CN1746297A - Placenta-derived mesenchymal stem cells and their preparation - Google Patents
Placenta-derived mesenchymal stem cells and their preparation Download PDFInfo
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Abstract
本发明涉及胎盘来源间充质干细胞及其制备，属于干细胞和组织工程领域。胎盘从子宫娩出后立即放血、灌注。然后采用持续灌注或胶原酶消化法获得单细胞悬液，后者经密度梯度离心法或免疫磁珠分离法获得间充质干细胞。所获得的间充质干细胞具有与骨髓来源的间充质干细胞类似的体外增殖潜力和多向分化潜力。该细胞群还具有免疫原性较低的特点，是组织工程和细胞治疗潜在的种子细胞来源，具有重要的临床意义。The invention relates to placenta-derived mesenchymal stem cells and preparation thereof, belonging to the field of stem cells and tissue engineering. The placenta was exsanguinated and perfused immediately after delivery from the uterus. Then, the single cell suspension was obtained by continuous perfusion or collagenase digestion, and the mesenchymal stem cells were obtained by density gradient centrifugation or immunomagnetic bead separation. The obtained mesenchymal stem cells have in vitro proliferation potential and multilineage differentiation potential similar to bone marrow-derived mesenchymal stem cells. The cell group also has the characteristics of low immunogenicity, and is a potential source of seed cells for tissue engineering and cell therapy, and has important clinical significance.
Description
Invention field
The present invention relates to placenta derived mesenchymal stem cell and preparation thereof, belong to stem cell and field of tissue engineering technology.
Background technology
The seed cell source is the matter of utmost importance of organizational project and cell therapy.Owing to autologous tissue's cell is originated less, the amplification difficulty, lose phenotype during vitro culture easily, therefore people bet attention on stem cell gradually in recent years.These cells have certain self ability external, can under specific inductive condition, be divided into a kind of, multiple, even all cell types of human body, thereby shown the good clinical application prospect.Separate the stem cell that obtains so far and comprise two kinds, be i.e. embryonic stem cell and adult stem cell.Embryonic stem cell has all-round differentiation capability and unlimited multiplication capacity, but be subjected to ethics with this in the deficiency of separating, cultivate, inducing differentiation and aspect human knowledge such as immunogenicity and controlling, can't be applied to clinical in a short time.Adult stem cell is to separate a class stem cell that obtains from the tissue of adult, and these cells have certain in-vitro multiplication ability, can be divided into one or more cell types under certain conditions in external or body.What research was maximum at present is the mescenchymal stem cell of derived from bone marrow, and this cell is drawn materials comparatively easy.Its hetero-organization comprises in the adult tissues such as fat, muscle, nerve, dental pulp and to find that also the existence of stem cell is arranged, but draws materials and separate comparatively speaking comparatively difficulty.And these cells all can cause wound to the patient when drawing materials, thereby are not easy to be accepted by the patient; The amplification in vitro condition also is not easy control, cost is big.The problem that another one is not allowed to ignore is that these cells all have certain antigenicity, can bring the immunological rejection problem during heteroplastic transplantation, therefore can be restricted when clinical application.Based on this, seek the source of human stem cell that a kind of harvest yield is big, immunogenicity is little and obtain easily and have more actual application value.
Placenta is the appurtenant of fetus, also is a temporary organ, has promptly finished historic mission after fetus is given birth to, and all abandoned is handled usually.The complex structure of placenta own is made up of vascular system, mesenchyme composition and nurse cell.Except that having female youngster's exchange of substance function, still an important secretory is being carried out extremely complex physiological functions.In recent years studies show that to have in the placenta tissue and the similar attached cell of mesenchymal stem cells MSCs.Report such as external Jaroscak (2000) separation and Culture from frozen placenta tissue goes out heterogeneous attached cell.Report such as domestic Zhang Yi adopts enzyme digestion to obtain the sustenticular cell of attached cell as hemopoietic stem cell from placenta tissue.Therefore, placenta tissue is expected to become the abundant source of potential of mescenchymal stem cell.The a small amount of that only has at present both at home and abroad fragmentary document to relate to placenta source attached cell is separated, and is mainly used in hematopoiesis support research, the technical data that mass-producing separation, purifying and the amplification method of placenta derived mesenchymal stem cell then lacked.The objective of the invention is to disclose the mescenchymal stem cell in a kind of placenta source and the method for from placenta tissue, obtaining mescenchymal stem cell on a large scale, be specifically related to separation, purifying, evaluation and the amplification of cell.
Summary of the invention
The present invention is the mescenchymal stem cell in a kind of placenta source and preparation method thereof.This cell is taken from the healthy placenta tissue of depleted in postpartum, and this cell mass has and the similar biological characteristics of the mescenchymal stem cell of derived from bone marrow, is cell therapy and Tissue Engineering Study and application potential cell source.Its preparation method is as follows:
1) the fresh placenta of giving birth to is carried out bloodletting, perfusion immediately, to remove hemocyte and fragment of tissue;
2) continue perfusion, or placental lobules is shredded, use collagenase digesting, the collecting cell composition;
3) adopt immunomagnetic beads absorption method or Percoll density gradient centrifugation to collect mononuclearcell;
4) adopt flow cytometer or external evoked differential method to identify the phenotype and the cell cycle feature of the cell mass that obtains;
5) measure cell doubling time and growth curve;
6) adopt conventional external evoked system, the mescenchymal stem cell in external evoked placenta source is divided into the ability of scleroblast, chondrocyte, adipocyte.
7) mescenchymal stem cell and the allochthonous peripheral blood T-lymphocyte co-cultivation that placenta is originated, whether the mescenchymal stem cell that detects the placenta source stimulates the T-lymphopoiesis, whether suppress the mediation of allogeneic dendritic cell, do not rely on the T-lymphopoiesis that cell mechanism produces that passes through of excreted factor; The mescenchymal stem cell in placenta source is injected subcutaneous rat, detect the immune response situation, to determine the immunogenicity of this cell.
8) adopt bio-reactor that the cell that is obtained is carried out the mass-producing amplification.
Beneficial effect
The present invention extracts mescenchymal stem cell from postpartum the depleted placenta tissue.This cell is drawn materials easily, the output height, and external easy amplification can keep cell phenotype and multidirectional differentiation capability in the amplification procedure.And this cell mass also has lower immunogenicity, thereby is cell therapy, gene therapy, and bone and cartilage tissue engineered ideal seed cell source, has broad clinical application prospect.
Embodiment
Embodiment 1 perfusion obtains placenta derived mesenchymal stem cell
Place aseptic pallet or container after placenta is given birth to immediately, inject through Umbilical artery and contain antithrombotics and antibiotic balanced salt solution, bloodletting, and begin perfusion.Antithrombotics can be the heparin of 1 ~ 100u/mL, and microbiotic can be the mycillin of 100u/mL.Collect perfusion liquid, centrifugal acquisition cellular constituent suspends cellular constituent more again, with density is the percoll of 1.073g/mL, with 500g centrifugal force centrifugal 30 minutes, and results interface cellular layer, PBS washing back inoculation culture bottle or culture plate, or be directly used in other purpose.Also can separate the mescenchymal stem cell that has certain special marking with the immunomagnetic beads of D7-FIB or NGFR mark.
Embodiment 2 enzyme digestions obtain placenta derived mesenchymal stem cell
Place aseptic pallet or container after placenta is given birth to immediately, inject through Umbilical artery and contain antithrombotics and antibiotic balanced salt solution, bloodletting, and begin perfusion.Antithrombotics can be the heparin of 1 ~ 100u/mL, and microbiotic can be the mycillin of 100u/mL.The aseptic clip placental lobules in preliminary perfusion back, with trypsinase or collagenase digesting placenta tissue, obtain cell suspension, again cell suspension is crossed the cell sieve, remove fragment of tissue and hemocyte, the cell that obtains suspends again, with density is the percoll of 1.073g/mL, with 500g centrifugal force centrifugal 30 minutes, and results interface cellular layer, PBS washing back inoculation culture bottle or culture plate, or be directly used in other purpose.Also can separate the mescenchymal stem cell that has certain special marking with the immunomagnetic beads of D7-FIB or NGFR mark.
The evaluation of embodiment 3 placenta derived mesenchymal stem cells
Induce the mescenchymal stem cell in placenta source to be divided into scleroblast, chondrocyte or adipocyte according to the method for the external evoked mesenchymal stem cells MSCs differentiation of bibliographical information.Adopt the phenotype of flow cytometer detection placenta derived mesenchymal stem cell, as CD29, CD34, CD44, CD45, CD105, CD166, HLA-DR etc.Detect the cell cycle simultaneously, determine to be in G
0-G
1The ratio of phase cell.
The result shows that the mescenchymal stem cell of originating through the placenta after external evoked can be divided into scleroblast, chondrocyte, adipocyte.Fluidic cell shows this cell CD29, CD44, CD105, the CD166 positive with detection, CD34, CD45, HLA-DR feminine gender.The cell that is in the G0-G1 phase accounts for more than 95%.
Claims (9)
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| Application Number | Priority Date | Filing Date | Title |
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| CNA2004100095347A CN1746297A (en) | 2004-09-09 | 2004-09-09 | Placenta-derived mesenchymal stem cells and their preparation |
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| CNA2004100095347A CN1746297A (en) | 2004-09-09 | 2004-09-09 | Placenta-derived mesenchymal stem cells and their preparation |
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| CN1746297A true CN1746297A (en) | 2006-03-15 |
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Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2010040262A1 (en) * | 2008-10-10 | 2010-04-15 | 深圳市嘉天源生物科技有限公司 | Methods for isolating animal embryonic mesenchymal stem cells and extracting secretion substance thereof |
| CN101492654B (en) * | 2008-03-17 | 2011-02-16 | 协和干细胞基因工程有限公司 | Method for using umbilical stalk placenta to prepare mesenchyma stem cell |
| CN102146359A (en) * | 2011-01-13 | 2011-08-10 | 王泰华 | Method for extracting original mesenchymal stem cells from placenta and serum-free amplification |
| CN102433301A (en) * | 2011-12-02 | 2012-05-02 | 上海安集协康生物技术有限公司 | Method for extracting and amplifying monoclonal mesenchymal stem cells and culture solution for same |
| CN102550543A (en) * | 2012-02-10 | 2012-07-11 | 四川新生命干细胞科技股份有限公司 | Liquid for preserving umbilical cord tissue |
| CN103124493A (en) * | 2010-08-14 | 2013-05-29 | 齐托内两合公司 | Process for the preparation of disinfected human cell suspensions |
| CN103555664A (en) * | 2013-11-19 | 2014-02-05 | 武汉道培胎盘干细胞生物技术有限公司 | Method for cultivating human placenta mesenchymal stem cells |
| CN104818264A (en) * | 2015-05-29 | 2015-08-05 | 广州赛莱拉干细胞科技股份有限公司 | Digestive enzyme composition, and preparation and application thereof |
| CN104928234A (en) * | 2015-07-07 | 2015-09-23 | 河南中科干细胞基因工程有限公司 | Method for extracting various placenta stem cells |
| CN106754680A (en) * | 2015-12-31 | 2017-05-31 | 中国人民解放军军事医学科学院放射与辐射医学研究所 | A kind of separation method of placenta derived stem cells and its application |
| CN116144583A (en) * | 2023-04-19 | 2023-05-23 | 北京雪颜博润生物科技有限公司 | Method for obtaining human placenta embryo-derived stem cells and application |
-
2004
- 2004-09-09 CN CNA2004100095347A patent/CN1746297A/en active Pending
Cited By (14)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101492654B (en) * | 2008-03-17 | 2011-02-16 | 协和干细胞基因工程有限公司 | Method for using umbilical stalk placenta to prepare mesenchyma stem cell |
| WO2010040262A1 (en) * | 2008-10-10 | 2010-04-15 | 深圳市嘉天源生物科技有限公司 | Methods for isolating animal embryonic mesenchymal stem cells and extracting secretion substance thereof |
| CN103124493A (en) * | 2010-08-14 | 2013-05-29 | 齐托内两合公司 | Process for the preparation of disinfected human cell suspensions |
| CN102146359A (en) * | 2011-01-13 | 2011-08-10 | 王泰华 | Method for extracting original mesenchymal stem cells from placenta and serum-free amplification |
| CN102433301A (en) * | 2011-12-02 | 2012-05-02 | 上海安集协康生物技术有限公司 | Method for extracting and amplifying monoclonal mesenchymal stem cells and culture solution for same |
| CN102550543A (en) * | 2012-02-10 | 2012-07-11 | 四川新生命干细胞科技股份有限公司 | Liquid for preserving umbilical cord tissue |
| CN103555664A (en) * | 2013-11-19 | 2014-02-05 | 武汉道培胎盘干细胞生物技术有限公司 | Method for cultivating human placenta mesenchymal stem cells |
| CN103555664B (en) * | 2013-11-19 | 2015-11-04 | 武汉道培胎盘干细胞生物技术有限公司 | A kind of method of cultivator placenta mesenchyma stem cell |
| CN104818264A (en) * | 2015-05-29 | 2015-08-05 | 广州赛莱拉干细胞科技股份有限公司 | Digestive enzyme composition, and preparation and application thereof |
| CN104928234A (en) * | 2015-07-07 | 2015-09-23 | 河南中科干细胞基因工程有限公司 | Method for extracting various placenta stem cells |
| CN104928234B (en) * | 2015-07-07 | 2019-01-01 | 河南中科干细胞基因工程有限公司 | A method of extracting a variety of stem cells of placenta |
| CN106754680A (en) * | 2015-12-31 | 2017-05-31 | 中国人民解放军军事医学科学院放射与辐射医学研究所 | A kind of separation method of placenta derived stem cells and its application |
| CN116144583A (en) * | 2023-04-19 | 2023-05-23 | 北京雪颜博润生物科技有限公司 | Method for obtaining human placenta embryo-derived stem cells and application |
| CN116144583B (en) * | 2023-04-19 | 2023-08-25 | 北京雪颜博润生物科技有限公司 | Method for obtaining human placenta embryo-derived stem cells and application |
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