CN109251890A - A kind of method and its application of high efficiency extraction endometrium mescenchymal stem cell - Google Patents
A kind of method and its application of high efficiency extraction endometrium mescenchymal stem cell Download PDFInfo
- Publication number
- CN109251890A CN109251890A CN201811226089.8A CN201811226089A CN109251890A CN 109251890 A CN109251890 A CN 109251890A CN 201811226089 A CN201811226089 A CN 201811226089A CN 109251890 A CN109251890 A CN 109251890A
- Authority
- CN
- China
- Prior art keywords
- stem cell
- menses
- mescenchymal stem
- endometrium
- culture
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0662—Stem cells
- C12N5/0668—Mesenchymal stem cells from other natural sources
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P15/00—Drugs for genital or sexual disorders; Contraceptives
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Developmental Biology & Embryology (AREA)
- Biomedical Technology (AREA)
- General Health & Medical Sciences (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Cell Biology (AREA)
- Organic Chemistry (AREA)
- Biotechnology (AREA)
- Immunology (AREA)
- Medicinal Chemistry (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Animal Behavior & Ethology (AREA)
- Wood Science & Technology (AREA)
- Pharmacology & Pharmacy (AREA)
- Genetics & Genomics (AREA)
- Epidemiology (AREA)
- Endocrinology (AREA)
- Hematology (AREA)
- Microbiology (AREA)
- Rheumatology (AREA)
- General Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Virology (AREA)
- Reproductive Health (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses Endometrium mescenchymal stem cell preparation and the methods applied in the treatment such as premature ovarian failure, endometrial impairment, including menstrual cup is used to collect menses as raw material；The endometrial tissue and menses liquid in menses are separated by the screen to filtrate；Then about 10-20ml digestive juice is added after collecting endometrial tissue chopping, digestion obtains single cell suspension, and carries out originally culture to it；Bacteria removing is carried out to menses liquid, intermediate film confluent monolayer cells are obtained using gradient centrifugation, and originally culture is carried out to it；Endometrium mescenchymal stem cell is obtained to the primary endometrium mescenchymal stem cell secondary culture of acquisition.The endometrium mescenchymal stem cell of acquisition is used for the treating correlative diseases such as premature ovarian failure, endometrial impairment.Using the extracting method for the endometrium mescenchymal stem cell that the invention patent technology provides, endometrium mescenchymal stem cell long-term cultivation process in vitro can be maintained, maintains cellular morphology, proliferative capacity, the expression of MSC surface markers, differentiation capability etc..Used method is simple and easy, easy to operate, and energy maximum obtains required stem cell, and cultivates success.
Description
Technical field
The present invention relates to stem cells technology fields, and endometrium mesenchyma is prepared from discarded menses more particularly to one kind
Stem cell and the method applied in the treatment such as premature ovarian failure, endometrial impairment.
Background technique
Mescenchymal stem cell (Mesenchymal Stem Cell, MSC) is a kind of adult stem cell, has self-renewing
Ability and multi-lineage potential, and immunogenicity is low, and heteroplastic transplantation is weaker without immunological rejection or reaction, therefore is regenerating
Medical domain has huge potential.The endometrium of people is a highly efficient regeneration tissue, and 400 are undergone in Women of Childbearing Age
Multiple proliferation breaks up and falls off.In recent years more and more researches show that, there are a small set of with powerful in endometrial tissue
Proliferation and multi-lineage potential stem cell, can continuous division growth, the terminally differentiated cells to fall off are replenished in time, make intrauterine
Film remains self-renewing and power of regeneration, and this adult stem cell is referred to as endometrium mescenchymal stem cell.The uterus
Inner membrance mescenchymal stem cell is with typical mescenchymal stem cell characteristic: having self-renewing and multi-lineage potential, and has
The special mark molecule expression of mescenchymal stem cell.Separating mesenchymal stem cell can avoid dispute of ethic, source from menses
It is abundant, turn waste into wealth, clinically can be used for the disease treatments such as premature ovarian failure, endometrial impairment, have a wide range of applications valence
Value.
Currently, the method for separating uterus inner membrance mescenchymal stem cell has the disadvantage in that (1) menstruation blood height in existing menses
Viscosity and high osmosis, the efficiency with water or ammonium salt lysed erythrocyte is very low, and cannot assist the endometrium group isolated and fallen off
It knits, endometrium mescenchymal stem cell easily sticks.(2) for large volume through blood sample, using in gradient centrifugation separating uterus
Film mescenchymal stem cell needs a large amount of lymphocyte separation mediums to be unfavorable for industrialization production so that separation costs are higher；And have bright
Aobvious red blood cell contamination and condensation is fast, these can all significantly reduce the extracted amount of endometrium mescenchymal stem cell.(3) for son
Endometrium mescenchymal stem cell method in terms of the disease treatments such as premature ovarian failure, endometrial impairment needs to be further improved.Cause
This, from menses there is at high cost, low efficiency in the technology of separating mesenchymal stem cell at present；To endometrium mesenchyma
Method of the stem cell in the diseases such as premature ovarian failure, endometrial impairment is left to be desired.
Summary of the invention
The object of the present invention is to provide a kind of efficient acquisition endometrium mescenchymal stem cell and in premature ovarian failure, uterus
The method applied in the treatment such as inner film injury.This method can isolate a large amount of, high quality son under conditions of lower cost
Endometrium mescenchymal stem cell is applied to industrialization production and clinical use.
Being separately cultured for people's endometrium mescenchymal stem cell provided by the present invention is trained with culture medium, including zooblast basis
Support base, fetal calf serum (FCS), epidermal growth factor (EGF) and platelet derived growth factor (PDGF)；The fetal calf serum
Final concentration of 1-200 mL/L, the final concentration of 0.1-150ng/ml of the epidermal growth factor, the platelet derived growth
The final concentration of 0.1-150ng/ml of the factor.
Wherein, the final concentration of the fetal calf serum is preferably 10-100 mL/L, especially preferably 10-30 ml/L；The epidermis
The final concentration of growth factor is preferably 5-50ng/ml；The final concentration of the platelet derived growth factor is preferably 5-50ng/
mll。
The zooblast basal medium can for DMEM/F12, MCDB-201, DMEM, MEM, RPMI1640, DMEM, M199,
In BME and IMEM etc. any one or any combination thereof.
The method provided by the present invention for being separately cultured people's endometrium mescenchymal stem cell is passed through using menstrual cup acquisition women
First day phase to the 4th day menses as raw material, by 100-200 μm of strainer by the endometrial tissue and menses in menses
Liquid separation, then separates and collects people's endometrial tissue from endometrial tissue and menses liquid respectively.It collects on strainer
Endometrial tissue, be cut into 1mm3Then size digests people's endometrial tissue, collect cell, collected cell is connect
Kind is added any of the above-described kind of culture medium and is cultivated in culture vessel (culture bottle, culture dish including different size etc.),
Adherent cell collecting；The attached cell is passed on 2 times or 2 times or more in the special culture media, obtains people's endometrium
Mescenchymal stem cell；The menses liquid for penetrating strainer is collected, cell precipitation is collected by centrifugation, the physiology salt of 1-5 times of precipitation capacity is added
Water mixes, and is slowly laid in the 50 ml sterile centrifugation tubes added with 5 times of human lymphocyte separating liquids.Intermediate thin is collected after centrifugation
Film layer cell passes on 2 times or 2 times or more in the special culture media, obtains people's endometrium mescenchymal stem cell.
In the above method, collected cell presses 4 × 105A/ml-1 × 107The inoculum density of a/ml is inoculated in culture vessel
In；Preferred inoculum density is 2 × 106A/ml culture medium.
In the above method, the environmental condition of the culture is 50ml/L CO2, 37 DEG C and 95% relative air humidity.
Endometrium mescenchymal stem cell established by the present invention repair it is a kind of efficiently obtain endometrium mescenchymal stem cell and
It is that it is subjected to Regeneration and Repair with the compound implantation lesions position of biomaterial in the methods of premature ovarian failure, endometrial impairment.
Isolated culture method of the invention is suitable for separating uterus inner membrance mescenchymal stem cell, the side of being separately cultured from menses
Method is simple, at low cost, high-efficient.Cultivating obtained endometrium mescenchymal stem cell has CD34- and HLA-DR-, and
The phenotype of CD44+, CD90+, CD105+；It can be induced to differentiate into osteocyte, lipocyte, cartilage cell in vitro.The present invention
The endometrium mescenchymal stem cell that method obtains can Applied experimental study and clinical research and application, mentioned for cellular transplantation therapy
New approach is supplied.
Detailed description of the invention
Fig. 1 is the endometrium mescenchymal stem cell figure of originally culture
Fig. 2 is flow cytometer showed endometrium mescenchymal stem cell surface markers figure
Fig. 3 is growth of mesenchymal stem cells curve graph
Fig. 4 is into rouge, Osteoinductive differentiation figure.A, Osteoblast Differentiation；B, break up at rouge
Claims (14)
1. a kind of from preparing endometrium mescenchymal stem cell in menses and answered in the treatment such as premature ovarian failure, endometrial impairment
Method, which comprises the following steps:
1) use first day to the 4th day in menstrual cup acquisition female menstrual period menses as raw material；Collected menses are poured into
Equipped in the sterile tube for saving liquid, 4 DEG C are saved.
2.2) it will be immersed in the menses that menses save in liquid in 0-72 hours and is transported to experiment, by 100-200 μm of sieve by menses
In endometrial tissue separated with menses liquid, then respectively from collector's intrauterine in endometrial tissue and menses liquid
Membrane tissue.
3.3) from extracting mescenchymal stem cell in endometrial tissue: be added in the endometrium shredded about 1-4 times volume by
The digestive juice of the clostridiopetidase A composition of the trypsase and 0.05-2% of 0.05-2.5%, 25 DEG C -37 DEG C digestion 15-60min are obtained
Single cell suspension.
4.4) the separating mesenchymal stem cell from filtered menses liquid: 1-5 times of blueness/strepto- is added in filtered menses liquid
Element and anphotericin, are placed in 2-8 DEG C of processing 8-24 h of refrigerator.
5.5) isometric physiological saline is added in filtered cell precipitation to mix, is slowly laid in human lymphocyte separating liquid
Sterile centrifugation tube, 1500-3000 rpm are centrifuged 10-30min, collect intermediate tunica albuginea confluent monolayer cells.
6. being seeded in culture bottle, it is placed in 37 DEG C, 50ml/LCO2It is cultivated in 95% relative air humidity incubator.
7.6) by step 3) handle 8-24h filtrate using ficoll partition method obtain intermediate film confluent monolayer cells, and to its into
Row originally culture.
8.7) by step 5) and step 6) separation obtain karyocyte be placed in endometrium mescenchymal stem cell culture medium into
Row selectivity culture, shuttle shape attached cell is the endometrium mescenchymal stem cell.
9.8) the endometrium mescenchymal stem cell obtained in step 7) is used for the diseases such as premature ovarian failure, endometrial impairment
Treatment.
10. the method as described in claim 1, which is characterized in that 1-5 times of volume is added in step 1), in menses and contains 0.1%-
The physiological saline of 5% penicillin, 0.1% -5% streptomysin and 1-4 μ g/ml amphotericin B, mixing are filtered.
11. the method as described in claim 1, which is characterized in that in step 2), the endometrium impregnated in 0-72 hours is used
100-200 μm of sieve is filtered separation, collects and obtains endometrial tissue；
The method as described in claim 1, which is characterized in that in step 3), the 5- of 1-4 times of volume is added in the endometrium shredded
100U/ml DNA enzymatic, the trypsase of 0.05-2.5% and 0.01-0.5% clostridiopetidase A composition digestive juice, be placed in 25 DEG C-
37 DEG C of digestion 15-60min obtain single cell suspension.
12. the method as described in claim 1, which is characterized in that 1-5 times of volume is added in step 4), in supernatant fluid filtrate and contains
0.5% -25% penicillin, 0.5% -25% streptomysin and 1-4 μ g/ml amphotericin B are placed in 4 DEG C of refrigerator processing for 24 hours.
13. the method as described in claim 1, which is characterized in that in step 5), collected using density gradient centrifugation method white
Film layer collects process using 1500-3000 rpm and is centrifuged 10-30min, is centrifuged by the way of slowly stopping, after collection, kind
It plants in sterile culture consumptive material；
The method as described in claim 1, which is characterized in that in step 6), the filtrate that 8-24h is handled in step 3) is applied
Ficoll partition method obtains intermediate film confluent monolayer cells, and carries out originally culture to it.
14. the method as described in claim 1, which is characterized in that karyocyte merging is selected in step 5) and step 6)
Property culture, obtain the adherent cell of shuttle shape and carry out secondary culture.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811226089.8A CN109251890A (en) | 2018-10-22 | 2018-10-22 | A kind of method and its application of high efficiency extraction endometrium mescenchymal stem cell |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811226089.8A CN109251890A (en) | 2018-10-22 | 2018-10-22 | A kind of method and its application of high efficiency extraction endometrium mescenchymal stem cell |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN109251890A true CN109251890A (en) | 2019-01-22 |
Family
ID=65045697
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201811226089.8A Pending CN109251890A (en) | 2018-10-22 | 2018-10-22 | A kind of method and its application of high efficiency extraction endometrium mescenchymal stem cell |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109251890A (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111040992A (en) * | 2019-12-16 | 2020-04-21 | 杭州恩格生物医疗科技有限公司 | Separation culture method of endometrial stem cells |
| CN114717185A (en) * | 2022-03-31 | 2022-07-08 | 秦岭大熊猫研究中心(陕西省珍稀野生动物救护基地) | Separation and culture method of giant panda placenta mesenchymal stem cells |
| CN115386538A (en) * | 2021-05-24 | 2022-11-25 | 上海长一干细胞研究中心有限公司 | Method for separating, extracting and culturing endometrial stem cells from menstrual blood and special culture medium |
| CN116515745A (en) * | 2023-04-27 | 2023-08-01 | 陕西朗泰生物科技有限公司 | Preparation method of stem cells for female premature ovarian failure reproduction repair |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103966162A (en) * | 2014-05-29 | 2014-08-06 | 成都清科生物科技有限公司 | Novel menstrual blood-derived mesenchymal stem cell separation method |
| CN104560871A (en) * | 2014-12-29 | 2015-04-29 | 深圳市北科生物科技有限公司 | Culturing method of mesenchymal stem cells of menstrual blood |
| CN105586308A (en) * | 2016-02-04 | 2016-05-18 | 杭州易文赛生物技术有限公司 | Stem cell culture medium and method for culturing endometrium stem cells |
| CN107629998A (en) * | 2017-08-24 | 2018-01-26 | 浙江大学 | A kind of stem cell in vitro reparation with menstrual blood source is damaged the model of endometrium |
-
2018
- 2018-10-22 CN CN201811226089.8A patent/CN109251890A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103966162A (en) * | 2014-05-29 | 2014-08-06 | 成都清科生物科技有限公司 | Novel menstrual blood-derived mesenchymal stem cell separation method |
| CN104560871A (en) * | 2014-12-29 | 2015-04-29 | 深圳市北科生物科技有限公司 | Culturing method of mesenchymal stem cells of menstrual blood |
| CN105586308A (en) * | 2016-02-04 | 2016-05-18 | 杭州易文赛生物技术有限公司 | Stem cell culture medium and method for culturing endometrium stem cells |
| CN107629998A (en) * | 2017-08-24 | 2018-01-26 | 浙江大学 | A kind of stem cell in vitro reparation with menstrual blood source is damaged the model of endometrium |
Non-Patent Citations (2)
| Title |
|---|
| HAIYAN ZHU ET AL.: "Human menstrual blood-derived stem cells promote the repair of impaired endometrial stromal cells by activating the p38 MAPK and AKT signaling pathways", 《REPRODUCTIVE BIOLOGY》 * |
| 乔联桥 等: "从子宫内膜间充质干细胞(eMSC)角度看妇科再生医疗", 《转化医学电子杂志》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111040992A (en) * | 2019-12-16 | 2020-04-21 | 杭州恩格生物医疗科技有限公司 | Separation culture method of endometrial stem cells |
| CN115386538A (en) * | 2021-05-24 | 2022-11-25 | 上海长一干细胞研究中心有限公司 | Method for separating, extracting and culturing endometrial stem cells from menstrual blood and special culture medium |
| CN114717185A (en) * | 2022-03-31 | 2022-07-08 | 秦岭大熊猫研究中心(陕西省珍稀野生动物救护基地) | Separation and culture method of giant panda placenta mesenchymal stem cells |
| CN116515745A (en) * | 2023-04-27 | 2023-08-01 | 陕西朗泰生物科技有限公司 | Preparation method of stem cells for female premature ovarian failure reproduction repair |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN109251890A (en) | A kind of method and its application of high efficiency extraction endometrium mescenchymal stem cell | |
| CN106701672B (en) | Human adipose-derived mesenchymal stem cell factor and preparation method and application thereof | |
| CN106754674B (en) | The method and its application of amnion mesenchymal stem cell are prepared from Human plactnta amnion | |
| CN109880797A (en) | A method of preparing human umbilical cord mesenchymal stem cells excretion body | |
| CN103074298B (en) | A kind of Human fat mesenchymal stem cell bank and construction process thereof | |
| CN107746829B (en) | Method for separating and primary culturing dog placenta-derived mesenchymal stem cells | |
| CN107164319B (en) | Method for primary culture of canine umbilical cord-derived mesenchymal stem cells | |
| CN102676452A (en) | Culture medium containing human umbilical cord mesenchymal stem cell exudates and preparation method and applications thereof | |
| CN106085952A (en) | A kind of placental villi plate mescenchymal stem cell and extracting method thereof | |
| CN103667187A (en) | Isolated culture method of human adipose-derived stem cells and construction method of stem cell bank | |
| CN104450611A (en) | Primary separation and culture method of human amniotic mesenchymal stem cells | |
| CN113249317A (en) | Isolated culture and amplification method and system for human umbilical cord mesenchymal stem cells | |
| CN103223194A (en) | Cartilage graft for cartilage injury repair and preparation method thereof | |
| CN105434468A (en) | Preparation method of skin cell damage repairing reagent | |
| CN105238749A (en) | Method for resuscitating mesenchymal stem cells | |
| CN112553155A (en) | Umbilical cord blood mesenchymal stem cell culture method | |
| CN105861432A (en) | In vitro efficient amplification method for human umbilical cord blood hematopoietic stem cells | |
| CN103215223B (en) | Method for constructing in vitro model of interaction of human intervertebral disc nucleus pulposus cell and immune cell | |
| CN106367346A (en) | Mesenchymal stem cell extraction and perfusion culture system | |
| CN100564518C (en) | Placenta amnion cell extract and induce application in the differentiation at mescenchymal stem cell | |
| CN107354130B (en) | Human placenta chorion mesenchymal stem cell separation method | |
| CN109576216A (en) | Urinate the extraction and amplification cultivation method of derived mesenchymal stem cell | |
| CN110628706B (en) | Method for extracting and culturing embryonic neural stem cells in vitro and preparation of culture medium | |
| CN115820548A (en) | Preparation method and application of apoptotic vesicles derived from animal tissues | |
| CN109666633A (en) | A method of improving brown fat stem cell myocardiac differentiation efficiency |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20190122 |
|
| RJ01 | Rejection of invention patent application after publication |