JP5035737B2 - Cell preparation for treating prostate cancer containing adipose tissue-derived mesenchymal stem cells - Google Patents

Description

  The present invention relates to cell preparations. Specifically, the present invention relates to a cell preparation effective for treating prostate cancer. This application claims the priority based on the Japan patent application 2009-277437 for which it applied on December 7, 2009, The whole content of the said patent application is used by reference.

  Attempts have been made worldwide to reconstruct damaged tissues using pluripotent stem cells that can differentiate into various cells. For example, mesenchymal stem cells (MSCs), which are one of pluripotent stem cells, have the ability to differentiate into various cells such as bone cells, chondrocytes and cardiomyocytes, and their clinical application is attracting attention. On the other hand, some research groups have reported that adipose tissue is promising as a source of pluripotent stem cells (Non-patent Document 1). Kitagawa et al. Reported that it is possible to prepare a large number of cell populations showing pluripotency from adipose tissue by a simple operation, and that the obtained cells have the ability to differentiate into adipose tissue. It has been shown to be effective for the reconstruction of adipose tissue (Patent Document 1). In our previous patent application, our research group also disclosed Adipose-derived stem cells (ASC, Adipose) for ischemic diseases, renal dysfunction, wounds, urinary incontinence and osteoporosis. -derived regeneration cells: ADRC, Adipose-derived mesenchymal stem cells: called AT-MSC, AD-MSC, etc.) were reported to be effective (Patent Document 2).

International Publication No. 2006/006692 Pamphlet International Publication No. 2008/018450 Pamphlet

Secretion of Angiogenic and Antiapoptotic Factors by Human Adipose Stromal Cells. Circulation 109: 1292-1298, 2004 Tokuda Y, Satoh Y, Fujiyama C, Toda S, Sugihara H, Masaki Z. Prostate cancer cell growth is modulated by adipocyte-cancer cell interaction.BJU Int. 2003 May; 91 (7): 716-20. Micke P, Ostman A. Exploring the tumour environment: cancer-associated fibroblasts as targets in cancer therapy. Expert Opin Ther Targets. 2005 Dec; 9 (6): 1217-33.

  An object of the present invention is to provide a new medical use of adipose tissue-derived mesenchymal stem cells (ASC).

  While studying for clinical application of ASC, we experienced radical prostatectomy for prostate cancer, and transplanted ASC around the paraurethral in patients with intractable cases who had sustained stress urinary incontinence for more than 1 year after surgery However, an interesting phenomenon was observed in which the blood PSA concentration, which is a sensitive marker for prostate cancer, decreased. Focusing on this phenomenon, we hypothesized that "ASC has an antitumor effect and is effective in treating prostate cancer" and decided to conduct various experiments. First, as a preliminary experiment, PSA producing ability of PC-3 cells, DU145 cells, and LNCaP cells was compared in order to select cell lines having high PSA producing ability from currently available prostate cancer cells. Since LNCaP cells showed high PSA production ability, the cells were used in the subsequent experiments. Next, LNCaP cells were cultured in a medium supplemented with ASC culture supernatant under the expectation that the humoral component secreted by ASC exhibits antitumor activity, and changes in PSA production were examined. As a result, contrary to expectations, there was no change in the amount of PSA produced by the addition of the ASC culture supernatant. That is, the involvement of humoral components secreted by ASC was denied. Therefore, we decided to investigate the effect of co-culturing LNCaP cells and ASC. As a result of the experiment, surprisingly, PSA production was suppressed by co-culture with ASC. This suggests that ASC suppresses the growth of LNCaP cells or decreases the activity by direct action, that is, cell-to-cell interaction. As a result of further studies, it was confirmed that the same effect was not observed against colorectal cancer cell lines, and that the antitumor action of ASC was highly cancer-specific.

  As described above, the present inventors have found that ASC is effective in suppressing the proliferation of prostate cancer cells and / or reducing the activity, and have shown that ASC can be applied to the treatment of prostate cancer. In addition, a part of the mechanism of action was clarified. When mature adipocytes and prostate cancer cells are co-cultured, the proliferation of cancer cells is promoted (Non-patent Document 2), and the proliferation of cancer cells is suppressed by stroma fibroblasts (Non-patent Document 3). Has been reported. The above findings clarified after the study by the present inventors are not predictable from these reports, but also indicate facts that may be contradictory, and their academic and clinical significance is extremely great. .

The present invention described below is mainly based on the above results or knowledge.
[1] A cell preparation for treating prostate cancer, comprising adipose tissue-derived mesenchymal stem cells.
[2] The cell preparation according to [1], wherein the adipose tissue-derived mesenchymal stem cell is positive for a cell surface marker CD44.
[3] The adipose tissue-derived mesenchymal stem cell is
(1) Adherent cells or passage cells thereof included in a sedimented cell population that settles when a cell population separated from adipose tissue is centrifuged.
(2) cells grown when the precipitated cell population is cultured under low serum conditions;
(3) cells that proliferate when the cell population isolated from adipose tissue is cultured under low serum conditions;
(4) A set of precipitated cells collected as a precipitate by subjecting the adipose tissue to protease treatment, and then subjecting the filtrate to centrifugation, or (5) Filtration treatment after treating the adipose tissue with protease. Sedimented cell population recovered as sediment by centrifugation without
The cell preparation according to [1].
[4] The cell preparation according to [3], wherein the low serum condition is a condition where the serum concentration in the culture solution is 5% (V / V) or less.
[5] The cell preparation according to any one of [1] to [4], wherein the adipose tissue is human adipose tissue.
[6] The cell preparation according to any one of [1] to [5], further comprising a substance that promotes contact between cells.
[7] Use of adipose tissue-derived mesenchymal stem cells for producing a cell preparation for treating prostate cancer.
[8] A method for treating prostate cancer, comprising a step of administering the cell preparation according to any one of [1] to [6] to a patient with prostate cancer.
[9] The treatment method according to [8], wherein the cell preparation is administered to or around a lesion site of prostate cancer.
[10] A method for treating prostate cancer, comprising administering a therapeutically effective amount of adipose tissue-derived mesenchymal stem cells to a patient with prostate cancer.
[11] The method according to [10], wherein the adipose tissue-derived mesenchymal stem cells are administered to or around a lesion site of prostate cancer.

Results of measuring surface markers of stem cells (ASC) prepared from adipose tissue. It turns out that it is CD29 and CD44 positive. Results of culturing stem cells (ASC) prepared from adipose tissue. Differentiation was induced by culturing in a fat differentiation medium. Results of immunostaining of commercially available adipose tissue-derived stem cells (ASC). a: Immunostained image using anti-CD44 antibody (red fluorescence observed in original image), b: Hoechst 33342 stained image (blue fluorescence observed in original image), c: synthesis of a and b, d : Phase contrast microscope image, synthesis of e: a, b, d. All cells in the visual field are stained with anti-CD44 antibody. Magnification (× 40) Immunostaining results of stem cells (ASC) prepared from patient adipose tissue. a: Immunostained image using anti-CD44 antibody (red fluorescence observed in original image), b: Hoechst 33342 stained image (blue fluorescence observed in original image), c: synthesis of a and b, d : Phase contrast microscope image, synthesis of e: a, b, d. All cells in the visual field are stained with anti-CD44 antibody. Magnification (× 40) Antitumor action of ASC culture supernatant. LNCaP cells were cultured for 20 hours in a medium supplemented with ASC culture supernatant and examined for changes in PSA production. No antitumor activity was observed in the culture supernatant of ASC. Lane 1 shows PSA concentration (ng / ml) when cultured in medium supplemented with 10% (v / v) inactivated FCS (Fetalcalf serum), and Lane 2 shows inactivated FCS (10% (v / v)) and PSA concentration (ng / ml) when cultured in medium supplemented with ASC culture supernatant, Lane 3 is medium supplemented with 20% (v / v) KSR (Knockout serum replacement) PSA concentration (ng / ml) when cultured (control), lane 4 is PSA concentration (ng / ml) when cultured in a medium supplemented with KSR (20% (v / v)) and ASC culture supernatant . PSA was detected by EIA method. PSA: prostate specific antigen. Anti-tumor effect by co-culture of ASC. The PSA concentration when ASC and LNCaP cells were co-cultured (48 hours) was compared with the PSA concentration when LNCaP cells were cultured alone. From left to right, PSA concentration when only ASC is cultured, PSA concentration when only LNCaP cells are cultured, PSA concentration when LNCaP cells and ASC are co-cultured (cell number ratio 1: 1), and LNCaP cells PSA concentration when ASC is co-cultured (cell number ratio 1: 2), LNCaP cells and ASC are co-cultured (cell ratio 2: 1. The number of LNCaP cells is twice that of the former two) ) Is the PSA concentration. The culture was performed using 10% (v / v) inactivated FCS-containing knockout DMEM (KODMEM, Invitrogen). The PSA was detected by the EIA method. PSA: prostate specific antigen. N.D .: Not detected. Anti-tumor effect by co-culture of ASC. The state of the cells when ASC and LNCaP cells were co-cultured (48 hours) was observed with a phase contrast microscope. a: Phase contrast microscopic image when only ASC is cultured, b: Phase contrast microscopic image when only LNCaP cells are cultured, c, d: LNCaP cells and ASC were co-cultured (cell ratio 1: 1) Phase contrast microscopic image, e, f: Phase contrast microscopic image when LNCaP cells and ASC are co-cultured (cell number ratio 1: 2), g, h: Co-culture of LNCaP cells and ASC (cell number (2: 1 ratio) Phase contrast microscope image when the number of LNCaP cells is twice that of the former. The culture was performed using 10% (v / v) inactivated FCS-containing knockout DMEM (KODMEM, Invitrogen). Changes in tumor markers by co-culture with ASC. Whether colorectal cancer cell lines (LoVo cells and LS180 cells) and prostate cancer cell lines (LNCaP cells) were co-cultured with ASC was examined to determine whether the tumor marker value was changed. a: Experimental results of LoVo cells (48 hours culture). Lane 1 is when ASC alone is cultured (cell count 1.8 × 10 ⁵ ), Lane 2 is when only LoVo cells are cultured (cell count 1.8 × 10 ⁵ ), and Lane 3 is co-cultured with LoVo cells and ASC (cell count) When the number of cells is 1.8 × 10 ⁵ ), lane 4 is co-cultured with LoVo cells and ASC (ratio of cell number is 1: 2. LoVo cell number is 1.8 × 10 ⁵ ). 5 shows a case where only LoVo cells are cultured (cell number 3.6 × 10 ⁵ ), and lane 6 shows a co-culture of LoVo cells and ASC (ratio of cell number 2: 1; LoVo cell number 3.6 × 10 ⁵ ). b: Experimental results of LoVo cells (96 hours culture). The conditions for each lane are the same as a. c: Experimental results of LS180 cells (cultured for 48 hours). Lane 1 shows the case where only ASC is cultured (number of cells: 1.8 × 10 ⁵ ), Lane 2 shows the case where only LS180 cells are cultured (number of cells: 1.8 × 10 ⁵ ), and Lane 3 shows co-culture of LS180 cells and ASC (number of cells) When the number of cells is 1.8 × 10 ⁵ ), lane 4 is when LS180 cells and ASC are co-cultured (cell number ratio is 1: 2, the number of LS180 cells is 1.8 × 10 ⁵ ). 5 shows a case where only LS180 cells were cultured (cell number 3.6 × 10 ⁵ ), and lane 6 shows a co-culture of LS180 cells and ASC (ratio of cell numbers 2: 1, LS180 cell number 3.6 × 10 ⁵ ). d: Experimental results of LS180 cells (96 hours culture). The conditions for each lane are the same as for c. e: Experimental results of LNCaP cells (cultured for 48 hours). Lane 1 is when only ASC is cultured (cell count 1.8 × 10 ⁵ ), Lane 2 is when only LNCaP cells are cultured (cell count 1.8 × 10 ⁵ ), and Lane 3 is co-cultured with LNCaP cells and ASC (cell count) The ratio of each cell number was 1.8 × 10 ⁵ ), lane 4 was cultured with LNCaP cells only (cell number 3.6 × 10 ⁵ ), and lane 5 was co-cultured with LNCaP cells and ASC (cell number The ratio is 2: 1. The number of LNCaP cells is 3.6 × 10 ⁵ ). f: LNCaP cell experimental results (96 hours culture). The conditions for each lane are the same as e. CEA: Carcinoembryonic antigen. PSA: prostate specific antigen. ND: Not detected. Changes in tumor markers by co-culture with ASC. Whether or not the tumor marker value was changed by co-culturing a colon cancer cell line (LoVo cell) with ASC (commercially available ASC (Invitrogen) and patient-derived ASC) was examined. a: Experimental results of commercially available ASC (culture for 48 hours). Lane 1 is when ASC alone is cultured (cell count 1.8 × 10 ⁵ ), Lane 2 is when only LoVo cells are cultured (cell count 1.8 × 10 ⁵ ), and Lane 3 is co-cultured with LoVo cells and ASC (cell count) The ratio is 1: 1, each cell number is 1.8 × 10 ⁵ ), lane 4 is when only LoVo cells are cultured (cell number 3.6 × 10 ⁵ ), lane 5 is co-cultured with LoVo cells and ASC (number of cells) The ratio is 2: 1. The number of LoVo cells is 3.6 × 10 ⁵ ). b: Results of commercial ASC experiment (96 hours culture). The conditions for each lane are the same as a. c: Experimental results of patient-derived ASC (cultured for 48 hours). Lane 1 is when ASC alone is cultured (cell count 1.8 × 10 ⁵ ), Lane 2 is when only LoVo cells are cultured (cell count 1.8 × 10 ⁵ ), and Lane 3 is co-cultured with LoVo cells and ASC (cell count) The ratio is 1: 1, each cell number is 1.8 × 10 ⁵ ), lane 4 is when only LoVo cells are cultured (cell number 3.6 × 10 ⁵ ), lane 5 is co-cultured with LoVo cells and ASC (number of cells) The ratio is 2: 1. The number of LoVo cells is 3.6 × 10 ⁵ ). d: Experimental results of patient-derived ASC (96 hours culture). The conditions for each lane are the same as for c. Changes in tumor markers by co-culture with ASC. It was examined whether or not the tumor marker value was changed by co-culturing a colon cancer cell line (LS180 cell) with ASC (commercially available ASC (Invitrogen) and patient-derived ASC). a: Experimental results of commercially available ASC (culture for 48 hours). Lane 1 shows the case where only ASC is cultured (number of cells: 1.8 × 10 ⁵ ), Lane 2 shows the case where only LS180 cells are cultured (number of cells: 1.8 × 10 ⁵ ), and Lane 3 shows co-culture of LS180 cells and ASC (number of cells) The ratio is 1: 1, each cell number is 1.8 × 10 ⁵ ), lane 4 is when only LS180 cells are cultured (cell number 3.6 × 10 ⁵ ), lane 5 is LS180 cells and ASC co-cultured (cell number The ratio is 2: 1. The number of LS180 cells is 3.6 × 10 ⁵ ). b: Results of commercial ASC experiment (96 hours culture). The conditions for each lane are the same as a. c: Experimental results of patient-derived ASC (cultured for 48 hours). Lane 1 shows the case where only ASC is cultured (number of cells: 1.8 × 10 ⁵ ), Lane 2 shows the case where only LS180 cells are cultured (number of cells: 1.8 × 10 ⁵ ), and Lane 3 shows co-culture of LS180 cells and ASC (number of cells) The ratio is 1: 1, each cell number is 1.8 × 10 ⁵ ), lane 4 is when only LS180 cells are cultured (cell number 3.6 × 10 ⁵ ), lane 5 is LS180 cells and ASC co-cultured (cell number The ratio is 2: 1. The number of LS180 cells is 3.6 × 10 ⁵ ). d: Experimental results of patient-derived ASC (96 hours culture). The conditions for each lane are the same as for c. Changes in tumor markers by co-culture with ASC. It was examined whether or not the tumor marker value was changed by co-culturing prostate cancer cell lines (LNCaP cells) with ASC (commercially available ASC (Invitrogen) and patient-derived ASC). a: Experimental results of commercially available ASC (culture for 48 hours). Lane 1 is when only ASC is cultured (cell count 1.8 × 10 ⁵ ), Lane 2 is when only LNCaP cells are cultured (cell count 1.8 × 10 ⁵ ), and Lane 3 is co-cultured with LNCaP cells and ASC (cell count) The ratio of each cell number was 1.8 × 10 ⁵ ), lane 4 was cultured with LNCaP cells only (cell number 3.6 × 10 ⁵ ), and lane 5 was co-cultured with LNCaP cells and ASC (cell number The ratio is 2: 1. The number of LNCaP cells is 3.6 × 10 ⁵ ). b: Results of commercial ASC experiment (96 hours culture). The conditions for each lane are the same as a. c: Experimental results of patient-derived ASC (cultured for 48 hours). Lane 1 is when only ASC is cultured (cell count 1.8 × 10 ⁵ ), Lane 2 is when only LNCaP cells are cultured (cell count 1.8 × 10 ⁵ ), and Lane 3 is co-cultured with LNCaP cells and ASC (cell count) The ratio of each cell number was 1.8 × 10 ⁵ ), lane 4 was cultured with LNCaP cells only (cell number 3.6 × 10 ⁵ ), and lane 5 was co-cultured with LNCaP cells and ASC (cell number The ratio is 2: 1. The number of LNCaP cells is 3.6 × 10 ⁵ ). d: Experimental results of patient-derived ASC (96 hours culture). The conditions for each lane are the same as for c. Results of transplantation experiment using nude rats. The tumor size after transplantation was compared between the LNCaP single transplant group (left bar) and the LNCaP and ASC transplant group (right bar). n = 6

  The present invention relates to cell preparations applied to specific diseases and uses thereof. The cell preparation of the present invention contains adipose tissue-derived mesenchymal stem cells (sometimes abbreviated as “ASC” in the present specification). In the present invention, “adipose tissue-derived mesenchymal stem cell (ASC)” refers to a somatic stem cell contained in an adipose tissue. As long as pluripotency is maintained, the somatic stem cell culture ( Cells obtained by subculture) also correspond to “adipose tissue-derived mesenchymal stem cells (ASC)”. Usually, ASC is prepared in an “isolated state” as a cell constituting a cell population (including cells other than ASC derived from adipose tissue) using adipose tissue separated from a living body as a starting material. The “isolated state” as used herein means a state extracted from its original environment (that is, a state constituting a part of a living body), that is, a state different from the original existence state by an artificial operation. Means that The adipose tissue-derived mesenchymal stem cells are also referred to as ADRC (Adipose-derived regeneration cells), AT-MSC (Adipose-derived mesenchymal stem cells), AD-MSC (Adipose-derived mesenchymal stem cells), and the like. In the present specification, the following terms, ie, adipose tissue-derived mesenchymal stem cells, ASC, ADRC, AT-MSC, and AD-MSC are used interchangeably.

(ASC preparation method)
ASC is prepared through steps such as separation, washing, concentration, and culture of stem cells from adipose matrix. A method for preparing ASC is not particularly limited. For example, a known method (Fraser JK et al. (2006), Fat tissue: an underappreciated source of stem cells for biotechnology. Trends in Biotechnology; Apr; 24 (4): 150-4. Epub 2006 Feb 20. Review .; Zuk PA et al. (2002), Human adipose tissue is a source of multipotent stem cells.Molecular Biology of the Cell; Dec; 13 (12): 4279-95 .; Zuk PA et al. (2001), Multilineage cells from human Adipose tissue: implications for cell-based therapies. Tissue Engineering; Apr; 7 (2): 211-28. In addition, devices for preparing ASC from adipose tissue (for example, Celution (registered trademark) device (Cytori Therapeutics, Inc., San Diego, USA)) are also commercially available, and ASC is prepared using the device. You may decide. By using this apparatus, a cell population containing ASC can be separated from adipose tissue (K. Lin. Et al. Cytotherapy (2008) Vol. 10, No. 4, 417-426). Moreover, as shown in the below-mentioned Example, the present inventors performed the characterization of the cell isolate | separated with the said apparatus. Hereinafter, specific examples of the preparation method of ASC are shown.

(1) Preparation of cell population from adipose tissue Adipose tissue is collected from animals by means such as excision and suction. The term “animal” herein includes humans and non-human mammals (pet animals, domestic animals, laboratory animals. Specifically, for example, mice, rats, guinea pigs, hamsters, monkeys, cows, pigs, goats, sheep, Dogs, cats, etc.). In order to avoid the problem of immune rejection, it is preferable to collect adipose tissue (self-adipose tissue) from the same individual as the subject (patient) to which the cell preparation of the present invention is applied. However, this does not preclude the use of adipose tissue of the same species (other family) or adipose tissue of different species.

  Examples of adipose tissue include subcutaneous fat, visceral fat, intramuscular fat, and intermuscular fat. Among these, subcutaneous fat can be collected very easily under local anesthesia, so that the burden on the patient at the time of collection is small and it can be said that it is a preferable cell source. Usually, one type of adipose tissue is used, but two or more types of adipose tissue can be used in combination. In addition, adipose tissue collected in multiple times (not necessarily the same type of adipose tissue) may be mixed and used for subsequent operations. The amount of adipose tissue collected can be determined in consideration of the type of donor, the type of tissue, or the amount of ASC required, for example, about 0.5 to 500 g. When humans are used as donors, the amount collected at a time is preferably about 10 to 20 g or less in consideration of the burden on the donor. The collected adipose tissue is subjected to the following enzyme treatment after removal of blood components adhering to it and fragmentation as necessary. The blood component can be removed by washing the adipose tissue in an appropriate buffer or culture solution.

  Enzymatic treatment is performed by digesting adipose tissue with enzymes such as collagenase, trypsin, dispase and the like. Such enzyme treatment may be performed by techniques and conditions known to those skilled in the art (see, for example, RI Freshney, Culture of Animal Cells: A Manual of Basic Technique, 4th Edition, A John Wiley & Sones Inc., Publication). . Preferably, the enzyme treatment here is performed according to the methods and conditions described in the Examples described later. The cell population obtained by the above enzyme treatment includes pluripotent stem cells, endothelial cells, stromal cells, blood cells, and / or precursor cells thereof. The type and ratio of the cells constituting the cell population depend on the origin and type of the adipose tissue used.

(2) Acquisition of sedimented cell population (SVF fraction: stroma vascular fractions) The cell population is subsequently subjected to centrifugation. The sediment by centrifugation is collected as a sedimented cell population (also referred to herein as “SVF fraction”). The conditions for the centrifugation process vary depending on the type and amount of cells, but are, for example, 1 to 10 minutes and 800 to 1500 rpm. Prior to centrifugation, the cell population after the enzyme treatment is preferably subjected to filtration or the like, and the enzyme undigested tissue contained therein is preferably removed.

  The “SVF fraction” obtained here contains ASC. Therefore, the cell preparation of the present invention can be prepared using the SVF fraction. That is, in one embodiment of the cell preparation of the present invention, the SVF fraction is contained. The type and ratio of cells constituting the SVF fraction depend on the origin and type of the adipose tissue used, the conditions for enzyme treatment, and the like. In addition, the characteristics of the SVF fraction are shown in the pamphlet of International Publication No. 2006 / 006692A1.

(3) Selective culture of adherent cells (ASC) and cell recovery
In addition to ASC, the SVF fraction contains other cell components (endothelial cells, stromal cells, blood cells, progenitor cells thereof, etc.). Therefore, in one embodiment of the present invention, the following selective culture is performed to remove unnecessary cell components from the SVF fraction. The resulting cells are used as ASC in the cell preparation of the present invention.

  First, the SVF fraction is suspended in an appropriate medium, seeded on a culture dish, and cultured overnight. Suspension cells (non-adherent cells) are removed by medium exchange. Thereafter, the culture is continued while appropriately changing the medium (for example, once every 2 to 4 days). Subculture as necessary. The number of passages is not particularly limited, but from the viewpoint of maintaining pluripotency and proliferation ability, it is not preferable to repeat the passages excessively (preferably to be kept to about 5 passages). As the culture medium, a normal animal cell culture medium can be used. For example, Dulbecco's modified Eagle's Medium (DMEM) (Nissui Pharmaceutical Co., Ltd.), α-MEM (Dainippon Pharmaceutical Co., Ltd.), DMEM: Ham's F12 mixed medium (1: 1) (Dainippon Pharmaceutical Co., Ltd.), Ham's F12 medium (Dainippon Pharmaceutical Co., Ltd.), MCDB201 medium (Functional Peptide Research Institute), etc. can be used. A medium supplemented with serum (fetal calf serum, human serum, sheep serum, etc.) or a serum substitute (Knockout serum replacement (KSR), etc.) may be used. The addition amount of serum or serum replacement can be set, for example, within a range of 5% (v / v) to 30% (v / v).

  By the above operation, adherent cells selectively survive and proliferate. Subsequently, the proliferated cells are collected. The collection operation may be carried out in accordance with a conventional method. For example, the cells after enzyme treatment (trypsin or dispase treatment) can be easily collected by detaching them with a cell scraper or pipette. In addition, when sheet culture is performed using a commercially available temperature-sensitive culture dish or the like, it is also possible to recover the cells as they are without performing enzyme treatment. By using the cells (ASC) collected in this manner, a cell preparation containing ASC with high purity can be prepared.

(4) Low-serum culture (selective culture in low-serum medium) and cell recovery In one embodiment of the present invention, the following low-serum culture is performed instead of or after the operation of (3) above. I do. The resulting cells are used as ASC in the cell preparation of the present invention.

  In low serum culture, the SVF fraction (when using this step after (3), the cells collected in (3) are used) are cultured under low serum conditions, and the target pluripotent stem cell (ie ASC ) Selectively. Since a small amount of serum is used in the low serum culture method, it is possible to use the serum of the subject (patient) to whom the cell preparation of the present invention is administered. That is, culture using autoserum becomes possible. By using autologous serum, a cell preparation is provided that is capable of excluding foreign animal material from the manufacturing process, and is expected to have high safety and high therapeutic effect. Here, “under low serum conditions” is a condition containing 5% or less of serum in the medium. The cells are preferably cultured in a culture solution containing 2% (V / V) or less of serum. More preferably, the cells are cultured in a culture solution containing 2% (V / V) or less of serum and 1 to 100 ng / ml of fibroblast growth factor-2 (bFGF).

  Serum is not limited to fetal bovine serum, and human serum, sheep serum and the like can be used. Preferably, human serum, more preferably, serum of a subject to which the cell preparation of the present invention is applied (that is, autoserum) is used.

  As a medium, a normal medium for animal cell culture can be used on the condition that the amount of serum contained in use is low. For example, Dulbecco's modified Eagle's Medium (DMEM) (Nissui Pharmaceutical Co., Ltd.), α-MEM (Dainippon Pharmaceutical Co., Ltd.), DMEM: Ham's F12 mixed medium (1: 1) (Dainippon Pharmaceutical Co., Ltd.), Ham's F12 medium (Dainippon Pharmaceutical Co., Ltd.), MCDB201 medium (Functional Peptide Research Institute), etc. can be used.

  By culturing by the above method, pluripotent stem cells (ASC) can be selectively proliferated. In addition, since pluripotent stem cells (ASC) that proliferate under the above culture conditions have high proliferative activity, the number of cells required for the cell preparation of the present invention can be easily prepared by subculture. . In addition, International Publication No. 2006 / 006692A1 pamphlet shows the characteristics of cells that selectively proliferate by culturing the SVF fraction in low serum.

  Subsequently, the cells selectively proliferated by the low serum culture are collected. The collection operation may be performed in the same manner as in the above (3). By using the collected cells (ASC), a cell preparation containing ASC with high purity can be prepared.

(5) Formulation
The cells obtained from the SVF fraction, the cells obtained as a result of the selective culture (3), or the cells obtained as a result of the low serum culture (4) are subjected to physiological saline or an appropriate buffer (for example, a phosphate buffer). A cell preparation can be obtained by suspending in, for example. For example, 1 × 10 ⁶ to 1 × 10 ¹⁰ cells may be contained as a single dose so that a therapeutically effective amount of cells is administered. The content of the cells can be appropriately adjusted in consideration of the purpose of use, the target disease, the sex of the application target (recipient), age, weight, the state of the affected area, the state of the cells, and the like.

  Dimethyl sulfoxide (DMSO), serum albumin, etc. for the purpose of cell protection, antibiotics, etc. for the purpose of preventing bacterial contamination, and various components for the purpose of cell activation, proliferation or differentiation induction, etc. (Vitamins, cytokines, growth factors, steroids, etc.) may be contained in the cell preparation of the present invention. Examples of cytokines are interleukin (IL), interferon (IFN), colony stimulating factor (CSF), granulocyte colony stimulating factor (G-CSF) and erythropoietin (EPO), activin, oncostatin M (OSM). CSF, G-CSF, EPO, etc. are also growth factors. Examples of growth factors are hepatocyte growth factor (HGF), basic fibroblast growth factor (bFGF, FGF2), epidermal growth factor (EGF), platelet-derived growth factor (PDGF), insulin-like growth factor (IGF) Transforming growth factor (TGF), nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF). In addition, other pharmaceutically acceptable ingredients (for example, carriers, excipients, disintegrants, buffers, emulsifiers, suspensions, soothing agents, stabilizers, preservatives, preservatives, physiological saline, etc.) You may make it contain in the cell formulation of this invention.

  In the above method, the cell preparation is composed of cells grown by low serum culture of the SVF fraction, but the cell population obtained from the adipose tissue is directly (via centrifugation to obtain the SVF fraction). Alternatively, a cell preparation may be prepared using cells grown by low serum culture as ASC. That is, in one embodiment of the present invention, cells that proliferate when a cell population obtained from adipose tissue is cultured in low serum are used as ASC. In addition, a cell preparation should be constructed using the SVF fraction (containing adipose tissue-derived mesenchymal stem cells) as it is, instead of the pluripotent stem cells obtained by selective culture (above (3) and (4)). It may be. The cell preparation of this embodiment comprises (a) a precipitated cell population (SVF fraction) recovered as a sediment by subjecting adipose tissue to protease treatment, followed by filtration, and then centrifuging the filtrate, or (b) fat After the tissue is treated with protease, it contains a sedimented cell population (SVF fraction) that is collected as a sediment by centrifuging without filtration. Here, “use as it is” means to use it as an active ingredient of a cell preparation without undergoing selective culture.

(Combination of substances that promote contact between cells)
Experiments shown in the examples described below have shown that ASC exerts its effects (proliferation suppression and / or activity reduction of prostate cancer cells) by cell-to-cell interaction. Therefore, in order for the cell preparation of the present invention to exert a therapeutic effect, it is necessary and important that the active ingredient, ASC, comes into contact with cancer cells. In view of this, in one embodiment of the present invention, a substance that promotes contact between cells (hereinafter abbreviated as “cell-cell contact promoting substance”) is used in combination with ASC in order to enhance the therapeutic effect. That is, the cell preparation of this embodiment contains an intercellular contact promoting substance in addition to ASC. The cell-cell contact promoting substance may be any substance that can directly or indirectly promote contact between cells. Examples of intercellular contact promoters include cells such as extracellular matrix, collagen, atelocollagen (eg, Koken Co., Ltd.), Matrigel (trademark, BD Biosciences), fibronectin, vitronectin, laminin, cadherin, integrin, selectin, RGD peptide Mention may be made of adhesion peptides. Two or more kinds of intercellular contact promoting substances may be used in combination.

(Applicable disease)
The disease to be treated with the cell preparation of the present invention is prostate cancer. In other words, the cell preparation of the present invention is used for the treatment of prostate cancer. Therefore, the cell preparation of the present invention is usually administered to a patient with prostate cancer. However, the cell preparation of the present invention can also be used for experiments or research purposes such as confirmation and verification of the effect.

  “Prostate cancer” is a cancer that develops in the prostate gland, often accompanied by symptoms such as dysuria (difficulty urinating, frequent urination, residual urine sensation, urgency), and lower discomfort. Prostate cancer is known to easily metastasize to lymph nodes and bone. Age (elderly), race, family history of prostate cancer, etc. have been reported as risk factors for prostate cancer. There is a sensitive tumor marker called prostate specific antigen (PSA), which is used for early detection of prostate cancer. PSA values correlate with tumor volume (Matthew A. Uhlman et al .: Tumor Volume, Tumor Percentage Involvement, or Prostate Volume: Which Is Predictive of Prostate-specific Antigen Recurrence ?, The Journal of Urology, April 2009, Vol. 181, Issue 4, Page 272), an index to evaluate the growth rate of tumor cells. The stage of prostate cancer is generally classified according to the TNM classification. In the TNM classification, the stage is represented by a combination of three indicators consisting of the size and progression of the primary lesion (T), the status of metastasis to regional lymph nodes (N), and the presence or absence of distant metastasis (M). In the treatment of prostate cancer, “surgical therapy”, “radiotherapy”, “hormone therapy” and the like are adopted in consideration of the stage and age of the patient.

(Applicable)
The subject to which the cell preparation of the present invention is administered is typically a human male. However, cell preparations for mammals other than humans (including pet animals, domestic animals, laboratory animals, specifically mice, rats, guinea pigs, hamsters, monkeys, cows, pigs, goats, sheep, dogs, cats, etc.) It is also possible to configure.

(Method of administration)
The administration route of the cell preparation of the present invention is not particularly limited. For example, the cell preparation of the present invention is administered by intravenous injection, intraarterial injection, intraportal injection, intradermal injection, subcutaneous injection, intramuscular injection, or intraperitoneal injection. Preferably, the cell preparation of the present invention is administered by local injection into the affected area. That is, the cell preparation of the present invention is particularly suitable for local treatment (focal therapy). The injection site in this case is typically at or around the prostate cancer lesion. If metastasis is observed, it may be administered locally to or around the metastatic lesion. Examples of the dosage of the cell preparation are 0.1 ml to 20 ml, preferably 0.5 ml to 10 ml, for example. You may decide to administer to two or more places.

  The administration schedule may be prepared in consideration of the subject's (patient) sex, age, weight, disease state, and the like. In addition to single administration, multiple administration may be performed continuously or periodically. The administration interval when administering multiple times is not particularly limited, and is, for example, 1 day to 1 month. Moreover, the frequency | count of administration is not specifically limited. Examples of the number of administrations are 2 to 10 times.

In addition, you may decide to administer the cell in any one of the following (1)-(5) directly to a therapeutically effective amount and the patient of an autoimmune disease.
(1) Adherent cells or passage cells thereof included in a sedimented cell population that settles when a cell population separated from adipose tissue is centrifuged.
(2) Cells that proliferate when the precipitated cell population is cultured under low serum conditions.
(3) Cells grown when a cell population separated from adipose tissue is cultured under low serum conditions.
(4) A sedimented cell population recovered as a sediment by subjecting adipose tissue to protease treatment, followed by filtration, and then centrifuging the filtrate.
(5) A sedimented cell population recovered as a sediment by subjecting adipose tissue to protease treatment and centrifugation without passing through filtration treatment.
In addition, the conditions for the centrifugation (1) are preferably 800 to 1500 rpm and 1 to 10 minutes.

1. Isolation and characterization of adipose tissue-derived mesenchymal stem cells (1) Method After extracellular fluid was injected subcutaneously, subcutaneous fat (human) was aspirated finely with a liposuction tube. Next, of 300 g of the collected adipose tissue, 260 g for cell separation was injected into an adipose-derived stem cell separation device (Celution (registered trademark) device), and then operated according to the instruction manual to separate stem cells. The separated stem cells were collected from the device using a syringe. The Celution (registered trademark) apparatus is equipped with a disposable set and can treat adipose tissue for each case. The adipose-derived stem cell separation apparatus performs a process of separating the stem cells from the fat matrix, and at that time, the stem cells are separated and then washed and concentrated. This real-time processing is performed in a closed environment to minimize the risk of contact with contaminants and is completed within the time of a single surgical procedure.

(2) Results
The properties of cells prepared from human adipose tissue were evaluated using a Celution® device. First, the sample immediately after FACS separation was hemolyzed by adding ammonium chloride, and then mesenchymal stem cell markers CD29 and CD44 were measured. As a result, it was shown that CD29 and CD44 were positive (FIG. 1). On the other hand, cells prepared using a Celution (registered trademark) apparatus were cultured in an adipose differentiation medium containing insulin, dexamethasone, indomethacin and 3-isobutyl-1-methyl-xanthine (IBMX). The primary cells showed a fibroblast-like morphology and were 70-80% confluent on the fourth day. On the seventh day of induction of fat differentiation, formation of lipid droplets was confirmed and stained with Oil-Red (FIG. 2). In this way, differentiation into adipocytes was confirmed. In addition, it was confirmed by immunostaining that both commercially available adipose-derived stem cells (Invitrogen) and cells prepared by the Celution (registered trademark) apparatus were CD44 positive (FIGS. 3 and 4).

2. Examination of the therapeutic effect of ASC After undergoing radical prostatectomy for prostate cancer and transplanting ASC around the paraurethral in patients with intractable cases who have sustained stress urinary incontinence for more than 1 year after surgery, blood PSA concentration An interesting phenomenon was observed, which declined (results not shown). ASC was prepared and transplanted as follows ((1) to (3)).

(1) Collection of subcutaneous adipose tissue Under general anesthesia or local and lumbar anesthesia, mixed solution in patient abdomen or buttocks subcutaneous adipose tissue [component: saline 1000ml + 1% lidocaine (xylocaine) 2ml + 0.1% adrenaline (bosmin) ) 1.5ml + 8.4% Meyron 10ml] was injected in an appropriate amount and fully inflated. A suspension containing adipose tissue was aspirated by a negative pressure with a dedicated syringe usually used in the plastic surgery field. When performed under lumbar anesthesia, additional anesthesia was added for analgesia when additional pain occurred for the patient.

(2) Subcutaneous adipose tissue treatment (ASC separation)
From about 250 to 300 g of the adipose tissue obtained above, ASC was separated and concentrated (about 1 × 10 ⁶ to 1 × 10 ⁸ pieces / 5 ml) using an ASC separation device (Celution (registered trademark) device). First, the collected adipose tissue was injected into a sterilized disposable set and washed. Thereafter, the adipose tissue was thawed, digested by adding an enzyme (Celase ^™ ) for cell separation, and the cell suspension was automatically centrifuged and the enzyme washed in a closed circuit. The collection, adjustment, and transplantation were performed in the operating room of the Nagoya University Hospital. Its cleanliness was class 100-10000, and a clean environment was maintained in all procedures.

(3) ASC transplantation around the paraurethra
Two kinds of injected cell solutions using ASC (about 1 × 10 ⁶ to 1 × 10 ⁸ cells) separated into 5 ml, (a) 0.5 to 1.0 ml of ASC cell solution and (b) 20 g of self fat and ASC A cell solution mixed with 4.0-4.5 ml was prepared. For each patient, an endoscope was inserted from the urethra under general anesthesia or local and lumbar anesthesia, and the above two types of injected cell solutions were injected under the endoscope using an 18G needle syringe. That is, into the external urethral sphincter, (a) 0.5 to 1.0 ml of the solution is placed on the left and right sides, and (b) the solution is placed under the membrane-like urethral mucosa (4, 8, 6 o'clock) depending on the case (bulking The urethral lumen was closed due to the effect).

3. Effect of ASC on Prostate Cancer In view of the above findings, the following experiments were conducted under the hypothesis that “ASC is effective in treating prostate cancer”.

3-1. Preliminary experiment (cell line selection)
In order to select the optimal cells for verifying the therapeutic effect of ASC, PC-3 cells (ATCC, ATCC number: CRL-1435) and DU145 cells (ATCC, ATCC number: HTB-81) and LNCaP cells (ATCC. ATCC number: CRL-1740) were compared for their ability to produce PSA. As a result, high PSA production ability was observed in LNCaP cells. Therefore, we decided to use LNCaP cells for the following experiments.

3-2. Effects of liquid components
It was predicted that the PSA level was reduced by ASC transplantation because the humoral component secreted by ASC suppressed the growth or activity of cancer cells. Under this expectation, the following culture experiment was conducted.

(1) Method
ASC and LNCaP cells were seeded on a 12-well plate, and the cell culture supernatant of ASC was collected 24 hours later. Next, the culture solution of LNCaP cells was divided into two groups, ASC supernatant and normal culture solution, and replaced. After 24 hours, two groups of cell culture supernatants were collected and the PSA concentration was measured. In the experiment, the above 2. ASC prepared by the method shown in 1. was used.

(2) Results As shown in FIG. 5, even when cultured in a medium supplemented with ASC culture supernatant, no substantial change was observed in the amount of PSA produced by LNCaP cells. That is, contrary to the initial expectation, the result of denying the involvement of the humoral component secreted by ASC.

3-3. Effect of co-culture of ASC As a result of the above, it is possible that ASC directly acts on LNCaP cells. Therefore, we decided to investigate the effect of co-culturing ASC and LNCaP cells.

(1) Method
ASC alone, LNCaP cells alone and ASC / LNCaP cell mixture (multiple conditions with different mixing ratios) were seeded on a 12-well plate, and cell culture supernatants after 48 hours and 96 hours were collected and PSA Concentration was measured.

(2) Results FIG. 6 shows the results of measuring the PSA concentration in the culture medium after culture. Moreover, the phase-contrast microscope image is shown in FIG. 7 about each cell after culture | cultivation. When LNCaP cells were cultured in the presence of ASC, the PSA concentration in the medium was significantly reduced (FIG. 6). The decrease in PSA concentration due to ASC was dependent on the number of ASC cells (concentration dependency). On the other hand, observation with a phase contrast microscope showed that ASC surrounded LNCaP cells when ASC was co-cultured (FIGS. 7e, f, g, h). From the above results, it was found that ASC suppresses the growth of LNCaP cells or decreases the activity by a direct action by cell-to-cell interaction. In other words, the surprising fact that ASC has an antitumor effect on prostate cancer was found, and part of its mechanism of action became clear. This finding strongly suggests that ASC is effective in the treatment of prostate cancer and is noteworthy.

4). Effect on other cancer types
It was examined whether ASC exerts an antitumor effect on other cancer types.

(1) Method
According to the LNCaP cell experimental system, ASC alone, LoVo cell alone, LS180 cell alone, ASC / LoVo cell mixture, and ASC / LS180 cell mixture (multiple conditions with different mixing ratios) were seeded on 12-well plates. Then, the cell culture supernatants after 48 hours and 96 hours were collected and the CEA concentration was measured.

(2) Results
In the case of LNCaP cells, a decrease in the marker value (PSA concentration) was observed by co-culture with ASC, consistent with the above results, while for LoVo cells and LS180 cells, an increase in the marker value was obtained by co-culture with ASC. Recognized (FIG. 8). This result confirms the validity of the above experimental results and the findings derived therefrom (ASC suppresses the proliferation or decreases the activity of prostate cancer cells by cell-to-cell interaction). In addition, it shows that the antitumor action by ASC is specific to a specific cancer type (high cancer type specificity).

  The same experiment was performed again using patient-derived ASC, and the same experiment using commercially available ASC was performed in parallel. As a result, reproducible results were obtained (FIGS. 9 to 11).

5. Transplant experiment
LNCaP alone (3 × 10 ⁶ ) or LNCaP (3 × 10 ⁶ ) and ASC (6 × 10 ⁶ ) were transplanted subcutaneously into male nude rats at 8 weeks of age, and changes in tumor size over time were compared. . A commercially available ASC (Invitrogen) was used. As shown in FIG. 12, the tumor size of the transplanted group (LNCaP + ASC) was clearly smaller on the 28th day of transplantation compared to the LNCaP single transplanted group, indicating that ASC inhibits the increase in tumor size.

6). Summary As described above, it was found that ASC exerts an antitumor effect on prostate cancer cells. In other words, the effectiveness of ASC in the treatment of prostate cancer has been experimentally demonstrated. In addition, a part of the mechanism of action of ASC's antitumor action was clarified. Furthermore, it was also clarified that the antitumor action of ASC is highly specific to cancer types.

  The cell preparation of the present invention is used for the treatment of prostate cancer. Application to patients of various stages is envisaged. In addition, application to treatment targeting metastatic lesions is also envisaged. Furthermore, application to the treatment of other cancer types is also expected.

The present invention is not limited to the description of the embodiments and examples of the invention described above. Various modifications may be included in the present invention as long as those skilled in the art can easily conceive without departing from the description of the scope of claims.
The contents of papers, published patent gazettes, patent gazettes, and the like specified in this specification are incorporated by reference in their entirety.

Claims (7)

  A cell preparation for treating prostate cancer, comprising adipose tissue-derived mesenchymal stem cells and exhibiting an antitumor action by cell-cell interaction.   The cell preparation according to claim 1, wherein the adipose tissue-derived mesenchymal stem cell is positive for a cell surface marker CD44. The adipose tissue-derived mesenchymal stem cells are
(1) Adherent cells or passage cells thereof included in a sedimented cell population that settles when a cell population separated from adipose tissue is centrifuged.
(2) cells grown when the precipitated cell population is cultured under low serum conditions;
(3) cells that proliferate when the cell population isolated from adipose tissue is cultured under low serum conditions;
(4) A set of precipitated cells collected as a precipitate by subjecting the adipose tissue to protease treatment, and then subjecting the filtrate to centrifugation, or (5) Filtration treatment after treating the adipose tissue with protease. Sedimented cell population recovered as sediment by centrifugation without
The cell preparation according to claim 1, wherein   The cell preparation according to claim 3, wherein the low serum condition is a condition where the serum concentration in the culture solution is 5% (V / V) or less.   The cell preparation according to any one of claims 1 to 4, wherein the adipose tissue is human adipose tissue.   The cell preparation according to any one of claims 1 to 5, further comprising a substance that promotes contact between cells.   Use of adipose tissue-derived mesenchymal stem cells for producing a cell preparation for treating prostate cancer that exhibits an antitumor action by cell-cell interaction.

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