METHOD FOR OBTAINING ENDOMETRIAL MESENCHYMAL STEM CELLS FROM HUMAN MENSTRUAL BLOOD

Abstract

Provided are a method for obtaining endometrial mesenchymal stem cells from human menstrual blood and the endometrial mesenchymal stem cells prepared thereby. The method include: 1) subjecting an aliquot of menstrual blood to cascade filtration through graded cell strainers of decreasing mesh sizes to separate tissue debris, wherein said cell strainers include a filter-through cell strainer for collecting filtrate and a retention cell strainer for collecting retentate, wherein the mesh size of the retention cell strainer is smaller than that of the filter-through cell strainer; 2) collecting the retentate on the retention cell strainer as the tissue debris; 3) culturing the tissue debris in a mesenchymal stem cells medium; and 4) collecting adherent cells as the endometrial mesenchymal stem cells.

Claims

1-10. (canceled)

11. A method for obtaining endometrial mesenchymal stem cells from human menstrual blood, comprising: 1) subjecting an aliquot of menstrual blood to cascade filtration through graded cell strainers of decreasing mesh sizes to separate tissue debris, wherein said cell strainers include a filter-through cell strainer for collecting filtrate and a retention cell strainer for collecting retentate, wherein the mesh size of the retention cell strainer is smaller than that of the filter-through cell strainer; 2) collecting the retentate on the retention cell strainer as the tissue debris; 3) culturing the tissue debris in a mesenchymal stem cells medium; and 4) collecting adherent cells as the endometrial mesenchymal stem cells.

12. The method of claim 11, wherein, the retention cell strainer has a mesh size ranging from 70 mesh to 160 mesh, preferably from 80 mesh to 160 mesh.

13. The method of claim 11, wherein, the filter-through cell strainer has a mesh size for collecting tissue debris smaller than 30 mesh, smaller than 35 mesh or smaller than 40 mesh; or, the filter-through cell strainer has a mesh size ranging from 18 mesh to 60 mesh, preferably from 36 mesh to 60 mesh, more preferably 36 mesh to 50 mesh, and even more preferably 36 mesh to 40 mesh.

14. The method of claim 11, wherein, in said step 1), said cascade filtration includes passing through at least three grades of cell strainers, preferably passing through at least four grades of cell strainers; or, said cascade filtration includes passing through one or two or more grades of filter-through cell strainers, and/or passing through one or two or more grades of retention cell strainers; preferably, said cascade filtration includes two or more grades of retention cell strainers, and said step 2) of collecting the retentate on the retention cell strainer includes pooling the tissue debris retained on the multiple grades of retention cell strainers.

15. The method of claim 11, wherein, said step 2) of collecting the retentate on the retention cell strainer includes releasing the tissue debris retained on the retention cell strainer(s) via back-flush with a washing fluid.

16. The method of claim 11, wherein, said step 2) of collecting the retentate on the retention cell strainer further includes washing the tissue debris with a washing fluid.

17. The method of claim 15, wherein, the washing fluid is a phosphate buffer; preferably a phosphate buffer comprising an antibiotic preferably selected from the group consisting of Gentamicin, Penicillin, Streptomycin, Amphotericin B, or a combination thereof; Optionally, the concentration of Gentamicin is 50-100 U/mL, preferably 100 U/mL; the concentration of Penicillin is 180-200 U/mL, preferably 200 U/mL; the concentration of Streptomycin is 0.15-0.2 mg/mL, preferably 0.2 mg/mL; and/or the concentration of Amphotericin B is 4-5 μg/mL, preferably 5 μg/mL.

18. The method of claim 16, wherein, the washing fluid is a phosphate buffer; preferably a phosphate buffer comprising an antibiotic preferably selected from the group consisting of Gentamicin, Penicillin, Streptomycin, Amphotericin B, or a combination thereof; Optionally, the concentration of Gentamicin is 50-100 U/mL, preferably 100 U/mL; the concentration of Penicillin is 180-200 U/mL, preferably 200 U/mL; the concentration of Streptomycin is 0.15-0.2 mg/mL, preferably 0.2 mg/mL; and/or the concentration of Amphotericin B is 4-5 μg/mL, preferably 5 μg/mL.

19. The method of claim 11, wherein, the mesenchymal stem cell medium is selected from the group consisting of MSC primary cell serum-free medium and Amniocyte Culture Medium.

20. The method of claim 11, wherein, in step 3), tissue debris is cultured for 4-6 days, preferably 5 days.

21. Endometrial mesenchymal stem cells obtained by a method according to claim 11.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0014] FIG. 1 shows fold expansion of EMSCs prepared by the mononuclear method and EMSCs prepared by the method of the invention in comparison.

[0015] FIG. 2 shows doubling time for EMSCs prepared by the mononuclear method and for EMSCs prepared by the method of the invention in comparison.

[0016] FIG. 3A shows staining of the EMSCs prepared by the method of the invention for osteogenic differentiation potential; FIG. 3B shows staining of EMSCs prepared by the method of the invention for chondrogenic differentiation potential.

DETAILED DESCRIPTION OF THE INVENTION

[0017] In the present disclosure, components and preferred components can be recombined into alternative embodiments of the invention, unless otherwise specified expressively.

[0018] In the present disclosure, embodiments and preferred embodiments can be reorganized with one another into alternative embodiments of the invention, unless otherwise specified expressively.

[0019] In the present disclosure, features and preferred features can be recombined into alternative embodiments of the invention, unless otherwise specified expressively.

[0020] As used herein, “a” and “an” means “at least one”, unless otherwise specified expressively.

[0021] In the present disclosure, ranges are each defined by a lower limit and an upper limit, wherein either limit may have more than one values of option. A given rang is defined in boundary by a selected lower limit and a selected upper limit, and all ranges defined thereby are inclusive and combinable to encompass ranges defined by anyone of the lower limits and anyone of the upper limits.

[0022] Specifically, in a first aspect, provided is a method for obtaining endometrial mesenchymal stem cells from human menstrual blood, comprising:

[0023] 1) subjecting an aliquot of menstrual blood to cascade filtration through graded cell strainers of decreasing mesh sizes to separate tissue debris, wherein said cell strainers include a filter-through cell strainer for collecting filtrate and a retention cell strainer for collecting retentate, wherein the mesh size of the retention cell strainer is smaller than that of the filter-through cell strainer;

[0024] 2) collecting the retentate on the retention cell strainer as the tissue debris;

[0025] 3) culturing the tissue debris in a mesenchymal stem cells medium; and

[0026] 4) collecting adherent cells as the endometrial mesenchymal stem cells.

[0027] In an embodiment, the method comprises the followings:

[0028] 1) Tissue separation: subjecting an aliquot of menstrual blood to cascade filtration through graded cell strainers of decreasing mesh sizes;

[0029] 2) Tissue back-flushing: releasing the tissue debris retained on cell strainer via back-flush with a washing fluid;

[0030] 3) Tissue washing: resuspending and washing the tissue debris in the washing fluid, followed by centrifugation to recover the pellet of tissue debris;

[0031] 4) Tissue culturing: resuspending the pellet of tissue debris into a suspension in culture medium, which was then inoculated into a culture flask, followed by addition of culture medium and culturing in an incubator;

[0032] 5) Collecting adherent cells as endometrial mesenchymal stem cells.

[0033] In some embodiments, the collected endometrial mesenchymal stem cells may be further expanded.

[0034] For the present invention, the cell strainers may be of whatever a porous structure that is capable of separating particles based on differentia in size without contaminating the material such as tissues and cells. Cascade filtration is a process of sifting by passing through graded cell strainers of decreasing mesh sizes in sequence to obtain graded retentates of different size distributions, or graded filtrates of different size distributions.

[0035] In some embodiments of the invention, first, a filter-through cell strainer of larger mesh size is used to retain and remove oversize tissue debris, and then a retention cell strainer of smaller mesh size is used to retain and collect the tissue debris for culturing. In the present disclosure, the term “tissue debris” refers to tissues in the form of debris or fragments, with no intention to limit to a product of some specific means of disruption or fragmentation.

[0036] In some embodiments, a retention cell strainer may have a mesh size ranging from 70 mesh to 160 mesh, preferably from 80 mesh to 160 mesh. The present invention involves at least one grade of retention cell strainer, and optionally and preferably two or more grades of retention cell strainers of decreasing mesh sizes. When multiple grades of retention cell strainers were used, tissue debris retained by different grades of cell strainers can be pooled for use in subsequent steps. This may involve, for example, releasing the retained debris from the strainer via back-flush with a washing fluid followed by pooling the washes, or scraping and collecting the debris followed by resuspension, washing, etc.

[0037] In the present invention, a filter-through cell strainer is used to remove oversize tissue debris and to collect the filtrate. Preferably, tissue debris smaller than the mesh size of 30 mesh, 35 mesh, 40 mesh or even a lower mesh size can be collected thereby, and correspondingly, the filter-through cell strainers may include those having a mesh size not exceeding 30 mesh, 35 mesh or 40 mesh or a even lower mesh size. For example, to collect tissue debris smaller than the mesh size of 36 mesh, the filter-through cell strainer(s) may include at least a grade of cell strainer of 36 mesh. Oversize tissue debris may be removed using one or more grades of strainers. Accordingly, the present invention involves at least one grade of filter-through cell strainer, and optionally and preferably two or more grades of filter-through cell strainers of decreasing mesh sizes. A filter-through cell strainer may have a mesh size ranging from 18 mesh to 60 mesh, preferably 36 mesh to 60 mesh, more preferably 36 mesh to 50 mesh, and even more preferably 36 mesh to 40 mesh.

[0038] In some embodiments, the step of cascade filtration includes passing through at least three grades of cell strainers, preferably passing through at least four grades of cell strainers. For example, the step of cascade filtration may include passing through at least two grades of filter-through cell strainers and at least one grade of retention cell strainer, or passing through at least one grade of filter-through cell strainer and two grades of retention cell strainers, or passing through at least two grades of filter-through cell strainers and two grades of retention cell strainers.

[0039] Retained tissue debris can be released from a retention cell strainer by any of suitable methods as will be a selection of sound judgment by an artisan in the art, including but not limited to scraping, picking, and back-flush. In some embodiments, the tissue debris retained on a cell strainer was washed down by back-flush with a washing fluid. In the present disclosure, the terms “tissue back-flushing” and “back-flush” refer to the operation of flushing down the retentate from a strainer using a flow of washing fluid in the direction opposite to the flow of filtration through the cell strainer. For a maximum recovery of retentates, the endpoint of flushing may be set as disappearance of any tissues visible to the naked eye.

[0040] For example, said back-flush may include the following steps: collecting the cell strainer(s) with tissue debris retained thereon; placing one or more of the cell strainer(s) with tissue debris retained thereon in a culture dish, wherein the number of strainer(s) depends on the size of the dish (e.g., five at the most for a dish of 10 cm.sup.2); adding into the culture dish washing fluid to just submerge the strainer(s) with tissue debris retained thereon; aspirating the washing fluid from the culture dish using a Pasteur pipette to wash the strainer(s) via back-flush to release the tissue debris retained thereon until there is no tissue visible to the naked eye on the strainer(s).

[0041] In some embodiments, the method of the invention may further include a step of washing to clean the tissue, i.e., the tissue debris harvested from the retention cell strainer(s), which may be, for example, resuspending the tissue debris in a washing fluid, optionally followed by solid-liquid separation (e.g., filtration or centrifugation) to obtain the pellet of washed (i.e., cleaned) tissue debris. For instance, when tissue debris is harvested from the strainer(s) by means of back-flush, tissue washing may include collecting the washes from the back-flush, collecting the pellet of tissue debris by means of solid-liquid separation (e.g., filtration or centrifugation, with the supernatant being discarded) and adding washing fluid to the pellet, wherein the specified steps may be repeated to obtain a pellet of washed and cleaned tissue debris. For centrifugation, it may be at the force of 400 g for 10 minutes at 20-22° C., preferably 20° C.

[0042] For the present invention, the “washing fluid” may be a buffer that is conventionally used in cell manipulation, such as a phosphate buffer-based washing fluid. The washing fluid may include one or more supplements as needed, such as an antibiotic. In some embodiments of the invention, the washing fluid is a phosphate buffer, and preferably a phosphate buffer comprising an antibiotic. Preferably, the antibiotic may be selected from the group consisting of Gentamicin, Penicillin, Streptomycin, Amphotericin B, or a combination thereof. Preferably, in the washing fluid, if present, the concentration of Gentamicin may be 50-100 U/mL, preferably 100 U/mL; if present, the concentration of Penicillin may be 180-200 U/mL, preferably 200 U/mL; if present, the concentration of Streptomycin may be 0.15-0.2 mg/mL, preferably 0.2 mg/mL; if present, the concentration of Amphotericin B may be 4-5 μg/mL, preferably 5 μg/mL.

[0043] In the step 3) culturing the tissue debris in a mesenchymal stem cells medium, i.e., “tissue culturing”, wherein the tissue debris was cultured in a mesenchymal stem cell medium, the tissue debris is inoculated into the mesenchymal stem cell medium and cultured under a condition such that adherent mesenchymal stem cells grow from the tissue debris. In some embodiments, the step of inoculation may include resuspending the pellet of tissue debris into a suspension in the mesenchymal stem cell medium for inoculation. The tissue may be cultured in a culture flask. Optionally, after ensuring that the endometrial mesenchymal stem cells are tightly adhered to the vessel wall (e.g., after tissue-culturing for 48 hours), there may be included a step of removing non-adherent contaminant cells (e.g., erythrocytes) to preclude contamination. The culturing may then be continued, and the culture is ready for passaging when reaching 90%-100% confluence.

[0044] The present invention has no limits on usable mesenchymal stem cell medium. It may be any culture medium that is known as applicable to mesenchymal stem cells, and it may be either purchased or in-house prepared. Examples of mesenchymal stem cell medium include the primary MSC serum-free medium available from Beijing Youkang Technology Co., Ltd and the Amniocyte Culture Medium available from Guangzhou Baiyunshan Baidi Biopharmaceutical Co., Ltd.

[0045] In some embodiments, the tissue is cultured under a condition typical for mesenchymal stem cell culturing, such as at 5% CO.sub.2, 37° C. The culturing may last for 4-6 days, preferably 5 days.

[0046] In some embodiments, the method may further include, after said tissue culturing, a step of separating the adherent endometrial mesenchymal stem cells for expansion, and optionally a step of expansion.

[0047] In some embodiments, there may be optionally included before step 1) an examination on the collected menstrual blood for contamination. Specifically, a sample of the collected menstrual blood is mixed with a washing fluid, preferably a phosphate buffer comprising an antibiotic, such as a phosphate buffer comprising Gentamicin and Amphotericin B; an aliquot of the mixture is added into the culture medium and incubated in an incubator; and the culture is monitored for contamination. Therein, the sample of menstrual blood may be mixed with the washing fluid at the ratio of 1:1.

[0048] In another aspect, provided is endometrial mesenchymal stem cells prepared by the method of the invention. The endometrial mesenchymal stem cells obtained thereby advantageously have increased proliferation rate and fold expansion in passaging as well as desirable differentiate potentials, compared with those prepared by prior art techniques such as the mononuclear method.

Examples

[0049] The present invention will be further described in details with reference to the examples below. It should be understood that these examples are merely provided for the purpose of illustration, with no intention to limit the scope of the invention in any sense.

Example 1: Preparation of Endometrial Mesenchymal Stem Cells

[0050] Endometrial mesenchymal stem cells were prepared according to the method of the invention. The process is detailed as follows:

[0051] I. Tissue separation:

[0052] Menstrual blood in a 50 mL spin tube was sequentially filtered through an 18 mesh stainless cell strainer, a 36 mesh stainless cell strainer, an 80 mesh stainless cell strainer and a 160 mesh stainless cell strainer.

[0053] II. Tissue back-flushing:

[0054] 1) The 80 mesh and the 160 mesh stainless cell strainers though which the menstrual blood was passed through in step I were placed in 10 cm.sup.2 culture dishes, 5 strainers per dish at the most. To each 10 cm.sup.2 culture dish, 40 mL washing fluid was added to just submerge the strainers with tissue debris retained thereon, wherein the washing fluid was phosphate buffer containing 100 U/mL Gentamicin and 5 μg/mL Amphotericin B;

[0055] 2) The tissue debris retained on the 80 mesh and the 160 mesh cell strainers were released via back-flush with the washing fluid in the culture dish using a 3 mL Pasteur pipette.

[0056] III. Tissue washing:

[0057] 1) After the back-flush, the obtained washes comprising the tissue debris were collected into 50 mL spin tubes, 40 mL per tube, and centrifuged at 400 g, 20° C., for 10 minutes, the supernatant being discarded then;

[0058] 2) To each spin tube, 10 mL washing fluid was added using a pipette to resuspend the pellet; the resuspensions were pooled in a 50 mL spin tube, centrifuged at 400 g, 20° C., for 10 minutes, the supernatant being discarded then;

[0059] 3) To the spin tube, 30 mL washing fluid was added using a pipette to resuspend the pellet; the resuspension was centrifuged at 400 g, 20° C., for 10 minutes, the supernatant being discarded then.

[0060] IV. Tissue culturing:

[0061] 1) The pellet was resuspended in 1 mL medium (Amniocyte Culture Medium, also referred to as “YS” herein below, Guangzhou Baiyunshan Baidi Biopharmaceutical Co., Ltd.). The resuspensions were inoculated into T25 culture flasks, 500 μL/flask, and resuspended by adding 10 mL medium;

[0062] 2) The cells were mixed by drawing “X” or drawing “8”, and then cultured in an incubator at 37° C., 5% CO.sub.2;

[0063] 3) After culturing for 48 hours, non-adherent contaminant cells (e.g., erythrocytes) were washed off via medium exchange, and the culturing continues. The cells were ready for passaging when reaching 90% confluence.

Example 2: Growth Profiles of Endometrial Mesenchymal Stem Cells Prepared by Different Methods

[0064] Endometrial mesenchymal stem cells were prepared using the mononuclear method as described in “Isolation, Culture And Identification Of Menstrual Blood-Derived Mesenchymal stem cells”, Zhou Yun-fang, Yang Bo, Hu Xiang et al., (Chinese Journal of Clinical Rehabilitative Tissue Engineering Research, 2010, 14(32):5952-5956).

[0065] An aliquot of the endometrial mesenchymal stem cells prepared in Example 1 and an aliquot of the endometrial mesenchymal stem cells prepared using the mononuclear method were subcultured. Each passage was subcultured via inoculation at a density of 5.0×10.sup.3 cells/cm.sup.2 into a T75 culture flask containing the Amniocyte Culture Medium (also referred to as “YS”, available from Guangzhou Baiyunshan Baidi Biopharmaceutical Co., Ltd.). At around 90% confluence, the medium was discarded, and the cells were gently washed with phosphate buffer solution (PBS). Then, the washing fluid was discarded and the cells were digested with 3 ml 0.25% trypsin for 4 minutes. 6 ml PBS containing 5% fetal calf serum was added to end the digestion. The cell suspensions were collected into two spin tubes separately, centrifuged at 1500 r/min for 5 min. The supernatants was discarded, and 1 mL Amniocyte Culture Medium (also referred to as “YS”, available from Guangzhou Baiyunshan Baidi Biopharmaceutical Co., Ltd.) was added into each tube to resuspend the cells. For each passage, the harvest was quantitated using the AO/PI (AO: Acridine Orange, PI: Propidium Iodide) Double-Stain Apoptosis Detection Kit (a DNA probe-based nuclei double-stain detection, available from Shanghai Ruiyu Biotechnology Co., Ltd.), and fold expansion and doubling time were calculated. The results were as summarized in Table 1 and exhibited in FIGS. 1 and 2.

TABLE-US-00001 TABLE 1 Growth Profile of Endometrial Mesenchymal Stem Cells Prepa- P1 P2 P3 P4 P5 P6 Sample ration Fold Doubling Fold Doubling Fold Doubling Fold Doubling Fold Doubling Fold No. Method expansion Time expansion Time expansion Time expansion Time expansion Time expansion 0001 FC 7.09 50.95 13.04 38.87 7.49 49.56 14.56 37.27 9.33 37.24 7.04 0031 FC 3.36 82.36 7.44 49.74 5.17 50.63 19.49 33.61 8.29 47.19 12.16 0026 FC 8.27 39.37 12.4 33.04 9.28 44.80 11.15 41.39 8.48 46.69 4.88 0001 DH 5.98 55.81 7.49 49.57 8.4 46.90 11.52 40.84 6.16 45.75 3.01 0031 DH 3.09 88.47 8.4 46.90 13.28 38.59 3.12 87.72 8.37 46.98 5.44 0026 DH 4.9 73.27 11.2 41.31 6.32 54.14 4.05 71.36 9.41 37.10 5.57 P6 P7 P8 P9 P10 P11 Sample Doubling Fold Doubling Fold Doubling Fold Doubling Fold Doubling Fold Doubling No. Time expansion Time expansion Time expansion Time expansion Time expansion Time 0001 42.62 8.11 31.80 4.4 44.91 3.33 82.97 13.52 25.55 1.92 102.01  0031 33.30 9.44 29.64 6.29 45.23 11.7 33.78 2.27 121.80 4.32 45.48 0026 62.97 3.68 76.61 3.79 74.91 8.32 47.11 3.01 90.58 3.8  74.77 0001 75.48 7.24 25.21 1.87 106.61 3.44 53.86 2.56 88.49 — — 0031 58.93 3.47 80.23 2.5 109.00 2.28 161.48 1.95 199.79 — — 0026 58.12 6.13 55.05 2.384 114.89 5.6 57.94 3.79 74.91 3.04 89.77 Notes: 1) P1: The first passage of subculturing; 2) FC: The method of the present invention; DH: the mononuclear method.

[0066] Results: The mesenchymal stem cells prepared by the method of the invention largely exhibited a higher fold expansion and a reduced doubling time than those prepared by the mononuclear method across passages. Furthermore, the doubling time fluctuated prominently between passages for the MSCs prepared by the mononuclear method while appeared more stable for the MSCs prepared by the method of the invention. Accordingly, the method of the invention is advantageously capable of providing endometrial mesenchymal stem cells with improved doubling time and fold expansion, as compared with the mononuclear method.

Example 3: Differentiation Potency of Endometrial Mesenchymal Stem Cells Prepared by the Method of the Invention

[0067] Methods: The endometrial mesenchymal stem cells 0031 prepared in Example 1 were plated into wells of a 12-well plate containing the Amniocyte Culture Medium (also referred to as “YS”, available from Guangzhou Baiyunshan Baidi Biopharmaceutical Co., Ltd.) at 1.0×10.sup.5 cells/well, and cultured in an incubator at 5% CO.sub.2, 37° C. for 3-4 day until reaching 90%-100% confluence. On a clean bench, the medium in each well was aspirated and discarded, then 1 mL/well the Induced Osteogenic Differentiation Complete Medium (available from Cyagen Biotechnology Co., Ltd.) was added. The medium was refreshed every 3 days. At day 21 of the osteogenic differentiation, the cells were stained with Alizarin Red S for osteogenic differentiation. The result was shown in FIG. 3A.

[0068] The endometrial mesenchymal stem cells 0031 prepared in Example 1 were resuspended in the Amniocyte Culture Medium at 1.0×10.sup.7/ml. The suspension was added evenly in drops onto a 10 cm.sup.2 petri dish cover, and incubated in incubator at 5% CO.sub.2, 37° C. for 24 hours. On a clean bench, spheric drops were pipetted into wells of a 6-well plate, into which was added 2 mL/well the Chondrogenic Differentiation Medium (available from STEMCELL). The medium was refreshed every 3 days. At day 28 of the chondrogenic differentiation, cell sections were stained with Alcian blue for the chondrogenic differentiation. The result was shown in FIG. 3B.

[0069] As seen from FIG. 3A, the endometrial mesenchymal stem cells prepared according to Example 1 were prominently visible in orange-red as stained with Alizarin Red S, which indicates a great potential of osteogenic differentiation. As seen from FIG. 3B, the endometrial mesenchymal stem cells prepared according to Example 1 were visible in the tissues in blue as stained with Alcian blue, which indicates the chondrogenic potential. In conclusion, the endometrial mesenchymal stem cells prepared by the method of the invention have sound differentiation potentials.

[0070] Despite of the described with specific examples of the invention, it will be apparent to those skilled in the art that various changes and modifications can be made in the present invention without departing from the spirit and scope of the invention, which all fall within the scope of the invention as defined by the appended claims.