CN104480533A - Placenta stem cell bank construction method and placenta tissue resuscitation method - Google Patents
Placenta stem cell bank construction method and placenta tissue resuscitation method Download PDFInfo
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Abstract
The invention discloses a placenta stem cell bank construction method and a placenta tissue resuscitation method, and relates to placenta stem cell bank construction methods. The placenta stem cell bank construction method comprises the following steps: after non-bacterial cleaning treatment to placenta tissues of at least three parts, the steps of tissue peeling, further sterilizing, shearing to be thin, protecting using a refrigerant, programmed freezing, cryogenic temperature temporary storage, long term storage with nitrogen canisters, and the like are carried out, and the placenta tissue with biological activities can be preserved for a long term. The placenta tissue preserved through the method can be used for the construction of a stem cell bank, resuscitation, enzymic digestion, cultivation, expansion, quality inspection and the like are performed after selective tissue part resuscitation according to required cell types, so as to obtain placenta amniotic membrance epithelial cells, placenta amniotic membrance mesenchyme, placenta chorion mesenchymal stem cell, placenta decidua serotina mesenchymal stem cells and the like. The method provided by the invention has the advantages that operation steps are simplified, manual intervention errors are reduced, the efficiency is improved, more stem cell resources are preserved, and a basis is provided for further personalized stem cell customization.
Description
Technical field
The present invention relates to a kind of construction process and placenta tissue method for resuscitation of placenta stem-cell storehouse.
Background technology
Placenta is the tie of the various nutritive substance conveyings that baby depends on for existence, is made up of amnion, chorion and decidua basalis.Amnion and chorion derive from fetus, and decidua basalis then originates from parent.Placenta except playing a significant role in fetation, nutritional support and maintenance tolerance, or the stem cell warehouse of a body.
Have multiple multipotential stem cell in placenta, known at present have three class placenta stem-cells at least.In placenta, first kind multipotential stem cell is mescenchymal stem cell (Mesenchymal stem cells, MSCs), MSCs is that a class can self, has the multipotential stem cell of Multidirectional Differentiation ability, can be divided into the histocytes such as bone, cartilage, fat, nerve and liver cell.
The characteristic of placenta MSCs energy Immunosuppression and promotion hematopoietic reconstitution, makes its GVHD after hematopoietic stem cell transplantation and transplanting have a good application prospect in preventing and treating.Because placenta source MSCs has tissue-derived wide and advantage that ability of cell proliferation is strong compared with derived from bone marrow MSCs, make it have better potential applicability in clinical practice.Placenta MSCs at least can be divided into 3 kinds, chorion MSCs, amnion MSCs, also comprise the MSCs of the decidua basalis tissue from parent, by the immune labeled detection to tapetum and decidua basalis MSCs, find that they all express typical MSCs surface antigen CD13, CD29, CD44, CD54, CD73, CD105, do not express hemopoietic stem cell surface antigens c D3, CD14, CD34, CD45 and surface endothelial cell antigens CD31.Find by detecting, chorion MSCs is weak expression embryonic stem cell class surface antigen S SEA-4 also, and amnion MSCs expresses outside typical MSCs surface antigen, also expresses the surface marker Oct-4 of embryonic stem cell class.And decidua basalis is as one of the integral part of Materno-fetal interface, be parent face, mean that the MSCs deriving from decidua basalis can be used as the one of the stem cell in mother source.
Equations of The Second Kind multipotential stem cell in placenta is amniotic epithelial cells (human amniotic epithelial cells, hAECs).People hAECs, as a kind of stem cell, expresses multiple embryonic stem cell class mark, comprises SSEA-4, SSEA-3, TRA-1-60, TRA-1-80, and hAECs expresses the specific transcription factor OCT-4 of pluripotent stem cell and Nanog gene.There are the potentiality of the polyphyly differentiation such as Cardiomyocytes, liver, neurocyte and islet cells simultaneously, because this person hAECs is a middle-bracket stem cell between embryonic stem cell and adult stem cell.Show in research, hAECs does not express the human leucocyte antigens such as HLA-A, B, C, DR and has immune privilege, the generation of immunological rejection can be avoided after transplanting, have potential applicability in clinical practice widely, Collection and preservation amniotic epithelial cells also becomes and will carry out clinical application future and create condition in recent years.
The 3rd class stem cell in placenta is placental hematopoietic stem cell (human placenta-derived hemopoietic stem cells, hPD-HSCs).HPD-HSCs is the tissue block being organized into 0.5mm3 by shearing placental fetal surface, carry out tissue digestion through neutral protease, trypsinase and collagenase etc., or add perfusion liquid perfusion acquisition middle layer mononuclearcell through CXCR-4 receptor-blocking agent AMD3100.The hemopoietic stem cells such as its CD34, CD133 show that antigen presentation is the positive.Compared with the Cord blood of flowing, in hPD-HSCs in placenta tissue gap, CD34 positive cell is the several times in Cord blood, and the granulocyte monocyte colony that placenta CD34+CD38+hPD-HSCs cell subset is differentiated to form, quick-fried red type colony, mixed type colony number are all apparently higher than umbilical cord blood hematopoietic stem cell.The clinical value of hPD-HSCs is similar to umbilical cord blood hematopoietic stem cell, can at treatment hematologic disease, as there is good effect the aspects such as acute and chronic leukemia, aplastic anemia, thalassemia and lymphoma, it is an important stem cell resource can supplementing umbilical cord blood hematopoietic stem cell quantity not sufficient.
At present, both at home and abroad for placenta stem-cell storing mode be single position sampling, then cultured and amplified in vitro, carries out Cryopreserved after reaching some amount.This mode has only carried out amplification, the cultivation of a kind of stem cell in placenta tissue, and most of stem cell does not have to be preserved.The invention provides a kind of placenta stem-cell base construction method, present method saves at least 3 placenta tissue positions (including but not limited to amnion, chorion, placenta decidua basalis), also multiple stem cell known is at present saved, simultaneously, to in placenta tissue at present unknown, may also preserve by the larger stem cell of development and application potentiality in the future, this method simplify the operation step, raise the efficiency and save multiple stem cell resource, for further customization stem cell be separated, cultivate, amplification lay a good foundation.
Summary of the invention
The present invention is for the different sites preservation of placenta tissue provides reliable method, this method is used for placenta stem-cell storehouse and sets up, be separated the stem cell of difference in functionality, extend the shelf time of placenta tissue, guarantee organizes inner cell unaffected, ensure histiocytic activity, working method is simple, can be directly used in stem cell separation and Culture after recovery.
The cryopreservation methods of the present invention's application placenta tissue, cuts little of 0.5 ~ 1mm respectively after being peeled off by placenta different sites tissue
3size, add configure and meet in advance cold frozen storing liquid resuspended after, be sub-packed in cryopreservation tube, 30min is balanced at 4 DEG C of temperature, proceed to-80 DEG C of fridge overnight, be finally placed in the medium-term and long-term preservation of liquid nitrogen, carry out respectively recovering for cell cultures according to required cell category.
A kind of placenta stem-cell base construction method of the present invention, it carries out according to following steps:
One, placenta collection: gather the complete placenta leaving 3 ~ 5cm umbilical cord, for subsequent use;
Two, placenta tissue pre-treatment: the placenta tissue getting step one at least 3 positions adopts tissue shear respectively, pincers are carefully peeled off, collected, after collection organize respectively with physiological saline clean blood, cut little of 0.5 ~ 1mm
3size, respectively with physiological saline cleaning 2 ~ 3 times, wherein, after each cleaning under 1300rpm rotating speed centrifugal 6min, collecting precipitation, continues to clean next time; After cleaning terminates, collecting precipitation is for subsequent use;
Three, tissue block freezing liquid preparation: the every 1mL of described frozen storing liquid is made up of 0.68 ~ 0.72mL DMEM, 0.07 ~ 0.12mL foetal calf serum, 0.01 ~ 0.02mL L-glutaminate and 0.2mL dimethyl sulfoxide (DMSO), preserves in 4 DEG C of precoolings after preparing; Or tissue block freezing liquid adopts serum free medium to prepare;
Four, organization procedure thaws and deposits: the tissue block that step 2 precooling is preserved is made into tissue suspension with the substratum of 0.5 times of tissue block volume is resuspended, add the frozen storing liquid of the step 3 preparation of 0.5 ~ 1.5 times of tissue suspension volume again, then divide and be filled in cryopreservation tube, first 4 DEG C of balance 30min in Programmed freezing box, then proceed to-80 DEG C and spend the night temporary;
Five, the antifreeze bar code of low temperature is established to each cryopreservation tube, manage;
Six, anerobe, aerophil, mould, hepatitis B virus, hepatitis C virus, virus of AIDS and Lues Assay are carried out to the pretreated placenta tissue sample of step 2 and the placenta tissue of step 5 recovery; Be all that negative placenta tissue proceeds to-150 DEG C ~-180 DEG C medium-term and long-term preservations of liquid nitrogen container gas phase by detected result, namely construct placenta stem-cell storehouse.
The placenta tissue method for resuscitation in a kind of placenta stem-cell storehouse of the present invention, it carries out according to following steps:
One, first Accessory Right requires that taking out cryopreservation tube in the 1 placenta stem-cell storehouse built is placed on 1 ~ 2min in 37 DEG C ~ 40 DEG C water-baths and thaws;
Two, the placenta tissue after step one being thawed puts into clean centrifuge tube, add the DMEM mixing of 2 ~ 3 times of volumes, then centrifugal 6min under 4 DEG C of temperature, rotating speed are the condition of 1300 revs/min, go supernatant to collect solid formation, repeated centrifugation operation 2 ~ 3 times, collects solid formation;
Three, the solid formation that step 2 is collected is inoculated in T75 culturing bottle, adds DMEM nutrient solution and be placed on CO
2cultivate in incubator after 2 days, calculate adherent tissue number of blocks and suspended tissue's number of blocks;
The adherent surviving rate of computation organization's block in the following way:
Adherent tissue block quantity/(suspend in the adherent tissue block quantity+culturing bottle survived in culturing bottle non-adherent tissue number of blocks) * 100% survived in adherent surviving rate=culturing bottle;
Namely the placenta tissue recovery in placenta stem-cell storehouse is completed.
The placenta tissue method for resuscitation in a kind of placenta stem-cell storehouse of the present invention, it carries out according to following steps:
One, first Accessory Right requires that taking out cryopreservation tube in the 1 placenta stem-cell storehouse built is placed on 1 ~ 2min in 37 DEG C ~ 40 DEG C water-baths and thaws;
Two, the placenta tissue after step one being thawed puts into clean centrifuge tube, add the DMEM mixing of 2 ~ 3 times of volumes, then centrifugal 6min under 4 DEG C of temperature, rotating speed are the condition of 1300 revs/min, go supernatant to collect solid formation, repeated centrifugation operation 2 ~ 3 times, collects solid formation;
Three, after the solid formation collected step 2 carries out enzymolysis 30 ~ 50min, centrifugal 6min under 4 DEG C of temperature, rotating speed are the condition of 1300 revs/min, remove the cell of supernatant collecting precipitation, sampling and measuring Cell viability after DMEM suspends also calculates recovery Cell viability in the following way: Cell viability=viable cell quantity/(viable cell quantity+dead cell quantity) * 100%; And the total cellular score quantity calculated after recovery; Namely the placenta tissue recovery in placenta stem-cell storehouse is completed.
The present invention comprises following beneficial effect:
1) when setting up stem cell bank, only efficient tissue freezing is carried out, easy and simple to handle, save time, save space resources, but save a large amount of stem cell resources.If build storehouse to carry out cell amplification at the very start, multiple amplification cultivation system, cultivation preserve various kinds of cell will be set up, part placenta stem-cell resource known at present could be preserved like this.
2) at least 3 placenta tissue positions (placenta amnion, Chorionic villi of placenta, placenta decidua basalis) are first peeled off by the present invention, according to different sites (the fetus face of placenta, parent face) carry out frozen respectively, the tissue block of recovery different sites is carried out according to patient demand;
3) the present invention be tissue block is cut respectively little of 0.5 ~ 1mm
3size, in frozen process, frozen protection liquid can in infiltrating tissues cell, make intracellular moisture deviate from, and in frozen process, the effective formation reducing ice crystal in frozen process, reduces the damage of cell;
4) different sites of freezen protective liquid protection placenta tissue of the present invention, carries out the placenta tissue of recovery different sites, has effectively saved placenta tissue block, add the access times of patient according to the user demand of patient;
5) the inventive method can be applicable to newborn infant be born after the preservation at placenta different tissues position, extend the shelf time of tissue by least 20 years, can preserve for a long time in liquid nitrogen, according to the tissue block of mother, fetal disease selectivity recovery different sites, thus the cultivation of different types of stem cell is applied to clinical treatment.
6) present method saves whole cells at least 3 placenta tissue positions, this not only saves placenta stem-cell known at present, can when needs Clinical practice, carry out amplification cultivation selectively, and, also current the unknown is saved, but the placenta cells that likely application prospect is wider in the future, this kind of cell does not likely know its function due to the restriction of current our human-subject test, and may its using value just be found after 10-20, if adopt common banking process, this kind of cell is starting will to increase and preserve by not understood because of us most.
7) though present method also ensure that in placenta tissue some at present we understand its following function there is no good cultivation, the stem cell of amplification method is able to complete preservation.After development in science and technology, after amplification and the means of cultivation are constantly improved, then from the placenta stem-cell storehouse that present method is set up, these cells are bred in a large number, thus carry out clinical study or application better.
8) if adopt tissue block method to carry out placenta stem-cell cultivation, after the inventive method recovery, the adherent surviving rate of placenta tissue block is up to more than 98%.According to enzyme digestion culturing stem cells, after the recovery of cryopreserved tissue block, enzymolysis, digestion, cytoactive is more than 90%, can successfully prepare the stem cells such as placenta mesenchyma stem cell, amnion mesenchymal stem cell, amniotic epithelial cells.
Accompanying drawing explanation
Fig. 1 is placenta stem-cell storehouse Establishing process schematic diagram;
Fig. 2 is placenta mesenchyma stem cell aspect graph;
Fig. 3 is amnion mesenchymal stem cell aspect graph;
Fig. 4 is amniotic epithelial cells aspect graph.
Embodiment
Embodiment one: a kind of placenta stem-cell base construction method of present embodiment, it carries out according to following steps:
One, placenta collection: gather the complete placenta leaving 3 ~ 5cm umbilical cord, for subsequent use;
Two, placenta tissue pre-treatment: the placenta tissue getting step one at least 3 positions adopts tissue shear respectively, pincers are carefully peeled off, collected, after collection organize respectively with physiological saline clean blood, cut little of 0.5 ~ 1mm
3size, respectively with physiological saline cleaning 2 ~ 3 times, wherein, after each cleaning under 1300rpm rotating speed centrifugal 6min, collecting precipitation, continues to clean next time; After cleaning terminates, collecting precipitation is for subsequent use;
Three, tissue block freezing liquid preparation: the every 1mL of described frozen storing liquid is made up of 0.68 ~ 0.72mL DMEM, 0.07 ~ 0.12mL foetal calf serum, 0.01 ~ 0.02mL L-glutaminate and 0.2mL dimethyl sulfoxide (DMSO), preserves in 4 DEG C of precoolings after preparing; Or tissue block freezing liquid adopts serum free medium to prepare;
Four, organization procedure thaws and deposits: the tissue block that step 2 precooling is preserved is made into tissue suspension with the substratum of 0.5 times of tissue block volume is resuspended, add the frozen storing liquid of the step 3 preparation of 0.5 ~ 1.5 times of tissue suspension volume again, then divide and be filled in cryopreservation tube, first 4 DEG C of balance 30min in Programmed freezing box, then proceed to-80 DEG C and spend the night temporary;
Five, the antifreeze bar code of low temperature is established to each cryopreservation tube, manage;
Six, anerobe, aerophil, mould, hepatitis B virus, hepatitis C virus, virus of AIDS and Lues Assay are carried out to the pretreated placenta tissue sample of step 2 and the placenta tissue of step 5 recovery; Be all that negative placenta tissue proceeds to-150 DEG C ~-180 DEG C medium-term and long-term preservations of liquid nitrogen container gas phase by detected result, namely construct placenta stem-cell storehouse.
Embodiment two: present embodiment and embodiment one unlike: described placenta stem-cell storehouse is including but not limited to Chorionic villi of placenta mescenchymal stem cell, placenta amnion mescenchymal stem cell, placenta decidua basalis mescenchymal stem cell and placenta amnion epithelial cell four kinds of stem cells.Other is identical with embodiment one.
Embodiment three: present embodiment and embodiment one unlike: the placenta stroke-physiological saline solution after gathering in step one is rinsed surperficial blood and is placed in sterilized bio specimen sample bag, transport in 4 ~ 25 DEG C of collector tanks, collector tank transportation requires that control temperature is constant, prevent from jolting, the placenta of collection need process in 72 hours.Other is identical with embodiment one.
Embodiment four: present embodiment and embodiment one unlike: the temperature of collector tank is 4 ~ 10 DEG C.Other is identical with embodiment one.
Embodiment five: present embodiment and embodiment one unlike: the placenta of collection need process in 24 hours.Other is identical with embodiment one.
Embodiment six: during present embodiment and embodiment one are set with unlike the collection of: collector tank, can place but be not limited to the conserving liquid such as physiological saline, DMEM, volume was as the criterion to cover placenta, and required suit favorable sealing property.Other is identical with embodiment one.
Embodiment seven: present embodiment and embodiment one unlike: the donor of the placenta gathered in step one is the donor infecting history, malignant tumour history, history of drug abuse, chromosome abnormalty history, diabetic history, history of venereal disease and other hereditary medical history without blood system medical history, immunity system medical history, neural system medical history, endocrine system medical history, HTLV, HBV, HCV, HIV, syphilis etc.Other is identical with embodiment one.
Embodiment eight: present embodiment and embodiment one unlike: the every 1mL of described frozen storing liquid is made up of 0.70mL DMEM, 0.10mL foetal calf serum, 0.15mL L-glutaminate and 0.2mL dimethyl sulfoxide (DMSO).Other is identical with embodiment one.
Embodiment nine: present embodiment and embodiment one unlike: the substratum in step 4 is DMEM, DMEM/F12, AMEM or serum free medium.Other is identical with embodiment one.
Embodiment ten: present embodiment and embodiment one unlike: the placenta tissue at 3 described positions comprises amnion, Chorionic villi of placenta and placenta decidua basalis.Other is identical with embodiment one.
Embodiment 11: the placenta tissue method for resuscitation in a kind of placenta stem-cell storehouse of present embodiment, it carries out according to following steps:
One, first Accessory Right requires that taking out cryopreservation tube in the 1 placenta stem-cell storehouse built is placed on 1 ~ 2min in 37 DEG C ~ 40 DEG C water-baths and thaws;
Two, the placenta tissue after step one being thawed puts into clean centrifuge tube, add the DMEM mixing of 2 ~ 3 times of volumes, then centrifugal 6min under 4 DEG C of temperature, rotating speed are the condition of 1300 revs/min, go supernatant to collect solid formation, repeated centrifugation operation 2 ~ 3 times, collects solid formation;
Three, the solid formation that step 2 is collected is inoculated in T75 culturing bottle, adds DMEM nutrient solution and be placed on CO
2cultivate in incubator after 2 days, calculate adherent tissue number of blocks and suspended tissue's number of blocks;
The adherent surviving rate of computation organization's block in the following way:
Adherent tissue block quantity/(suspend in the adherent tissue block quantity+culturing bottle survived in culturing bottle non-adherent tissue number of blocks) * 100% survived in adherent surviving rate=culturing bottle;
Namely the placenta tissue recovery in placenta stem-cell storehouse is completed.
Embodiment 12: the placenta tissue method for resuscitation in a kind of placenta stem-cell storehouse of present embodiment, it carries out according to following steps:
One, first Accessory Right requires that taking out cryopreservation tube in the 1 placenta stem-cell storehouse built is placed on 1 ~ 2min in 37 DEG C ~ 40 DEG C water-baths and thaws;
Two, the placenta tissue after step one being thawed puts into clean centrifuge tube, add the DMEM mixing of 2 ~ 3 times of volumes, then centrifugal 6min under 4 DEG C of temperature, rotating speed are the condition of 1300 revs/min, go supernatant to collect solid formation, repeated centrifugation operation 2 ~ 3 times, collects solid formation;
Three, after the solid formation collected step 2 carries out enzymolysis 30 ~ 50min, centrifugal 6min under 4 DEG C of temperature, rotating speed are the condition of 1300 revs/min, remove the cell of supernatant collecting precipitation, sampling and measuring Cell viability after DMEM suspends also calculates recovery Cell viability in the following way: Cell viability=viable cell quantity/(viable cell quantity+dead cell quantity) * 100%; And the total cellular score quantity calculated after recovery; Namely the placenta tissue recovery in placenta stem-cell storehouse is completed.
Content of the present invention is not limited only to the content of the respective embodiments described above, and the combination of one of them or several embodiment equally also can realize the object of inventing.
Beneficial effect of the present invention is verified by following examples:
Embodiment one
The placenta stem-cell storehouse establishment method of the present embodiment is as follows:
One, placenta collection: gather the complete placenta leaving 3 ~ 5cm umbilical cord, placenta stroke-physiological saline solution after collection is rinsed surperficial blood and is placed in sterilized bio specimen sample bag, transport in 4 ~ 25 DEG C of collector tanks (optimum control is at 4-10 DEG C), gather in suit, can place but be not limited to the conserving liquid such as physiological saline, DMEM, volume was as the criterion to cover placenta, and required suit favorable sealing property.Transportation requires control temperature, prevents from jolting, haulage time in 72 hours, the best is no more than in 24 hours and is prepared;
Wherein, placenta donor requires to infect history, malignant tumour history, history of drug abuse, chromosome abnormalty history, diabetic history, history of venereal disease and other hereditary medical history without blood system medical history, immunity system medical history, neural system medical history, endocrine system medical history, HTLV, HBV, HCV, HIV, syphilis etc.; The donor pregnancy period requires that fetus avoids as premature infant in 38-42 week;
Two, placenta tissue pre-treatment: the placenta tissue getting step one at least 3 positions includes but not limited to amnion, chorion, placenta decidua basalis) adopt tissue shear respectively, pincers carefully peel off, collect, each portion of tissue is at least peeled off, collect 5 ~ 20 grams of weight in wet bases, after collection organize respectively with physiological saline clean blood, cut little of 0.5 ~ 1mm
3size, respectively with physiological saline cleaning 2 ~ 3 times, wherein, after each cleaning under 1300rpm rotating speed centrifugal 6min, collecting precipitation, continues to clean next time; After cleaning terminates, collecting precipitation is for subsequent use;
Three, tissue block freezing liquid preparation: be formulated as example with 10ml frozen storing liquid, it is characterized in that described frozen storing liquid is made up of 6.8 ~ 7.2mLDMEM, 0.7 ~ 1.2mL foetal calf serum, 0.1 ~ 0.2mL L-glutaminate and 2mL dimethyl sulfoxide (DMSO), preserve in 4 DEG C of precoolings after preparing, serum free medium also can be adopted to prepare.
Four, organization procedure thaws and deposits: the tissue block substratum (substratum can be but be not limited to DMEM, DMEM/F12, AMEM or serum free medium) of 0.5 times of volume step 2 precooling preserved is resuspended is made into tissue suspension (1ml organizes: 0.5mlDMEM substratum), then in re-suspension liquid, add the frozen storing liquid 0.5 ~ 1.5 times (1ml tissue suspension: 0.5ml ~ 1.5ml frozen storing liquid) of step 3 preparation, then be dispensed in cryopreservation tube by every pipe 1mL, the frozen 10-30 pipe of every site tissue.Cryopreservation tube proceeds to-80 degree Ultralow Temperature Freezers and spends the night temporary after being placed in Programmed freezing box 4 DEG C of balance 30min, proceed to the medium-term and long-term preservation of liquid nitrogen after every detected result is qualified;
Five, sample bar code management:
Placenta stem-cell storehouse process of establishing can manage according to bar code, and the placenta tissue at each position can have unique bar code corresponding, and the bar code on corresponding cryopreservation tube is the antifreeze bar code of low temperature;
Six, anerobe, aerophil, mould detection and hepatitis B virus, hepatitis C virus, virus of AIDS and Lues Assay are carried out to the placenta tissue of the placenta tissue sample of step 2 and the recovery of step 5;-150 DEG C ~-180 DEG C medium-term and long-term preservations of liquid nitrogen container gas phase are proceeded to the placenta tissue that detected result is total negative, namely constructs placenta stem-cell storehouse.
The foundation in the present embodiment placenta stem-cell storehouse mainly comprises Chorionic villi of placenta mescenchymal stem cell, placenta amnion mescenchymal stem cell, placenta decidua basalis mescenchymal stem cell, placenta amnion epithelial cell four kinds of stem cells, by detecting the immunophenotype of often kind of cell, expressed by the surface marker of flow cytomery placenta stem-cell and bone marrow stem cell similar, positive expression mescenchymal stem cell surface marker: CD90, CD105, CD166, CD29, CD44 and CD73, amnion-derived stem cell also expresses the surface marker SSEA-4 of embryonic stem cell class, the distinctive protein ingredient OCT-4 of its high expression level embryonic stem cell is found by the stem cell that Immunofluorescence test is amnion-derived, the positive expression of these factors illustrates that amnion-derived stem cell is the embryonic stem cell-like with versatility between embryonic stem cell and adult stem cell.In addition, can carry out cellular identification from morphology, placenta mesenchyma stem cell, amnion mesenchymal stem cell all have fusiform and swirling growthhabit, and amniotic epithelial cells has cobblestone-shaped cells form.
Placenta tissue in the placenta stem-cell storehouse build embodiment one carries out recovery process:
When thawing, first cryopreservation tube is taken out from liquid nitrogen, be placed in 37 DEG C ~ 40 DEG C water-baths to complete rapidly in 1 ~ 2min and thaw; With transfer pipet, the tissue block of recovery is sucked the centrifuge tube putting into cleaning, the DMEM adding 2 ~ 3 times of volumes mixes gently, and then centrifugal (4 DEG C, 1300 revs/min, 6 minutes) remove supernatant liquor, repeatedly cleans 2-3 time, and removes supernatant liquor; Tissue block is directly inoculated in T75 culturing bottle, adds nutrient solution and be placed on CO
2cultivate in incubator, cultivate after 2 days, in each culturing bottle, calculate adherent tissue number of blocks and suspended tissue's number of blocks, the adherent surviving rate of computation organization's block (in adherent surviving rate=culturing bottle adherent tissue block quantity/(suspend in the adherent tissue block quantity+culturing bottle survived in culturing bottle non-adherent tissue number of blocks) * 100% survived); Or employing enzymolysis process, adopts but is not limited to after the digestive ferment such as collagenase I, II, IV, pancreatin, pancreatin surrogate carries out digestion 30-50 minute, 4 DEG C, 1300 revs/min, 6 minutes centrifugal, remove supernatant liquor, collect unicellular particle.Calculate recovery Cell viability (Cell viability=viable cell quantity/(viable cell quantity+dead cell quantity) * 100%) and total cellular score after Trypan Blue, and carry out stem cell qualification.Qualification result as shown in Table 1 and Table 2,
Form 1: recovery is cultivated, increase rear flow cytometer and Immunofluorescence test qualification
Note: ("+": represent positive expression, "-": represent negative expression)
Form 2: cell, tissue culture result after placenta tissue recovery
After embodiment one builds storehouse, to the adherent surviving rate of placenta tissue block after placenta tissue recovery up to more than 98%.According to enzyme digestion separation and Culture cell, after the recovery of cryopreserved tissue block, enzymolysis, digestion, cytoactive is more than 90%.
This shows, the placenta stem-cell storehouse method adopting this method to set up is simple, if organize adherent amplification Shuai Gao road 98% digestion method culturing cell, after digestion, Cell viability is more than 90%, illustrate after after the processes such as most tissues, cell are thin through tissue shear of the present invention, frozen storing liquid permeates, sequencing is frozen, rapid fluid resuscitation, maintain original cytoactive, its original Growth of Cells trend is not subject to any change, be one reliably and easy placenta stem-cell storage method.
Frozen, after cell cultures after recovery, can with normally turn out all kinds of placenta multipotential stem cell, comprise amniotic epithelial cells, amnion mesenchymal stem cell, placenta mesenchyma stem cell etc., the result of cultivating out with flesh tissue form is consistent, from flow cytometry and fluorescence immunoassay Epidemiological Analysis result, all kinds of multipotential stem cell also expresses corresponding surface antigen and specific proteins mark, illustrate that this kind sets up the stem cell bank method also fundamental property of multiple stem cell and the prospect of following application from the placenta tissue guaranteed, by this kind of stem cell bank establishment method, variform can be obtained, the multipotential stem cell of function excellence, at present not available for other banking process.
Claims (8)
1. a placenta stem-cell base construction method, is characterized in that it carries out according to following steps:
One, placenta collection: gather the complete placenta leaving 3 ~ 5cm umbilical cord, for subsequent use;
Two, placenta tissue pre-treatment: the placenta tissue getting step one at least 3 positions adopts tissue shear respectively, pincers are carefully peeled off, collected, after collection organize respectively with physiological saline clean blood, cut little of 0.5 ~ 1mm
3size, respectively with physiological saline cleaning 2 ~ 3 times, wherein, after each cleaning under 1300rpm rotating speed centrifugal 6min, collecting precipitation, continues to clean next time; After cleaning terminates, collecting precipitation is for subsequent use;
Three, tissue block freezing liquid preparation: the every 1mL of described frozen storing liquid is made up of 0.68 ~ 0.72mL DMEM, 0.07 ~ 0.12mL foetal calf serum, 0.01 ~ 0.02mL L-glutaminate and 0.2mL dimethyl sulfoxide (DMSO), preserves in 4 DEG C of precoolings after preparing; Or tissue block freezing liquid adopts serum free medium to prepare;
Four, organization procedure thaws and deposits: the tissue block that step 2 precooling is preserved is made into tissue suspension with the substratum of 0.5 times of tissue block volume is resuspended, add the frozen storing liquid of the step 3 preparation of 0.5 ~ 1.5 times of tissue suspension volume again, then divide and be filled in cryopreservation tube, first 4 DEG C of balance 30min in Programmed freezing box, then proceed to-80 DEG C and spend the night temporary;
Five, the antifreeze bar code of low temperature is established to each cryopreservation tube, manage;
Six, anerobe, aerophil, mould, hepatitis B virus, hepatitis C virus, virus of AIDS and Lues Assay are carried out to the pretreated placenta tissue sample of step 2 and the placenta tissue of step 5 recovery; Be all that negative placenta tissue proceeds to-150 DEG C ~-180 DEG C medium-term and long-term preservations of liquid nitrogen container gas phase by detected result, namely construct placenta stem-cell storehouse.
2. a kind of placenta stem-cell base construction method according to claim 1, is characterized in that described placenta stem-cell storehouse comprises Chorionic villi of placenta mescenchymal stem cell, placenta amnion mescenchymal stem cell, placenta decidua basalis mescenchymal stem cell and placenta amnion epithelial cell four kinds of stem cells.
3. a kind of placenta stem-cell base construction method according to claim 1, it is characterized in that the placenta stroke-physiological saline solution after gathering in step one is rinsed surperficial blood and is placed in sterilized bio specimen sample bag, transport in 4 ~ 25 DEG C of collector tanks, collector tank transportation requires that control temperature is constant, prevent from jolting, the placenta of collection need process in 72 hours.
4. a kind of placenta stem-cell base construction method according to claim 1, is characterized in that the every 1mL of described frozen storing liquid is made up of 0.70mL DMEM, 0.10mL foetal calf serum, 0.15mL L-glutaminate and 0.2mL dimethyl sulfoxide (DMSO).
5. a kind of placenta stem-cell base construction method according to claim 1, is characterized in that the substratum in step 4 is DMEM, DMEM/F12, AMEM or serum free medium.
6. a kind of placenta stem-cell base construction method according to claim 1, is characterized in that the placenta tissue at 3 described positions comprises amnion, Chorionic villi of placenta and placenta decidua basalis.
7. the placenta tissue method for resuscitation in placenta stem-cell storehouse, is characterized in that it carries out according to following steps:
One, first Accessory Right requires that taking out cryopreservation tube in the 1 placenta stem-cell storehouse built is placed on 1 ~ 2min in 37 DEG C ~ 40 DEG C water-baths and thaws;
Two, the placenta tissue after step one being thawed puts into clean centrifuge tube, add the DMEM mixing of 2 ~ 3 times of volumes, then centrifugal 6min under 4 DEG C of temperature, rotating speed are the condition of 1300 revs/min, go supernatant to collect solid formation, repeated centrifugation operation 2 ~ 3 times, collects solid formation;
Three, the solid formation that step 2 is collected is inoculated in T75 culturing bottle, adds DMEM nutrient solution and be placed on CO
2cultivate in incubator after 2 days, calculate adherent tissue number of blocks and suspended tissue's number of blocks;
The adherent surviving rate of computation organization's block in the following way:
Adherent tissue block quantity/(suspend in the adherent tissue block quantity+culturing bottle survived in culturing bottle non-adherent tissue number of blocks) * 100% survived in adherent surviving rate=culturing bottle;
Namely the placenta tissue recovery in placenta stem-cell storehouse is completed.
8. the placenta tissue method for resuscitation in placenta stem-cell storehouse, is characterized in that it carries out according to following steps:
One, first Accessory Right requires that taking out cryopreservation tube in the 1 placenta stem-cell storehouse built is placed on 1 ~ 2min in 37 DEG C ~ 40 DEG C water-baths and thaws;
Two, the placenta tissue after step one being thawed puts into clean centrifuge tube, add the DMEM mixing of 2 ~ 3 times of volumes, then centrifugal 6min under 4 DEG C of temperature, rotating speed are the condition of 1300 revs/min, go supernatant to collect solid formation, repeated centrifugation operation 2 ~ 3 times, collects solid formation;
Three, after the solid formation collected step 2 carries out enzymolysis 30 ~ 50min, centrifugal 6min under 4 DEG C of temperature, rotating speed are the condition of 1300 revs/min, remove the cell of supernatant collecting precipitation, sampling and measuring Cell viability after DMEM suspends also calculates recovery Cell viability in the following way: Cell viability=viable cell quantity/(viable cell quantity+dead cell quantity) * 100%; And the total cellular score quantity calculated after recovery; Namely the placenta tissue recovery in placenta stem-cell storehouse is completed.
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