CN109182260A - A kind of method of in vitro culture fetal membrane mescenchymal stem cell - Google Patents
A kind of method of in vitro culture fetal membrane mescenchymal stem cell Download PDFInfo
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- CN109182260A CN109182260A CN201811057204.3A CN201811057204A CN109182260A CN 109182260 A CN109182260 A CN 109182260A CN 201811057204 A CN201811057204 A CN 201811057204A CN 109182260 A CN109182260 A CN 109182260A
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- 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
Abstract
The invention discloses a kind of methods of in vitro culture fetal membrane mescenchymal stem cell, comprising the following steps: the acquisition and model foundation of step 1, people's fetal membrane sample；Step 2, fetal membrane mesenchymal stem cell transplantation；Step 3, the preparation of fetal membrane mescenchymal stem cell.The present invention uses state-of-the-art stem cells technology, blocks fetal membrane cut using biomembrane, and effect becomes apparent from, and is more advantageous to clinical application.
Description
Technical field
The invention belongs to medicine technology fields, and in particular to a kind of method of in vitro culture fetal membrane mescenchymal stem cell.
Background technique
Premature rupture of membranes (Preterm premature rupture ofmembranes, PPROM) refer to gestation
The just before giving birth preceding spontaneous rupture of membranes occurred in 37 weeks.Premature rupture of fetal membranes incidence after gestation is 37 weeks full is 10%, and gestation is discontented
37 weeks prematures rupture of fetal membranes, that is, premature rupture of membranes incidence is 2. 0~3. 5%, and it is early to be discontented with the mid-term fetal membrane occurred for 28 weeks
Broken incidence is 0.4% -0.7%.Although nowadays pole survival rate of premature infants has had clear improvement under medical condition, overall survival
Rate is still lower, generally abnormal with body development short-term and at a specified future date, and economic drain is high.It is pregnant to improve premature rupture of membranes pregnant woman
Final result of being pregnent is clinical hot spot, and repairing fetal membrane cut is even more world-famous puzzle.
The ideal method for treating PPROM is amniotic cavity block therapy, so that amniotic cavity is in closed state again, has both reduced pregnant
Woman's infection of amniotic cavity rate, and amniotic fluid volume can be made to gradually increase and restore normal, baby sends out caused by reducing due to hapamnion
Educate it is slow with it is lopsided.
Currently used closed material mainly has:
1. Fibrin Glue: natural macromolecular material is derived from mammal ox and the serum of people.Fibrin Glue can simulate
The last one step of blood coagulation chain forms a semirigid fibrin clot, and functional closing is presented in amniotic cavity, rather than dissects
Learn closing.
2. amnion sticking patch: the fibrin bracket of external structure, main component are blood platelet and cryoprecipitate, and amnion is broken
The fibr tissue of mouth exposure can excite the aggregation, adherency and activation of blood platelet, be partially formed embolus blocking cut.Its mechanism of action
It is considered blood platelet and collagen mud, the mixture of Fibrin Glue can form a kind of physiological embolism and be adhered to chorion decidua
Basal surface simultaneously forms a kind of mixture for promoting healing, and clinical trial proves that intra-amniotic injection can effectively treat doctor source
PPROM, and it is also indefinite for spontaneous PPROM effect.
3. gelfoam:, there is scholar to carry out embolotherapy on the basis of cerclage of cervix, improves curative effect.It is external real
< fetal membrane the cut of 7mm can be closed by issuing after examination and approval existing gelfoam bolt.
4. collagen plugs: a kind of natural macromolecular material similar with Fibrin Glue, being partially formed collagenous fibres can close
The curative effect that defective tissue, collagen plugs and matrix sticking patch are used to treat iatrogenic PPROM has also obtained the card of animal model experiment
It is real.
5. biomembrane: thering is part article to report novel membrane-biological membrane, but belong to case, be only limitted to experiment in vitro, correlative study is still
It is less
Currently, the closing mode used mainly has:
1. cervical injection: generally selection Fibrin Glue.Baumgarten is through cervical injection Fibrin Glue and is equipped with uterine neck ring
It pricks art and treats 26 26-33 weeks patients, the pregnancy period extends 3-172 days, and 17 are obtained baby living.
2. through amniotic cavity injection: Quincy injection blood platelet and cryoprecipitate have treated 7 iatrogenic morning and have broken patient, pregnant age
16-20 weeks, wherein 4 patient's amniotic cavities reclose, leakage of amniotic fluid stopped, and index of amniotic fluid increases in various degree, 2 gestation
Extremely mature, 4 are obtained health baby living, but amnion sticking patch fails to close spontaneous premature rupture of membranes patient.
3. injecting through fetoscope: Young is one have been implemented under fetoscope secondary to the postoperative patient of amniocentesis
Amniotic cavity block technique, when art, are age 20 weeks pregnant.It is accurately positioned behind fetal membrane cut position and injects pregnant woman's autologous platelet, fibre at the position
Fibrillarin glue, postoperative leakage of amniotic fluid stop.
Presently, there are the problem of: such as the side reaction of closed material, closed material is as a kind of heterologous substance, fibrin
The chemical component and cow's serum of glue can increase the probability of intrauterine infection；There are heterologous for allosome blood platelet used by amnion sticking patch
The danger of reaction, and, somewhat expensive many and diverse from donor method program, limit clinical use.It furthermore include Fibrin Glue, gelatin
Closed material including sponge all exists accidentally to be inhaled by fetus, is influenced to swallow, is caused the undesirable danger of development of fetus.In injecting pathway
Aspect needs a kind of wound smaller and exposes the injecting method for understanding fetal membrane cut position.Closed performance conveniently prefers to reach
Anatomy sealing effect.
It is an emerging technology, the biological characteristics of mescenchymal stem cell that mescenchymal stem cell, which repairs premature rupture of fetal membranes technology,
Property: mescenchymal stem cell (MSC) is a kind of adult stem cell with self-renewing, multi-lineage potential.MSC is early derived from development
The mesoderm of phase belongs to non-terminally differentiated cells.Its abundance can use a variety of derived from marrow, Cord blood, fat, placenta etc.
Tissue.Its existing interstitial cell, and have the feature of endothelial cell and epithelial cell.In addition, it is there are also tissue repair and is immunized
The characteristic of inhibition.
Mescenchymal stem cell due to its can Multidirectional Differentiation be purpose cell, secrete cytokines promote tissue repair；It is immune
The characteristic of adjusting and hematopoiesis support is applied to the treatment of a variety of diseases.With the development of regenerative medicine and to mescenchymal stem cell life
The further investigation of object characteristic, MSC are widely used in injury repair as seed cell.A large amount of zooperies and face early period
Bed experiments have shown that, MSC can by directed differentiation substitute denaturation or missing dopaminergic neuron and secretion it is various have mind
Bioactie agent through nutrition, anti-apoptotic, anti-inflammatory effect improves the symptom of Parkinson's disease.There are also animal experiment study tables
Bright, MSC has the function of promoting hematopoiesis.Placenta mesenchyma is dry to have lower immunogenicity, does not cause immunological rejection, also has and exempt from
The effect of epidemic disease regulation.
Currently, being badly in need of a kind of method of in vitro culture fetal membrane mescenchymal stem cell in the prior art.
Summary of the invention
The present invention provides a kind of method of in vitro culture fetal membrane mescenchymal stem cell.
Its technical solution are as follows:
A kind of method of in vitro culture fetal membrane mescenchymal stem cell, comprising the following steps:
The acquisition and model foundation of step 1, people's fetal membrane sample:
People's fetal membrane of term birth healthy fetus is obtained, sterile tissue pincers remove female decidua, cut square sample, be placed on 24 orifice plates
In, sample is incubated for disinfection with 70% ethyl alcohol of 3 milliliters of 3mL in distilled water, then by sample with phosphate buffered saline (PBS) (PBS)
It washs three times, and internal diameter is that 1 centimetre of becket is fixed to shaft bottom, to form the culture well for being used for cell inoculation.
Step 2, fetal membrane mesenchymal stem cell transplantation
With the DMEM culture medium inoculated MSC of 5ml on every tunic, 37 DEG C, 5% CO2Under, after culture 2 hours, to allow cell attached
, becket is removed, and the DMEM of 2ML is added in condition of culture, and changes weekly three times.With 5x106The cell of/ml is close
Degree drops evenly stem cell suspension until surface is hydraulically full on the surface ADM, plants again after 1 hour, continuous plantation 3 times, kind
Enough Amino.MAX II culture medium+20%FBS are added in plant after finishing 1 hour, be placed in standard incubation case and cultivate 3 days.
Step 3, the preparation of fetal membrane mescenchymal stem cell.
Further, in step 1, the side length of the square sample is 1.5~1.5 centimetres.
Further, in step 1, the model of foundation includes: model 1: low concentration and high concentration MMP-9 handle 12 samples respectively
Product；No. 2:7 and No. 9 syringe needles of model handle 12 samples respectively.
Further, step 3 specifically:
It collects: using the brine people's fetal membrane for containing 200U/ml dual anti-(penicillin and streptomysin), and it is solidifying to wash tissue surface
Solid clot, continuous washing three times, until people's fetal membrane surface has no that obvious blood clot, cleaning solution are limpider.It will with tissue shear
People's fetal membrane is cut into small pieces tissue, and size is about lcmxlcm.The fritter people's fetal membrane shredded is clamped, 50ml centrifuge tube, every pipe dress are put into
About 15-20ml people's fetal membrane.
Culture: 0.1% neutral proteinase of 10ml is added in every pipe fetal membrane, is placed in 37 DEG C of processing 10-15 minutes, draws cell
Suspension, is added enough 0.25% pancreatin and 0.02%EDTA solution continues digestion 20 minutes, and continuous digestion 4 times is collected all thin
Born of the same parents' suspension, 1200 rpm are centrifuged 5 minutes, are washed twice with the dual anti-PBS containing 200u/m1, and Amino-Max 1I culture is added
Base is placed in standard incubation case culture.It passes on according to a conventional method.
Identification: collecting the 5th generation single cell suspension, and the antibody of PE or the FITC label of saturated concentration is added according to cell quantity
CD34, CD45, CD73, CD90,4 DEG C are protected from light incubation 30--50 minutes, and PBS is washed 2 times, to wash away unmarked antibody；4% poly
Formaldehyde is the same as fixed；With BD vantage flow cytomery.
Beneficial effects of the present invention:
It is previous bad using the material result of machine plugging, it is not used widely.The present invention uses state-of-the-art stem cell
Technology blocks fetal membrane cut using biomembrane, and effect becomes apparent from, and is more advantageous to clinical application.
Specific embodiment
Present invention will be explained in further detail With reference to embodiment.
A kind of method of in vitro culture fetal membrane mescenchymal stem cell, comprising the following steps:
The acquisition and model foundation of step 1, people's fetal membrane sample:
People's fetal membrane of term birth healthy fetus is obtained, sterile tissue pincers remove female decidua, cut (1.5~1.5 lis of square sample
Rice), it is placed in 24 orifice plates, sample is incubated for disinfection with 70% ethyl alcohol of 3 milliliters of 3mL in distilled water, then by sample phosphoric acid
Salt buffer salt water (PBS) washs three times, and internal diameter is that 1 centimetre of becket is fixed to shaft bottom, to form the training for being used for cell inoculation
Support well.
Model 1: low concentration and high concentration MMP-9 handle 12 samples respectively；
No. 2:7 and No. 9 syringe needles of model handle 12 samples respectively.
Step 2, fetal membrane mesenchymal stem cell transplantation
The DMEM culture medium inoculated MSC of 5ml is used on every tunic, and after culture 2 hours (37 DEG C, 5% CO2) to allow cell attached
, becket is removed, and the DMEM of 2ML is added in condition of culture, and changes weekly three times.With 5x106The cell of/ml is close
Degree drops evenly stem cell suspension until surface is hydraulically full on the surface ADM, plants again after 1 hour, continuous plantation 3 times, kind
Enough Amino.MAX II culture medium+20%FBS are added in plant after finishing 1 hour, be placed in standard incubation case and cultivate 3 days.
Step 3, the preparation of fetal membrane mescenchymal stem cell:
It collects: using the brine people's fetal membrane for containing 200U/ml dual anti-(penicillin and streptomysin), and it is solidifying to wash tissue surface
Solid clot, continuous washing three times, until people's fetal membrane surface has no that obvious blood clot, cleaning solution are limpider.It will with tissue shear
People's fetal membrane is cut into small pieces tissue, and size is about lcmxlcm.The fritter people's fetal membrane shredded is clamped, 50ml centrifuge tube, every pipe dress are put into
About 15-20ml people's fetal membrane.
Culture: 0.1% neutral proteinase of 10ml is added in every pipe fetal membrane, is placed in 37 DEG C of processing 10-15 minutes, draws cell
Suspension is added enough 0.25% pancreatin, one 0.02%EDTA solution and continues digestion 20 minutes, and continuous digestion 4 times is collected all thin
Born of the same parents' suspension, 1200 rpm are centrifuged 5 minutes, are washed twice with the dual anti-PBS containing 200u/m1, and Amino-Max 1I culture is added
Base is placed in standard incubation case culture.It passes on according to a conventional method.
Identification: collecting the 5th generation single cell suspension, and the antibody of PE or the FITC label of saturated concentration is added according to cell quantity
CD34, CD45, CD73, CD90,4 DEG C are protected from light incubation 30--50 minutes, and PBS is washed 2 times, to wash away unmarked antibody；4% poly
Formaldehyde is the same as fixed；With BD vantage flow cytomery.
(attached cell in people's fetal membrane source expresses mescenchymal stem cell surface marker CD73, CD90, and it is thin not express hematopoiesis
Cellular surface marker CD34, CD45 show that the cell is mescenchymal stem cell.)
As a result:
It is checked after culture 24 hours and sees that fetal membrane cut is closed.
The foregoing is only a preferred embodiment of the present invention, the scope of protection of the present invention is not limited to this, it is any ripe
Know those skilled in the art within the technical scope of the present disclosure, the letter for the technical solution that can be become apparent to
Altered or equivalence replacement are fallen within the protection scope of the present invention.
Claims (4)
1. a kind of method of in vitro culture fetal membrane mescenchymal stem cell, which comprises the following steps:
The acquisition and model foundation of step 1, people's fetal membrane sample；
People's fetal membrane of term birth healthy fetus is obtained, sterile tissue pincers remove female decidua, cut square sample, be placed on 24 orifice plates
In, sample is incubated for disinfection with 70% ethyl alcohol of 3 milliliters of 3mL in distilled water, and sample is then washed three with phosphate buffered saline (PBS)
It is secondary, and internal diameter is that 1 centimetre of becket is fixed to shaft bottom, to form the culture well for being used for cell inoculation；
Step 2, fetal membrane mesenchymal stem cell transplantation；
With the DMEM culture medium inoculated MSC of 5ml on every tunic, 37 DEG C, 5% CO2Under, after culture 2 hours, to allow cell attached
, becket is removed, and the DMEM of 2ML is added in condition of culture, and changes weekly three times；With 5x106The cell of/ml is close
Degree drops evenly stem cell suspension until surface is hydraulically full on the surface ADM, plants again after 1 hour, continuous plantation 3 times, kind
Enough Amino.MAX II culture medium+20%FBS are added in plant after finishing 1 hour, be placed in standard incubation case and cultivate 3 days；
Step 3, the preparation of fetal membrane mescenchymal stem cell.
2. the method for in vitro culture fetal membrane mescenchymal stem cell according to claim 1, which is characterized in that in step 1, institute
The side length for stating square sample is 1.5~1.5 centimetres.
3. the method for in vitro culture fetal membrane mescenchymal stem cell according to claim 1, which is characterized in that in step 1, build
Vertical model includes: model 1: low concentration and high concentration MMP-9 handle 12 samples respectively；No. 2:7 and No. 9 syringe needles of model point
Manage 12 samples in other places.
4. the method for in vitro culture fetal membrane mescenchymal stem cell according to claim 1, which is characterized in that step 3 is specific
Are as follows:
It collects: with the dual anti-brine people's fetal membrane of penicillin containing 200U/ml and streptomysin, and washing tissue surface solidification
Clot, continuous washing three times, until people's fetal membrane surface has no that obvious blood clot, cleaning solution are limpid；It will be by people's tire with tissue shear
Film is cut into small pieces tissue, size lcmxlcm；The fritter people's fetal membrane shredded is clamped, 50ml centrifuge tube is put into, every pipe fills 15-
20ml people's fetal membrane；
Culture: 0.1% neutral proteinase of 10ml is added in every pipe fetal membrane, is placed in 37 DEG C of processing 10-15 minutes, draws cell suspension,
Enough 0.25% pancreatin, one 0.02%EDTA solution is added and continues digestion 20 minutes, it is outstanding to collect all cells for continuous digestion 4 times
Liquid, 1200 rpm are centrifuged 5 minutes, are washed twice with the dual anti-PBS containing 200u/m1, and Amino-Max 1I culture medium is added, sets
In standard incubation case culture；It passes on according to a conventional method；
Identification: collecting the 5th generation single cell suspension, and the antibody of PE or the FITC label of saturated concentration is added according to cell quantity
CD34, CD45, CD73, CD90,4 DEG C are protected from light incubation 30--50 minutes, and PBS is washed 2 times, to wash away unmarked antibody；4% poly
Formaldehyde is the same as fixed；With BD vantage flow cytomery.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN115252646A (en) * | 2022-08-30 | 2022-11-01 | 重庆医科大学附属第一医院 | Application of umbilical cord mesenchymal stem cell exosome and LXA4 in repair of premature rupture of fetal membranes |
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