Separation culture method

Abstract

The invention provides an isolated culture method, and belongs to the field of biology. The isolated culture method comprises the following steps: adding hydroxyethyl starch into the uterine blood sample and uniformly mixing to obtain a mixed solution; standing the mixed solution; when red blood cells are layered, sucking supernate; centrifuging the supernate to obtain a precipitate; resuspending the precipitate by using a complete medium containing platelet lysate to obtain a primary cell suspension; and culturing the primary cell suspension to obtain the uterine blood mesenchymal stem cells. According to the isolated culture method, hydroxyethyl starch is added into the uterine blood sample, erythrocytes in the uterine blood sample can be settled by the hydroxyethyl starch, the extraction rate of leukocytes in the uterine blood sample can be increased by combining with an optimized centrifugal process, the cells can be better centrifugally cleaned, and the separation time is short.

Description

Technical Field

[0001] The present disclosure relates to the biological field, and in particular to a separation and culture method. Background Art

[0002] Endometrial stromal cells (mesenchymal stem cells, MSCs) are responsible for the endometrium's high regenerative capacity. They are shed during menstruation and are incorporated into menstrual blood. MSCs derived from menstrual blood are called menstrual mesenchymal stem cells (MenSCs). MenSCs are derived from menstrual blood collected non-invasively (via a menstrual cup) from healthy female donors. Current methods for extracting uterine stem cells from menstrual blood involve density gradient centrifugation.

[0003] The existing Ficoll density gradient centrifugation method uses existing M4 medium for primary cell culture, followed by separation based on differences in cell specific gravity. It is primarily used to isolate mononuclear cells from blood, and can be used to isolate mononuclear cells from uterine blood samples. However, when using M4 medium for primary cell culture, the primary cells have a low adhesion rate. Furthermore, this method typically takes more than 2.5 hours to separate, resulting in a long separation time and unstable white blood cell extraction rates. Therefore, the current Ficoll density gradient centrifugation method is not conducive to the isolation and extraction of uterine blood stem cells from menstrual blood.

[0004] Public content In order to solve the problems of the prior art, the present disclosure provides a separation medium and a separation method. The technical solution is as follows: The present disclosure provides a separation and culture method, which comprises: adding hydroxyethyl starch to the uterine blood sample and mixing to obtain a mixed solution; Allowing the mixed solution to stand; When the red blood cells are separated, the supernatant is aspirated; Centrifuging the supernatant to obtain a cell pellet; resuspending the cell pellet in complete culture medium containing platelet lysate to obtain a primary cell suspension; The primary cell suspension is cultured to obtain uterine blood mesenchymal stem cells.

[0005] Specifically, the complete culture medium for culturing the platelet lysate includes: a basic culture medium DMEM / F12 and the platelet lysate in a volume ratio of 2% to 5%.

[0006] Specifically, when the primary cell suspension is cultured to obtain uterine blood mesenchymal stem cells, the culture conditions are as follows: the primary cell suspension is cultured in an incubator at 37°C and containing 5% CO2, and the medium is replaced every 2 to 3 days. When the degree of fusion reaches 70 to 90%, it is digested with a recombinant trypsin solution to obtain the uterine blood mesenchymal stem cells.

[0007] Specifically, the concentration of the recombinant trypsin is 1.2 mg / mL.

[0008] Specifically, the uterine blood sample is filtered through a blood transfusion filter, and a 6% hydroxyethyl starch solution is added to the filtered uterine blood sample. The solvent of the 6% high molecular weight hydroxyethyl starch solution is sodium chloride, and the molecular weight of the high molecular weight hydroxyethyl starch ranges from 500 kDa to 800 kDa. The volume ratio of the filtered uterine blood sample to the 6% high molecular weight hydroxyethyl starch is 1:1.

[0009] Further, let it stand for 30 to 60 minutes.

[0010] Furthermore, the supernatant is centrifuged using countercurrent centrifugation to obtain the cell pellet.

[0011] Specifically, the uterine blood sample is filtered through a blood transfusion filter, and a 6% hydroxyethyl starch solution is added to the filtered uterine blood sample. The solvent of the 6% medium molecular hydroxyethyl starch solution is sodium chloride, and the molecular weight of the medium molecular hydroxyethyl starch ranges from 110 kDa to 230 kDa. The volume ratio of the filtered uterine blood sample to the 6% medium molecular hydroxyethyl starch solution is 1:1.

[0012] Further, let it stand for 40 to 90 minutes.

[0013] Further, the supernatant is subjected to a first centrifugation, wherein the parameters of the first centrifugation are 600-1000g and 5 minutes to obtain a first precipitate; adding PBS buffer containing antibiotics to the first precipitate to resuspend the precipitate to obtain a first resuspension; The first resuspension was centrifuged for the second time at 600-1000 g for 5 min to obtain a second precipitate; adding PBS buffer containing antibiotics to the second precipitate to resuspend the precipitate to obtain a second resuspension; The second resuspension was centrifuged for the third time at a speed of 600-1000 g and 5 min to obtain the precipitate.

[0014] The technical solution provided by the embodiments of the present disclosure has the following beneficial effects: the present disclosure provides a separation and culture method, in which hydroxyethyl starch is added to a uterine blood sample. The hydroxyethyl starch can precipitate the red blood cells in the uterine blood sample. Combined with the optimized centrifugation process, the extraction rate of white blood cells in the uterine blood sample can be improved, the cells can be better centrifuged and cleaned, and the separation time is short. Moreover, uterine blood mesenchymal stem cells with a high expansion multiple and excellent cell morphology can be obtained. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to more clearly illustrate the technical solutions in the embodiments of the present disclosure, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present disclosure. For ordinary technicians in this field, other drawings can be obtained based on these drawings without any creative work.

[0016] Figure 1 This is a morphological diagram of uterine blood mesenchymal stem cells P0 provided in Example 1 of the present disclosure; Figure 2 This is a cell morphology diagram at P1 provided in Example 1 of the present disclosure; Figure 3 This is a cell morphology diagram at P2 provided in Example 1 of the present disclosure; Figure 4 This is a cell morphology diagram at P3 provided in Example 1 of the present disclosure; Figure 5 This is a morphological diagram of uterine blood mesenchymal stem cells P0 provided in the comparative example of the present disclosure; Figure 6 This is a cell morphology diagram at P1 provided in the comparative example of the present disclosure; Figure 7 This is a cell morphology diagram at P2 provided in the comparative example of the present disclosure; Figure 8 This is a cell morphology diagram at P3 provided in the comparative example of the present disclosure; Figure 9 This is a morphological diagram of uterine blood mesenchymal stem cells P0 provided in Example 2 of the present disclosure; Figure 10 This is a cell morphology diagram at P1 provided in Example 2 of the present disclosure; Figure 11 This is a cell morphology diagram at P2 provided in Example 2 of the present disclosure; Figure 12 This is a cell morphology diagram at P3 provided in Example 2 of the present disclosure; Figure 13 This is a morphological diagram of uterine blood mesenchymal stem cells P0 provided in the comparative example of the present disclosure; Figure 14 This is a cell morphology diagram at P1 provided in the comparative example of the present disclosure; Figure 15 This is a cell morphology diagram at P2 provided in the comparative example of the present disclosure; Figure 16 This is a cell morphology diagram at P3 provided in the comparative example of the present disclosure; Figure 17 This is a morphological diagram of uterine blood mesenchymal stem cells P0 provided in Example 3 of the present disclosure; Figure 18 This is a cell morphology diagram at P1 provided in Example 3 of the present disclosure; Figure 19 This is a cell morphology diagram at P2 provided in Example 3 of the present disclosure; Figure 20 This is a cell morphology diagram at P3 provided in Example 3 of the present disclosure; Figure 21 This is a morphological diagram of uterine blood mesenchymal stem cells P0 provided in Example 4 of the present disclosure; Figure 22 This is a cell morphology diagram at P1 provided in Example 4 of the present disclosure; Figure 23 This is a cell morphology diagram at P2 provided in Example 4 of the present disclosure; Figure 24 This is a cell morphology diagram at P3 provided in Example 4 of the present disclosure. DETAILED DESCRIPTION

[0017] In order to make the objectives, technical solutions and advantages of the present disclosure more clear, the embodiments of the present disclosure will be further described in detail below with reference to the accompanying drawings.

[0018] Example 1 The present disclosure provides a separation and culture method, which includes: adding hydroxyethyl starch to a uterine blood sample and mixing it to obtain a mixed solution; in this embodiment, the uterine blood sample is obtained from a qualified donor of appropriate age who meets the requirements of a health examination, and menstrual blood is collected using a menstrual cup and stored in a collection solution at 2°C to 8°C.

[0019] Let the mixture stand; When red blood cells were observed to separate, the supernatant was aspirated; The supernatant was centrifuged to obtain a cell pellet; Resuspend the cell pellet in complete medium containing platelet lysate to obtain primary cell suspension; The primary cell suspension was cultured to obtain uterine blood mesenchymal stem cells.

[0020] Specifically, the complete culture medium for culturing platelet lysate includes: a basic culture medium DMEM / F12 and a platelet lysate at a volume ratio of 5%.

[0021] Specifically, the concentration of recombinant trypsin was 1.2 mg / mL.

[0022] Specifically, a uterine blood sample was filtered through a blood transfusion filter, and a 6% high-molecular-weight hydroxyethyl starch solution was added to the filtered uterine blood sample. The solvent of the 6% high-molecular-weight hydroxyethyl starch solution was sodium chloride, and the molecular weight of the high-molecular-weight hydroxyethyl starch ranged from 500 kDa to 800 kDa. The volume ratio of the filtered uterine blood sample to the 6% high-molecular-weight hydroxyethyl starch was 1:1. In this embodiment, the average molecular weight of the high-molecular-weight hydroxyethyl starch was approximately 670 kDa.

[0023] Furthermore, the mixture is allowed to stand for 30 to 60 minutes, preferably 40 minutes. In this embodiment, the mixture is allowed to stand for 40 minutes.

[0024] Furthermore, the supernatant was centrifuged using countercurrent centrifugation to obtain a cell pellet.

[0025] Specifically, when the primary cell suspension is cultured to obtain uterine blood mesenchymal stem cells, the culture conditions are as follows: the primary cell suspension is cultured in an incubator at 37°C and containing 5% CO2, and the medium is changed every 2 to 3 days for a total of 12 days. When the confluence reaches 70 to 90%, it is digested with a recombinant trypsin solution for 5 minutes to obtain P0 uterine blood mesenchymal stem cells, and the collected cell amount of uterine blood mesenchymal stem cells is 2.3×10 6 cells, the morphology of uterine blood mesenchymal stem cells is as follows Figure 1 As shown. It can be adjusted to 1~1.5×10 6 cells / flask for subculture, and the inoculation volume for each T175 flask was 1×10 6 cells, cultured for 7 days, digested for 8 min, and collected cells of 4.56 × 10 6 cells, the expansion times is 4.56, and the cell morphology at P1 is as follows Figure 2 As shown, the cells were frozen at the P1 generation and stored in a liquid nitrogen tank.

[0026] Continue to subculture, the inoculum volume for each T175 bottle is 1×10 6 cells, cultured for 5 days, digested for 6 min, and collected cells of 5.28 × 10 6 cells, the expansion times is 5.28, and the cell morphology at P2 is as follows Figure 3 shown.

[0027] Continue to subculture, the inoculum volume for each T175 bottle is 1×10 6 cells, cultured for 6 days, digested for 4 min, and collected cells of 4.78 × 10 6cells, the expansion times is 4.78, and the cell morphology at P3 is as follows Figure 4 The cells were subsequently passaged to P5 for use in cell preparation. Flow cytometry data revealed that the CD73 concentration in this example was 100%, CD90 concentration was 99.99%, and CD105 concentration was 99.99%. The cells isolated and cultured using the isolation method provided in this example met the requirements for stem cell characteristics.

[0028] Comparative Example This comparative example provides a separation and culture method. The difference between this comparative example and Example 1 is that the uterine blood sample is filtered through a blood transfusion filter, the filtrate is centrifuged to obtain a precipitate, and a PBS buffer containing antibiotics is added to the precipitate for centrifugal washing twice, with the centrifugation parameters set to 600g and 5 minutes. Density gradient centrifugation purification is performed at a volume ratio of 2:1, with the centrifugation parameters set to 700g and 30 minutes. After centrifugation, the buffy coat layer (mononuclear cells) is aspirated, and the buffy coat layer is added to a PBS buffer containing antibiotics for three centrifugal washings. The first centrifugation parameter is 800g and 6 minutes, and the second and third centrifugation parameters are both 300g and 6 minutes to obtain a precipitate. The remaining parameters were the same as those in Example 1. The primary cell suspension was cultured to obtain uterine blood mesenchymal stem cells. The culture conditions were as follows: the primary cell suspension was cultured in an incubator at 37°C and containing 5% CO2, and the medium was changed every 2 to 3 days for a total of 12 days. When the confluence reached 70 to 90%, the cells were digested with a recombinant trypsin solution for 5 minutes to obtain P0 uterine blood mesenchymal stem cells, and the collected cell amount of uterine blood mesenchymal stem cells was 1.44×10 6 cells, the morphology of uterine blood mesenchymal stem cells is as follows Figure 5 As shown. It can be adjusted to 1~1.5×10 6 cells / flask for subculture, and the inoculation volume for each T175 flask was 1×10 6 cells, cultured for 7 days, digested for 8 min, and collected cells of 2.94 × 10 6 cells, the expansion times is 2.94, and the cell morphology at P1 is as follows Figure 6 As shown, the cells were frozen at the P1 generation and stored in a liquid nitrogen tank.

[0029] Continue to subculture, the inoculum volume for each T175 bottle is 1×10 6 cells, cultured for 5 days, digested for 6 min, and collected cells of 4.94 × 10 6 cells, the expansion times is 4.94, and the cell morphology at P2 is as follows Figure 7 shown.

[0030] Continue to subculture, the inoculum volume for each T175 bottle is 1×10 6cells, cultured for 6 days, digested for 4 min, and collected cells of 4.54 × 10 6 cells, the expansion times is 4.54, and the cell morphology at P3 is as follows Figure 8 The cells were subsequently passaged to P5 for the preparation of cell preparations.

[0031] Compared with Example 1, the method provided in Example 1 collected more cells than the comparative example. Furthermore, the white blood cell (WBC) extraction rates obtained by separating cells in Example 1 and the comparative example were tested. The WBC extraction rate provided by Example 1 was 82%, while that of the comparative example was 37%. Comparison of flow cytometry data revealed that the comparative example had CD73: 100%, CD90: 99.99%, and CD105: 100%.

[0032] Example 2 The present disclosure provides a separation and culture method, which includes: adding hydroxyethyl starch to a uterine blood sample and mixing it to obtain a mixed solution; in this embodiment, the uterine blood sample is obtained from a qualified donor of appropriate age who meets the requirements of a health examination, and menstrual blood is collected using a menstrual cup and stored in a collection solution at 2°C to 8°C.

[0033] Let the mixture stand; When red blood cells were observed to separate, the supernatant was aspirated; The supernatant was centrifuged to obtain a cell pellet; Resuspend the cell pellet in complete medium containing platelet lysate to obtain primary cell suspension; The primary cell suspension was cultured to obtain uterine blood mesenchymal stem cells.

[0034] Specifically, the complete culture medium for culturing platelet lysate includes: a basic culture medium DMEM / F12 and a platelet lysate at a volume ratio of 5%.

[0035] Specifically, the concentration of recombinant trypsin was 1.2 mg / mL.

[0036] Specifically, a uterine blood sample was filtered through a blood transfusion filter, and a 6% high-molecular-weight hydroxyethyl starch solution was added to the filtered uterine blood sample. The solvent of the 6% high-molecular-weight hydroxyethyl starch solution was sodium chloride, and the molecular weight of the high-molecular-weight hydroxyethyl starch ranged from 500 kDa to 800 kDa. The volume ratio of the filtered uterine blood sample to the 6% high-molecular-weight hydroxyethyl starch was 1:1. In this embodiment, the average molecular weight of the high-molecular-weight hydroxyethyl starch was approximately 670 kDa.

[0037] Furthermore, the mixture is allowed to stand for 30 to 60 minutes, preferably 40 minutes. In this embodiment, the mixture is allowed to stand for 40 minutes.

[0038] Furthermore, the supernatant was centrifuged using countercurrent centrifugation to obtain a cell pellet.

[0039] Specifically, when the primary cell suspension was cultured to obtain uterine blood mesenchymal stem cells, the culture conditions were as follows: the culture product was cultured in an incubator at 37°C and containing 5% CO2, and the medium was changed every 2 to 3 days for a total of 8 days. When the confluence reached 70 to 90%, the cells were digested with a recombinant trypsin solution for 4 minutes to obtain P0 uterine blood mesenchymal stem cells, and the collected cell amount of uterine blood mesenchymal stem cells was 1.52×10 6 cells, the morphology of uterine blood mesenchymal stem cells is as follows Figure 9 As shown. It can be adjusted to 1~1.5×10 6 cells / bottle for subculture, and the inoculation volume for each T175 bottle was 1.2×10 6 cells, cultured for 6 days, digested for 2 min, and collected cells of 5.64 × 10 6 cells, the expansion times is 4.7, and the cell morphology at P1 is as follows Figure 10 As shown, the cells were frozen at the P1 generation and stored in a liquid nitrogen tank.

[0040] Continue to subculture, the inoculum volume for each T175 bottle is 1.2×10 6 cells, cultured for 5 days, digested for 2 min, and collected cells of 6.22 × 10 6 cells, the expansion times is 5.18, and the cell morphology at P2 is as follows Figure 11 shown.

[0041] Continue to subculture, the inoculum volume for each T175 bottle is 1.2×10 6 cells, cultured for 5 days, digested for 2 min, and collected cells of 5.3 × 10 6 cells, the expansion times is 4.42, and the cell morphology at P3 is as follows Figure 12 The cells were subsequently passaged to P5 for use in cell preparation. Flow cytometry data revealed that the CD73 concentrations in this example were 99.99%, CD90 concentrations were 100%, and CD105 concentrations were 97.99%. The cells isolated and cultured using the isolation method provided in this example met the requirements for stem cell characteristics.

[0042] Comparative Example This comparative example provides a separation and culture method. The difference between this comparative example and Example 2 is that the uterine blood sample is filtered through a blood transfusion filter, the filtrate is centrifuged to obtain a precipitate, and PBS buffer containing antibiotics is added to the precipitate for centrifugal washing twice, with the centrifugation parameters set to 600g and 5 minutes. Density gradient centrifugation purification is performed at a volume ratio of 2:1, with the centrifugation parameters set to 700g and 30 minutes. After centrifugation, the buffy coat layer (mononuclear cells) is aspirated, and the buffy coat layer is added to a PBS buffer containing antibiotics for three centrifugal washings. The first centrifugation parameter is 800g and 6 minutes, and the second and third centrifugation parameters are 300g and 6 minutes. Centrifugation obtains a precipitate. The remaining parameters were the same as those in Example 2. When the primary cell suspension was cultured to obtain uterine blood mesenchymal stem cells, the culture conditions were as follows: the primary cell suspension was cultured in an incubator at 37°C and containing 5% CO2, and the medium was changed every 2 to 3 days for a total of 11 days. When the confluence reached 70 to 90%, the cells were digested with a recombinant trypsin solution for 3 minutes to obtain P0 generation uterine blood mesenchymal stem cells, and the collected cell amount of uterine blood mesenchymal stem cells was 8.65×10 5 cells, the morphology of uterine blood mesenchymal stem cells is as follows Figure 13 As shown. It can be adjusted to 1~1.5×10 6 cells / bottle for subculture, and the inoculation volume for each T175 bottle was 1.2×10 6 cells, cultured for 6 days, digested for 2 min, and collected cells of 4.14 × 10 6 cells, the expansion times is 3.45, and the cell morphology at P1 is as follows Figure 14 As shown, the cells were frozen at the P1 generation and stored in a liquid nitrogen tank.

[0043] Continue to subculture, the inoculum volume for each T175 bottle is 1.2×10 6 cells, cultured for 5 days, digested for 2 min, and collected cells of 5.74 × 10 6 cells, the expansion times is 4.78, and the cell morphology at P2 is as follows Figure 15 shown.

[0044] Continue to subculture, the inoculum volume for each T175 bottle is 1.2×10 6 cells, cultured for 4 days, digested for 3 min, and collected cells of 4.46 × 10 6 cells, the expansion times is 3.72, and the cell morphology at P3 is as follows Figure 16 The cells were subsequently passaged to P5 for the preparation of cell preparations.

[0045] Compared with Example 2, the method provided in Example 2 collected more cells than the comparative example. Furthermore, the white blood cell (WBC) extraction rates obtained by separating cells in Example 2 and the comparative example were tested. The WBC extraction rate provided by Example 2 was 79%, while that of the comparative example was 50%. Comparison of flow cytometry data showed that the comparative example had CD73: 100%, CD90: 100%, and CD105: 100%.

[0046] Example 3 The present disclosure provides a separation and culture method, which includes: adding hydroxyethyl starch to a uterine blood sample and mixing it to obtain a mixed solution; in this embodiment, the uterine blood sample is obtained from a qualified donor of appropriate age who meets the requirements of a health examination, and menstrual blood is collected using a menstrual cup and stored in a collection solution at 2°C to 8°C.

[0047] Let the mixture stand; When the red blood cells are separated, the supernatant is aspirated; The supernatant was centrifuged to obtain a cell pellet; Resuspend the cell pellet in complete medium containing platelet lysate to obtain primary cell suspension; The primary cell suspension was cultured to obtain uterine blood mesenchymal stem cells.

[0048] Specifically, the complete culture medium for culturing platelet lysate includes: a basic culture medium DMEM / F12 and a platelet lysate in a volume ratio of 2.5%.

[0049] Specifically, when the primary cell suspension is cultured to obtain uterine blood mesenchymal stem cells, the culture conditions are as follows: the culture product is cultured in an incubator at 37°C and containing 5% CO2, and the medium is changed every 2 to 3 days. When the degree of fusion reaches 70 to 90%, it is digested with a recombinant trypsin solution to obtain uterine blood mesenchymal stem cells.

[0050] Specifically, the concentration of recombinant trypsin was 1.2 mg / mL.

[0051] Specifically, a uterine blood sample is filtered through a blood transfusion filter, and a 6% hydroxyethyl starch solution is added to the filtered uterine blood sample. The solvent of the 6% medium-molecular-weight hydroxyethyl starch solution is sodium chloride, and the molecular weight of the medium-molecular-weight hydroxyethyl starch ranges from 110 kDa to 230 kDa. The volume ratio of the filtered uterine blood sample to the 6% medium-molecular-weight hydroxyethyl starch solution is 1:1. In this embodiment, the average molecular weight of the medium-molecular-weight hydroxyethyl starch is approximately 130 kDa.

[0052] Furthermore, the mixture is allowed to stand for 40 to 90 minutes, preferably 60 minutes. In this embodiment, the mixture is allowed to stand for 60 minutes.

[0053] Furthermore, the supernatant was centrifuged at a centrifugal force ranging from 600 g to 1000 g to obtain a cell pellet.

[0054] Further, the supernatant was subjected to a first centrifugation at a speed of 600-1000 g and 5 min to obtain a first cell pellet; Adding PBS buffer containing antibiotics to the first precipitate to resuspend the cell pellet to obtain a first resuspension; The first resuspension was centrifuged for the second time at 600-1000 g and 5 min to obtain a second cell pellet; Adding PBS buffer containing antibiotics to the second precipitate to resuspend the cell pellet to obtain a second resuspension; The second resuspension was centrifuged for the third time at 600-1000 g and 5 min to obtain a third cell pellet.

[0055] The centrifugation parameters for the three times in this example were all 600 g and 5 min.

[0056] Specifically, when the primary cell suspension is cultured to obtain uterine blood mesenchymal stem cells, the culture conditions are as follows: the primary cell suspension is cultured in an incubator at 37°C and containing 5% CO2, and the medium is changed every 2 to 3 days for a total of 6 days. When the confluence reaches 70 to 90%, it is digested with a recombinant trypsin solution for 12 minutes to obtain P0 uterine blood mesenchymal stem cells, and the collected cell amount of uterine blood mesenchymal stem cells is 1.32×10 6 cells, the morphology of uterine blood mesenchymal stem cells is as follows Figure 17 As shown. It can be adjusted to 1~1.5×10 6 cells / flask for subculture, and the inoculation volume for each T175 flask was 1×10 6 cells, cultured for 8 days, digested for 5 min, and collected cells of 4.35 × 10 6 cells, the expansion factor is 4.35, and the cell morphology at P1 is as follows Figure 18 As shown, the cells were frozen at the P1 generation and stored in a liquid nitrogen tank.

[0057] Continue to subculture, the inoculum volume for each T175 bottle is 1.2×10 6 cells, cultured for 5 days, digested for 12 min, and collected cells of 4.63 × 10 6 cells, the expansion times is 3.85, and the cell morphology at P2 is as follows Figure 19 shown.

[0058] Continue to subculture, the inoculum volume for each T175 bottle is 1.2×10 6cells, cultured for 6 days, digested for 12 min, and collected cells of 4.23 × 10 6 cells, the expansion times is 3.5, and the cell morphology at P3 is as follows Figure 20 The cells were subsequently passaged to P5 for the preparation of cell preparations.

[0059] The extraction rate of white blood cells (WBC) obtained by separation in Example 3 was tested. The WBC extraction rate provided in Example 3 was 98%.

[0060] The surface markers of the mesenchymal stem cells from uterine blood obtained in Example 3 were detected as CD73: 99.99%, CD90: 100%, and CD105: 99.52%. The cells isolated and cultured using the separation method provided in this example meet the requirements for stem cell characteristics.

[0061] Example 4 The present disclosure provides a separation and culture method, which includes: adding hydroxyethyl starch to a uterine blood sample and mixing it to obtain a mixed solution; in this embodiment, the uterine blood sample is obtained from a qualified donor of appropriate age who meets the requirements of a health examination, and menstrual blood is collected using a menstrual cup and stored in a collection solution at 2°C to 8°C.

[0062] Let the mixture stand; When the red blood cells are separated, the supernatant is aspirated; The supernatant was centrifuged to obtain a cell pellet; Resuspend the cell pellet in M4 complete medium to obtain primary cell suspension; The primary cell suspension was cultured to obtain uterine blood mesenchymal stem cells.

[0063] Specifically, the M4 complete culture medium includes: a basic culture medium DMEM / F12 and a serum replacement (Ultroser G) with a volume ratio of 2%.

[0064] Specifically, when the primary cell suspension is cultured to obtain uterine blood mesenchymal stem cells, the culture conditions are as follows: the culture product is cultured in an incubator at 37°C and containing 5% CO2, and the medium is changed every 2 to 3 days. When the degree of fusion reaches 70 to 90%, it is digested with a recombinant trypsin solution to obtain uterine blood mesenchymal stem cells.

[0065] Specifically, the concentration of recombinant trypsin was 1.2 mg / mL.

[0066] Further, the supernatant is centrifuged at a centrifugal force ranging from 600 g to 1000 g to obtain a precipitate.

[0067] Specifically, a uterine blood sample is filtered through a blood transfusion filter, and a 6% hydroxyethyl starch solution is added to the filtered uterine blood sample. The solvent of the 6% medium-molecular-weight hydroxyethyl starch solution is sodium chloride, and the molecular weight of the medium-molecular-weight hydroxyethyl starch ranges from 110 kDa to 230 kDa. The volume ratio of the filtered uterine blood sample to the 6% medium-molecular-weight hydroxyethyl starch solution is 1:1. In this embodiment, the average molecular weight of the medium-molecular-weight hydroxyethyl starch is approximately 130 kDa.

[0068] Furthermore, the mixture is allowed to stand for 40 to 90 minutes, preferably 60 minutes. In this embodiment, the mixture is allowed to stand for 60 minutes.

[0069] Further, the supernatant was subjected to a first centrifugation at a speed of 600-1000 g and 5 min to obtain a first cell pellet; Adding PBS buffer containing antibiotics to the first precipitate to resuspend the cell pellet to obtain a first resuspension; The first resuspension was centrifuged for the second time at 600-1000 g and 5 min to obtain a second cell pellet; Adding PBS buffer containing antibiotics to the second precipitate to resuspend the cell pellet to obtain a second resuspension; The second resuspension was centrifuged for the third time at 600-1000 g and 5 min to obtain a third cell pellet.

[0070] The centrifugation parameters for the three times in this example were all 600 g and 5 min.

[0071] Specifically, when the primary cell suspension is cultured to obtain uterine blood mesenchymal stem cells, the culture conditions are as follows: the primary cell suspension is cultured in an incubator at 37°C and containing 5% CO2, and the medium is changed every 2 to 3 days for a total of 12 days. When the confluence reaches 70 to 90%, it is digested with a recombinant trypsin solution for 8 minutes to obtain P0 uterine blood mesenchymal stem cells, and the collected cell amount of uterine blood mesenchymal stem cells is 8.16×10 5 cells, the morphology of uterine blood mesenchymal stem cells is as follows Figure 21 As shown. It can be adjusted to 0.8~1.2×10 6 cells / bottle for subculture, and the inoculation volume for each T175 bottle was 0.8×10 6 cells, cultured for 6 days, digested for 10 min, and collected cells of 5.23 × 10 6 cells, the expansion times is 6.4, and the cell morphology at P1 is as follows Figure 22 As shown, the cells were frozen at the P1 generation and stored in a liquid nitrogen tank.

[0072] Continue to subculture, the inoculum volume for each T175 bottle is 1×10 6 cells, cultured for 5 days, digested for 4 min, and collected cells of 1.06 × 10 7 cells, the expansion times is 10.6, and the cell morphology at P2 is as follows Figure 23 shown.

[0073] Continue to subculture, the inoculum volume for each T175 bottle is 1×10 6 cells, cultured for 5 days, digested for 4 min, and collected cells of 1.05 × 10 7 cells, the expansion times is 10.5, and the cell morphology at P3 is as follows Figure 24 The cells were subsequently passaged to P5 for the preparation of cell preparations.

[0074] The white blood cell (WBC) extraction rate obtained in Example 4 was detected. The WBC extraction rate provided in Example 4 was 75%.

[0075] The surface markers of the uterine blood mesenchymal stem cells obtained in Example 4 were detected to be 100% for CD73, 100% for CD90, and 98.93% for CD105. The cells isolated and cultured using the separation method provided in this example meet the requirements for stem cell characteristics.

[0076] The above description is merely an optional embodiment of the present disclosure and is not intended to limit the present disclosure. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present disclosure shall be included in the scope of protection of the present disclosure.

Claims

1. A separation and culture method, characterized in that: The separation and culture method comprises: adding hydroxyethyl starch to the uterine blood sample and mixing to obtain a mixed solution; Allowing the mixed solution to stand; When the red blood cells are separated, the supernatant is aspirated; Centrifuging the supernatant to obtain a cell pellet; resuspending the cell pellet in complete culture medium containing platelet lysate to obtain a primary cell suspension; The primary cell suspension is cultured to obtain uterine blood mesenchymal stem cells.

2. The separation and culture method according to claim 1, wherein The complete culture medium for culturing the platelet lysate includes: a basic culture medium DMEM / F12 and the platelet lysate in a volume ratio of 2% to 5%.

3. The separation and culture method according to claim 1, wherein When the primary cell suspension is cultured to obtain uterine blood mesenchymal stem cells, the culture conditions are as follows: the primary cell suspension is cultured in an incubator at 37°C and containing 5% CO2, and the medium is replaced every 2 to 3 days. When the confluence reaches 70 to 90%, it is digested with a recombinant trypsin solution to obtain the uterine blood mesenchymal stem cells.

4. The separation and culture method according to claim 1, wherein The concentration of the recombinant trypsin is 1.2 mg / mL.

5. The separation and culture method according to claim 1, wherein The uterine blood sample is filtered through a blood transfusion filter, and a 6% high molecular weight hydroxyethyl starch solution is added to the filtered uterine blood sample. The solvent of the 6% high molecular weight hydroxyethyl starch solution is sodium chloride, and the molecular weight of the high molecular weight hydroxyethyl starch ranges from 500 kDa to 800 kDa. The volume ratio of the filtered uterine blood sample to the 6% high molecular weight hydroxyethyl starch is 1:

1.

6. The separation and culture method according to claim 5, characterized in that Let it sit for 30 to 60 minutes.

7. The separation and culture method according to claim 5, wherein The supernatant is centrifuged using a countercurrent centrifugation method to obtain the precipitate.

8. The separation and culture method according to claim 1, wherein The uterine blood sample is filtered through a blood transfusion filter, and a 6% medium molecular weight hydroxyethyl starch solution is added to the filtered uterine blood sample. The solvent of the 6% medium molecular weight hydroxyethyl starch solution is sodium chloride, and the molecular weight of the medium molecular weight hydroxyethyl starch ranges from 110 kDa to 230 kDa. The volume ratio of the filtered uterine blood sample to the 6% medium molecular weight hydroxyethyl starch solution is 1:

1.

9. The separation and culture method according to claim 8, wherein Let it sit for 40 to 90 minutes.

10. The separation and culture method according to claim 8, characterized in that The supernatant is subjected to a first centrifugation at a speed of 600-1000 g for 5 min to obtain a first precipitate; adding PBS buffer containing antibiotics to the first precipitate to resuspend the precipitate to obtain a first resuspension; The first resuspension was centrifuged for the second time at 600-1000 g for 5 min to obtain a second precipitate; adding PBS buffer containing antibiotics to the second precipitate to resuspend the precipitate to obtain a second resuspension; The second resuspension was centrifuged for the third time at a speed of 600-1000 g and 5 min to obtain the precipitate.

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