysis was determined to be a high purity CD31 positive cell population (vascular endothelial progenitor cells).

[0136] For amplification, the highly purified CD31 positive cell population was seeded in a plastic container for culture (culture flask, etc.). The seeding density was 5x10 4 Nucleated cells / cm 2 (5×10 3 ~5×10 5 Nucleated cells / cm 2 ) and the medium was 0.2 mL / cm 2 (0.02-20 mL / cm 2 A vascular endothelial cell culture medium (e.g., EGM-2 or EGM2MV, Lonza) was used. The cells were cultured for 5 days (2 to 10 days is acceptable), and the highly purified CD31 positive cell population was passaged. The seeding density was 2.5 × 10 3 Nucleated cells / cm 2 (2.5×10 2 ~2.5×10 5 Nucleated cells / cm 2 ) and the medium was 0.2 mL / cm 2 (0.02-20 mL / cm 2 ) vascular endothelial cell culture medium (e.g., EGM-2 or EGM2MV, Lonza) was used. As a result of the above, purified adipose tissue-derived vascular endothelial (progenitor) cells were obtained (FIG. 11).

Claims

1. (1) treating the aspirated fat tissue with an enzyme to obtain a cell population containing at least vascular endothelial (precursor) cells and adipose stem cells; (2) obtaining a cell population containing CD45-negative and CD31-positive cells by selecting CD45-negative and CD31-positive cells from the cell population obtained in the step (1); (3) culturing the cell population containing CD45-negative and CD31-positive cells obtained in the step (2) for 2.0 to 6.0 days; and (4) obtaining a cell population containing CD31-positive cells by selecting CD31-positive cells from the cell population obtained in the step (3); A method for producing a cell population containing vascular endothelial (precursor) cells, comprising:

2. The method according to claim 1, further comprising, between the step (1) and the step (2), a step of culturing the cell population obtained in the step (1) for 1.0 hour to 5.0 days.

3. The method according to claim 1 or 2, wherein in step (1), the enzyme is collagenase.

4. 4. The method according to claim 3, wherein the collagenase concentration during the enzyme treatment in step (1) is 0.02% to 0.5%.

5. 4. The method according to claim 3, wherein the collagenase concentration during the enzyme treatment in step (1) is 0.1% to 0.3%.

6. The method according to claim 3 , wherein the enzyme treatment solution used in the enzyme treatment in step (1) further contains DNase I.

7. The method according to any one of claims 3 to 6, wherein the enzyme treatment solution used in the enzyme treatment in step (1) does not contain poloxamer or pronase.

8. The method according to any one of claims 1 to 7, wherein in step (2), CD45-negative and CD31-positive cells are selected using magnetic beads labeled with an anti-CD45 antibody and magnetic beads labeled with an anti-CD31 antibody.

9. The method according to any one of claims 1 to 8, wherein in the step (4), CD31-positive cells are selected using magnetic beads labeled with an anti-CD31 antibody.

10. The method according to any one of claims 1 to 9, further comprising a step of culturing a cell population containing CD31-positive cells selected in step (4).

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