WO2022217878A1 - 一种壁蜕膜间充质干细胞的制备方法、复苏方法 - Google Patents

一种壁蜕膜间充质干细胞的制备方法、复苏方法 Download PDF

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WO2022217878A1
WO2022217878A1 PCT/CN2021/125480 CN2021125480W WO2022217878A1 WO 2022217878 A1 WO2022217878 A1 WO 2022217878A1 CN 2021125480 W CN2021125480 W CN 2021125480W WO 2022217878 A1 WO2022217878 A1 WO 2022217878A1
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mesenchymal stem
stem cells
parietal
decidual
cells
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French (fr)
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刘小翠
许峻荣
唐淑艳
邓燕莲
蒙燕瑶
杨景利
王进辉
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广东唯泰生物科技有限公司
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Priority to US18/009,473 priority Critical patent/US20230227786A1/en
Publication of WO2022217878A1 publication Critical patent/WO2022217878A1/zh

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Definitions

  • the invention relates to the field of biotechnology, in particular to a preparation method and a recovery method of parietal decidua mesenchymal stem cells.
  • MSCs Mesenchymal stem cells
  • PMSCs pariduval Mesenchymal stem cells
  • PMSCs Decidual parietal mesenchymal stem cells
  • PMSCs have the characteristics of simple in vitro proliferation and culture, no ethics, good migration ability, homing to tumor cells and low immunogenicity, and avoid the bone marrow.
  • Mesenchymal stem cells are difficult to obtain and involve risks such as medical ethics and ethics, which make them a new carrier to replace bone marrow-derived mesenchymal stem cells as a new carrier for tumor biotherapy.
  • the effects of maternal-derived mesenchymal stem cells on the biological behavior of malignant tumor cells reported in different studies are different. Studying the adaptability and selectivity of PMSCs to various malignant tumors is the basis for whether PMSCs can be used as carriers for malignant tumor treatment.
  • PMSCs Existing methods for obtaining PMSCs: separate and crush placental wall decidual tissue, and adhere to culture in complete medium (DMEM with 10% FBS) for 30 days; conventional cryopreservation method: after programmed cooling in complete medium containing 10% DMSO Freeze in liquid nitrogen.
  • the technical problem to be solved by the present invention is to provide a method for preparing parietal decidual mesenchymal stem cells, the cells are easy to climb out of the tissue to adhere to the wall, the selection medium can effectively promote the growth of PMSCs, the cell concentration is high, the quality is good, and the effective suppress tumor cells.
  • the technical problem to be solved by the present invention is to provide a method for resuscitating parietal decidual mesenchymal stem cells, which can effectively maintain cell viability and biological function, and ensure that each batch of resuscitated cells has similar cell activity.
  • the technical problem to be solved by the present invention is to provide a preparation method of parietal decidual mesenchymal stem cells, comprising the following steps:
  • tissue digestion solution contains 40-60% Tryple-EDTA enzyme and 8-12 mg/ml type II Collagenase in high glucose DMEM medium;
  • the selection medium contains 8-12% serum substitute by volume, 0.5-1mol/ml L-glutamine, 18-25ng/ml basic fibroblast growth factor, 16-22ng /ml epidermal growth factor and 6-12ng/ml stem cell growth factor in DMEM serum-free medium.
  • the tissue washing solution is prepared from the following raw materials by volume percentage: 0.8-1.5% penicillin-streptomycin mixture, 50-55% red blood cell lysate, 44-49% normal saline, The mass fraction of the physiological saline is 0.8-1%.
  • step S12 the tissue digestion solution is vibrated with the tissue block at a temperature of 36 to 39° C. for 1.5 to 4 hours, and the vibration speed is 150 to 200 rpm/min;
  • the centrifugation speed is 1200 ⁇ 1400rpm/min, and the centrifugation time is 5 ⁇ 7min.
  • step S15 when the cell confluence is greater than 80%, the cell surface is washed with PBS buffer at least twice;
  • the centrifugation speed is 1200 ⁇ 1400rpm/min, and the centrifugation time is 5 ⁇ 7min.
  • the cell digestion solution includes trypsin with a mass percentage of 0.1-0.15% and EDTA with a mass percentage of 0.003-0.005%.
  • the method before collecting the parietal decidual mesenchymal stem cells, the method further includes: performing surface antibody marker detection on the parietal decidual mesenchymal stem cells, when the positive indicators of CD73, CD90 and CD105 are >99 at the same time % before collecting the parietal decidual mesenchymal stem cells.
  • step S16 the parietal decidual mesenchymal stem cells of the P3 generation are collected, the parietal decidual mesenchymal stem cells of the P3 generation are digested with trypsin, centrifuged, the supernatant is removed, and the cryopreserved solution is added to the pellet , program cooling and cryopreservation in a liquid nitrogen tank.
  • the cryopreservation solution is a serum-free complete medium containing 18-25% Cryosure-DEX-40 by volume;
  • the density of cryopreserved cells is 1.5 ⁇ 10 6 to 2.5 ⁇ 10 6 cells/ml.
  • the present invention also provides a recovery method for parietal decidual mesenchymal stem cells, comprising the following steps:
  • step S22 Resuspend the wall decidual mesenchymal stem cells in step S21 with a selective medium, centrifuge, wash the pellet with PBS buffer, centrifuge, add the selective medium to the pellet, and transfer it to a culture bottle for culture.
  • the present invention adopts the high-glucose DMEM medium containing 40-60% Tryple-EDTA enzyme and 8-12 mg/ml type II collagenase as the tissue digestion solution to digest tissue blocks, which is conducive to the crawling of parietal decidual mesenchymal stem cells out of the tissue for adherent growth.
  • the present invention adopts serum substitute containing 8-12% volume concentration, 0.5-1mol/ml L-glutamine, 18-25ng/ml basic fibroblast growth factor, 16-22ng/ml epidermal growth factor and 6 DMEM serum-free medium containing ⁇ 12ng/ml stem cell growth factor is used as a selective medium to terminate digestion, and resuspend decidual-derived mesenchymal stem cells from the parietal decidua, which is beneficial to improve the purity of parietal decidual mesenchymal stem cells and accelerate the Mesenchymal stem cell growth to achieve rapid in vitro expansion of parietal decidual mesenchymal stem cells.
  • the resuscitation method of the present invention uses a selective medium to resuscitate the parietal decidual mesenchymal stem cells, so that the parietal decidual mesenchymal stem cells can quickly recover and grow.
  • the present invention effectively maintains the viability and biological function of parietal decidual mesenchymal stem cells through the cooperation of the cryopreservation method and the recovery method, and ensures that each batch of recovered cells has similar cell activity.
  • Fig. 1 is the cell appearance diagram of the electron microscope after the P5 generation PMSCs are cultured in the upper chamber of the Transwell chamber for 3 days in Example 2 of the present invention
  • Fig. 2 is the cell appearance diagram of the electron microscope after Hela cells are cultured and stabilized in the lower chamber of the Transwell chamber in Example 3 of the present invention
  • Fig. 3 is the cell appearance diagram of the electron microscope after the co-culture of PDB-MSCs and Hela cells in the low concentration group in Example 3 of the present invention
  • Fig. 4 is the cell appearance diagram of the electron microscope after the co-culture of PDB-MSCs in the middle concentration group and Hela cells in Example 3 of the present invention
  • Fig. 5 is the cell appearance diagram of the electron microscope after the co-culture of PDB-MSCs and Hela cells in the high concentration group in Example 3 of the present invention
  • FIG. 6 is a graph showing the inhibition of the proliferation of Hela cells after the co-culture of PDB-MSCs and Hela cells in different concentration groups in Example 3 of the present invention
  • Fig. 7 is the cell appearance diagram of the electron microscope after the MCF-7 cells are cultured and stabilized in the lower chamber of the Transwell chamber in Example 4 of the present invention.
  • Fig. 8 is the cell appearance diagram of the electron microscope after the co-culture of PDB-MSCs and MCF-7 cells in the low concentration group in Example 4 of the present invention.
  • Fig. 9 is the cell appearance diagram of the electron microscope after the co-culture of PDB-MSCs and MCF-7 cells in the middle concentration group in Example 4 of the present invention.
  • Fig. 10 is the cell appearance diagram of the electron microscope after the co-culture of PDB-MSCs and MCF-7 cells in the high concentration group in Example 4 of the present invention.
  • Figure 11 is a graph showing the inhibition of the proliferation of MCF-7 cells after the co-culture of PDB-MSCs and MCF-7 cells in different concentration groups in Example 4 of the present invention.
  • the method for preparing parietal decidua mesenchymal stem cells includes the following steps:
  • tissue washing solution to wash blood and blood clots on the placental tissue of healthy full-term neonates, use surgical peeling instruments to separate the parietal decidual tissue, and then use surgical scissors to cut into 1-4 mm 3 tissue blocks And wash with tissue washing solution;
  • the tissue washing solution is prepared from the following raw materials by volume percentage: 1% penicillin-streptomycin mixture, 51.1% red blood cell lysate, 47% physiological saline, and the mass fraction of the physiological saline is 0.9% ;
  • Digestive wall decidua tissue block after adding tissue digestion solution to the chopped tissue blocks, digest with constant temperature shaking at 37° C. for 2 hours, and the rotation speed is 200 rpm/min; the tissue digestion solution contains 50% Tryple- High glucose DMEM medium with EDTA enzyme and 10mg/ml type II collagenase;
  • Termination of digestion add 3 times the volume of selective medium to terminate digestion, filter (filter mesh size is 100 ⁇ m), centrifuge at 1300 rpm/min for 5 min, and retain the precipitate;
  • the selective medium contains 10% serum substitute, DMEM serum-free medium containing 0.8mol/ml L-glutamine, 20ng/ml basic fibroblast growth factor, 20ng/ml epidermal growth factor, and 10ng/ml stem cell growth factor;
  • Cultivate PMSCs inoculate the obtained cell suspension into a T75 culture flask, place it in an incubator at 37°C, 5% CO 2 , and let it stand for primary culture, record it as P0 generation, and observe the change of the medium. , change the fluid every 2-3 days;
  • Cryopreservation use trypsin to digest the P3 generation PMSCs, add the cryopreservation solution to the precipitate after centrifugation, count and calculate the survival rate, program the cooling and cryopreservation in a liquid nitrogen tank for preservation; the S6 cryopreservation solution contains Serum-free complete medium with a volume concentration of 20% Cryosure-DEX-40; cryopreserved cell density is 2 ⁇ 10 6 cells/ml.
  • Methods of recovery of parietal decidual mesenchymal stem cell culture medium including:
  • step S22 Resuspend the wall decidual mesenchymal stem cells in step S21 with selective medium, centrifuge at 1300 rpm for 6 min, wash the cell surface twice with PBS buffer, centrifuge at 1300 rpm for 6 min, add selective medium to resuspend the pellet for subculture;
  • Co-culture test of parietal decidual mesenchymal stem cell medium and cervical cancer cells including:
  • the complete medium is a high-glucose DMEM serum-free medium containing 10% fetal bovine serum and 1% anti-penicillin by volume;
  • S36 Detection of proliferation inhibition: set a blank group, and collect Hela cells after co-cultivation in different groups to form a low-concentration co-culture group, a medium-concentration co-culture group, and a high-concentration co-culture group; The cells were plated into 96-well plates, and their proliferation was detected by CCK-8 method after 24 hours of growth.
  • Figure 3 is the cell morphology diagram of co-culture of PMSCs and Hela cells in the low concentration group
  • Figure 4 is the cell morphology diagram of the co-culture of PMSCs and Hela cells in the medium concentration group
  • Figure 5 is the cell morphology diagram of the co-culture of PMSCs and Hela cells in the high concentration group ;
  • Figure 3 takes the overlay images from left to right according to the distribution of Hela cells in the well plate (with the upper chamber as the occupied area as the distribution center of Hela cells), and the left one is the low concentration group after co-cultivation for 3 days.
  • Morphology of the outer edge of the well plate is the morphological map of Hela cells in the transition circle of the well plate after co-culture for 3 days in the low concentration group; the third from the left is the morphological map of Hela cells in the center of the well plate after co-culture for 3 days in the low concentration group; Figure 4 from the left To the right, the overlay images are taken in order according to the distribution of Hela cells in the well plate (with the upper chamber as the occupied area as the center of cell distribution). The left one is the morphological map of Hela cells on the outer edge of the well plate after co-cultivation for 3 days in the medium concentration group.
  • the second is the morphological diagram of Hela cells in the transition circle of the well plate after 3 days of co-culture in the medium concentration group;
  • the third from the left is the morphological diagram of Hela cells in the center of the well plate after 3 days of co-culture in the medium concentration group;
  • the distribution in the inner chamber (with the upper chamber as the occupied area as the cell distribution center), the superimposed images were taken sequentially.
  • the left one is the morphological diagram of Hela cells on the outer edge of the well plate after co-cultivation for 3 days in the high concentration group, and the second left is the high concentration group co-cultured for 3 days Morphological diagram of Hela cells in the transition circle of the orifice plate after 3 days; the third from the left is the morphological diagram of Hela cells in the center of the orifice plate after 3 days of co-culture in the high concentration group.
  • Figure 3- Figure 5 shows the cell morphology after co-culture It is obvious that there are significant differences in the inhibition effect of different concentrations of PMSCs on the proliferation of the same Hela cell. Within the concentration range of PMSCs selected in this example, the higher the concentration of PMSCs, the more obvious the inhibition of Hela cell proliferation.
  • Figure 6 shows the proliferation inhibition of Hela cells after co-cultivation with different concentrations of parietal decidua mesenchymal stem cell medium and Hela cells.
  • the blank group did not add parietal decidual mesenchymal stem cell medium to co-culture with Hela cells, so the growth inhibition rate of Hela cells in the blank group was zero. The higher the growth inhibition rate.
  • the complete medium is a high-glucose DMEM serum-free medium containing 10% fetal bovine serum and 1% anti-penicillin-resistant streptomycin by volume;
  • S32, MCF-7 cell passage observe the cell morphology and medium changes after resuscitation, replace the complete medium every 2 days, when the cell growth and fusion is greater than 80%, collect the cell suspension, count and calculate the survival rate, microbial detection ,pass on;
  • MCF-7 cells observe the growth of MCF-7 cells after co-cultivation for 3 days, and take pictures to record their growth state;
  • S36 Detection of proliferation inhibition: set a blank group, and collect MCF-7 cells after co-cultivation in different groups to form a low-concentration co-cultivation group, a medium-concentration co-cultivation group, and a high-concentration co-cultivation group; 5000 cells were plated into 96-well plates, and their proliferation was detected by CCK-8 method after 24 hours of growth.
  • Figure 8 is the cell morphology diagram of co-culture of PMSCs and MCF-7 cells in the low concentration group
  • Figure 9 is the cell morphology diagram of the co-culture of PMSCs and MCF-7 cells in the medium concentration group
  • Figure 10 is the high concentration group of PMSCs and MCF-7 cells
  • the morphological diagram of co-cultured cells; among them, Figure 8 is the order of the distribution of MCF-7 cells in the well plate (with the upper chamber as the occupied area as the center of cell distribution) from left to right, and the left one is the low concentration group.
  • Morphology of MCF-7 cells on the outer edge of the well plate after 3 days of co-culture is the morphological map of MCF-7 cells in the transition circle of the well plate after 3 days of co-culture in the low concentration group; the third from the left is the MCF- 7.
  • Morphological diagram of cells in the center of the well plate Figure 9 is the order of the distribution of MCF-7 cells in the well plate from left to right (with the upper chamber as the occupied area as the center of cell distribution), and the left one is the middle concentration group.
  • Morphology of MCF-7 cells on the outer edge of the well plate after 3 days of co-culture is the morphological map of MCF-7 cells in the transition circle of the well plate after 3 days of co-culture in the medium concentration group; the third from the left is the MCF-7 cells in the medium concentration group after 3 days of co-culture 7.
  • Morphological diagram of cells in the center of the well plate; Figure 10 is the order of the distribution of MCF-7 cells in the well plate from left to right (with the upper chamber as the occupied area as the center of cell distribution), and the left one is the high concentration group.
  • Morphology of MCF-7 cells on the outer edge of the well plate after 3 days of co-culture is the morphological map of the transition circle of MCF-7 cells in the well plate after 3 days of co-culture in the high-concentration group; the third from the left is the MCF- 7 Morphological diagram of cells in the center of the well plate.
  • Figure 11 shows the inhibition of proliferation of MCF-7 cells after co-culture of MCF-7 cells with different concentrations of parietal decidua mesenchymal stem cell medium.
  • the blank group did not add the parietal decidua mesenchymal stem cell medium to co-culture with MCF-7 cells, so the blank group had zero growth inhibition rate of MCF-7 cells, and the higher the concentration of parietal decidual mesenchymal stem cells was cultured The higher the rate of growth inhibition of MCF-7 cells.
  • parietal decidual mesenchymal stem cell medium has a significant inhibitory effect on the proliferation ability of cervical cancer Hela cells and breast cancer MCF-7 cells.
  • the inhibitory effect of HeLa cells was significantly higher than that of breast cancer MCF-7 cells; it can be seen that parietal decidual mesenchymal stem cell culture medium has selective and adaptive inhibitory effect on the proliferation ability of different gynecological tumors.
  • the selection medium contains 10% serum replacement by volume, 0.8mol/ml L-glutamine, 0.8mg/ml feverfew extract, 20ng/ml basic fibroblasts Growth factors, 20ng/ml epidermal growth factor, 10ng/ml stem cell growth factor in DMEM serum-free medium.
  • the feverfew extract is extracted by the existing extraction method, and the main active ingredient is feverfew lactone.
  • Example 5 The difference from Example 5 is that the selection medium contains 10% serum replacement by volume concentration, 0.8mol/ml L-glutamine, 0.8mg/ml feverfew water extract, 20ng/ml basic fibroblasts. Cell growth factor, 20ng/ml epidermal growth factor, 10ng/ml stem cell growth factor in DMEM serum-free medium.
  • the preparation method of described feverfew water extract is:
  • feverfew powder into deionized water, leaching at 75° C. for 90 minutes, filtering, and drying to obtain a feverfew water extract.
  • the weight ratio of the feverfew powder and deionized water is 1:9.
  • Example 5 The difference from Example 5 is that the selection medium contains 10% serum replacement by volume, 0.8mol/ml L-glutamine, 0.8mg/ml parthenol extract, 20ng/ml basic fibroblasts Cell growth factor, 20ng/ml epidermal growth factor, 10ng/ml stem cell growth factor in DMEM serum-free medium.
  • the preparation method of the parthenol alcohol extract is:
  • feverfew powder added to a 60wt% ethanol solution, leaching at 75° C. for 90 minutes, filtering, and drying to obtain a feverfew alcoholic extract.
  • the weight ratio of the feverfew powder to the ethanol solution is 1:9.
  • Example 1 The allogeneic wall decidual mesenchymal stem cells prepared in Example 1, Example 5, Comparative Example 1, and Comparative Example 2 were stained with trypan blue and counted, and counted with a CountStar cell counter.
  • Cell viability number of viable cells/ The total number of cells ⁇ 100%, the test results are shown in Table 1.
  • the addition of the feverfew extract described in Example 5 can significantly improve the cell viability, and compared with the water extract of feverfew and the alcoholic extract of feverfew, it can be seen that the extraction methods of different feverfew It can significantly affect the effect of improving the cell viability, and the feverfew extract obtained by the steam distillation method described in Example 5 can significantly improve the cell viability.

Abstract

提供了一种壁蜕膜间充质干细胞的制备方法和复苏方法，以及用于制备壁蜕膜间充质干细胞的组织消化液和组合试剂。还提供了壁蜕膜间充质干细胞在抑制癌细胞增殖能力中的应用。还提供了小白菊提取物在提高壁蜕膜间充质干细胞活率中的应用。

Description

一种壁蜕膜间充质干细胞的制备方法、复苏方法
本申请要求于2021年04月13日提交中国专利局、申请号为202110392680.6、发明名称为“一种壁蜕膜间充质干细胞的制备方法、复苏方法”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明涉及生物技术领域，尤其涉及一种壁蜕膜间充质干细胞的制备方法、复苏方法。
背景技术
间充质干细胞(mesenchymal stem cell，MSC)是具有趋化肿瘤能力的多能干祖细胞，在妇科肿瘤方面的研究有着显著的进展。但子宫来源间充质干细胞对妇科恶性肿瘤细胞生物学行为的影响报道不一，研究壁蜕膜MSCs(Pariduval Mesenchymal stem cell，PMSCs)与妇科肿瘤的适配性和选择性成为临床上治疗妇科肿瘤的新方法。
壁蜕膜间充质干细胞(Pariduval Mesenchymal stem cells，PMSCs)具有体外增殖培养简单、不触及道德伦理学、具有良好的迁移能力、向肿瘤细胞归巢和低免疫原性等特点，并规避了骨髓间充质干细胞获取困难、涉及医学道德伦理等风险，使其可以成为替代骨髓间充质干细胞成为肿瘤生物治疗的新载体。但研究报道的母源间充质干细胞对恶性肿瘤细胞生物学行为的影响不一，研究PMSCs与多种恶性肿瘤的适配性和选择性，是PMSCs能否成为恶性肿瘤治疗载体的基础。
现有的PMSCs获取方法：分离粉碎胎盘壁蜕膜组织，在完全培养基(10％FBS的DMEM)中贴壁培养30天；常规的冻存方法：含10％DMSO完全培养基中程序降温后冻存在液氮中。
现有PMSCs的制备方法，细胞难以爬出组织贴壁生长，且现有的培养基不能有效促进PMSCs的生长，以至于细胞的浓度低，质量差，从而影响PMSCs对肿瘤细胞的抑制效果。此外，现有PMSCs的冻存方法和复苏方法不能有效地保持细胞活性和生物学功能，且不能保证每一批复苏的细胞均具有相似地细胞活性。
发明内容
本发明所要解决的技术问题在于，提供了一种壁蜕膜间充质干细胞的制备方法，细胞便于爬出组织贴壁生长，选择培养基有效促进PMSCs的生长，细胞浓度高，质量好，有效抑制肿瘤细胞。
本发明所要解决的技术问题在于，提供了一种壁蜕膜间充质干细胞的复苏方法，有效保持细胞活率和生物学功能，并保证每一批复苏细胞均具有相似地细胞活性。
本发明还要解决的技术问题在于，提供了一种壁蜕膜间充质干细胞的制备方法，包括以下步骤：
S11、将壁蜕膜组织剪成1～4mm 3的组织块，用组织清洗液清洗组织块；
S12、清洗后用组织消化液在恒温振荡下消化组织块，终止消化，过滤，离心，保留沉淀，所述组织消化液为含有体积浓度40～60％Tryple-EDTA酶和8～12mg/mlⅡ型胶原酶的高糖DMEM培养基；
S13、用生理盐水清洗沉淀并将沉淀重悬，离心，去除上清液，加入选择培养基重悬，得到PMSCs悬液；
S14、将PMSCs悬液接种至培养瓶中，在培养箱中进行原代培养，计为P0代；
S15、当细胞融合度大于80％时，消化，过滤、离心，采用选择培养基重悬沉淀进行传代培养；
S6、收集Pn代的壁蜕膜间充质干细胞，消化，离心，去除上清液，将冻存液加入沉淀中，程序降温冻存置于液氮罐中保藏，n≥2。
作为上述方案的改进，所述选择培养基为含有体积浓度8～12％血清替代物、0.5～1mol/ml L-谷氨酰胺、18～25ng/ml碱性成纤维细胞生长因子、16～22ng/ml表皮生长因子和6～12ng/ml干细胞生长因子的DMEM无血清培养基。
作为上述方案的改进，步骤S11中，所述组织清洗液由以下体积百分比原料配制而成：0.8～1.5％青链霉素合剂，50～55％红细胞裂解液，44～49％的生理盐水，所述生理盐水为质量分数为0.8～1％。
作为上述方案的改进，步骤S12中，组织消化液在温度为36～39℃下与组织块振动1.5～4h，振荡速度为150～200rpm/min；
采用选择培养基来终止消化，选择培养基的体积为消化液体积的3～6 倍；
采用孔径为100μm的滤网来过滤；
离心速度为1200～1400rpm/min，离心时间为5～7min。
作为上述方案的改进，步骤S15中，当细胞融合度大于80％时，采用PBS缓冲液洗涤细胞表面至少2次；
采用细胞消化液消化3～6min，采用选择培养基来终止消化；
采用孔径为100μm的滤网来过滤；
离心速度为1200～1400rpm/min，离心时间为5～7min。
作为上述方案的改进，所述细胞消化液包括质量百分数为0.1～0.15％的胰蛋白酶和质量百分数为0.003～0.005％的EDTA。
作为上述方案的改进，步骤S16中，在收集壁蜕膜间充质干细胞之前，还包括：对壁蜕膜间充质干细胞进行表面抗体标记检测，当CD73、CD90和CD105的阳性指标同时>99％时，才收集壁蜕膜间充质干细胞。
作为上述方案的改进，步骤S16中，收集P3代的壁蜕膜间充质干细胞，采用胰酶消化P3代的壁蜕膜间充质干细胞，离心，去除上清液，将冻存液加入沉淀中，程序降温冻存置于液氮罐中保藏。
作为上述方案的改进，步骤S16中，所述冻存液为含有体积浓度18～25％Cryosure-DEX-40的无血清完全培养基；
冻存细胞密度为1.5×10 6～2.5×10 6个/ml。
相应地，本发明还提供了一种壁蜕膜间充质干细胞的复苏方法，包括以下步骤：
S21、将上述冻存的壁蜕膜间充质干细胞置于36～39℃的水浴中溶解；
S22、采用选择培养基重悬步骤S21的壁蜕膜间充质干细胞，离心，采用PBS缓冲液洗涤沉淀，离心，将选择培养基加入沉淀，转移至培养瓶中培养。
实施本发明，具有如下有益效果：
1、本发明采用含有体积浓度40～60％Tryple-EDTA酶和8～12mg/mlⅡ型胶原酶的高糖DMEM培养基作为组织消化液来消化组织块，有利于壁蜕膜间充质干细胞爬出组织进行贴壁生长。
2、本发明采用含有体积浓度8～12％血清替代物、0.5～1mol/ml L-谷氨酰胺、18～25ng/ml碱性成纤维细胞生长因子、16～22ng/ml表皮生长因 子和6～12ng/ml干细胞生长因子的DMEM无血清培养基作为选择培养基来终止消化，以及重悬从壁蜕膜间充质干细胞，有利于提高壁蜕膜间充质干细胞纯度，加速壁蜕膜间充质干细胞生长，实现壁蜕膜间充质干细胞的体外快速扩增。
3、本发明的复苏方法采用选择培养基来复苏壁蜕膜间充质干细胞，可以使得壁蜕膜间充质干细胞快速恢复生长。
4、本发明通过冻存方法和复苏方法的相互配合，有效保持壁蜕膜间充质干细胞的活率和生物学功能，并保证每一批复苏的细胞均具有相似的细胞活性。
说明书附图
图1是本发明实施例2中P5代PMSCs在Transwell小室的上室中培养3天后的电子显微镜的细胞外观图；
图2是本发明实施例3中Hela细胞在Transwell小室下室中培养稳定后的电子显微镜的细胞外观图；
图3是本发明实施例3中低浓度组PDB-MSCs与Hela细胞共培养后的电子显微镜的细胞外观图；
图4是本发明实施例3中中浓度组PDB-MSCs与Hela细胞共培养后的电子显微镜的细胞外观图；
图5是本发明实施例3中高浓度组PDB-MSCs与Hela细胞共培养后的电子显微镜的细胞外观图；
图6是本发明实施例3中不同浓度组PDB-MSCs与Hela细胞共培养后的，Hela细胞的增殖受抑制的情况图表；
图7是本发明实施例4中MCF-7细胞在Transwell小室下室中培养稳定后的电子显微镜的细胞外观图；
图8是本发明实施例4中低浓度组PDB-MSCs与MCF-7细胞共培养后的电子显微镜的细胞外观图；
图9是本发明实施例4中中浓度组PDB-MSCs与MCF-7细胞共培养后的电子显微镜的细胞外观图；
图10是本发明实施例4中高浓度组PDB-MSCs与MCF-7细胞共培养后的电子显微镜的细胞外观图；
图11是本发明实施例4中不同浓度组PDB-MSCs与MCF-7细胞共 培养后的，MCF-7细胞的增殖受抑制情况图表。
具体实施方式
为使本发明的目的、技术方案和优点更加清楚，下面将结合附图对本发明作进一步地详细描述。仅此声明，本发明在文中出现或即将出现的上、下、左、右、前、后、内、外等方位用词，仅以本发明的附图为基准，其并不是对本发明的具体限定。
实施例1
壁蜕膜间充质干细胞的制备方法，包括以下步骤：
S11、分离壁蜕膜组织：使用组织清洗液清洗健康足月新生儿胎盘组织上的血液和血块，使用手术剥离器械分离得到壁蜕膜组织，再用手术剪剪成1～4mm 3的组织块并用组织清洗液清洗；所述组织清洗液由以下体积百分比原料配制而成：1％青链霉素合剂，51.1％红细胞裂解液，47％的生理盐水，所述生理盐水为质量分数为0.9％；
S12、消化壁蜕膜组织块：往剪碎的组织块中加入组织消化液后，于37℃下恒温振荡消化2h，转速为200rpm/min；所述组织消化液为含有体积浓度50％Tryple-EDTA酶和10mg/mlⅡ型胶原酶的高糖DMEM培养基；
S13终止消化：加入3倍体积的选择培养基终止消化，过滤(滤网大小为100μm)，以1300rpm/min转速离心5min，保留沉淀；所述选择培养基为含有体积浓度10％血清替代物、0.8mol/ml L-谷氨酰胺、20ng/ml碱性成纤维细胞生长因子、20ng/ml表皮生长因子、10ng/ml干细胞生长因子的DMEM无血清培养基；
S14、分离得到PMSCs：采用生理盐水清洗并将沉淀重悬，得到重悬液，离心弃去上清液，加入选择培养基重悬，即得到PMSCs悬液；
S15、培养PMSCs：将得到的细胞悬液接种至T75培养瓶中，置于37℃、5％CO 2、饱和湿度的培养箱中静置原代培养，记作P0代，观察培养基变化情况，每隔2-3天换液一次；
S16、换液与传代：观察细胞增殖融合大于80％后，PBS缓冲液洗涤贴壁细胞表面分泌物，使用细胞消化液消化细胞5min，用新鲜完全培养基终止消化，用100μm滤网过滤，离心，细胞培养液重悬沉淀，进行计 数和计算存活率，检测无菌等指标，进行传代培养，每隔2～3天传代一次；所述细胞消化液包括质量分数为0.125％的胰蛋白酶和质量分数为0.004％的EDTA；
S17、所述传代培养传至P3时，进行PMSCs阳性指标检测，CD73、CD90、CD105阳性指标均>99％时，停止传代，收集细胞；
S18、冻存：采用胰酶来消化P3代的PMSCs，离心后往沉淀中加入冻存液，计数和计算存活率，程序降温冻存置于液氮罐中保藏；所述S6冻存液为含有体积浓度20％Cryosure-DEX-40的无血清完全培养基；冻存细胞密度为2×10 6个/ml。
实施例2
壁蜕膜间充质干细胞培养基的复苏方法，包括：
S21、将实施例1中冻存的壁蜕膜间充质干细胞置于37℃的水浴中溶解；
S22、采用选择培养基重悬步骤S21的壁蜕膜间充质干细胞，1300rpm离心6min，采用PBS缓冲液洗涤细胞表面2次，1300rpm离心6min，加入选择培养基重悬沉淀进行传代培养；
S23、收集P5代的PMSCs，加入选择培养基将细胞重悬成低浓度组、中浓度组和高浓度组，其中，低浓度组的PMSCs浓度为1×10 5个/孔，中浓度组的PMSCs浓度为2×10 5个/孔，高浓度组的PMSCs浓度为4×10 5个/孔，每组以每孔200μL的体积分别铺到不同Transwell小室的上室中，培养3天，PMSCs形态图如图1所示。
实施例3
壁蜕膜间充质干细胞培养基与宫颈癌细胞的共培试验，包括：
S31、将宫颈癌细胞株Hela于37℃水浴中快速溶解后，加入完全培养基重悬细胞，离心，用PBS缓冲液洗涤2次，1000rpm离心3min，加入新鲜的完全培养基重悬细胞，转移至T25培养瓶中培养，记作P1代；所述完全培养基为含有体积浓度10％胎牛血清和体积浓度1％抗青链霉素的高糖的DMEM无血清培养液；
S32、Hela细胞传代：观察复苏后的细胞形态与培养基变化情况，2天更换一次完全培养基，待细胞生长融合大于80％，收集细胞悬液，进 行计数和计算存活率，微生物检测，传代；
S33、Transwell小室下室中培养Hela细胞：弃去原培养基，PBS缓冲液洗涤2次，采用1ml的胰酶消化细胞2min，用10ml完全培养基终止消化，收集P3代的Hela细胞，加入完全培养基将Hela细胞重悬，按照每孔2×10 4个细胞(24孔板)铺板到实施例2每组的Transwell小室下室中，待细胞生长稳定(12h)，Hela的细胞形态如图2所示；
S34、共同培养：待Transwell小室下室中Hela细胞生长稳定后，将载有PMSCs的上室放入孔板内与下室Hela细胞共培养3天；
S35、Hela细胞的生长：观察共培养3天后的Hela细胞的生长情况，拍照记录其生长状态；
S36、增殖抑制作用检测：设置空白组，以及收集不同组共培养后的Hela细胞，形成低浓度共培组、中浓度共培组和高浓度共培组；具体的，每组按照每孔5000个细胞铺板至96孔板中，生长24h后使用CCK-8法检测其增殖情况。
每组试验至少重复3次，结果取平均值。
图3为低浓度组PMSCs与Hela细胞共培养的细胞形态图，图4为中浓度组PMSCs与Hela细胞共培养的细胞形态图，图5为高浓度组PMSCs与Hela细胞共培养的细胞形态图；其中，图3从左到右按照Hela细胞在孔板内的分布(以小室上室作占据区域作为Hela细胞分布中心)顺序拍摄叠加图，左一是低浓度组共培养3天后Hela细胞在孔板外沿形态图，左二是低浓度组共培养3天后Hela细胞在孔板过渡圈形态图；左三是低浓度组共培养3天后Hela细胞在孔板中心形态图；图4从左到右按照Hela细胞在孔板内的分布(以小室上室作占据区域作为细胞分布中心)顺序拍摄叠加图，左一是中浓度组共培养3天后Hela细胞在孔板外沿形态图，左二是中浓度组共培养3天后Hela细胞在孔板过渡圈形态图；左三是中浓度组共培养3天后Hela细胞在孔板中心形态图；图5从左到右按照Hela细胞在孔板内的分布(以小室上室作占据区域作为细胞分布中心)顺序拍摄叠加图，左一是高浓度组共培养3天后Hela细胞在孔板外沿形态图，左二是高浓度组共培养3天后Hela细胞在孔板过渡圈形态图；左三是高浓度组共培养3天后Hela细胞在孔板中心形态图。
从图3-图5中可以看到，越接近载有PMSCs小室的Hela细胞数量越稀疏，同时出现外缘Hela细胞向小室中心迁移的现象；图3-图5的共培养后的细胞形态图显著表明不同浓度的PMSCs对同一种Hela细胞增殖作用的抑制存在显著差异，在该实施例选取的PMSCs浓度范围内表现出，PMSCs浓度越高，抑制Hela细胞增殖作用越明显。
图6是不同浓度的壁蜕膜间充质干细胞培养基与Hela细胞共培养后，Hela细胞的增殖抑制情况。其中，空白组没有加入壁蜕膜间充质干细胞培养基与Hela细胞共培，因此空白组对Hela细胞的增长抑制率为零，浓度越高的壁蜕膜间充质干细胞培养基对Hela细胞的增长抑制率越高。
实施例4
壁蜕膜间充质干细胞培养基与乳腺癌细胞的共培试验，包括：
S31、将乳腺癌细胞株MCF-7于37℃水浴中快速溶解后，加入完全培养基重悬细胞，离心，用PBS缓冲液洗涤2次，1000rpm离心3min，加入新鲜的完全培养基重悬细胞，转移至T25培养瓶中培养，记作P1代；所述完全培养基为，含有体积浓度10％胎牛血清和体积浓度1％抗青链霉素的高糖的DMEM无血清培养液；
S32、MCF-7细胞传代：观察复苏后的细胞形态与培养基变化情况，2天更换一次完全培养基，待细胞生长融合大于80％，收集细胞悬液，进行计数和计算存活率，微生物检测，传代；
S33、Transwell小室下室中培养MCF-7细胞：弃去原培养基，PBS缓冲液洗涤2次，采用1ml的胰酶消化细胞2min，用10ml完全培养基终止消化，收集P3代的MCF-7细胞，加入完全培养基将MCF-7细胞重悬，按照每孔2×10 4个细胞(24孔板)铺板到实施例2每组的Transwell小室下室中，待细胞生长稳定(12h)，MCF-7的细胞形态如图7所示；
S34、共同培养：待Transwell小室下室中MCF-7细胞生长稳定后，将载有PMSCs的上室放入孔板内与下室MCF-7细胞共培养3天；
S35、MCF-7细胞的生长：观察共培养3天后的MCF-7细胞的生长情况，拍照记录其生长状态；
S36、增殖抑制作用检测：设置空白组，以及收集不同组共培养后的MCF-7细胞，形成低浓度共培组、中浓度共培组和高浓度共培组；具体 的，每组按照每孔5000个细胞铺板至96孔板中，生长24h后使用CCK-8法检测其增殖情况。
每组试验至少重复3次，结果取平均值。
图8为低浓度组PMSCs与MCF-7细胞共培养的细胞形态图，图9为中浓度组PMSCs与MCF-7细胞共培养的细胞形态图，图10为高浓度组PMSCs与MCF-7细胞共培养的细胞形态图；其中，图8从左到右按照MCF-7细胞在孔板内的分布(以小室上室作占据区域作为细胞分布中心)顺序拍摄叠加图，左一是低浓度组共培养3天后MCF-7细胞在孔板外沿形态图，左二是低浓度组共培养3天后MCF-7细胞在孔板过渡圈形态图；左三是低浓度组共培养3天后MCF-7细胞在孔板中心形态图；图9从左到右按照MCF-7细胞在孔板内的分布(以小室上室作占据区域作为细胞分布中心)顺序拍摄叠加图，左一是中浓度组共培养3天后MCF-7细胞在孔板外沿形态图，左二是中浓度组共培养3天后MCF-7细胞在孔板过渡圈形态图；左三是中浓度组共培养3天后MCF-7细胞在孔板中心形态图；图10从左到右按照MCF-7细胞在孔板内的分布(以小室上室作占据区域作为细胞分布中心)顺序拍摄叠加图，左一是高浓度组共培养3天后MCF-7细胞在孔板外沿形态图，左二是高浓度组共培养3天后MCF-7细胞在孔板过渡圈形态图；左三是高浓度组共培养3天后MCF-7细胞在孔板中心形态图。
从图8-图10中可以看到，越接近载有PMSCs小室的MCF-7细胞数量越稀疏，同时出现外缘MCF-7细胞向小室中心迁移的现象；图8-图10的共培养后的细胞形态图显著表明不同浓度的PMSCs对同一种MCF-7细胞增殖作用的抑制存在显著差异，在该实施例选取的PMSCs浓度范围内表现出，PMSCs浓度越高，抑制MCF-7细胞增殖作用越明显。
图11是不同浓度的壁蜕膜间充质干细胞培养基与MCF-7细胞共培养后，MCF-7细胞的增殖抑制情况。其中，空白组没有加入壁蜕膜间充质干细胞培养基与MCF-7细胞共培，因此空白组对MCF-7细胞的增长抑制率为零，浓度越高的壁蜕膜间充质干细胞培养基对MCF-7细胞的增长抑制率越高。
从图6和图11中可以看出，壁蜕膜间充质干细胞培养基对宫颈癌Hela 细胞的增殖能力、乳腺癌MCF-7细胞的增殖能力都具有显著的抑制作用，其中，对于宫颈癌Hela细胞的抑制作用明显高于乳腺癌MCF-7细胞；由此可知，壁蜕膜间充质干细胞培养基对不同妇科肿瘤增殖能力的抑制作用具有选择性和适配性。
实施例5
与实施例1不同的是，所述选择培养基为含有体积浓度10％血清替代物、0.8mol/ml L-谷氨酰胺、0.8mg/ml小白菊提取物、20ng/ml碱性成纤维细胞生长因子、20ng/ml表皮生长因子、10ng/ml干细胞生长因子的DMEM无血清培养基。
小白菊提取物采用现有的提取方法来提取，主要有效成分为小白菊内酯。
实施例6
与实施例5不同的是，所述选择培养基为含有体积浓度10％血清替代物、0.8mol/ml L-谷氨酰胺、0.8mg/ml小白菊水提取物、20ng/ml碱性成纤维细胞生长因子、20ng/ml表皮生长因子、10ng/ml干细胞生长因子的DMEM无血清培养基。
所述小白菊水提物的制备方法为：
S41、将小白菊粉碎至100目，得到小白菊粉末；
S42、将小白菊粉末加入到去离子水中，在75℃下浸提90min，过滤，干燥，即得小白菊水提物，所述小白菊粉末与去离子水重量比为1：9。
实施例7
与实施例5不同的是，所述选择培养基为含有体积浓度10％血清替代物、0.8mol/ml L-谷氨酰胺、0.8mg/ml小白菊醇提取物、20ng/ml碱性成纤维细胞生长因子、20ng/ml表皮生长因子、10ng/ml干细胞生长因子的DMEM无血清培养基。
所述小白菊醇提物的制备方法为：
S51、将小白菊粉碎至100目，得到小白菊粉末；
S52、将小白菊粉末加入到60wt％乙醇溶液中，在75℃下浸提90min，过滤，干燥，即得小白菊醇提物，所述小白菊粉末与乙醇溶液重量比为1：9。
将实施例1、实施例5、对比例1、对比例2制备所得的异体壁蜕膜间充质干细胞用台盼蓝染色后计数，采用CountStar细胞计数仪计数，细胞活率＝活细胞数/总细胞数×100％，测试结果见表1。
表1测试结果
组别 实施例1 实施例5 实施例6 实施例7
细胞活率(％) 85 95.9 86.5 86.1
从表1中可看出，实施例5所述的小白菊提取物的加入能够显著提高细胞活率，且对比于小白菊水提物、小白菊醇提物，可见，不同小白菊的提取方法能够显著影响对于细胞活率的提高效果，实施例5所述的水蒸气蒸馏法提取得到的小白菊提取物能够著提高细胞活率。
尽管上述实施例对本发明做出了详尽的描述，但它仅仅是本发明一部分实施例，而不是全部实施例，人们还可以根据本实施例在不经创造性前提下获得其它实施例，这些实施例都属于本发明保护范围。

Claims (17)

  1. 用于制备壁蜕膜间充质干细胞的组织消化液，为含有体积浓度40～60％Tryple-EDTA酶和8～12mg/mlⅡ型胶原酶的高糖DMEM培养基。
  2. 用于制备壁蜕膜间充质干细胞的组合试剂，包括组织消化液和选择培养基，所述组织消化液为含有体积浓度40～60％Tryple-EDTA酶和8～12mg/mlⅡ型胶原酶的高糖DMEM培养基；
    所述选择培养基为含有体积浓度8～12％血清替代物、0.5～1mol/ml L-谷氨酰胺、18～25ng/ml碱性成纤维细胞生长因子、16～22ng/ml表皮生长因子和6～12ng/ml干细胞生长因子的DMEM无血清培养基。
  3. 根据权利要求2所述的组合试剂，其特征在于，还包括组织清洗液，所述组织清洗液由以下体积百分比原料配制而成：0.8～1.5％青链霉素合剂，50～55％红细胞裂解液，44～49％的生理盐水，所述生理盐水的质量分数为0.8～1％。
  4. 根据权利要求2或3所述的组合试剂，其特征在于，还包括细胞消化液，所述细胞消化液包括质量百分数为0.1～0.15％的胰蛋白酶和质量百分数为0.003～0.005％的EDTA。
  5. 根据权利要求2～4任意一项所述的组合试剂，其特征在于，还包括冻存液，所述冻存液为含有体积浓度18～25％Cryosure-DEX-40的无血清完全培养基。
  6. 一种壁蜕膜间充质干细胞的制备方法，其特征在于，包括以下步骤：
    S11、将壁蜕膜组织剪成1～4mm 3的组织块，用组织清洗液清洗组织块；
    S12、清洗后用组织消化液在恒温振荡下消化组织块，终止消化，过滤，离心，保留沉淀，所述组织消化液为含有体积浓度40～60％Tryple-EDTA酶和8～12mg/mlⅡ型胶原酶的高糖DMEM培养基；
    S13、用生理盐水清洗沉淀并将沉淀重悬，离心，去除上清液，加入选择培养基重悬，得到PMSCs悬液；
    S14、将PMSCs悬液接种至培养瓶中，在培养箱中进行原代培养，计为P0代；
    S15、当细胞融合度大于80％时，消化，过滤、离心，采用选择培养 基重悬沉淀进行传代培养；
    S6、收集Pn代的壁蜕膜间充质干细胞，消化，离心，去除上清液，将冻存液加入沉淀中，程序降温冻存置于液氮罐中保藏，n≥2。
  7. 如权利要求6所述的壁蜕膜间充质干细胞的制备方法，其特征在于，所述选择培养基为含有体积浓度8～12％血清替代物、0.5～1mol/ml L-谷氨酰胺、18～25ng/ml碱性成纤维细胞生长因子、16～22ng/ml表皮生长因子和6～12ng/ml干细胞生长因子的DMEM无血清培养基。
  8. 如权利要求6所述的壁蜕膜间充质干细胞的制备方法，其特征在于，步骤S11中，所述组织清洗液由以下体积百分比原料配制而成：0.8～1.5％青链霉素合剂，50～55％红细胞裂解液，44～49％的生理盐水，所述生理盐水的质量分数为0.8～1％。
  9. 如权利要求6所述的壁蜕膜间充质干细胞的制备方法，其特征在于，步骤S12中，组织消化液在温度为36～39℃下与组织块恒温振动1.5～4h，振荡速度为150～200rpm/min；
    采用选择培养基终止消化，选择培养基的体积为消化液体积的3～6倍；
    所述过滤为采用孔径为100μm的滤网过滤；
    离心速度为1200～1400rpm/min，离心时间为5～7min。
  10. 如权利要求6所述的壁蜕膜间充质干细胞的制备方法，其特征在于，步骤S15中，当细胞融合度大于80％时，采用PBS缓冲液洗涤细胞表面至少2次；
    采用细胞消化液消化3～6min，采用选择培养基来终止消化。
  11. 如权利要求6所述的壁蜕膜间充质干细胞的制备方法，其特征在于，所述细胞消化液包括质量百分数为0.1～0.15％的胰蛋白酶和质量百分数为0.003～0.005％的EDTA。
  12. 如权利要求6所述的壁蜕膜间充质干细胞的制备方法，其特征在于，步骤S16中，在收集Pn代的壁蜕膜间充质干细胞之前，还包括：对Pn代的壁蜕膜间充质干细胞进行表面抗体标记检测，当CD73、CD90和CD105的阳性指标同时>99％时，才收集壁蜕膜间充质干细胞。
  13. 如权利要求6或11所述的壁蜕膜间充质干细胞的制备方法，其特征在于，步骤S16中，收集P3代的壁蜕膜间充质干细胞，采用胰酶消化 P3代的壁蜕膜间充质干细胞，离心，去除上清液，将冻存液加入沉淀中，程序降温冻存置于液氮罐中保藏。
  14. 如权利要求6所述的壁蜕膜间充质干细胞的制备方法，其特征在于，步骤S16中，所述冻存液为含有体积浓度18～25％Cryosure-DEX-40的无血清完全培养基；
    冻存细胞密度为1.5×10 6～2.5×10 6个/ml。
  15. 一种壁蜕膜间充质干细胞的复苏方法，其特征在于，包括以下步骤：
    S21、将权利要求6～14所述制备方法得到的冻存壁蜕膜间充质干细胞置于36～39℃的水浴中溶解；
    S22、采用选择培养基重悬步骤S21的壁蜕膜间充质干细胞，离心，采用PBS缓冲液洗涤沉淀，离心，将选择培养基加入沉淀，转移至培养瓶中培养。
  16. 壁蜕膜间充质干细胞在抑制癌细胞增殖能力中的应用；所述癌细胞包括宫颈癌细胞和/或乳腺癌细胞。
  17. 小白菊提取物在提高壁蜕膜间充质干细胞活率中的应用，所述小白菊提取物的主要有效成分为小白菊内酯，所述小白菊提取物包括小白菊水提物、小白菊醇提物和水蒸气蒸馏法提取得到的小白菊提取物中的一种或两种以上。
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