WO2016049986A1 - Procédé de séparation de cellules souches mésenchymateuses de cordon ombilical - Google Patents

Procédé de séparation de cellules souches mésenchymateuses de cordon ombilical Download PDF

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WO2016049986A1
WO2016049986A1 PCT/CN2014/094957 CN2014094957W WO2016049986A1 WO 2016049986 A1 WO2016049986 A1 WO 2016049986A1 CN 2014094957 W CN2014094957 W CN 2014094957W WO 2016049986 A1 WO2016049986 A1 WO 2016049986A1
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umbilical cord
mesenchymal stem
cells
medium
stem cells
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PCT/CN2014/094957
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English (en)
Chinese (zh)
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钟立武
赖真阳
范兆心
伍明俊
张博
陈强
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四川新生命干细胞科技股份有限公司
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Publication of WO2016049986A1 publication Critical patent/WO2016049986A1/fr

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  • tissue block attachment method enzymatic digestion method has high cost, easy damage to cells and even cell mutation, and there is great risk in clinical application
  • tissue block adherence method has The utility model has the advantages of simple operation, low cost and small cell damage, and thus is more suitable for clinical application, but the conventional tissue block adherence method obtains a longer cell cycle and low efficiency of the umbilical cord mesenchymal stem cells, resulting in the number of primary cells obtained. Less, and the purity is not high, easy to be mixed with endothelial cells, can not meet the international standard of clinical use of MSC, resulting in clinical application is more difficult.
  • the shortening is a small fragment cut to a length of 1 to 1.5 cm.
  • the concentration of the gelatin is 0.1 to 0.5% (w/v); and the concentration of the Cellstart matrix is 1% to 2% (w/v).
  • the concentration of the gelatin is 0.2% (w/v); the concentration of the Cellstart matrix is 2% (w/v).
  • the culture time before the addition of the medium is 2 h.
  • the present invention also provides umbilical cord mesenchymal stem cells prepared by the aforementioned method.
  • Figure 7 shows the results of flow cytometry
  • Tissue block method for separation of umbilical cord mesenchymal stem cells Cut the fresh umbilical cord into 1cm length with ophthalmic scissors, and clean the umbilical cord blood stain with PBS; cut the umbilical cord to 2 ⁇ 3mm 3 size tissue block every 1cm length, and evenly spread it in the culture dish.
  • the cells were cultured for 2 h at 37 ° C under 5% CO 2 , then 10 ml of mesenchymal stem cell medium (STEMPRO MSC SFM, purchased from Life Technologies) was added to continue the culture under the same conditions; on the 5th day, the medium was half-exchanged; On day 8 all changes were made and all tissue pieces were removed; cells were harvested at a cell fusion of 30-40%.
  • STEMPRO MSC SFM mesenchymal stem cell medium
  • Petri dish pretreatment 10 cm cell culture dishes were coated with 1 ml of 1% Cellstart matrix, 0.2% Cellstart matrix, 0.2% gelatin overnight to remove residual matrix; the control group did not coat the matrix.
  • Tissue block method for separation of umbilical cord mesenchymal stem cells Cut the fresh umbilical cord into 1cm length with ophthalmic scissors, and clean the umbilical cord blood stain with PBS; place the umbilical cord 1cm long in a gelatin-coated petri dish, add 1ml fetal bovine serum and cut the umbilical cord Broken to 2 ⁇ 3mm 3 size tissue block and evenly spread in the culture dish; placed at 37 ° C 5% CO 2 for 2h, then add mesenchymal stem cell medium (LG-DMEM / F12 and 10% fetal bovine serum 10 ml continued to be cultured under the same conditions; medium half-liquid change was performed on day 5; all tissue pieces were removed on day 8 and all tissue blocks were removed; cells were harvested on day 11 and cell counts were performed.
  • mesenchymal stem cell medium LG-DMEM / F12 and 10% fetal bovine serum
  • the number of primary cells obtained by the control group was (104 ⁇ 55) ⁇ 10 4 per dish per 1 cm length of umbilical cord; the number of primary cells obtained by gelatin coating was (179 ⁇ 99) ⁇ 10 4 per dish; The primary cells obtained by the experimental group were (287 ⁇ 98) ⁇ 10 4 / dish;
  • Example 1 The primary cells of umbilical cord mesenchymal stem cells were prepared in Example 1. The medium in the culture dish was removed and rinsed twice with PBS; 1 ml of 0.05% trypsin (containing 0.02% EDTA) was placed in each dish and placed.
  • Chondrogenic induction experiment The umbilical cord mesenchymal stem cells were adjusted to a density of 2 ⁇ 10 7 cells/ml, and the cell pellet was inoculated into a 6-well plate to add a chondrogenic induction medium (high glucose DMEM, 10% fetal bovine serum, ascorbic acid 50 ⁇ g/ Ml, 100 nmol/L dexamethasone, 40 ⁇ g/ml proline, 50 mg/ml ITS and TGF- ⁇ 110 ng/ml); the induction medium was changed every three days and stained with toluidine blue 14 days later.
  • a chondrogenic induction medium high glucose DMEM, 10% fetal bovine serum, ascorbic acid 50 ⁇ g/ Ml, 100 nmol/L dexamethasone, 40 ⁇ g/ml proline, 50 mg/ml ITS and TGF- ⁇ 110 ng/ml

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  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

L'invention concerne un procédé de séparation de cellules mésenchymateuses de cordon ombilical, comprenant les étapes suivantes, consistant à : (1) prendre un cordon ombilical frais, le découper et enlever les taches de sang; (2) découper en blocs de tissu d'un volume de 2-3 mm3, les étaler uniformément sur un récipient de culture revêtu d'une matrice extracellulaire et cultiver dans des conditions de 37°C et 5 % de CO2 pendant 1-4 h puis ajouter un milieu de culture pour les cellules souches mésenchymateuses et continuer la culture dans des conditions de 37°C et 5 % de CO2 et (3) le cinquième jour de culture, changer la moitié du milieu de culture; le huitième jour de culture, retirer tous les blocs de tissu et changer complètement le milieu de culture; et récolter les cellules à une confluence cellulaire de 30-40 %.
PCT/CN2014/094957 2014-09-29 2014-12-25 Procédé de séparation de cellules souches mésenchymateuses de cordon ombilical WO2016049986A1 (fr)

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CN201410515345.0A CN105524877A (zh) 2014-09-29 2014-09-29 一种脐带间充质干细胞的分离方法
CN201410515345.0 2014-09-29

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CN110438071A (zh) * 2019-08-26 2019-11-12 广东唯泰生物科技有限公司 人脐带华通氏胶间充质干细胞及其制备方法
CN110577929A (zh) * 2019-09-18 2019-12-17 安徽科门生物科技有限公司 一种脐带间充质干细胞的分离培养方法
CN110628713A (zh) * 2019-11-07 2019-12-31 安徽科门生物科技有限公司 一种脂肪源干细胞提取制备方法
CN113528344A (zh) * 2021-07-22 2021-10-22 青岛思拓新源细胞医学有限公司 一种脐带组织间充质干细胞培养组件及使用方法
CN113604428A (zh) * 2021-08-19 2021-11-05 上海纳米技术及应用国家工程研究中心有限公司 一种人脐带来源间充质干细胞的原代分离方法
CN113980895A (zh) * 2020-07-27 2022-01-28 山东荆卫生物科技有限公司 一种改良人脐带间充质干细胞培养基和人脐带间充质干细胞分离纯化方法
CN114921408A (zh) * 2022-05-25 2022-08-19 河北三臧生物科技有限公司 一种体外规模化培养扩增间充质干细胞的方法

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CN105970300A (zh) * 2016-05-11 2016-09-28 黑龙江天晴干细胞股份有限公司 一种采用血清替代物建立人脐带间充质干细胞库的方法
CN108660108A (zh) * 2017-03-31 2018-10-16 四川新生命干细胞科技股份有限公司 一种可增强脐带间充质干细胞免疫调节能力的方法
CN107236702A (zh) * 2017-05-24 2017-10-10 北京再生生物科技研究院有限公司 一种围产期脐带源间充质干细胞主细胞库的制备方法
CN111235102A (zh) * 2020-03-26 2020-06-05 苏州梅赛生物技术有限公司 一种脐带间充质干细胞原代培养方法
CN113652397A (zh) * 2020-05-12 2021-11-16 辽宁医学诊疗科技研发中心有限公司 一种无动物源性脐带间充质干细胞的培养方法
CN114438024A (zh) * 2022-02-21 2022-05-06 四川熙熙里生命健康研究中心 一种干细胞制剂的制备方法
CN117603909B (zh) * 2024-01-19 2024-04-12 四川新生命干细胞科技股份有限公司 高免疫抑制的UCMSCs及其培养方法、试剂与应用

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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110438071A (zh) * 2019-08-26 2019-11-12 广东唯泰生物科技有限公司 人脐带华通氏胶间充质干细胞及其制备方法
CN110577929A (zh) * 2019-09-18 2019-12-17 安徽科门生物科技有限公司 一种脐带间充质干细胞的分离培养方法
CN110628713A (zh) * 2019-11-07 2019-12-31 安徽科门生物科技有限公司 一种脂肪源干细胞提取制备方法
CN113980895A (zh) * 2020-07-27 2022-01-28 山东荆卫生物科技有限公司 一种改良人脐带间充质干细胞培养基和人脐带间充质干细胞分离纯化方法
CN113528344A (zh) * 2021-07-22 2021-10-22 青岛思拓新源细胞医学有限公司 一种脐带组织间充质干细胞培养组件及使用方法
CN113528344B (zh) * 2021-07-22 2023-12-01 青岛思拓新源细胞医学有限公司 一种脐带组织间充质干细胞培养组件及使用方法
CN113604428A (zh) * 2021-08-19 2021-11-05 上海纳米技术及应用国家工程研究中心有限公司 一种人脐带来源间充质干细胞的原代分离方法
CN114921408A (zh) * 2022-05-25 2022-08-19 河北三臧生物科技有限公司 一种体外规模化培养扩增间充质干细胞的方法

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