JP2019088279A - Cell population comprising vascular endothelial (progenitor) cells, method for producing the same, and pharmaceutical composition - Google Patents

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

  The present invention relates to cell populations comprising vascular endothelial (progenitor) cells. The invention further relates to methods of producing cell populations comprising vascular endothelial (progenitor) cells, as well as pharmaceutical compositions comprising said cell populations.

  Human-derived vascular endothelial (precursor) cells are expected as therapeutic tools in regenerative medicine because it can be expected that the therapeutic effect can be enhanced if vascular endothelial (precursor) cells are used in combination in transplantation treatment and the like. As a method of recovering human-derived vascular endothelial (precursor) cells from umbilical vein or large blood vessels, the lumen of the blood vessel is filled with an enzyme solution for cell dispersion, and the vascular endothelial (progenitor) cells are recovered and disseminated easily. It is possible to recover, purify and culture endothelial (progenitor) cells, and the method is established. However, because it is difficult to collect blood vessels as a cell source for regenerative medicine, vascular endothelial (progenitor) cells collected from umbilical vein or large blood vessels are used only in basic research and are used as a therapeutic tool. Not.

  On the other hand, a large amount (approximately 1 million cells / g of adipose tissue) of a large amount of subcutaneous adipose tissue with a small amount of sacrifice or invasiveness associated with the collection has a large amount of vascular endothelial (progenitor) cells, but its isolation and purification are difficult It is. Because, vascular endothelial (progenitor) cells are separated and collected as a mixture with other cells such as adipose stem cells (interstitial vascular cell group, hereinafter SVF (Stromal Vascular Fraction)), and in the subsequent culture process, Progenitor cells are destroyed by adipose stem cells.

  Patent Document 1 describes a method of preparing endothelial cells from adipose tissue. The method described in Patent Document 1 comprises the steps of washing adipose tissue obtained from a liposuction treated patient; recovering cells from the washed adipose tissue; enzymatically treating the cells with a purified collagenase preparation The process wherein the preparation lacks pepsin, trypsin and thermolysin; an antigen selected from the first group consisting of CD31, CD34, CD144 and CD146, or CD14, CD45 and F19. Sorting the treated cells by contacting with magnetic beads comprising a first antibody specific for an antigen selected from the second group consisting of: if the antibody is specific for the first group of antigens Recovering the cells bound to the magnetic beads, and recovering the cells not bound to the magnetic beads if the antibody is specific to the second group The method comprising.

Japanese Patent Application Publication No. 2009-528841

  As described above, production of vascular endothelial (precursor) cells from adipose tissue has a problem that vascular endothelial (progenitor) cells are destroyed by contamination with other cells such as adipose stem cells. In the method described in Patent Document 1, although the cells are purified using positive selection with CD31 microbeads, the percentage of endothelial (progenitor) cells after concentration is 17.2% to 86.7% ( See Table 1 in paragraph 0031 of Patent Document 1), the purity of endothelial cells is not sufficient.

  The present invention provides a cell population having a high content of vascular endothelial (progenitor) cells produced from adipose tissue, and a method for producing a cell population having a high content of vascular endothelial (progenitor) cells from adipose tissue. It was an issue to be solved. Furthermore, this invention made it the issue which should be solved to provide the pharmaceutical composition containing the cell population mentioned above.

  The cell population dispersed and collected from the aspirated and crushed adipose tissue through enzyme treatment contains a large number of other cells such as adipose stem cells and fibroblasts in addition to vascular endothelial (progenitor) cells. The proportion of precursor) cells is about 1 to several percent. In order to isolate only vascular endothelial (precursor) cells from such a cell population, the present inventors focused on the expression of two surface antigens (CD45 and CD31), and flow using magnetic beads. By performing cytometry, a cell population containing CD45 negative and CD31 positive cells was obtained. Thereafter, after culturing the obtained cell population for a certain period, CD31 positive cells were tried to be collected 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 in which the content of vascular endothelial (progenitor) cells is 92% or more. The present invention has been completed based on these findings.

That is, according to the present invention, the following inventions are provided.
<1> A cell population comprising vascular endothelial (precursor) cells and adipose stem cells, wherein the percentage of CD45 negative and CD31 positive vascular endothelial (precursor) cells in the cell population is 92% or more, and fat in the cell population A cell population in which the percentage of stem cells is 8% or less.

&Lt; 2 &gt; (1) A step of processing a fatty 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 sorting out 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 of (2) for 1 hour to 7.0 days; and (4) CD31 positive from the cell population obtained in the step of (3) above Obtaining a cell population containing CD31 positive cells by sorting out the cells of
A method of producing a cell population comprising vascular endothelial (progenitor) cells, comprising

<3> The step of (2) above obtains 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). The step (3) is performed for 2.0 to 6.0 days (or 3.0 to 6 days) a cell population containing CD45 negative and CD31 positive cells obtained in the step (2). The method according to <2>, which is a step of culturing for 0 days.
<4> Between the step of (1) and the step of (2), the cell population obtained in the step of (1) is 1.0 hour to 5.0 days (1.0 to 4 days) The method according to <2>, which comprises the step of culturing for 0 days.
<5> A cell population comprising vascular endothelial (precursor) cells is a cell population comprising vascular endothelial (precursor) cells and adipose stem cells, wherein the percentage of vascular endothelial (precursor) cells in the cell population is 92% or more The method according to any one of <2> to <4>, which is a cell population, wherein the percentage of adipose stem cells in the cell population is 8% or less.
<6> The method according to any one of <2> to <5>, wherein in the step (1), the enzyme is collagenase.
<7> The method according to <6>, wherein the collagenase concentration in the enzyme treatment in the step (1) is 0.02% to 0.5%.
<8> The method according to <6> or <7>, wherein the enzyme treatment liquid used for the enzyme treatment in the step (1) further comprises DNase I.
<9> The method according to any one of <6> to <8>, wherein the enzyme treatment solution used for the enzyme treatment in the step (1) does not contain poloxamer and pronase.

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

[14] Use of the cell population according to <1>, or the cell population produced by the method according to any one of <2> to <12>, for the production of a pharmaceutical composition.
[15] A cell population according to <1>, or a cell population produced by the method according to any one of <2> to <12>, for use in the treatment of a disease.
[16] A disease comprising administering the cell population according to <1> or the cell population produced by the method according to any one of <2> to <12> to a patient in need of treatment Treatment method.

  According to the present invention, a cell population having a high content of vascular endothelial (progenitor) cells can be produced from subcutaneous adipose tissue. Since the cell population of the present invention has a high content rate of vascular endothelial (progenitor) cells, vascular endothelial (progenitor) cells are not destroyed by other cells when cultured, It can be stably cultured in large quantities.

FIG. 1 shows the results of examining the optimal collagenase concentration. FIG. 2 shows the results of examining the optimal collagenase concentration. FIG. 3 shows the results of FACS analysis of the cell population recovered when the enzyme composition was changed. FIG. 4 shows the results of FACS confirmation of the effect of MACS purification. FIG. 5 shows cells over time when the second MACS was applied on the fourth day of culture and on the seventh day of culture. FIG. 6 shows cells after seeding and culturing SVF after hemolytic treatment. The comparative example which shows that the cell separation by 1st MACS is required is shown. FIG. 7 shows cells when CD45 negative and CD31 positive cell populations are seeded and cultured. The comparative example which shows that the cell isolation by 1st MACS is performed, and the cell isolation by 2nd MACS is also required is shown. FIG. 8 shows purified cultured endothelial (progenitor) cells. FIG. 9 shows the results of cell characterization analysis by network formation assay after cryopreservation. FIG. 10 shows the results of cell characterization by cytofluorescent immunostaining after cryopreservation. FIG. 11 shows purification of human adipose tissue-derived vascular endothelial precursor cells in Example 8.

Hereinafter, embodiments of the present invention will be specifically described.
[1] A cell population comprising vascular endothelial (precursor) cells and adipose stem cells The present invention relates to a cell population comprising vascular endothelial (precursor) cells and adipose stem cells, wherein the cell population is CD45 negative and CD31 positive in the cell population. The present invention relates to a cell population in which the percentage of endothelial (progenitor) cells is 92% or more and the percentage of adipose stem cells in the cell population is 8% or less.

  Vascular endothelial (progenitor) cells are cells that constitute the inner surface of a blood vessel and are cells in contact with the circulating lumen of blood. Vascular endothelial (progenitor) cells are a concept including vascular endothelial cells and vascular endothelial precursor cells. The vascular endothelial (precursor) cells in the present invention are vascular endothelial (progenitor) cells derived from adipose tissue, and retain the ability to proliferate. Vascular endothelial (progenitor) cells can be identified using CD45 negative and CD31 positive as an index, and can also be identified as CD146 positive and CD144 positive.

  Endothelial (progenitor) cells can be used to treat ischemic diseases of any organ through angiogenesis, and organ constituent cells, organ-specific precursor cells, stem cells (fetal) as regenerative medicine for organs (organ, tissue) Stem cells (including iPS cells) and cells derived from stem cells are cells required to regenerate organs in vitro or in the body. The cell population comprising vascular endothelial (progenitor) cells according to the invention is useful as a valuable cellular medicament that can be used to treat a wide range of diseases.

  An adipose stem cell is a pluripotent cell derived from fat, and can be differentiated into an adipocyte, an osteocyte, a chondrocyte, a myocyte, a myocyte, a muscle cell or a nerve cell, etc. It is.

  By cell population is meant a population comprising one or more multiple cells. A cell population is generally a non-pure cell population comprising one predominant cell type and one or more minor cell types. In the present invention, vascular endothelial (progenitor) cells correspond to the predominant cell types, and adipose stem cells correspond to a few cell types.

  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 recovered by magnetic cell separation (MACS) described later using CD45 antibody and CD31 antibody.

  In the cell population of the present invention, the proportion 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 (precursor) cells can be measured by FACS (fluorescence activated cell sorting).

  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 Yes, it may be 0%. The percentage of adipose stem cells can be confirmed as CD31 negative and CD146 negative cells and CD90 positive cells. The percentage of adipose stem cells can be further confirmed as CD45 negative, CD44 positive, CD29 positive, CD13 positive. The above markers can be measured by FACS (fluorescence activated cell sorting).

[2] A method for producing a cell population comprising vascular endothelial (progenitor) cells
(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 sorting out 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 of (2) for 1 hour to 7.0 days; and (4) CD31 positive from the cell population obtained in the step of (3) above Obtaining a cell population containing CD31 positive cells by sorting out the cells of
And a method of producing a cell population comprising vascular endothelial (progenitor) cells.
As “selecting CD31 positive cells” in step (2), it may be sorting by CD45 − / CD 31 + (ie, sorting of CD45 negative and CD 31 positive cells) or sorting by CD 31 + That is, cells may be sorted only by CD31 positive).
One example of the present invention is
(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 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 above step (2) for 2.0 to 6.0 days (or 3.0 to 6.0 days); A) obtaining a cell population containing CD31 positive cells by selecting CD31 positive cells from the cell population obtained in the step (3);
And a method of producing a cell population comprising vascular endothelial (progenitor) cells.
In addition, between the step (1) and the step (2), the cell population obtained in the step (1) may be 1.0 hour to 5.0 days (or 1.0 to 4 days). The step of culturing may be included.

  The method for producing a cell population containing vascular endothelial (precursor) cells according to the present invention separates, purifies and culture vascular endothelial (progenitor) cells which are cells useful for regenerative medicine from adipose tissue (subcutaneous adipose tissue) It relates to the method.

  In the present invention, adipose tissue which is a separation source of vascular endothelial (precursor) cells is a whole-body tissue mainly composed of fat cells, which is mainly present subcutaneously, has energy storage, and physical impact from the outside world. And protect the body against temperature changes, and secrete hormones and cytokines.

  Adipose tissue can be obtained, for example, by surgical excision from human or non-human mammals or birds. Examples of non-human mammals include dogs, cats, cattle, horses, pigs, goats, sheep, monkeys, ferrets, rabbits, mice, rats, gerbils, guinea pigs, hamsters and the like. As birds, a chicken etc. can be mentioned. During surgical resection, local anesthesia may be performed. Adipose tissue can also be obtained by aspiration by intubating a cannula into the abdomen, thighs, buttocks, or whole body subcutaneous adipose tissue. The amount of fat 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.

  Preferably, the obtained adipose tissue is visually confirmed to be free from neoplastic lesions and contamination. Adipose tissue may confirm that HBV, HCV, HIV, HTLV-1 and TPHA / RPR are all negative. Adipose tissue may be confirmed to have less than 128 times mycoplasma (PA method) and less than 320 times herpes simplex (CF method).

When using suctioned fat tissue, it is preferable to allow the suctioned fat tissue to stand and separate the fat layer and the water layer. Also, the aspirated fat tissue can be separated by a centrifuge to separate the fat layer and the water layer. After the fat layer and the aqueous layer are separated, only the fat layer can be isolated by recovering and removing the aqueous layer.
The obtained adipose tissue may be washed with, for example, physiological saline and then subjected to the enzyme treatment.

  It is preferable to warm the adipose tissue before being subjected to the enzyme treatment for 5 minutes to 15 minutes in a room temperature or 37 ° C. water bath before the enzyme treatment.

<About the step (1)>
The step (1) in the present invention is a step of treating the adipose tissue with an enzyme to obtain a cell population comprising at least vascular endothelial (progenitor) cells and adipose stem cells.

  The enzyme treatment can be carried out by adding an appropriate amount of the enzyme reaction solution to fat tissue in a tube, fixing the tube on a constant temperature 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 to 45 ° C, for example, 37 ° C. The shaking may be reciprocating shaking or rotational shaking. In the case of reciprocating shaking, the reciprocating shaking speed is not particularly limited, but 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.

Preferably, at least collagenase is used as the enzyme.
Collagenase is a recombinant protein produced by animal tissue cells, inflammatory cells, tumor cells, bacteria such as Clostridium histolyticum etc., or artificially produced by genetic recombination technology, and is type I, type II, III Refers to an enzyme that degrades type collagen.

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

  It is also preferable that the enzyme treatment solution used for the enzyme treatment further contains DNase I in addition to collagenase. When DNase I is used, the concentration of DNase I in the enzyme treatment solution is preferably 100 to 10000 U / mL, more preferably 200 to 5000 U / mL, and still more preferably 500 to 2000 U / mL.

  It is preferable that the enzyme treatment solution used for enzyme treatment does not contain poloxamer and pronase.

It is preferable that the enzyme treatment solution used for the enzyme treatment further contains CaCl ₂ . The concentration of CaCl ₂ in the enzyme-treated solution is preferably 1 mM to 10 mM, more preferably 2 mM to 5 mM, still more preferably 2 mM to 4 mM, for example, 3 mM.

  The enzyme treatment solution used for the enzyme treatment is preferably a buffer, and more preferably HBSS (Hanks' Balanced Salt Solution).

Preferred specific examples of the composition of the enzyme treatment solution used for the enzyme treatment are 0.2% collagenase, HBSS, 3 mM CaCl ₂ , and 1000 U / ml-DNase I.

<About 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 step (1).
Step (2) in the present invention 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 above step (1). May be

  There is no particular limitation on the method of selecting CD31 positive cells or CD45 negative and CD31 positive cells, and antibodies may be used to select and separate cells expressing marker proteins or cells not expressing marker proteins. Good.

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

  As a method of selecting and separating cells expressing marker protein or cells not expressing marker protein using an antibody, for example, fluorescence activated cell sorting (FACS), magnetic activated cell sorting (Magnetic activated cell sorting) : MACS) etc., and among the above, MACS is preferable.

  MACS can immobilize an antibody against a marker protein on magnetic beads, and use a strong magnet to separate target cells in the inner wall of a cylindrical container (column) or simply in a tube. As a magnetic bead reagent to be immobilized, a general one can be used. For example, MACS (made by Miltenyi Biotec), IMag (made by Japan BD) etc. are mentioned.

  In the step (2), CD31-positive cells or CD45-negative and CD31-positive cells are subjected to MACS by using magnetic beads labeled with anti-CD31 antibody and magnetic beads optionally labeled with anti-CD45 antibody. It can be sorted out.

  In FACS, using a flow cytometer having a cell sorter function, it is possible to sort out only specific cells that emit designated fluorescence. As such an apparatus, for example, FACSAria II (manufactured by Japan BD), JSAN (manufactured by Bay Biosciences), MoFlo XDP (manufactured by Beckman Coulter), etc. may be mentioned.

<About step (3)>
The step (3) in the present invention is a step of culturing the cell population containing the CD31 positive cells obtained in the step (2) for 1 hour to 7.0 days (may be 3.0 to 6.0 days). is there.

  It is one of the features of the present invention that the culture period of the cell population obtained in step (2) is 1 hour to 7.0 days, particularly 3.0 to 6.0 days. The culture period of the cell population obtained in step (2) is preferably 3.5 to 5.5 days, more preferably 3.5 to 5.0 days, and particularly preferably 4.0 to 4 .5 days.

  The culture conditions for culturing the cell population obtained in step (2) can be set according to the conditions suitable for culturing normal animal cells (preferably vascular endothelial (progenitor) cells).

  The medium is not particularly limited as long as it can culture vascular endothelial (progenitor) cells, and EGM-2 (Lonza), αMEM, Dulbecco's modified Eagle's medium (DMEM), Dulbecco's modified Eagle's medium / ham F-12 mixed medium DMEM / F12), RPMI 1640 and the like can be mentioned. Usually, various additives applicable to ordinary cell culture, such as serum, various vitamins, various antibiotics, various hormones, various growth factors, etc. may be added to these culture solutions. As the medium, particularly preferably, EGM-2 medium (Lonza), EGM-2MV (Lonza) or the like can be used.

The culture is preferably performed at 37 ° C., 5% CO ₂ , using a culture vessel such as a flask. Medium exchange may be performed, for example, every two days.

<About the 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).

There are no particular limitations on the method for selecting cell populations containing CD31 positive cells, and as in step (2), a method for selecting and separating cells expressing the marker protein CD31 using an antibody may be used. Just do it. Specifically, as in the case of the step (2), FACS, MACS and the like can be mentioned, and among the above, MACS is preferable.
In step (4), CD31 positive cells can be sorted using magnetic beads labeled with an anti-CD31 antibody.

<After Step (4)>
The method of the present invention may further include the step of culturing a cell population containing the CD31 positive cells selected in the step (4).

  The culture conditions for culturing the cell population obtained in the step (4) can be set according to the conditions suitable for culturing a normal animal cell (preferably, vascular endothelial (progenitor) cell).

  The medium is not particularly limited as long as it can culture vascular endothelial (progenitor) cells, and EGM-2 (Lonza), αMEM, Dulbecco's modified Eagle's medium (DMEM), Dulbecco's modified Eagle's medium / ham F-12 mixed medium DMEM / F12), RPMI 1640 and the like can be mentioned. Usually, various additives applicable to ordinary cell culture, such as serum, various vitamins, various antibiotics, various hormones, various growth factors, etc. may be added to these culture solutions. As the medium, particularly preferably, EGM-2 medium (Lonza), EGM-2MV (Lonza) or the like can be used.

The culture is preferably performed at 37 ° C., 5% CO ₂ , using a culture vessel such as a flask. Medium exchange may be performed, for example, every two days.
The culture period is not particularly limited, and for example, culture can be performed for 1 day to 14 days. After culturing for 1 to 6 days, cells may be passaged, and for example, culture for 3 to 6 days may be performed again. The number of passages and the number of cultures after step (4) are not particularly limited.

Although the seeding of the cell suspension is not particularly limited, for example, it can be performed with 1 × 10 ⁴ viable nucleated cells / cm ² and a medium volume of about 1 mL / 5 cm ² . From P3 on which subculture has become stable and possible, for example, 2500 viable nucleated cells / cm ² and the amount of medium can be 1 mL / 5 cm ² .

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

  The cell population of the present invention contains vascular endothelial (precursor) cells, and can be used as a graft material for treatment or cosmetic treatment of damage, degeneration, injury or malfunction of living tissues and organs.

The cell population of the present invention can promote angiogenesis in various pathological tissues having blood flow problems by using alone or in combination with stem cells and / or somatic cells.
By using the cell population of the present invention in combination with stem cells, various organs and tissues can be efficiently regenerated and engrafted at the transplantation destination.
By using the cell population of the present invention in combination with adipose tissue, it is possible to perform transplantation for the purpose of breast augmentation, breast reconstruction after removal of breast cancer, improvement of wrinkles and firmness of eyes and cheeks, and the like.
The cell population of the present invention can be used in combination with osteoblasts for transplantation for the treatment of bone fracture, bone distraction for the purpose of improving short stature, bone deformation, and leg length difference.
The cell population of the present invention can be used in combination with chondrocytes to treat diseases associated with degeneration, degeneration, deformation or other abnormalities of articular cartilage (eg, osteoarthritis, rheumatoid arthritis, shoulder joint arthritis, temporomandibular joint, etc. Transplants for the purpose of treatment of
The cell population of the present invention can be used in combination with smooth muscle cells to treat diseases caused by smooth muscle cell injury or abnormalities (eg, voiding disorder (eg, urine leak, frequent urination, urinary retention, etc.), leiomyoma, smooth Transplantation can be performed for the purpose of treatment of myeloma and the like.

The pharmaceutical composition of the present invention may be obtained by diluting the cell population of the present invention with an infusion preparation used as a pharmaceutically acceptable medium, or a culture solution. The infusion preparation is not particularly limited. For example, physiological saline, 5% glucose solution, Ringer solution, Ringer's lactate, Ringer's lactate, Ringer's acetate, starting solution (No. 1), dehydrated replacement solution (No. 2), maintenance infusion (No. 3) Solution), post-operative recovery solution (No. 4 solution), etc. can be mentioned. The number of cells upon dilution is not particularly limited, and can be, for example, 1 × 10 ³ to 1 × 10 ⁷ cells / mL.

  The pharmaceutical composition of the present invention can be added to various additives for increasing storage stability, sterility, isotonicity, absorption and / or viscosity, such as emulsifiers, dispersants, buffers, preservatives, wetting agents. It may contain an antibacterial agent, an antioxidant, a chelating agent, a thickener, a gelling agent, a pH adjuster and the like. Examples of the thickener include, but not limited to, HES, dextran, methylcellulose, xanthan gum, carboxymethylcellulose, hydroxypropyl cellulose and the like.

  The pH of the pharmaceutical composition of the present invention can be around neutral pH, for example, pH 6.5 or more or pH 7.0 or more, and can be pH 8.5 or less or pH 8.0 or less. It is not limited to these.

  The pharmaceutical composition of the present invention can be stored in a frozen state until immediately before use. When administering the pharmaceutical composition of the present invention to a patient or a subject, it can be used by rapidly thawing at 37 ° C.

The dose of the pharmaceutical composition of the present invention can be appropriately determined depending on the administration form, administration method, purpose of use, age, body weight, symptoms and the like of the patient or subject. The dose of vascular endothelial (progenitor) cells is not particularly limited, and is, for example, 10 ⁴ cells / kg body weight or more, 10 ⁵ cells / kg body weight or more, or 10 ⁶ cells / kg body weight or more. The dose of vascular endothelial (progenitor) cells is not particularly limited, and is, for example, 10 ⁹ cells / kg body weight or less, 10 ⁸ cells / kg body weight or less, or 10 ⁷ cells / kg body weight or less.

  The administration method of the pharmaceutical composition of the present invention is not particularly limited, and for example, subcutaneous injection, intralymphatic injection, intravenous injection, intraarterial injection, intraperitoneal injection, intrathoracic injection, or direct local injection may be used. Or directly implanted locally by a catheter or the like.

Also, the pharmaceutical composition of the present invention is not limited to administration as a cell suspension,
(1) Adhere to or mix with other tissues such as fat and skin;
(2) Adhere or mix with other cells or scaffolds and administer;
(3) Adhering to or mixing with other cells or scaffolds, and administering it as a fabricated cell product;
Other forms of administration are also possible.

  The administration target of the pharmaceutical composition of the present invention is typically a human but may be other animals. Other animals include mammals or birds. Examples of mammals include dogs, cats, cattle, horses, pigs, goats, sheep, monkeys, ferrets, rabbits, mice, rats, gerbils, guinea pigs, hamsters and the like. As birds, a chicken etc. can be mentioned.

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

<Reagent, equipment used>
HBSS without Ca ++, without Mg ++ (Gibco, # 14175-095)
Coranagenase crude purified, from Clostridium histolyticum (Wako, # 032-22364)
Centrifuge tube (500 mL, Corning, # 431123; 250 mL, Corning # 430776; 50 mL, Falcon, # 352070, 15 mL, FALCON # 352096)
Desktop centrifuge (Kubota, main unit # S700T, post # RS-7504M, bucket # 053-0100)
Isothermal shaker (Yamato, # 100)
Electronic scale Callus pipette 10 mL (Iwaki, # K-PIPET-LT10)
Pipette sterilizer square type (Sanyo, # 92-0655-4)
For dropper silicone rubber 10 mL (As One, # 6-356-04)
Cell culture dish (Falcon, # 353025, 150 mm dish, growth area 156.36 cm2)
Cell strainer φ100 μm, FALCON # 352360
Cell strainer φ 40 μm, FALCON # 352340
DNase I crude purification (Worthington, # LS 0021 38)
Blood cell lysis kit (Milteny, # 130-094-183)
BSA fatty acid free, low endotoxin, lyophilized powder, BioReagent, suitable for cell culture, 96 96% (Agarose gel electrophoresis) (Sigma, # A8806)
EDTA-2Na (Dojindo, # N001)
MS column (Milteny, # 120-000-472)
MACS separator (Milteny)
CD45 Microbeads (Milteny, # 130-045-801)
CD31 Microbeads kit (human) (Milteny, # 130-091-935)
Culture sure CaCl ₂ (wako, # 037-24031, MW 110. 98)
0.22 μmφ Syringe Filter (Millipore, Millex-GV, # 2LGV-33RS)
Endothelial (progenitor) cell culture medium (EGM-2, Lonza # CC-3162)
TrypLE express (Gibco, # 12604-021)
Dimethyl sulfoxide for molecular biology (DMSO) (Wako Pure Chemical Industries, # 047-29353)
Nonfunctional Fetal Bovine Serum (FBS)
Freeze storage unit (Thermo Fisher Scientific, # 5100-0001)
-80 ° C deep freezer, vapor phase liquid nitrogen cryogenic freezer sonicator (Branson, # M2800J)
Disposable Pipettes (Corning, Coaster, 5 mL # 4487, 10 mL # 4488, 25 mL # 4489, 50 mL # 4490)
Matrigel basement membrane matrix phenol red free (Corning, # 356237)
96 well plate, SpectraPlate-96 TC (Perkin Elmer, # 6005650)
4% paraformaldehyde phosphate buffer solution (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)

<Preparation of Reagents>
(Preparation of 1M CaCl2 stock)
Culture sure CaCl2 (wako, # 037-24031, MW 110.98) 11.1 g
MiliQ water 100 mL
The above was mixed well and autoclaved (121 ° C., 20 minutes).

(Preparation of collagenase basal 2x mix)

  The collagenase was dissolved in HBSS to 0.4%. In order to completely dissolve and dissolve, it was dissolved by a rotator for 15 minutes. In the safety cabinet, 0.4% collagenase / HBSS was applied to a syringe filter and sterile filtered. Thereafter, 1 M CaCl2 was added to prepare a collagenase basal 2x mix containing 6 mM CaCl2.

(Preparation of enzyme reaction solution)

Preparation of Enzyme Reaction Solution of Example 3 With regard to the enzyme reaction solution under condition B, the collagenase concentration was 0.0%, 0.02%, 0.1%, 0.2%, 0.4%, 0.8. An enzyme reaction solution was also prepared which was changed to 10%, 1.0% or 2.0%.

  The callus pipette was placed in a sterile tube and dried by heat (180 ° C., 2 hours).

(Preparation of 100 mM EDTA stock)
0.37 g of EDTA-2Na was dissolved in 10 mL of MiliQ water. It was sterile filtered with a syringe filter of 0.22 μmφ.

(Preparation of MACS buffer)
It was dissolved in 0.5% BSA / 2 mM EDTA / HBSS, and sterile filtered with a syringe filter 0.22 μmφ. Store for 10 minutes in a sonicator at 4 ° C. for degassing.

Example 1: Enzymatic treatment <dilution of aspirated adipose tissue>
The conical tube weight was weighed.
Allow the aspirated adipose tissue to settle and allow the fat and water layers to separate. After separation, the disposable pipette was inserted into the water layer under positive pressure using a dropper silicone rubber, and the water layer was gently recovered and removed.
The tip of the 10 mL disposable pipette was folded to widen the mouth.
The aspirated adipose tissue was stirred with the above pipette to make the tissue as homogeneous as possible and dispensed into conical tubes.

Conical tube dispensing volume:
50 mL conical tube: around 5 g of suctioned fat (small scale)
250 mL conical tube: around 25 g of suctioned fat (medium scale)
500 mL conical tube: around 50 g of suctioned fat (large scale)

The weight of the aspirated fat was weighed according to the following.
"Weight of suctioned fat" = "weight after tissue dispensing"-"weight of empty conical tube"

  The aspirated adipose tissue which had been dispensed was previously warmed in a 37 ° C. water bath for 5 minutes to 30 minutes, or left at room temperature.

<Enzyme treatment>
The enzyme reaction solution was prepared under the conditions described above, and was previously warmed at 37 ° C. for 10 minutes or left at room temperature.
An equal volume of enzyme reaction solution (conditions A, B, C, D, E or F) was added to aspirated adipose tissue. The conical tube was fixed sideways on a thermostat and shaken at 37 ° C. and 120 rpm for 30 minutes for enzyme reaction. The conical tube cap wrapped parafilm to prevent contamination. The tube was removed and centrifuged at 800G for 10 minutes. The tissue-enzyme reaction solution after centrifugation was separated from the top of the centrifuge tube into an oil layer, a residual fat layer, an emulsion layer, an aqueous layer, and a cell pellet. The oil layer, fat layer and emulsion layer were removed from the tube. The medium and large scale was removed with a callus pipette, and the small scale with a 50 mL disposable pipette. Care was taken that the precipitated cell mass did not collapse. The aqueous layer was aspirated off with an aspirator using 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 and medium scale to obtain a cell suspension.

  The φ100 um cell strainer was placed in a new 50 mL conical tube, and the milky fiber mass was pipetted along with the cell suspension (about 10 mL) onto the cell strainer to pass the cell suspension. The fibrous mass trapped on the cell strainer was scraped with a 5 mL disposable pipette tip to recover the cells adhering to the fibrous material. The remaining cell suspension (about 35 mL) was passed through a cell strainer. The cell suspension was sufficiently performed (15 times) with a 50 mL disposable pipette (3.15 mm internal diameter of the pipette tip of Corning # 4490). (The cells were suspended 15 times through a hole of 3.15 mm internal diameter).

  The φ40 um cell strainer was placed in a new 50 mL conical tube, and the cell suspension passed through a 100 μm cell strainer was further passed through a φ40 um cell strainer.

  Centrifugation operation, it was centrifuged at 4 ° C. for 5 minutes at 800G. The supernatant was aspirated and re-suspended to 45 mL with HBSS (4 ° C.). Centrifuge at 800 ° C for 5 minutes at 4 ° C. The supernatant was aspirated and resuspended in HBSS (4 ° C.). Suspended at 0.5 mL / g of fat.

  The number of nucleated viable cells was counted using a Luna-stem. The number of nucleated viable cells was determined by co-staining with Acridine Orange (AO) and Propidium Iodide (PI). This nucleated viable cell number was defined as the number of cells of SVF (stromal vascular fraction).

  Hemolysis was performed with a Miltenyi red blood cell lysis solution kit (all were performed according to the protocol of the kit). The supernatant was aspirated and resuspended in HBSS (4 ° C.). Suspended at 1 mL per 5 g of fat. The number of nucleated viable cells was counted by Luna-stem. The number of nucleated viable cells was taken as the number of SVF cells after hemolysis treatment.

  With respect to SVF after hemolysis treatment, the ratio investigation of cell population included in SVF after hemolysis treatment by FACS (Example 2), ratio investigation of cell population included in SVF after hemolysis treatment by MACS (Example 3), and fat by MACS Purification of tissue-derived vascular endothelial (precursor) cells (EC cells) (Example 4) was performed.

Example 2: Ratio investigation of cell population included in SVF after hemolytic treatment by FACS In the enzyme reaction solution under condition B, collagenase concentration is 0.0%, 0.02%, 0.1%, 0.2%, The ratio of CD45, CD34 and CD31 was confirmed by FACS for the case where the enzyme reaction was carried out using 0.4%, 0.8%, 1.0% or 2.0% of the enzyme reaction solution.

(Preparation of FACS buffer)
It was dissolved in 0.5% BSA / 2 mM EDTA / DPBS, and sterile filtered with a syringe filter 0.22 μmφ. Store for 10 minutes in a sonicator at 4 ° C. for degassing.

(Preparation of FcR block reaction solution)
^{BD Fc block TM Reagent for Human (} BD, # 564220) was FcR block reaction solution was added to FACS buffer at a concentration of 2.5 [mu] g / mL.
After 4 × 10 ⁵ hemolytic treatments, SVF was suspended in 80 μL of FcR block reaction solution and incubated in ice for 10 minutes. The antibody or FACS buffer was added according to the following experimental sequence, mixed well, and incubated in ice for 10 minutes. After antibody reaction, 0.5 mL of FACS buffer was added, allowed to stand in ice, and subjected to sequential FACS. In the series # 01-05, the Voltage setting and the Compensation setting were performed, the setting conditions were fixed, and the sample group series # 06-13 was applied to FACS to acquire data.

Experiment series

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

Population judgment:
CD45- / CD34 + / CD31 +: population containing a large number of vascular endothelial (progenitor) cells
CD45- / CD34 + / CD31-: population containing a large number of adipose stem cells
CD45 +: population containing a large number of blood cells

  Moreover, the ratio of CD45, CD34, and CD31 was confirmed by FACS about each enzyme reaction liquid (conditions A, B, C, D, E, or F). The method of FACS was performed in the same manner as described above in Example 2. The experimental sequence is shown below.

(Experimental series)

  The results are shown in FIG. The enzyme composition was found to change the proportion of recoverable cell populations. Condition B had the highest ratio of vascular endothelial (progenitor) cells.

Example 3: Probing ratio of cell population included in SVF after hemolytic treatment by MACS <Preparation of enzyme reaction solution of Example 3>
According to Table 1 (Preparation of Collagenase basal 2x mix), Collagenase basal 2x mix was prepared.
An enzyme reaction solution was prepared under the following conditions AD.

The results are shown in the following table.

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

Example 4 Purification of Adipose Tissue-Derived Vascular Endothelial (Progenitor) Cells (EC Cells) by MACS A Fat Tissue-Derived Vascular Endothelium by MACS Using SVF When the Enzyme Treatment Conditions Described in Example 1 Are Under the Following Conditions Purification of (progenitor) cells (EC cells) was performed. The SVF recovery time is Passage number P0.

The SVF recovery time is Passage number P0.
(1) Recovery of CD45- / CD31 + Cell Populations by MACS The following procedure was performed according to the protocol of Milteny MACS.
After counting the number of nucleated cells, cells to be subjected to MACS were aliquoted into 15 mL conical tubes. As a guide, 1 × 10 ^ 7 viable nucleated cells were applied to one column. Centrifugation (300 G, 3 minutes, 4 ° C.) was performed. The cell pellet was gently suspended in 80 μL of MACS buffer. 20 μL of CD45 microbeads were added and gently pipetted. The reaction with CD45 antibody was performed at 4 ° C. for 15 minutes. 1 mL of MACS buffer was added and mixed by tapping. Centrifugation (300 G, 3 minutes, 4 ° C.) was performed. The cell pellet was resuspended in 0.5 mL MACS buffer and allowed to stand at 4 ° C. The Milteny MS column was set as a separator, and the column was washed once with 0.5 mL MACS buffer. Immediately after column washing, CD45 microbead treated cell suspension was added to the column. Flow-through fractions were collected as CD45- cell populations. Fractions that were captured on a magnetic column and flashed out after separation from the separator were collected as a CD45 + cell population.

  The CD45-cell population was centrifuged (300 G, 3 min, 4 ° C.). The cell pellet was gently resuspended in 60 μL of MACS buffer. To the cell suspension, 20 μL of Fc receptor (FcR) block reagent was added and vortexed for 2-3 seconds. Add 20 μL of CD31 microbeads and pipet gently. The CD31 antibody reaction was performed at 4 ° C. for 15 minutes. Add 1 mL of MACS buffer and mix by tapping. Centrifugation (300 G, 3 minutes, 4 ° C.) was performed. The cell pellet was resuspended in 0.5 mL MACS buffer and allowed to stand at 4 ° C. A new Miltenyi MS column was set on the separator, and the column was washed once with 0.5 mL 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 fractions that were captured on the magnetic column and eluted in the flash after separation from the separator were collected as a CD45- / CD31 + cell population (containing a large number of vascular endothelial (progenitor) cells). The CD45- / CD31 + cell population was centrifuged (300 G, 3 minutes, 4 ° C.).

Immediately after the MACS operation, the following three cell populations were subjected to FACS with CD45 / CD31 / CD34 to confirm differences in the population of each cell population, in particular, the ratio of vascular endothelial (progenitor) cells to adipose stem cells.
(Cell population)
1. Post hemolysis treatment (MACS not processed) SVF
2. CD45- / CD31 + cell population
3. CD45- / CD31 + cell population
It carried out by the method of FACS and the same protocol as Example 2.
The experimental sequence is shown below.
(Experimental series)

  The result of having confirmed the effect of the above-mentioned purification by MACS by FACS is shown in FIG.

(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 with Luna-stem. The cells were suspended and seeded at 1 × 10 ⁴ viable nucleated cells / 0.25 mL EGM-2 medium / cm ₂ . A portion of the cell suspension of the CD45- / CD31 + cell population was FACS as Table 9 Condition B. Hereinafter, the condition AH corresponds to the culture schedule conditions shown in Table 9. Culturing (EGM-2 medium) (P0) was performed at 37 ° C., 5% CO _2, medium change every 2 days for 4 days or 7 days. In addition, a part of the cell suspension of SVF after hemolysis treatment was subjected to FACS as Condition A.
The cell culture schedule is shown in the table below.

In order to perform the second MACS on the 4th and 7th culture days, the following cell detachment and dispersion operations were performed on the 4th and 7th culture days. After washing the cells with HBSS (37 ° C.), an appropriate amount (3 mL TrypLE express / 25 cm ² ) of 1 × TrypLE express (37 ° C.) was added, and cell detachment was carried out at 37 ° C., 5% CO _{2 for} 5 minutes. The cell pellet was obtained by quenching with EGM-2 medium and centrifugation (300 G, 5 minutes, 4 ° C.). The number of nucleated cells was counted by Luna stem. Part of the cell suspension on day 4 of culture was subjected to condition C, and part of the cell suspension on day 7 of culture was subjected to FACS as condition F.

(3) Recovery of CD31 + Cell Population by MACS The cell suspension was centrifuged (300 G, 3 minutes, 4 ° C.). The cell pellet was gently resuspended in 60 μL of MACS buffer. To the cell suspension, 20 μL of Fc receptor (FcR) block reagent was added and vortexed for 2-3 seconds. Add 20 μL of CD31 microbeads and pipet gently. The CD31 antibody reaction was performed at 4 ° C. for 15 minutes. Add 1 mL of MACS buffer and mix by tapping. Centrifugation (300 G, 3 minutes, 4 ° C.) was performed. The cell pellet was resuspended in 0.5 mL MACS buffer and allowed to stand at 4 ° C. The Milteny MS column was set as a separator, and the column was washed once with 0.5 mL 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 CD31- cell population (rich in adipose stem cells). Fractions captured on a magnetic column and eluted in a flash after separation from the separator were collected as a CD31 + cell population (rich in vascular endothelial (progenitor) cells). The CD31 + cell population was centrifuged (300 G, 3 minutes, 4 ° C.). The cell suspension obtained by MACS of the cell suspension on day 4 of culture was subjected to FACS as a condition D, and part of the cell suspension on day 7 of the culture was subjected to condition G.

(4) Cell culture
The cells were suspended and seeded at 2500 viable nucleated cells / 0.2 mL EGM2 medium / cm ² . The culture (EGM-2 medium) (P1) was performed for 6 days at 37 ° C., 5% CO ₂ . Medium change was performed every two days. It was cultured to 80-90% confluence. Cell population obtained by culturing condition D with MACS cell suspension on the 4th day is defined as condition E, and cell population obtained by culturing condition G obtained by MACS cell suspension on the 7th day is defined as condition H in FIG. The mirror image is shown. Under condition E, purification of vascular endothelial (progenitor) cells is complete. On the other hand, under condition H, an increase in adipose stem cells could be confirmed, and vascular endothelial (progenitor) cells tended to be destroyed. Therefore, it is shown that purification of vascular endothelial (precursor) cells is completed only in the culture schedule that results in condition E.

  The analysis results by FACS at the time points of the conditions A to D and the conditions F and G and the photographs of the cells at the conditions E and H are shown in FIG.

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

Comparative example 2:
For comparison, in Example 4 above, after recovering the SVF cell population under the enzyme treatment condition B, the second MACS was not performed on the CD45 − / CD 31 + cell population recovered by the first MACS. A picture of the cells in the case is shown in FIG.

Example 5:
Example 5 is a cell culture and passaging step to proliferate and obtain large amounts of purified EC cells.

Example 4 Condition E cells were exfoliated and dispersed as described above. The cells were suspended and seeded at 2500 viable nucleated cells / 0.2 mL EGM2 medium / cm ₂ . The culture (EGM-2 medium) (P2) was performed for 5-6 days at 37 ° C., 5% CO ₂ . Medium change was performed every two days. It was cultured to 80-90% confluence.

The cells were detached and dispersed as described above. The cells were suspended and seeded at 2500 viable nucleated cells / 0.2 mL EGM2 medium / cm ₂ . Culture (EGM-2 medium) (P3) was performed for 5-6 days at 37 ° C., 5% CO ₂ . Medium change was performed every two days. It was cultured to 80-90% confluence.

  A photograph of the cells for the enzyme treatment condition B thus obtained is shown in FIG. 8A.

(result)
From the above results, it was found that it is preferable to recover the CD45- / CD31 + cell population by MACS and multiply the second MACS in culture for 4 days immediately after SVF recovery after hemolytic treatment.

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. Further, it was moved to a gas phase liquid nitrogen tank and stored at cryogenic temperature. The cells stored here are warmed in a 37 ° C. water bath for 2 minutes immediately after removal from the cryopreservation container, and 2500 viable nucleated cells / 0.2 mL EGM2 medium / cm in prewarmed EGM-2 medium _The cells were suspended at ₂ and seeded. The culture (EGM-2 medium) (P4) was performed for 5-6 days at 37 ° C., 5% CO ₂ . Medium change was performed every two days. It was cultured to 80-90% confluence. The cells were exfoliated 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 / cm ₂ . The culture (EGM-2 medium) (P5) was performed for 5-6 days at 37 ° C., 5% CO ₂ . Medium change was performed every two days. It was cultured to 80-90% confluence. The above steps of freezing, storage and sleep of the purified vascular endothelial (progenitor) cells are shown in FIG. 8B.

The cells were thawed and suspended and seeded at 2500 viable nucleated cells / 0.2 mL EGM2 medium / cm ₂ . Culture (EGM-2 medium) (P4) was performed for 5-6 days at 37 ° C., 5% CO ₂ . Medium change was performed every two days. By the above, vascular endothelial (progenitor) cells usable for various applications were obtained.

The results of cell property analysis after cryopreservation are shown in FIG. 9 and FIG.
As one of the characteristic properties of vascular endothelial (progenitor) cells, it is known that culture on Matrigel forms a tubular network structure. This is a commonly used method for cell characterization of umbilical vein endothelial (progenitor) cells and the like. Therefore, it was confirmed by network formation assay whether adipose tissue-derived vascular endothelial (progenitor) cells (P5) form a network structure. As a comparison condition, a fatty stem cell that has been reported to form a weak network structure with umbilical vein endothelial (precursor) cells (HUVEC) forming a network structure was set. Adipose stem cells were recovered as SVF, and cells obtained by subculturing in DMEM / F12 / penicillin streptomycin / 10% inactivated FBS medium were used.

The method of network formation assay is shown below.
The Matrigel was thawed at 4 ° C. overnight. Matrigel was added to a 96-well plate cooled on ice so as to be 50 mL / well. The Matrigel was gelled by standing for 30 minutes or more in an incubator at 37 ° C. and 5% CO ₂ . 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. Prepare cell suspension of fat-derived vascular endothelial (precursor) cells and umbilical vein vascular endothelial (precursor) cells at 5 × 10 ³ viable cells / 50 mL EGM-2 / well, add on Matrigel, and did. A cell suspension was prepared so that the adipose stem cells (P2) were 5 × 10 ³ viable cells / 50 mL DMEM / F12 / penicillin streptomycin / 10% inactivated FBS medium / well, and added onto Matrigel to obtain P3. . A photograph taken with an inverted phase contrast microscope after 6 hours of culture in a 37 ° C., 5% CO ₂ incubator is shown in FIG.

(result)
Adipose tissue-derived vascular endothelial (precursor) cells (P5) formed a tubular network structure equivalent to umbilical vein vascular endothelial (progenitor) cells.

The procedure of cytofluorescent immunostaining is shown below.
Preparation of PBS: It was prepared to be 137 mM NaCl / 2.7 mM KCl / 10 mM Na ₂ HPO ₄ /1.8 mM KH ₂ PO ₄ / MiliQ water. It was prepared to be 10 mM glycine / PBS. It was prepared to be 0.1% Triton X-100 / PBS. 3% BSA / PBS, 1% BSA / PBS, and 0.1% BSA / PBS were prepared. A primary antibody reaction solution was prepared by diluting CD31 antibody as a sample group and Normal Mouse IgG antibody as a control group at a concentration of 10 μg / mL in 1% BSA / PBS. Alexa-488 goat anti-mouse IgG1 antibody was diluted 1000 times with% BSA / PBS to prepare a secondary antibody reaction solution. DAPI was diluted 2000-fold with PBS to prepare a nuclear staining reaction solution.

Example 7
After dispersing the vascular endothelial (progenitor) cells (P4) and umbilical vein endothelial cells (P4) in the same manner as in Example 6, the cells were suspended in 2500 viable nucleated cells / 0.2 mL EGM2 medium / cm ₂ in a 4-well chamber slide. It became cloudy and sowed. The culture (EGM-2 medium) (P5) was performed for 4 days at 37 ° C., 5% CO ₂ . Medium change was performed every two days. The medium was removed and cells were fixed with 0.4 mL / well of 4% paraformaldehyde phosphate buffer for 10 minutes at room temperature. The cells were washed 3 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 permeabilized with 0.1% Triton X-100 / PBS 0.4 mL / well for 5 minutes at room temperature. Washed 3 times with PBS 1 mL / well for 5 minutes at room temperature. The PBS was removed and blocked with 3% BSA / PBS 0.4 mL / well for 10 minutes at room temperature. The 3% BSA / PBS was removed and washed with 1 mL PBS / well for 5 minutes at room temperature. A sample group of the primary antibody reaction solution and a control group were added at 0.25 mL / well, and incubated at 4 ° C. overnight in the dark, protected from light. The cells were washed three times for 5 minutes with 0.1% BSA / PBS 1 mL / well at room temperature. The secondary antibody reaction solution was added at 0.25 mL / well, and incubated for 20 minutes at room temperature, moist, and protected from light. The cells were washed three times for 5 minutes with 0.1% BSA / PBS 1 mL / well at room temperature. The nuclear staining reaction solution was added at 0.25 mL / well, and incubated for 5 minutes at room temperature, moist, and protected from light. The plate was washed once with PBS 1 mL / well for 5 minutes at room temperature. The VECTA shield was sealed and the mounting medium was allowed to solidify at room temperature for 24 hours. Images were taken with a confocal microscope. The results of imaging are shown in FIG.

(result)
Cell characterization by cytofluorescent immunostaining. The cellular morphology of vascular subendothelial (progenitor) cells (P5) subjected to repeated passage was confirmed by nuclear staining with CD31, which is a vascular endothelial (precursor) cell marker.

Example 8 Culture Method for Purifying Vascular Endothelial Progenitor Cells Derived from Human Adipose Tissue (New Method)
Twenty-five to two hundred grams of aspirated and crushed subcutaneous adipose tissue was collected.
Enzyme treatment (enzyme composition 1: 0.2% collagenase + 1000 U / mL DNase 1 + 3 mM CaCl ₂ + HBSS), and centrifugation from adipose tissue to interstitial vascular cell group (SVF, vascular endothelial progenitor cells, other cells such as adipose stem cells) (A mixture that also contains At this time, SVF obtains 2 to 10 × 10 ⁵ nucleated cells per 1 g of fat.
The cells were seeded in a culture plastic container (culture flask etc.). The seeding density is 5 × 10 ⁴ nucleated cells / cm ² (5 × 10 ^{3 to} 5 × 10 ⁵ nucleated cells / cm ² ), and the culture medium is 0.2 mL / cm ² (0.02 to 20 mL / cm ² ) A culture medium for vascular endothelial cell culture (eg, EGM-2 or EGM2MV, Lonza) was used.
SVF was cultured in the vascular endothelial cell culture medium for 2 days (1.0 to 5 days may be sufficient).

  Culture SVF was dispersed from the culture vessel using a cell dispersion to obtain a cell suspension of the SVF cell population. From the SVF cell population, a cell population containing CD31 positive cells (CD31 positive cell population) was obtained by sorting CD31 positive cells by MACS. At this time, the MACS operation can further increase the purity by repeating selection with CD31 microbeads twice (CD31 + / CD31 + cells are recovered).

The CD31 positive cell population was seeded in a plastic container for culture (such as a culture flask). The seeding density is 5 × 10 ⁴ nucleated cells / cm ² (5 × 10 ^{3 to} 5 × 10 ⁵ nucleated cells / cm ² ), and the culture medium is 0.2 mL / cm ² (0.02 to 20 mL / cm ^2). Medium for endothelial cell culture (eg, EGM-2 or EGM2MV, Lonza) was used. The CD31 positive cell population was cultured in the vascular endothelial cell culture medium for 3 days (one hour to 7 days may be sufficient).

  The cultured CD31 positive cell population was dispersed from the culture vessel using a cell dispersion to obtain a cell suspension of the CD31 positive cell population. By further sorting CD31 positive cells by MACS, a cell population (high purity CD31 positive cell population) containing CD31 positive cells with high efficiency was obtained. At this time, the MACS operation can further increase the purity by repeating the selection with CD31 microbeads twice. At this time, a cell population in which the ratio of CD45 negative / CD31 positive / CD146 positive cells is 90% or more by flow cytometry analysis was defined as a high purity CD31 positive cell population (blood vessel endothelial precursor cells).

For amplification, high purity CD31 positive cell populations were seeded in culture plastic containers (such as culture flasks). The seeding density is 5 × 10 ⁴ nucleated cells / cm ² (5 × 10 ^{3 to} 5 × 10 ⁵ nucleated cells / cm ² ), and the culture medium is 0.2 mL / cm ² (0.02 to 20 mL / cm ² ) A culture medium for vascular endothelial cell culture (eg, EGM-2 or EGM2MV, Lonza) was used. The cells were cultured for 5 days (only 2 days to 10 days), and the high purity CD31 positive cell population was passaged. The seeding density is 2.5 × 10 ³ nucleated cells / cm ² (2.5 × 10 ^{2 to} 2.5 × 10 ⁵ nucleated cells / cm ² ), and the culture medium is 0.2 mL / cm ² (0.02 to 20 mL / cm ^2). Medium for endothelial cell culture (eg, EGM-2 or EGM2MV, Lonza) was used.
Thus, the purified adipose tissue-derived vascular endothelial (progenitor) cells were obtained (FIG. 11).

Claims (13)

A cell population comprising vascular endothelial (precursor) cells and fat stem cells, wherein the percentage of CD45 negative and CD31 positive vascular endothelial (precursor) cells in the cell population is 92% or more, and fat stem cells in the cell population A cell population whose proportion is 8% or less. (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 sorting out 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 of (2) for 1 hour to 7.0 days; and (4) CD31 positive from the cell population obtained in the step of (3) above Obtaining a cell population containing CD31 positive cells by sorting out the cells of
A method of producing a cell population comprising vascular endothelial (progenitor) cells, comprising 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). 3. The method according to claim 2, wherein the step (3) is a step of culturing the cell population containing the CD45 negative and CD31 positive cells obtained in the step (2) for 2.0 to 6.0 days. the method of. The step of culturing the cell population obtained in the step (1) for 1.0 hour to 5.0 days, between the step (1) and the step (2). The method described in. A cell population comprising vascular endothelial (precursor) cells is a cell population comprising vascular endothelial (precursor) cells and adipose stem cells, wherein the percentage of vascular endothelial (precursor) cells in the cell population is 92% or more, The method according to any one of claims 2 to 4, which is a cell population in which the percentage of adipose stem cells in the cell population is 8% or less. The method according to any one of claims 2 to 5, wherein in the step (1), the enzyme is collagenase. The method according to claim 6, wherein the collagenase concentration in the enzyme treatment in the step (1) is 0.02% to 0.5%. The method according to claim 6 or 7, wherein the enzyme treatment solution used for the enzyme treatment in the step (1) further comprises DNase I. The method according to any one of claims 6 to 8, wherein the enzyme treatment solution used for the enzyme treatment in the step (1) does not contain poloxamer and pronase. The method according to any one of claims 2 to 9, wherein in the step (2), CD45 negative and CD31 positive cells are sorted using magnetic beads labeled with anti-CD45 antibody and magnetic beads labeled with anti-CD31 antibody. The method according to one item. The method according to any one of claims 2 to 10, wherein in the step (4), CD31 positive cells are sorted using magnetic beads labeled with an anti-CD31 antibody. The method according to any one of claims 2 to 11, further comprising the step of culturing a cell population comprising CD31 positive cells sorted in the step (4). A pharmaceutical composition comprising the cell population according to claim 1 or the cell population produced by the method according to any one of claims 2 to 12.