CN106754674B

CN106754674B - The method and its application of amnion mesenchymal stem cell are prepared from Human plactnta amnion

Info

Publication number: CN106754674B
Application number: CN201611129552.8A
Authority: CN
Inventors: 许晓椿; 王正; 肖海蓉
Original assignee: BOYA STEM CELL TECHNOLOGY Co Ltd
Current assignee: BOYA STEM CELL TECHNOLOGY Co Ltd
Priority date: 2016-12-09
Filing date: 2016-12-09
Publication date: 2019-09-20
Anticipated expiration: 2036-12-09
Also published as: CN106754674A

Abstract

The present invention relates to the method and its application that amnion mesenchymal stem cell is prepared from Human plactnta amnion.Method includes the following steps: being rinsed and disinfecting to placenta, amnion is rinsed and is disinfected and is fragmented, carry out tissue digestion, cell culture is carried out to filtrate portion and tissue block part, cell is harvested, counts and measure Cell viability, freeze.The invention further relates to the amnion mesenchymal stem cell being prepared by the method for the present invention and their therapeutical uses.Advantage described in specification is presented in the method for the present invention.

Description

The method and its application of amnion mesenchymal stem cell are prepared from Human plactnta amnion

Technical field

The invention belongs to stem-cell therapy technical field, it is related to preparing the side of amnion mesenchymal stem cell from Human plactnta amnion Method further relates to the amnion mesenchymal stem cell prepared by this method and their therapeutical uses.

Background technique

Stem cell is the basis of regenerative medicine development, and effective application is to realize long-lived human health, anti-resisting cancer and senility, forever Protect the effective ways of the ultimate dreams such as younger state, constantly improve quality of life.In recent years, embryonic stem cell and adult stem cell In cell biology and regenerative medicine research field by attention.However, although embryonic stem cell has other cells can not The totipotency reached, but the factors such as immunological rejection after oncogenicity, allograft and unavoidable dispute of ethic It is greatly limited in clinical application, and content is less in the tissue for adult stem cell, lacks the table of specificity The characteristics of face indicates also becomes the yoke of its clinical application.

Due to the multinomial differentiation potential of stem cell and its function of secretion cytokine profiles, by the favor of researcher. In recent years, stem cell tissue damage with repair in using more and more extensive, and the source of stem cell is always people's concern Focus.Mesenchymal stem cell is one of the adult stem cell being more early realized.But since bone marrow extraction is brought to patient Pain, and the content of stem cell is few, makes largely to be restricted.Other sources such as adipose tissue, skin histology Deng the human body that all needs to have drawn from is self or allosome tissue, also easy to drawing materials or ethics problem not may relate to.

People is amnion-derived in human placenta, no blood vessel, nerve and lymph, has certain elasticity, thick 0.02-0.5mm, Under Electronic Speculum, it is divided into 5 layers: epithelial layer, basilar memebrane, compacted zone, fibroblast layer and spongy layer.Amnion belongs to pregnant woman's production Waste afterwards, the amnion area about 600cm of a placenta², and amnion is easy to remove from placenta, so because of amnion It is convenient with materials, the feature of raw material abundance and be considered as current mescenchymal stem cell best source.

People's amnion is translucent film, and there are many nutritional ingredients for promoting cell Proliferation.Amnionic basement membrane and amnion base Matter layer contains a large amount of different glue members, the Collagen Type VI of predominantly I, III, IV, V, VII and fibronectin splicing variants, laminin etc. at Point.Fresh amnion can generate some growth factors, such as: basic fibroblast growth factor, hepatocyte growth factor and conversion Grouth factor beta etc..Currently, just in a variety of diseases of research and utilization amnion stem-cell therapy, including the nervous system disease, cardiac muscle stalk Extremely, muscular atrophy, diabetes and liver fibrosis etc..Some researches show that amnion mesenchymal stem cells by there is high angiogenesis energy Power and it is migrated to vigor, the healing of diabetes rat wound can be promoted.

The amnion-derived stem cell of people include human amnion membrane (humanamnioticepithelialcells, HAEC) and human amnion mesenchymal stem cell (humanamnioticmes-enchymalstromalcells, hAMSC), they are A variety of embryonic stem cell markers can be expressed, and all have more comprehensive multi-lineage potential.

Existing research discovery, the human amnion mesenchymal stem cell of culture is except the specificity marker protein for expressing neural stem cell Outside nestin, also expression eight aggressiveness of stem cell idiosyncratic transcription factor connection albumen 4 (octamer-binding protein4, Oct-4), and ability to express is more stronger than mesenchymal stem cell.Recently report confirms its oriented triploblastica cell differentiation Potential: ectoderm (nerve), mesoderm (skeletal muscle, cardiac muscle, endothelium etc.), entoderm (pancreas, liver).Some researches show that cultures 3 A large amount of adherent mescenchymal stem cell is obtained after~4 weeks, morphological category is similar to mesenchymal stem cell, and it compares derived from bone marrow MSCs have stronger proliferative capacity.The result of flow cytomery meets conclusions in test, illustrates amnion cell With surface marker similar with mescenchymal stem cell.It can at least pass to for the 15th generation in vitro and keep its form constant.Greatly The experimental study of amount also demonstrates that hMSCs has stronger proliferative capacity and stem cell properties than mesenchymal stem cell.

There are many reports about the preparation method for taking cell on amnion.Such as

CN102191218A (Chinese Patent Application No. 201110080968.6, Zun Yi) disclose a kind of complete medium and The cultural method of human amnion mesenchymal stem cell (hAMSCs).Complete medium is by volume to the low sugar-Dulbecco limit The self serum of umbilical cord blood of people that 3%-10% must be added in culture medium solution is formulated；Cultural method includes: (1) separation； (2) originally culture；(3) secondary culture.The advantages of present invention is cultivated using aforementioned complete medium for hAMSCs are as follows: avoid Using the risk of fetal calf serum, and without adding L-Glutamine, nonessential amino acid, 2 mercapto ethanol and pyruvic acid etc., still It can keep the preferable multiplication characteristic of hAMSCs and phenotypic characteristic and some stem cell multi-lineage potential marker gene and albumen table It reaches；HAMSCs adherent firmness is significantly lower than FBS condition of culture in secondary culture, and digestion time is obviously shortened, reduces Pancreatin is digested to the damage of cell and the loss of cell quantity.

CN101914490A (Chinese Patent Application No. 201010252201.2) is related to a kind of human amnion mesenchymal stem cell Serum free medium and its cultural method.The culture medium is that human serum albumins, people turn are added in DMEM/F12 basal medium Ferritin, actrapid monotard and sodium selenite；Its cultural method is that people's amnion is first used trypsin digestion, then uses clostridiopetidase A IV And single cell suspension is made in deoxyribonuclease I digestion, filtering；Pass through the training of the basis VDMEM: VF12=1: 1DMEM/F12 again It supports and adds human serum albumins, transferrins, insulin and sodium selenite in base, make human amnion mesenchymal stem cell in serum-free Under the conditions of, it is placed in 37 DEG C, saturated humidity, the CO that volume fraction is 5%₂In incubator, by changing liquid and passage, external training is realized Support and amplification, and maintain the potential of Multidirectional Differentiation, the cell after amplification can induce in vitro chondroblast, osteoblast and Fat cell.Have the characteristics that it is animal derived without other, from a wealth of sources, do not limited by ethics.

CN102559586A (Chinese Patent Application No. 201110404587.9, Zun Yi) discloses a kind of human amnion mesenchymal The isolation and purification method of stem cell, comprising the following steps: first, the mature Cesarean esction placenta of aseptic collection people, Mechanical Method is by amnion It is removed from placenta tissue, is rinsed with D-Hank ' s liquid and remain bloodstain for several times to remove, amnion is cut into fragment；Second, amnion The 0.05% trypsin digestion solution containing 0.02%EDTA is added in fragment, and in 37 DEG C, supernatant is abandoned in rotation digestion；It rejoins and disappears Change liquid, abandons supernatant in 37 DEG C of rotation digestion, leave and take indigested amnion fragment；Third, D-Hank ' s liquid rinse indigested sheep The clostridiopetidase A of the 0.75mg/ml containing 0.075mg/ml DNase I is added in film fragment, in 37 DEG C, rotation digestion about 2h to group Totally disappeared is casted off, cell filtrate is collected in the filtering of 300 mesh steel meshes, and centrifugation obtains original human amnion mesenchymal stem cell；4th, The original human amnion mesenchymal stem cell of separation is resuspended in LG-DMEM culture medium, cell density is inoculated in 6 holes Culture plate is placed in CO₂Incubator culture, removes the amniotic epithelial cells of completely non-adherent growth under inverted microscope, and the 3rd day The culture medium more renewed；After cell confluency degree up to after 80~90%, with trypsase-EDTA solution digestion, it is whole that culture medium is added Supernatant is abandoned in only trypsin acting, centrifugation, and cell precipitation is suspended again with culture medium, is passed on the cell density of 1 × 107/L.

CN104450610A (Chinese Patent Application No. 201410715000.X, Sai Laila) fills between disclosing a kind of people's amnion Matter stem cell secondary culture method, it is characterised in that the following steps are included: (1) raw material cleans: the people after originally culture separation After amnion mesenchymal stem cell degrees of fusion reaches 80~90%, the culture solution in culture dish is sucked, PBS cleaning is added, then inhales Remove the PBS in culture dish；(2) it digests: tryplE being added into culture dish, is transferred to CO₂After incubator is incubated for 1~2min, add Enter complete medium, blown and beaten repeatedly with pasteur pipet, until 80~90% cell is no longer adherent, terminates digestion；(3) it is centrifuged: will Step (2) obtains in cell suspension centrifuge tube, and 1000rpm/min is centrifuged 5min；(4) it counts: after centrifugation, discarding supernatant Cell precipitation is resuspended with complete medium in liquid, and piping and druming mixes, and obtained cell suspension is counted with cell counter；(5) bed board and Culture: by the cell suspension inoculation culture dish of step (4), adjustment cell density is 1.3 × 10⁴/cm², then it is added and contains Culture medium is gone to CO by the complete medium of the EGF of 10ng/ml₂It is cultivated in cell incubator.

CN103789258A (Chinese Patent Application No. 201210506430.1, Lu Hua) discloses a kind of human amnion mesenchymal The separation method of stem cell is applied to the process of enzyme digestion joint EGF culture human amnion mesenchymal stem cell, the culture Process includes the following steps: the separation of hAMSCs, the originally culture of hAMSCs, the secondary culture of hAMSCs and amplification, hAMSCs Freeze and recover and the detection of hAMSCs Immunophenotyping；It is characterized in that, the separating step packet of the hAMSCs Include: amnion is removed from placenta tissue using Mechanical Method, uses D-Hanks by the fresh human placenta for taking postpartum to throw aside under aseptic condition Liquid, which rinses, remains bloodstain for several times to remove, and the amnion after rinsing is cut into about 1mm with eye scissors³Trypsase 37 is added in fragment DEG C digestion 10 minutes；The DMEM containing calf serum is added and terminates digestion, and softly 200 mesh cell sieves filter after piping and druming mixing；It crosses II Collagenase Type digestive juice is added in amnion tissue after filter, 37 DEG C digest 30 minutes；The DMEM containing calf serum is added to terminate Digestion, 200 mesh cell sieves filter after soft piping and druming mixes, and collect cell；1000 revs/min are centrifuged 5 minutes；Platform expects blue dyeing meter Number living cells, adjustment cell density are 1 × 10⁶/ ml is seeded to culture bottle, and the DMEM culture medium containing bFGF and FBS is added；It sets 37 DEG C, the CO of saturated humidity, volume fraction 5%₂Culture in incubator；Primary and passage is observed under inverted phase contrast microscope daily The growing state and morphological feature of cell change liquid 1 time every other day, and take the photograph piece record；After cell confluency degree up to after 80%, with 0.25% Trypsase -0.02%EDTA solution in 37 DEG C digestion 2-3 minutes, be added culture medium terminate trypsin acting, 1000 revs/min Zhongli's heart 5 minutes, supernatant is abandoned, after cell precipitation is suspended again with culture medium, with 1 × 10⁷The cell density of/ml passes on；And In succeeding generations, the next day change liquid 1 time, to cell cover with 70% bottom of bottle repeat above step.

It is dry that CN1810959A (Chinese Patent Application No. 200610033007.9, northern section) discloses a kind of human amnion mesenchymal The isolation and culture method and medical composition of cell, isolation and culture method are by people's amnion trypsase, clostridiopetidase A, take off Oxygen ribalgilase digests in succession, and then single cell suspension is made in filtering, then uses added with volumn concentration as 10%-20% Fetal calf serum and final concentration of 10-20ng/ml Basic Fibroblast Growth Factor V_DMEM∶V_F12=1: 1 DMEM/F12 Culture medium is placed in 37 DEG C, saturated humidity, the CO that volume fraction is 5%₂It is cultivated in incubator, by changing liquid and passage, is made Human amnion mesenchymal stem cell is gradually expanded and is purified.It is believed that the inventive method have it is from a wealth of sources, do not limited by ethics Etc. superiority, have broad application prospects, medical composition of the invention can be used for neural replacement therapy, for myocardial infarction Treatment, for the treatment of diabetes, for bone tissue engineer, be applied to plastic surgery etc..

CN105420179A (Chinese Patent Application No. 201510963676.5, Si Tanmu) discloses a kind of from umbilical cord and tire The method that argali membrane tissue extracts epithelial cell and mescenchymal stem cell simultaneously, it is characterised in that: steps are as follows: (1) umbilical cord and tire Argali film materials and inoculation: 1. high-temperature sterilization surgical instrument and big glass dish, while disinfection by ultraviolet light superclean bench；2. preparing Culture medium: the composition of complete medium is: DMEM/F12 adds the fetal calf serum of complete medium total volume 5%, complete medium The mixing additive of the insulin of total volume 1%, siderophillin and selenium, final concentration of 20 nanogram/milli in complete medium Final concentration of 20 ngs/ml of basic fibroblast growth factor in the epidermal growth factor and complete medium risen, It is sterile filtered, 4 DEG C of preservations；The volume ratio of DMEM:F12 is 1:1 in the DMEM/F12；The mixing additive and add in mixing Add 5 mcg/ml of final concentration of insulin in object, 10 mcg/ml of siderophillin and 5 ngs/ml of sodium selenite； 3. strictly screening donor, blood testing excludes infectious disease；4. transporting umbilical cord and placenta: mechanical stripping placenta amnion in low temperature ice box Later, umbilical cord and placenta amnion are respectively placed in big glass dish, following steps all aseptically carry out, and umbilical cord is cut into Placenta amnion is cut into the square of about 5X5cm long by the segment of 1cm long, with the penicillin of phosphate buffered saline+total volume 1%+total The streptomysin cleansing tissue of volume 1% twice, the blood of wash clean umbilical cord；5. umbilical cord and placenta amnion tissue are placed on 4 DEG C In phosphate buffered saline, cell survival is influenced to prevent tissue from overheating when machinery shreds, is divided using tissue cutting machine It is not broken into the fragment of 1-3mm, using low speed disrupting tissue, while keeping cell activity, obtains broken umbilical cord and amnion group It knits；6. broken umbilical cord and amnion tissue are transferred to respectively in T175 Tissue Culture Flask, and tiled with tissue scraper In culture bottle bottom, be carefully added into a small amount of complete medium, make culture solution can cover broken umbilical cord and amnion tissue block and As for it is floated, it is placed in 37 DEG C, saturated humidity 5%CO₂It is cultivated in incubator；(2) mescenchymal stem cell and amnioic epithelium Cell is expanded and is passed in culture bottle: 1. being observed visible cell in incubation and gradually is vacillated out from tissue block, goes forward side by side one Step realizes adherent growth and proliferation, at the 3-4 days of culture, when cell growth merges in blocks, uses sterile phosphate buffered saline The tissue for washing away remaining, is all changed to fresh complete medium and continues to cultivate；2. hereafter every 4 days change a culture solution until cell 90-100% culture bottle floor space is covered with, pancreatin had digestive transfer culture is just carried out；3. pancreatin had digestive transfer culture: abandoning culture solution, washed with PBS Cell 2 times, every T175 is digested 10 minutes with the pancreas enzyme -EDTA that 10 milliliters of mass percents are 0.25% at 37 DEG C, outstanding to cell 1 milliliter of fetal calf serum is added to neutralize pancreatin reaction in each T175 after floating, and 1000 revs/min are centrifuged 10 minutes, is resuspended in completely In culture medium, in the passage of 1: 4 cell number ratio into new T175 culture bottle；(3) the mirror of mescenchymal stem cell and amniotic epithelial cells It is fixed: when 1. the third generation is passed in pancreatin digestion, with the purity of flow cytometry culture cell: the marker of amniotic epithelial cells It include: keratin CK19, CD29 and CD34；The marker of placenta and umbilical cord mesenchymal stem cells includes: CD73, CD90, and CD105；2. flow cytometer verifying cell purity reaches 90% or more, chemical examination excludes common infectious disease, and excludes bacterium, props up Mycoplasma contamination is the mixture complied with standard to get epithelial cell and mescenchymal stem cell.It is believed that the method cost of the invention It is low, simple and quick, umbilical cord and placenta amnion are shredded into minimum fragment using tissue cutting machine, are extremely conducive to directly be inoculated with Cell is vacillated to outside tissue after to culture bottle；Without using any protease reagent, so that cost be greatly reduced, system is reduced The standby time, so that short time mass cell is prepared into reality；This method uses addition growth factor and other nutrients Cell culture medium saves serum.

CN 103013913A (Chinese Patent Application No. 201210505486.5, Lu Hua) is disclosed for a kind of enzyme digestion The method that joint epidermal growth factor (EGF) cultivates human amnion mesenchymal stem cell (hAMSCs) comprising following steps: The separation of hAMSCs, the originally culture of hAMSCs, the secondary culture of hAMSCs and amplification, hAMSCs's freezes and recovers, and The detection of hAMSCs Immunophenotyping.Repeatedly digestion is thin to collect amnion using trypsase and clostridiopetidase A substep by the present invention Born of the same parents are combined adhere-wall culture and digestion time control by the way that 4ng/mlEGF is added, and obtain purity and the higher people's amnion of activity Mescenchymal stem cell (hAMSCs).Enzyme digestion joint joint EGF of the invention has many advantages, such as easy to operate, Fast Practical, It is a kind of method for more efficiently separating purifying hAMSCs.

CN 104450612A (Chinese Patent Application No. 201410741509.1, Ao Yunxia) is disclosed between a kind of people's amnion The separation of mesenchymal stem cells and identification method isolate amnion mesenchymal stem cell and secondary culture using tissue block adherent method, The observation for carrying out cellular morphology for cell to P1-P3, with flow cytomery cell membrane surface positive molecule and negative molecule And using six well plate methods detect cell proliferative conditions, it is hAMSCs that cell is isolated in this experiment from placenta amnion tissue, be into The biology and immunological characteristic, differentiation capability, karyotypic stability of one step research hAMSCs, and as clinical application Seed cell etc. provides the foundation.

CN103555663A (Chinese Patent Application No., road training) discloses a kind of side for cultivating human amnion mesenchymal stem cell Method.It includes the following steps: the people's amnion for taking fresh acquisition, cleaning, is put into amnion protection liquid and saves backup for 2-8 DEG C；Take leaching People's amnion in amnion protection liquid no more than 48 hours, cleaning are steeped, then successively sets the Benzylpenicillin sodium salt and sulfate chain faced with newly matching Mycin content is respectively the sodium chloride solution and every milliliter of injection containing 30-50U of 2500-3500U/mL and 3500-4500U/mL Embathed in the Metronidazule injection of amphotericin B, finally clean again be placed in disposable sterilized culture dish carry out it is spare, Then it separates, culture to cell fusion degree reaches 80-90% to obtain the final product.It is believed that the invention is avoided that the examination of animal blood serum and animal origin The potential risk of agent bring；The culture of amnion mesenchymal stem cell, strong operability are used equally in 48 hours；It can be significantly Mescenchymal stem cell is separately cultured underproof probability caused by reducing because of mould, anaerobic bacteria pollution.

CN103422176A (Chinese Patent Application No. 201310323661.3, Pang Xining) fills between disclosing a kind of people's amnion The construction method of matter stem cell bank.Its method is that people's amnion is taken to detect, and crushes after being washed with phosphate buffer flushing, adds phosphoric acid Buffer dilution is digested after trypsin digestion with clostridiopetidase A IV and deoxyribonuclease I, and filtering is made unicellular outstanding Liquid；Again by adding human serum albumins, transferrins, insulin in VDMEM:VF12=1:1DMEM/F12 basal medium And sodium selenite, make human amnion mesenchymal stem cell under serum-free condition, be placed in incubator, changes liquid and passage.It will be external Culture and amplification obtain mescenchymal stem cell and set liquid nitrogen frozen, save, build by neonatal sex and ABO/Rh parting and HLA parting Vertical cellular informatics archives, construct human amniotic mesenchymal stem cell bank.It is believed that the invention has, animal derived without other, source is wide General, the characteristics of not limited by ethics.It can provide mescenchymal stem cell and carry out cell therapy and other application.

CN104450611A (Chinese Patent Application No. 201410715077.7, Sai Laila) fills between disclosing a kind of people's amnion Matter stem cell primary isolation and culture method, comprising the following steps: human amniotic tissue is provided, is rinsed then with PBS buffer solution It shreds, the trypsase that mass concentration is 0.1~0.3% is added is digested, collagenase type I be added and DMEM in high glucose is trained substantially Base is supported to final concentration of 0.1~0.2% digestion of collagenase type I, centrifuge separation, taking precipitate, sediment are resuspended with PBS solution, It is centrifugated after filtering, discards supernatant liquid, obtain human amnion mesenchymal stem cell.Compared with prior art, it is believed that invention benefit Amnion tissue is pre-processed with a small amount of trypsase, keeps amnion tissue loose, and then utilizes the better collagenase type I of tissue specificity Amnion is handled, the step of substantially reducing the time of digestion, simplify primary separation, and higher stem cell can be obtained and produced Amount, the stem cell vigor of acquisition also greatly improve compared with prior art.

CN103642751A (Chinese Patent Application No. 201310650384.7, with pool) open one kind is produced from people's amnion The method of stem cell refers specifically to separate epithelial cell and mescenchymal stem cell method in a kind of amnion, is related to cell separation technology Field.It protects liquid to be acquired placenta by homemade sterile, efficient, stable placenta, utilizes digestion method and creep plate method Combination, amniotic epithelial cells and amnion mesenchymal stem cell are separated, respectively obtain the amniotic epithelial cells of high-purity with Amnion mesenchymal stem cell.The cell that the present invention solves after people's amnion acquisition pollution rate height, acquisition exists in the prior art is living The problem of property is poor, directly affects subsequent isolation and culture of cell.It is to be carried out to the method for separating stem cell from people's amnion Improvement and innovation, form a set of standardized separation scheme, can be more stable, make full use of the stem cell resource of amnion, nothing Pollution, purity is high, cell quantity is more, while keeping cell activity, ensure that the primary characteristic of stem cell.

CN105062959A (Chinese Patent Application No. 201510597698.4 is navigated) discloses a kind of human amnion mesenchymal The isolated culture method of stem cell sterilizes by the acquisition of Human plactnta amnion tissue, amnion tissue, washs, shreds, mixes collagen Enzymic digestion obtains mescenchymal stem cell, then carries out derived mesenchymal stem cells in vitro culture.The present invention can be obtained from human amnion tissue A large amount of phenotype stable homogeneous are obtained, proliferative capacity is strong, and has the mescenchymal stem cell of multi-lineage potential, extends out by medium body Increase, purifying cells, satisfaction freezes or the quantity of clinical use.Cell culture period is short, and repeatability is strong.The method obtains dry thin Born of the same parents have CD105⁺, CD90⁺, CD73⁺Phenotype.

However, this field is still expected to have new method to prepare human amnion mesenchymal stem cell, and expect this side Method shows one or more kinds of beneficial technical effects.

Summary of the invention

The purpose of the present invention is to provide a kind of method for preparing human amnion mesenchymal stem cell from placenta amnion, expecting should Method shows one or more kinds of beneficial technical effects.

First aspect present invention provides a kind of method for preparing human amnion mesenchymal stem cell from placenta amnion, this method The following steps are included:

(1) placenta is taken out from Placenta samples collecting cassette, be placed in ceramic whiteware disk, use basic balanced salt solution repeated flushing Surface carries out disinfection processing to placenta；

(2) it is slowly torn with surgical forceps and fetal membrane outer layer amnion is taken to be placed in 150mm glass dish, it is anti-using basic balanced salt solution Multiple clean the surface crimson blood shreds into the amnion tissue fritter that diameter is 5mm；It is filtered with 300 mesh filter screens, normal saline flushing is residual Blood, tissue block are transferred to 50ml centrifuge tube；

(3) tissue digestion: in 37 DEG C of constant-temperature tables, by tissue block with the mixed enzyme digestive juice of 1 times of tissue volume with 30~60min of 100rpm oscillation digestion；After digestion, adds 2ml fetal calf serum to mix and terminate；It is filtered and is used in combination with 200 mesh filter screens A large amount of physiological saline cleanings, collect filtrate；

(4) cell culture is carried out to filtrate portion and tissue block part respectively:

Filtrate portion: the filtrate of collection is centrifuged 5min with 1500rpm, removes supernatant, and it is heavy that cleaning cell is resuspended with physiological saline It forms sediment；It is centrifuged 5min with 1500rpm, supernatant is removed, cell precipitation is resuspended with complete medium；It counts instrument meter and takes number of nucleated cells simultaneously Blue dyeing measurement Cell viability is expected with platform；With every bottle 2 × 10⁶A cell inoculation is in 75cm²In culture bottle, addition 20ml is trained completely Base is supported, in 5%CO after rolling is even₂, cultivate in 37 DEG C of incubators；Periodically change liquid；Culture (usually can be 12 days left sides in 10~15 days It is right) after be passaged to P1 generation, continue with complete medium culture；

Tissue block part: the tissue block of collection is placed in 75cm²In culture bottle, tissue block is enable to cover one in culture bottle Half culture area, addition complete medium did not had tissue, in 5%CO after rolling is even₂, cultivate in 37 DEG C of incubators；Fluid infusion after 2d 10ml complete medium continues to cultivate；More mescenchymal stem cell removes tissue after climbing out of, periodically change liquid；Culture 10~15 days (logical Often can be 12 days or so) after be passaged to P1 generation, continue with complete medium culture；

(5) P1 is harvested for cell: the P1 after being merged two parts in previous step with pancreatin digestive juice is for cell dissociation Afterwards, cell is collected, counts and measures Cell viability, freeze to get the amnion mesenchymal stem cell in P1 generation, it is carried out when necessary Secondary culture.

The method of any embodiment according to a first aspect of the present invention, wherein including strepto- in the basis balanced salt solution Element and two kinds of antibiotic of penicillin.

The method of any embodiment according to a first aspect of the present invention, wherein the basis balanced salt solution is by as follows What mode was prepared: by the KH of KCl, 0.06g of 0.4g₂PO₄, 0.132g Na₂HPO₄.12H₂O, NaCl, 0.35g of 8g NaHCO₃, the D-Glucose of 1.0g, the streptomysin of 0.10g, 0.06g penicillin with water dissolve and constant volume at 1L solution.

The method of any embodiment according to a first aspect of the present invention, wherein including in the mixed enzyme digestive juice: The collagenase type I of 0.1mg/ml, the II Collagenase Type of 0.3mg/ml, in the hyaluronidase and 0.05mg/ml of 0.1mg/ml Property protease.

The method of any embodiment according to a first aspect of the present invention, wherein the complete medium includes: DMEM-F12 Culture medium, FBS, penicillin, streptomysin.

The method of any embodiment according to a first aspect of the present invention, wherein the DMEM-F12 culture medium is DMEM-F12 The culture medium prepared with volume ratio 1:1.

The method of any embodiment according to a first aspect of the present invention, wherein the complete medium includes: DMEM-F12 (1:1) culture medium, 10%FBS, 100 μ g/mL penicillin, 50 μ g/mL streptomysins.In one embodiment, described complete It also added 0.01~0.05% (such as 0.01~0.04%, such as 0.01~0.03%) thiamine mononitrate and 0.05 in culture medium ~0.1% (such as 0.06~0.1%, such as 0.07~0.1%) maltose.It has been unexpectedly discovered that into culture medium After adding both micro thiamine mononitrate and maltose, the yield of human amnion mesenchymal stem cell can be improved significantly, without This effect can not be obtained when using both of which or only increasing one of them.

The method of any embodiment according to a first aspect of the present invention wherein in step (4), is usually cultivated 10~15 days P1 generation is passaged to after (usually can be 12 days or so).

The method of any embodiment according to a first aspect of the present invention, wherein in step (5), pancreatin digestive juice is to include 0.25% trypsase and 0.02% EDTA digestive juice.

Further, second aspect of the present invention provides a kind of human amnion mesenchymal stem cell, is through the invention One side the method is prepared.

Human amnion mesenchymal stem cell according to a second aspect of the present invention is to be prepared by a method comprising the following steps It obtains:

Filtrate portion: the filtrate of collection is centrifuged 5min with 1500rpm, removes supernatant, and it is heavy that cleaning cell is resuspended with physiological saline It forms sediment；It is centrifuged 5min with 1500rpm, supernatant is removed, cell precipitation is resuspended with complete medium；It counts instrument meter and takes number of nucleated cells simultaneously Expect that blue dyeing measurement Cell viability (can usually obtain and be greater than 1 × 10 with platform⁷95%) cell, Cell viability are greater than；With every bottle 2 × 10⁶ A cell inoculation is in 75cm²In culture bottle, 20ml complete medium is added, in 5%CO after rolling is even₂, cultivate in 37 DEG C of incubators； Periodically change liquid；Culture is passaged to P1 generation after (usually can be 12 days or so) in 10~15 days, continues with complete medium culture；

Human amnion mesenchymal stem cell according to a second aspect of the present invention, wherein including chain in the basis balanced salt solution Two kinds of antibiotic of mycin and penicillin.

Human amnion mesenchymal stem cell according to a second aspect of the present invention, wherein the basis balanced salt solution is by such as What under type was prepared: by the KH of KCl, 0.06g of 0.4g₂PO₄, 0.132g Na₂HPO₄.12H₂O, NaCl, 0.35g of 8g NaHCO₃, the D-Glucose of 1.0g, the streptomysin of 0.10g, 0.06g penicillin with water dissolve and constant volume at 1L solution.

Human amnion mesenchymal stem cell according to a second aspect of the present invention, wherein including in the mixed enzyme digestive juice: The collagenase type I of 0.1mg/ml, the II Collagenase Type of 0.3mg/ml, in the hyaluronidase and 0.05mg/ml of 0.1mg/ml Property protease.

Human amnion mesenchymal stem cell according to a second aspect of the present invention, wherein the complete medium includes: DMEM- F12 culture medium, FBS, penicillin, streptomysin.

Human amnion mesenchymal stem cell according to a second aspect of the present invention, wherein the DMEM-F12 culture medium is DMEM- The culture medium that F12 is prepared with volume ratio 1:1.

Human amnion mesenchymal stem cell according to a second aspect of the present invention, wherein the complete medium includes: DMEM- F12 (1:1) culture medium, 10%FBS, 100 μ g/mL penicillin, 50 μ g/mL streptomysins.In one embodiment, described complete Also added in full culture medium 0.01~0.05% (such as 0.01~0.04%, such as 0.01~0.03%) thiamine mononitrate and 0.05~0.1% (such as 0.06~0.1%, such as 0.07~0.1%) maltose.

Human amnion mesenchymal stem cell according to a second aspect of the present invention wherein in step (4), usually cultivates 10~15 P1 generation is passaged to after its (usually can be 12 days or so).

Human amnion mesenchymal stem cell according to a second aspect of the present invention, wherein in step (5), pancreatin digestive juice is to include 0.25% trypsase and 0.02% EDTA digestive juice.

Further, third aspect present invention is filled between providing people's amnion described in second aspect of the present invention any embodiment Matter stem cell is preparing the purposes in the drug as cellular therapeutic agent.

The purposes of any embodiment according to a third aspect of the present invention, wherein the human amnion mesenchymal stem cell is to pass through What the method included the following steps was prepared:

The purposes of any embodiment according to a third aspect of the present invention, wherein including strepto- in the basis balanced salt solution Element and two kinds of antibiotic of penicillin.

The purposes of any embodiment according to a third aspect of the present invention, wherein the basis balanced salt solution is by as follows What mode was prepared: by the KH of KCl, 0.06g of 0.4g₂PO₄, 0.132g Na₂HPO₄.12H₂O, NaCl, 0.35g of 8g NaHCO₃, the D-Glucose of 1.0g, the streptomysin of 0.10g, 0.06g penicillin with water dissolve and constant volume at 1L solution.

The purposes of any embodiment according to a third aspect of the present invention, wherein including in the mixed enzyme digestive juice: The collagenase type I of 0.1mg/ml, the II Collagenase Type of 0.3mg/ml, in the hyaluronidase and 0.05mg/ml of 0.1mg/ml Property protease.

The purposes of any embodiment according to a third aspect of the present invention, wherein the complete medium includes: DMEM-F12 Culture medium, FBS, penicillin, streptomysin.

The purposes of any embodiment according to a third aspect of the present invention, wherein the DMEM-F12 culture medium is DMEM-F12 The culture medium prepared with volume ratio 1:1.

The purposes of any embodiment according to a third aspect of the present invention, wherein the complete medium includes: DMEM-F12 (1:1) culture medium, 10%FBS, 100 μ g/mL penicillin, 50 μ g/mL streptomysins.In one embodiment, described complete It also added 0.01~0.05% (such as 0.01~0.04%, such as 0.01~0.03%) thiamine mononitrate and 0.05 in culture medium ~0.1% (such as 0.06~0.1%, such as 0.07~0.1%) maltose.

The purposes of any embodiment according to a third aspect of the present invention wherein in step (4), is usually cultivated 10~15 days P1 generation is passaged to after (usually can be 12 days or so).

The purposes of any embodiment according to a third aspect of the present invention, wherein in step (5), pancreatin digestive juice is to include 0.25% trypsase and 0.02% EDTA digestive juice.

Any technical characteristic possessed by any embodiment of either side or the either side of the present invention is equally applicable Any embodiment of other any embodiments or other either sides, as long as they will not be conflicting, certainly mutual Between where applicable, if necessary can individual features be made with appropriate modification.Make to various aspects of the present invention with feature into one below The description of step.

All documents recited in the present invention, their full content are incorporated herein by reference, and if these are literary When offering expressed meaning and the inconsistent present invention, it is subject to statement of the invention.In addition, the various terms that use of the present invention and Phrase has that well known to a person skilled in the art general senses, nonetheless, the present invention remain desirable at this to these terms and Phrase is described in more detail and explains, the term and phrase referred to is if any inconsistent with common art-recognized meanings, with institute's table of the present invention Subject to the meaning stated.

It normally says, about 600 square centimeters of the area of a placenta amnion, amnion stem cell of the present invention is the placenta that has drawn from On amnion.

The whole flow process of the method for the present invention is easy to operate, and controllably, P1 generation can harvest a large amount of cells, for clinical application and grinds Study carefully and provides possibility.

With discovery, present invention gained human amnion mesenchymal stem cell is examined through immunocytochemistry and immunofluorescence dyeing It surveys, as a result shows, the characteristic feature of human amnion mesenchymal stem cell is presented in these cells.For example, being obtained for the present invention Flow cytometer detection show: CD73, CD90, CD105 positive are the positive, and CD34, CD45, HLADR, CD11b, CD19 are negative；Induction Differentiated result shows it: being the positive at cartilage, at rouge, skeletonization.Furthermore, it has been found that by using the method for the present invention, every 400 Square centimeter amnion available 10⁸A above human amnion mesenchymal stem cell.

Detailed description of the invention

Fig. 1 shows the present inventor's amnion mesenchymal stem cell cell microscopic (culture 3 days, 100 times of amplifications)；

Fig. 2 shows the present inventor's amnion mesenchymal stem cell cell microscopic (culture 10 days, 40 times of amplifications)；

Fig. 3 shows the present inventor's amnion mesenchymal stem cell cell microscopic (culture 10 days, 100 times of amplifications)；

Fig. 4 shows the skeletonization test result of the present inventor's amnion mesenchymal stem cell cell induction differentiation；

Fig. 5 show the present inventor's amnion mesenchymal stem cell cell induction differentiation at cartilage test result；

Fig. 6 show the present inventor's amnion mesenchymal stem cell cell induction differentiation at rouge test result.

Specific embodiment

The present invention can be further described by the following examples, however, the scope of the present invention and unlimited In following embodiments.One of skill in the art, can be with it is understood that under the premise of without departing substantially from the spirit and scope of the present invention Various change and modification are carried out to the present invention.The present invention carries out the material and test method arrived used in test general And/or specific description.Although to realize the present invention many materials and operating method used in purpose be it is known in the art that But the present invention still makees description as detailed as possible herein.

Material used in following experiments of the present invention for example wherein used in reagent be it is known in the art, can be with It is prepared using known method or is directly bought from market.

Embodiment 1: human amnion mesenchymal stem cell is prepared from placenta amnion

(1) placenta is taken out from Placenta samples collecting cassette, be placed in ceramic whiteware disk, using basic balanced salt solution (by 0.4g KCl, 0.06g KH₂PO₄, 0.132g Na₂HPO₄.12H₂O, the NaHCO of NaCl, 0.35g of 8g₃, 1.0g D- grape Sugar, the streptomysin of 0.10g, 0.06g penicillin with water dissolve and constant volume at the solution of 1L to get) repeated flushing surface, to tire Disk carries out disinfection processing；

(3) tissue digestion: in 37 degree of constant-temperature tables, tissue block is (interior with the mixed enzyme digestive juice of 1 times of tissue volume Contain: the collagenase type I of 0.1mg/ml, the II Collagenase Type of 0.3mg/ml, the hyaluronidase of 0.1mg/ml and 0.05mg/ml's Neutral proteinase) with 100rpm oscillation digestion 40min；After digestion, adds 2ml fetal calf serum to mix and terminate；With 200 mesh filter screens It filters and is cleaned with a large amount of physiological saline, collect filtrate；

Filtrate portion: the filtrate of collection is centrifuged 5min with 1500rpm, removes supernatant, and it is heavy that cleaning cell is resuspended with physiological saline It forms sediment；It is centrifuged 5min with 1500rpm, supernatant is removed, by cell precipitation complete medium (DMEM-F12 (1: 1) culture medium, 10% FBS, 100 μ g/mL penicillin, 50 μ g/mL streptomysins) it is resuspended；Instrument meter is counted to take number of nucleated cells and expect blue dyeing measurement with platform Cell viability (can usually obtain and be greater than 1 × 10⁷95%) cell, Cell viability are greater than；With every bottle 2 × 10⁶A cell inoculation in 75cm²In culture bottle, 20ml complete medium is added, in 5%CO after rolling is even₂, cultivate in 37 DEG C of incubators；Periodically change liquid；Culture It is passaged to P1 generation after 12 days, continues with complete medium culture；

Tissue block part: the tissue block of collection is placed in 75cm²In culture bottle, tissue block is enable to cover one in culture bottle Half culture area, addition complete medium did not had tissue, in 5%CO after rolling is even₂, cultivate in 37 DEG C of incubators；Fluid infusion after 2d 10ml complete medium continues to cultivate；More mescenchymal stem cell removes tissue after climbing out of, periodically change liquid；Culture is passed on after 12 days To P1 generation, continue with complete medium culture；

(5) P1 is harvested for cell: using (the digestion comprising 0.25% trypsase and 0.02% EDTA of pancreatin digestive juice Liquid) by previous step two parts merge after P1 for cell dissociation after, collect cell, count and measure Cell viability, freeze It deposits to get the amnion mesenchymal stem cell in P1 generation, secondary culture is carried out to it when necessary.

Embodiment 2: human amnion mesenchymal stem cell is prepared from placenta amnion

(3) tissue digestion: in 37 degree of constant-temperature tables, tissue block is (interior with the mixed enzyme digestive juice of 1 times of tissue volume Contain: the collagenase type I of 0.1mg/ml, the II Collagenase Type of 0.3mg/ml, the hyaluronidase of 0.1mg/ml and 0.05mg/ml's Neutral proteinase) with 100rpm oscillation digestion 30min；After digestion, adds 2ml fetal calf serum to mix and terminate；With 200 mesh filter screens It filters and is cleaned with a large amount of physiological saline, collect filtrate；

Filtrate portion: the filtrate of collection is centrifuged 5min with 1500rpm, removes supernatant, and it is heavy that cleaning cell is resuspended with physiological saline It forms sediment；It is centrifuged 5min with 1500rpm, supernatant is removed, by cell precipitation complete medium (DMEM-F12 (1:1) culture medium, 10% FBS, 100 μ g/mL penicillin, 50 μ g/mL streptomysins) it is resuspended；Instrument meter is counted to take number of nucleated cells and expect blue dyeing measurement with platform Cell viability (can usually obtain and be greater than 1 × 10⁷95%) cell, Cell viability are greater than；With every bottle 2 × 10⁶A cell inoculation in 75cm²In culture bottle, 20ml complete medium is added, in 5%CO after rolling is even₂, cultivate in 37 DEG C of incubators；Periodically change liquid；Culture It is passaged to P1 generation after 10 days, continues with complete medium culture；

Tissue block part: the tissue block of collection is placed in 75cm²In culture bottle, tissue block is enable to cover one in culture bottle Half culture area, addition complete medium did not had tissue, in 5%CO after rolling is even₂, cultivate in 37 DEG C of incubators；Fluid infusion after 2d 10ml complete medium continues to cultivate；More mescenchymal stem cell removes tissue after climbing out of, periodically change liquid；Culture is passed on after 15 days To P1 generation, continue with complete medium culture；

Embodiment 3: human amnion mesenchymal stem cell is prepared from placenta amnion

(3) tissue digestion: in 37 degree of constant-temperature tables, tissue block is (interior with the mixed enzyme digestive juice of 1 times of tissue volume Contain: the collagenase type I of 0.1mg/ml, the II Collagenase Type of 0.3mg/ml, the hyaluronidase of 0.1mg/ml and 0.05mg/ml's Neutral proteinase) with 100rpm oscillation digestion 60min；After digestion, adds 2ml fetal calf serum to mix and terminate；With 200 mesh filter screens It filters and is cleaned with a large amount of physiological saline, collect filtrate；

Filtrate portion: the filtrate of collection is centrifuged 5min with 1500rpm, removes supernatant, and it is heavy that cleaning cell is resuspended with physiological saline It forms sediment；It is centrifuged 5min with 1500rpm, supernatant is removed, by cell precipitation complete medium (DMEM-F12 (1:1) culture medium, 10% FBS, 100 μ g/mL penicillin, 50 μ g/mL streptomysins) it is resuspended；Instrument meter is counted to take number of nucleated cells and expect blue dyeing measurement with platform Cell viability (can usually obtain and be greater than 1 × 10⁷95%) cell, Cell viability are greater than；With every bottle 2 × 10⁶A cell inoculation in 75cm²In culture bottle, 20ml complete medium is added, in 5%CO after rolling is even₂, cultivate in 37 DEG C of incubators；Periodically change liquid；Culture It is passaged to P1 generation after 15 days, continues with complete medium culture；

Tissue block part: the tissue block of collection is placed in 75cm²In culture bottle, tissue block is enable to cover one in culture bottle Half culture area, addition complete medium did not had tissue, in 5%CO after rolling is even₂, cultivate in 37 DEG C of incubators；Fluid infusion after 2d 10ml complete medium continues to cultivate；More mescenchymal stem cell removes tissue after climbing out of, periodically change liquid；Culture is passed on after 10 days To P1 generation, continue with complete medium culture；

Method through the embodiment of the present invention 1~3 prepares the human amnion mesenchymal stem cell in P1 generation, for different placenta samples This, fifty-fifty says, every available (1.51~2.07) × 10 of 400 square centimeters of amnions⁸A human amnion mesenchymal stem cell.But It is the human amnion mesenchymal stem cell that the method through the embodiment of the present invention 4~6 prepares P1 generation, it is average for different placental samples Ground says, every available (8.83~10.77) × 10 of 400 square centimeters of amnions⁸A human amnion mesenchymal stem cell, significant ground are high In Examples 1 to 3 method.The present invention has been found that the nitre in complete medium in the complementary testing referring to embodiment 4~6 Both allithiamine and maltose only add one such, then the human amnion mesenchymal stem cell in P1 generation are prepared at this time, for not Same placental samples, fifty-fifty say, every available (1.58~1.91) × 10 of 400 square centimeters of amnions⁸A human amnion mesenchymal Stem cell, this show to be appended to one of thiamine mononitrate and maltose in complete medium can not effectively improve people's amnion between fill The yield of matter stem cell.In addition, the present inventor refers to other existing technical literatures, such as the patent document that the present invention has quoted In method preparation P1 generation (with P1 on behalf of comparing parameter, more comparativity) human amnion mesenchymal stem cell when, it is average every 400 square centimeters of available human amnion mesenchymal stem cells of amnion are less than 1.5 × 10⁸It is a.

Embodiment 4: human amnion mesenchymal stem cell is prepared from placenta amnion

Filtrate portion: the filtrate of collection is centrifuged 5min with 1500rpm, removes supernatant, and it is heavy that cleaning cell is resuspended with physiological saline It forms sediment；It is centrifuged 5min with 1500rpm, supernatant is removed, by cell precipitation complete medium (DMEM-F12 (1: 1) culture medium, 10% FBS, 100 μ g/mL penicillin, 50 μ g/mL streptomysins, 0.02% thiamine mononitrate, 0.085% maltose) it is resuspended；Instrument meter is counted to take Number of nucleated cells simultaneously expects that blue dyeing measurement Cell viability (can usually obtain and be greater than 1 × 10 with platform⁷Cell, Cell viability are greater than 95%)；With every bottle 2 × 10⁶A cell inoculation is in 75cm²In culture bottle, 20ml complete medium is added, in 5%CO after rolling is even₂、 It is cultivated in 37 DEG C of incubators；Periodically change liquid；Culture was passaged to P1 generation after 12 days, continued with complete medium culture；

Fig. 1 shows the present embodiment human amnion mesenchymal stem cell cell microscopic (culture 3 days, 100 times of amplifications)；Fig. 2 Show the present embodiment human amnion mesenchymal stem cell cell microscopic (culture 10 days, 40 times of amplifications)；Fig. 3 shows this implementation Example human amnion mesenchymal stem cell cell microscopic (culture 10 days, 100 times of amplifications)；Fig. 4 is shown between the present embodiment people's amnion The skeletonization test result of mesenchymal stem cells cell induction differentiation；Fig. 5 shows the present embodiment human amnion mesenchymal stem cell cell Induction differentiation at cartilage test result；Fig. 6 show the present embodiment human amnion mesenchymal stem cell cell induction differentiation at Rouge test result.

Embodiment 5: human amnion mesenchymal stem cell is prepared from placenta amnion

Filtrate portion: the filtrate of collection is centrifuged 5min with 1500rpm, removes supernatant, and it is heavy that cleaning cell is resuspended with physiological saline It forms sediment；It is centrifuged 5min with 1500rpm, supernatant is removed, by cell precipitation complete medium (DMEM-F12 (1:1) culture medium, 10% FBS, 100 μ g/mL penicillin, 50 μ g/mL streptomysins, 0.01% thiamine mononitrate, 0.1% maltose) it is resuspended；Instrument meter is counted to have taken Nucleus number simultaneously expects that blue dyeing measurement Cell viability (can usually obtain and be greater than 1 × 10 with platform⁷95%) cell, Cell viability are greater than； With every bottle 2 × 10⁶A cell inoculation is in 75cm²In culture bottle, 20ml complete medium is added, in 5%CO after rolling is even₂, 37 DEG C of trainings It supports and is cultivated in case；Periodically change liquid；Culture was passaged to P1 generation after 10 days, continued with complete medium culture；

Embodiment 6: human amnion mesenchymal stem cell is prepared from placenta amnion

Filtrate portion: the filtrate of collection is centrifuged 5min with 1500rpm, removes supernatant, and it is heavy that cleaning cell is resuspended with physiological saline It forms sediment；It is centrifuged 5min with 1500rpm, supernatant is removed, by cell precipitation complete medium (DMEM-F12 (1:1) culture medium, 10% FBS, 100 μ g/mL penicillin, 50 μ g/mL streptomysins, 0.03% thiamine mononitrate, 0.07% maltose) it is resuspended；Instrument meter is counted to take Number of nucleated cells simultaneously expects that blue dyeing measurement Cell viability (can usually obtain and be greater than 1 × 10 with platform⁷Cell, Cell viability are greater than 95%)；With every bottle 2 × 10⁶A cell inoculation is in 75cm²In culture bottle, 20ml complete medium is added, in 5%CO after rolling is even₂、 It is cultivated in 37 DEG C of incubators；Periodically change liquid；Culture was passaged to P1 generation after 15 days, continued with complete medium culture；

Test example 1: the verifying of amnion mesenchymal stem cell:

Use flow cytomery following items

CD73 detection: prepare two streaming pipes, number, wherein it is sample cell that one, which is Quality Control Guan Yizhi, by CD73's Control reagent shakes up, and 20ul is added into Quality Control pipe, then CD73 reagent is shaken up, and 20ul CD73 examination is added into sample cell Then sample concussion is shaken up 600ul sample is respectively added into two streaming pipes, then mixed by agent, be placed in 4 DEG C or so and be protected from light and incubate It educates 30 minutes, 1 milliliter of PBS is then separately added into Quality Control pipe and streaming pipe, be centrifuged 5 minutes for 1500 revs/min after mixing, after It discards supernatant, mixes upper machine after the PBS of 240ul is added, detection positive Jie Guo≤95% item is qualification, provides cell surface marker Analyte detection return on qualification.

CD90 detection: prepare two streaming pipes, number, wherein it is sample cell that one, which is Quality Control Guan Yizhi, by CD90's Control reagent shakes up, and 20ul is added into Quality Control pipe, then CD90 reagent is shaken up, and 10ul CD90 examination is added into sample cell Then sample concussion is shaken up 600ul sample is respectively added into two streaming pipes, then mixed by agent, be placed in 4 DEG C or so and be protected from light and incubate It educates 30 minutes, 1 milliliter of PBS is then separately added into Quality Control pipe and streaming pipe, be centrifuged 5 minutes for 1500 revs/min after mixing, after It discards supernatant, mixes upper machine after the PBS of 240ul is added, detection positive Jie Guo≤95% item is qualification, provides cell surface marker Analyte detection return on qualification.

CD105 detection: prepare two streaming pipes, number, wherein it is sample cell that one, which is Quality Control Guan Yizhi, by CD105's Control reagent shakes up, and 10ul is added into Quality Control pipe, then CD105 reagent is shaken up, and 5ulCD105 examination is added into sample cell Then sample concussion is shaken up 600ul sample is respectively added into two streaming pipes, then mixed by agent, be placed in 4 DEG C or so and be protected from light and incubate It educates 30 minutes, 1 milliliter of PBS is then separately added into Quality Control pipe and streaming pipe, be centrifuged 5 minutes for 1500 revs/min after mixing, after It discards supernatant, mixes upper machine after the PBS of 240ul is added, detection positive Jie Guo≤95% item is qualification, provides cell surface marker Analyte detection return on qualification.

Remarks: the amount of all cell phenotype detection agents useful for same is the usage amount determined after experiment is feasible.

CD45 detection: prepare two streaming pipes, number, wherein it is sample cell that one, which is Quality Control Guan Yizhi, by CD45's Control reagent shakes up, and 20ul is added into Quality Control pipe, then CD45 reagent is shaken up, and 20ul CD45 examination is added into sample cell Then sample concussion is shaken up 600ul sample is respectively added into two streaming pipes, then mixed by agent, be placed in 4 DEG C or so and be protected from light and incubate It educates 30 minutes, 1 milliliter of PBS is then separately added into Quality Control pipe and streaming pipe, be centrifuged 5 minutes for 1500 revs/min after mixing, after It discards supernatant, mixes upper machine after the PBS of 240ul is added, detection positive Jie Guo≤2% item is qualification, provides cell surface marker Analyte detection return on qualification.

HLA-DR detection: prepare two streaming pipes, number, wherein it is sample cell that one, which is Quality Control Guan Yizhi, by HLA-DR Control reagent shake up, 20ul is added into Quality Control pipe, then HLA-DR reagent is shaken up, is added into sample cell Then sample concussion is shaken up and 600ul sample is respectively added into two streaming pipes, then mixed, be placed in 4 by 20ulHLA-DR reagent DEG C or so be protected from light incubation 30 minutes, 1 milliliter of PBS is then separately added into Quality Control pipe and streaming pipe, 1500 revs/min after mixing Centrifugation 5 minutes, after discard supernatant, be added after the PBS of 240ul the upper machine that mixes, detection positive Jie Guo≤2% item be it is qualified, provide Cell surface marker analyte detection return on qualification.

Amnion mesenchymal stem cell breaks up to the induction of osteoblast: 1) alkaline phosphatase staining: osteoblast can generate A large amount of alkaline phosphatases, bluish violet dyeing are alkaline phosphatase positive.2) calcium deposition dyes: osteoblast can generate calcium salt Deposition, it is orange red to be colored as the positive.It is such the result is that typically to osteoblast induction differentiation positive findings.

Also method well known in the art can be used to carry out for other detection projects.

The human amnion mesenchymal stem cell as obtained by the test method of this test example 1 test embodiment of the present invention 1~6, warp Detection, each parameter is consistent with human amnion mesenchymal stem cell characteristic feature as the result is shown.For example, through flow cytometer detection, embodiment 1 Its CD73, CD90, CD105 positive of~6 gained human amnion mesenchymal stem cells be the positive, CD34, CD45, HLADR, CD11b, CD19 is negative；The induction differentiated result of these amnion mesenchymal stem cells shows it: being the positive at cartilage, at rouge, skeletonization.As a result Show that these markers or induction differentiation performance meet with human amnion mesenchymal stem cell.

Claims

1. the method for preparing human amnion mesenchymal stem cell from placenta amnion, method includes the following steps:

(1) placenta is taken out from Placenta samples collecting cassette, be placed in ceramic whiteware disk, use basic balanced salt solution repeated flushing table Face carries out disinfection processing to placenta；

(2) it is slowly torn with surgical forceps and fetal membrane outer layer amnion is taken to be placed in 150mm glass dish, it is repeatedly clear using basic balanced salt solution Surface crimson blood is washed, the amnion tissue fritter that diameter is 5mm is shredded into；It is filtered with 300 mesh filter screens, the residual blood of normal saline flushing, group It knits block and is transferred to 50ml centrifuge tube；

(3) tissue digestion: in 37 DEG C of constant-temperature tables, by tissue block with the mixed enzyme digestive juice of 1 times of tissue volume with 100rpm 30 ~ 60min of oscillation digestion；After digestion, adds 2ml fetal calf serum to mix and terminate；It is filtered with 200 mesh filter screens and uses a large amount of physiology Salt water cleaning, collects filtrate；

Filtrate portion: the filtrate of collection is centrifuged 5min with 1500rpm, removes supernatant, and cleaning cell precipitation is resuspended with physiological saline； It is centrifuged 5min with 1500rpm, supernatant is removed, cell precipitation is resuspended with complete medium；Counting instrument meter takes number of nucleated cells to be used in combination Platform expects blue dyeing measurement Cell viability；With every bottle 2 × 10⁶A cell inoculation is in 75cm²In culture bottle, addition 20ml is cultivated completely Base is cultivated in 5%CO2,37 DEG C of incubators after rolling is even；Periodically change liquid；Culture was passaged to P1 generation after 10 ~ 15 days, continued with complete Culture medium culture；The complete medium includes: DMEM-F12 culture medium, 10%FBS, 100 μ g/mL penicillin, 50 μ g/mL chains Mycin, 0.01 ~ 0.05% thiamine mononitrate, 0.05 ~ 0.1% maltose, the DMEM-F12 culture medium are DMEM-F12 with volume ratio The culture medium that 1:1 is prepared；

Tissue block part: the tissue block of collection is placed in 75cm²In culture bottle, tissue block is enable to cover half in culture bottle Culture area, addition complete medium did not had tissue, in 5%CO after rolling is even₂, cultivate in 37 DEG C of incubators；Fluid infusion 10ml is complete after 2d Full culture medium continues to cultivate；More mescenchymal stem cell removes tissue after climbing out of, periodically change liquid；Culture was passaged to P1 after 10 ~ 15 days In generation, continues with complete medium culture；

(5) P1 for cell harvest: with pancreatin digestive juice by previous step two parts merge after P1 for cell dissociation after, receive Collect cell, counts and measure Cell viability, freeze to get the amnion mesenchymal stem cell in P1 generation, it is passed on when necessary Culture.

2. the method according to claim 1, the basis balanced salt solution is prepared in the following way to be obtained: by 0.4g's The D- grape of NaHCO3,1.0g of NaCl, 0.35g of Na2HPO4.12H2O, 8g of KH2PO4,0.132g of KCl, 0.06g Sugar, the streptomysin of 0.10g, 0.06g penicillin with water dissolve and constant volume at 1L solution.

3. the method according to claim 1, include in the mixed enzyme digestive juice: the collagenase type I of 0.1mg/ml, 0.3mg/ml II Collagenase Type, the hyaluronidase of 0.1mg/ml and the neutral proteinase of 0.05mg/ml.

4. the method according to claim 1, in step (5), pancreatin digestive juice is the trypsase and 0.02% for including 0.25% The digestive juice of EDTA.