CN105969721A - Extraction preparation method of placenta hematopoietic stem cells - Google Patents
Extraction preparation method of placenta hematopoietic stem cells Download PDFInfo
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- CN105969721A CN105969721A CN201610570495.0A CN201610570495A CN105969721A CN 105969721 A CN105969721 A CN 105969721A CN 201610570495 A CN201610570495 A CN 201610570495A CN 105969721 A CN105969721 A CN 105969721A
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Abstract
The invention discloses an extraction preparation method of placenta hematopoietic stem cells. The method comprises the following steps of: collecting a healthy placenta of a woman after eutocia or cesarean in a sterile operating room environment, performing early cleaning treatment of the placenta using a cleaning solution, and collecting the treated liquid A; lavaging the blood in vein and artery of the placenta using a lavaging solution, and collecting liquid B after the lavaging; cutting the placenta into pieces, digesting the placenta tissue with enzymatic hydrolysate, and collecting liquid C after the enzymatic hydrolysis; mixing the collected liquid A, liquid B and liquid C to obtain mixed liquid, and centrifuging the mixed liquid and fetching the cell precipitate; and re-suspending the cell precipitate to obtain the placenta hematopoietic stem cells. In the invention, the preparation time is saved, and the maintaining of cell viability is facilitated; the whole operation process is simple and effective, the experimental operation time is short, the separation and extraction efficiency is remarkably improved, a large quantity of cells are obtained, the content of hematopoietic stem cells is greatly increased, and the cell number is relatively stable.
Description
Technical field
The invention belongs to technical field of biological extraction, relate to a kind of placental hematopoietic stem cell and extract preparation method.
Background technology
Hematopoietic stem cell, refers to a class cell with self renewal and Multidirectional Differentiation ability.Its fundamental characteristics is to have self-renewal capacity, i.e. after a cell cycle events, two hematopoietic stem cell identical with character before division can be produced, there is again Multidirectional Differentiation ability simultaneously, the most under certain environmental conditions, hematopoietic stem cell has the ability to the hemocyte differentiation of each system.
Hematopoietic stem cell transplantation is now widely used for malignant hematologic disease, non-malignant intractable hematopathy, hereditary and some treatment of solid tumors.After hematopoietic stem cell transplantation refers to patient is carried out total irradiation, chemotherapy and immunosuppressant pretreatment, by Normal donor or autologous hematopoietic stem cell intravascular infusion to patient, make the normal hemopoietic of reconstruction and immunologic function.
In general, hematopoietic stem cell is present in three positions, is bone marrow, peripheral blood and Cord blood respectively, is referred to as marrow hemopoietic stem cells, peripheral blood hematopoietic stem cells and umbilical cord blood hematopoietic stem cell according to its source.Along with medical science and the development of biotechnology, containing substantial amounts of hematopoietic stem cell in discovered in recent years Placenta Hominis, compared with the hematopoietic stem cell in above-mentioned three kinds of sources, in Placenta Hominis, the quantity of contained hematopoietic stem cell is the highest, and the distribution type transplanting placental hematopoietic stem cell requires to need not very strictly, and react relatively light after transplanting and need not use medicine.Additionally, as placental hematopoietic stem cell source-Placenta Hominis, wide material sources, anemia of pregnant woman often becomes garbage after producing, it gathers and will not cause the sensation of mother and neonate any discomfort or produce any bad impact.Plurality of advantages makes placental hematopoietic stem cell be expected to replace marrow hemopoietic stem cells, peripheral blood hematopoietic stem cells and umbilical cord blood hematopoietic stem cell in hematopoietic stem cell transplantation.
There is substantial amounts of hematopoietic stem cell in the mature Placenta Hominis of the mankind, has more hematopoietic stem cell than Cord blood, and these placental hematopoietic stem cell can be separated before and after stored frozen.The activity of placental hematopoietic stem cell colony-forming units (CFU) determines that, the transplantation experiments in Immune deficient mice has turned out placenta cells potentiality in transplanting.These results strongly suggest that, the mature Placenta Hominis of the mankind is likely to become the source of a kind of novel hematopoietic stem cell for transplanting.And Placenta Hominis has substantial amounts of hematopoietic stem cell, placental blood stem cell is stem cell more in early days, various cell can be divided in vivo, placental blood is contained within the hematopoietic stem cell in early days of various stages enriched, its content is about tens times of Cord blood, hematopoietic stem cell in one Placenta Hominis can fully meet requirements of two adult Man's Demands, if patient can be applied to together with cord blood cell, considerably increase the content of hematopoietic stem cell undoubtedly, make this hematopoietic stem cell can be entirely applied to all of applicable crowd.
But this area still expects to have new, more effective way to obtain hematopoietic stem cell, such as, expect to have new, the more effective method of separation and Extraction hematopoietic stem cell from Placenta Hominis.
Summary of the invention
It is an object of the invention to provide a kind of placental hematopoietic stem cell and extract preparation method.
The purpose of the present invention can be achieved through the following technical solutions:
A kind of placental hematopoietic stem cell extracts preparation method, and the method is produced as steps described below:
Step 1, gathers the healthy Placenta Hominis of normal natural labor or cesarean person under sterile operating room environment, then with cleanout fluid, Placenta Hominis carries out early stage cleaning treatment, collection process after liquid A；
Step 2, with irrigating solution lavation Placenta Hominis umbilicus blood quiet, endarterial respectively, collects the liquid B after lavation；
Step 3, shreds Placenta Hominis, digests placenta tissue with enzymolysis solution, the liquid C obtained after collecting enzymolysis；
Step 4, by the liquid A of above-mentioned collection, liquid B and liquid C and be mixed liquor, mixed liquor takes cell and precipitates after being centrifuged；
Step 5, obtains described placental hematopoietic stem cell by cell precipitation after resuspended.
Described cleanout fluid is mixed to prepare for 0.4-0.5:1-1.2 with normal saline in mass ratio by green grass or young crops-streptomycin is dual anti-, and concentration is 120-130mg/L.
Described irrigating solution is dual anti-by the green grass or young crops-streptomycin of 300mg/L, 0.05-0.1mg/mL phentolamine, and 1-3mg/mL vitamin C, 0.5-2mmol/L PBS and 2 heparin sodium injections prepare.
Containing 1L DEME culture fluid, 0.01-0.05mg/mL collagenase VIII, 0.01-0.05mg/mL pancreatin, 10-50U/mL heparin sodium, 0.01-0.1mg/mL phentolamine and 1-5mg/mL vitamin C in described enzymolysis solution.
In step 3, described Placenta Hominis shreds to 3-6cm, enzymolysis solution room temperature enzymolysis 0.5-1.5h.
In step 4, described mixed liquor at 6-10 DEG C, 1500r/min, centrifugal 15min, abandon supernatant, add DEME culture fluid, piping and druming mixing.
In step 5, described resuspended step is: precipitates with erythrocyte cracked liquid re-suspended cell successively, cracks the erythrocyte 5-15min of residual under room temperature, abandons supernatant after being centrifuged；Precipitate with hydroxyethyl starch solution re-suspended cell, left at room temperature 20-40min, take the centrifugal rear supernatant discarded of supernatant liquid；Precipitating with RPMI-1640 culture medium re-suspended cell, abandon supernatant after being centrifuged, gained cell precipitation is placental hematopoietic stem cell.
The holding of beneficial effects of the present invention: present invention saves preparation time, beneficially cell viability；Easy to operate, efficiency is high, be greatly shortened from the cycle being prepared into application；The inventive method lavation hematopoietic stem cell injuries out from Placenta Hominis is little, and quantity is many, and purity is high, and activity is high, can apply to the transplanting of the hematopoietic stem cell of autologous or allosome, and contributes to the reparation of damaged tissues；
Enzymolysis solution of the present invention is gentleer, use collagenase VIII and pancreatin simultaneously, not only reduce the damage to cell, it is also possible to the most preferably digest placenta tissue, discharge the hematopoietic stem cell inside tissue, improve quantity and the motility rate of the placental hematopoietic stem cell of isolated；Enzymolysis solution medium vessels expander phentolamine has good vasorelaxation action；
Whole operating process is simply effective, and the experimental implementation time is short, significantly improves separation and Extraction efficiency, it is thus achieved that cell quantity big, the content of hematopoietic stem cell is greatly improved, and cell quantity is more stable；Optimizing simultaneously and extract the condition separated, improve the survival rate of hematopoietic stem cell as far as possible, cell contamination rate is extremely low.
Detailed description of the invention
Below in conjunction with the embodiment of the present invention, technical scheme is clearly and completely described, it is clear that described embodiment is only a part of embodiment of the present invention rather than whole embodiments.Based on the embodiment in the present invention, all other embodiments that those of ordinary skill in the art are obtained under not making creative work premise, broadly fall into the scope of protection of the invention.
Embodiment 1:
The acquisition of placental hematopoietic stem cell, produces as steps described below:
Step 1, the healthy Placenta Hominis of normal natural labor or cesarean person is gathered under sterile operating room environment, then with cleanout fluid, Placenta Hominis is carried out early stage cleaning treatment, liquid A after collection process, cleanout fluid is mixed to prepare for 0.4:1.2 with normal saline in mass ratio by green grass or young crops-streptomycin is dual anti-, and concentration is 120mg/L；
Step 2, with irrigating solution lavation Placenta Hominis umbilicus blood quiet, endarterial respectively, collecting the liquid B after lavation, irrigating solution is dual anti-by the green grass or young crops-streptomycin of 300mg/L, 0.1mg/mL phentolamine, and 1mg/mL vitamin C, 2mmol/L PBS and 2 heparin sodium injections prepare；
Step 3, Placenta Hominis is shredded to 3cm, enzymolysis solution room temperature enzymolysis 1.5h, placenta tissue is digested with enzymolysis solution, the liquid C obtained after collecting enzymolysis, containing 1L DEME culture fluid, 0.01mg/mL collagenase VIII, 0.05mg/mL pancreatin, 10U/mL heparin sodium, 0.1mg/mL phentolamine and 1mg/mL vitamin C in enzymolysis solution；
Step 4, by the liquid A of above-mentioned collection, liquid B and liquid C and be mixed liquor, mixed liquor at 10 DEG C, 1500r/min, centrifugal 15min, abandon supernatant, add DEME culture fluid, piping and druming mixing, mixed liquor takes cell and precipitates after being centrifuged；
Step 5, precipitates cell by resuspended, and resuspended step is: precipitates with erythrocyte cracked liquid re-suspended cell successively, cracks the erythrocyte 5min of residual under room temperature, abandons supernatant after being centrifuged；Precipitate with hydroxyethyl starch solution re-suspended cell, left at room temperature 40min, take the centrifugal rear supernatant discarded of supernatant liquid；Precipitating with RPMI-1640 culture medium re-suspended cell, abandon supernatant after being centrifuged, gained cell precipitation is placental hematopoietic stem cell.
Irrigating solution component of the present invention is simple, in addition to dual anti-, PBS, containing phentolamine, vitamin C；Green grass or young crops-streptomycin is dual anti-has antibacterial action, prevents from polluting.PBS is used for maintaining intraor extracellular osmotic pressure.Phentolamine is identical with its effect in enzymolysis solution with ascorbic effect, and vitamin C, for strengthening the oxidation resistance of cell, improves the motility rate of cell；Phentolamine plays vasorelaxation action, utilizes the perfusion operation in experiment.
Embodiment 2:
The acquisition of placental hematopoietic stem cell, produces as steps described below:
Step 1, under sterile operating room environment, gather the healthy Placenta Hominis of normal natural labor or cesarean person, then with cleanout fluid, Placenta Hominis carried out early stage cleaning treatment, collection process after liquid A, cleanout fluid is mixed to prepare for 0.5:1 with normal saline in mass ratio by green grass or young crops-streptomycin is dual anti-, and concentration is 130mg/L；
Step 2, with irrigating solution lavation Placenta Hominis umbilicus blood quiet, endarterial respectively, collecting the liquid B after lavation, irrigating solution is dual anti-by the green grass or young crops-streptomycin of 300mg/L, 0.05mg/mL phentolamine, and 3mg/mL vitamin C, 0.5mmol/L PBS and 2 heparin sodium injections prepare；
Step 3, Placenta Hominis is shredded to 6cm, enzymolysis solution room temperature enzymolysis 0.5h, placenta tissue is digested with enzymolysis solution, the liquid C obtained after collecting enzymolysis, containing 1L DEME culture fluid, 0.05mg/mL collagenase VIII, 0.01mg/mL pancreatin, 50U/mL heparin sodium, 0.01mg/mL phentolamine and 5mg/mL vitamin C in enzymolysis solution；
Step 4, by the liquid A of above-mentioned collection, liquid B and liquid C and be mixed liquor, mixed liquor at 6 DEG C, 1500r/min, centrifugal 15min, abandon supernatant, add DEME culture fluid, piping and druming mixing, mixed liquor takes cell and precipitates after being centrifuged；
Step 5, precipitates cell by resuspended, and resuspended step is: precipitates with erythrocyte cracked liquid re-suspended cell successively, cracks the erythrocyte 15min of residual under room temperature, abandons supernatant after being centrifuged；Precipitate with hydroxyethyl starch solution re-suspended cell, left at room temperature 20min, take the centrifugal rear supernatant discarded of supernatant liquid；Precipitating with RPMI-1640 culture medium re-suspended cell, abandon supernatant after being centrifuged, gained cell precipitation is placental hematopoietic stem cell.
In the description of this specification, the description of reference term " embodiment ", " example ", " concrete example " etc. means that the specific features, structure, material or the feature that combine this embodiment or example description are contained at least one embodiment or the example of the present invention.In this manual, the schematic representation to above-mentioned term is not necessarily referring to identical embodiment or example.And, the specific features of description, structure, material or feature can combine in any one or more embodiments or example in an appropriate manner.
Present invention disclosed above preferred embodiment is only intended to help to illustrate the present invention.Preferred embodiment does not has all of details of detailed descriptionthe, is not intended to the detailed description of the invention that this invention is only described yet.Obviously, according to the content of this specification, can make many modifications and variations.These embodiments are chosen and specifically described to this specification, is to preferably explain the principle of the present invention and actual application, so that skilled artisan can be best understood by and utilize the present invention.The present invention is only limited by claims and four corner thereof and equivalent.
Claims (7)
1. a placental hematopoietic stem cell extracts preparation method, it is characterised in that the method is as steps described below
Produce:
Step 1, gathers the healthy Placenta Hominis of normal natural labor or cesarean person under sterile operating room environment, then uses
Cleanout fluid carries out early stage cleaning treatment, the liquid A after collection process to Placenta Hominis；
Step 2, with irrigating solution lavation Placenta Hominis umbilicus blood quiet, endarterial respectively, collects the liquid B after lavation；
Step 3, shreds Placenta Hominis, digests placenta tissue with enzymolysis solution, the liquid C obtained after collecting enzymolysis；
Step 4, by the liquid A of above-mentioned collection, liquid B and liquid C and be mixed liquor, after mixed liquor is centrifuged
Take cell precipitation；
Step 5, obtains described placental hematopoietic stem cell by cell precipitation after resuspended.
A kind of placental hematopoietic stem cell the most according to claim 1 extracts preparation method, it is characterised in that
Described cleanout fluid is mixed system for 0.4-0.5:1-1.2 by green grass or young crops-streptomycin is dual anti-in mass ratio with normal saline
, concentration is 120-130mg/L.
A kind of placental hematopoietic stem cell the most according to claim 1 extracts preparation method, it is characterised in that
Described irrigating solution is dual anti-by the green grass or young crops-streptomycin of 300mg/L, 0.05-0.1mg/mL phentolamine, 1-3mg/mL
Vitamin C, 0.5-2mmol/L PBS and 2 heparin sodium injections prepare.
A kind of placental hematopoietic stem cell the most according to claim 1 extracts preparation method, it is characterised in that
In described enzymolysis solution containing 1L DEME culture fluid, 0.01-0.05mg/mL collagenase VIII,
0.01-0.05mg/mL pancreatin, 10-50U/mL heparin sodium, 0.01-0.1mg/mL phentolamine and 1-
5mg/mL vitamin C.
A kind of placental hematopoietic stem cell the most according to claim 1 extracts preparation method, it is characterised in that
In step 3, described Placenta Hominis shreds to 3-6cm, enzymolysis solution room temperature enzymolysis 0.5-1.5h.
A kind of placental hematopoietic stem cell the most according to claim 1 extracts preparation method, it is characterised in that
In step 4, described mixed liquor at 6-10 DEG C, 1500r/min, centrifugal 15min, abandon supernatant, add DEME
Culture fluid, piping and druming mixing.
A kind of placental hematopoietic stem cell the most according to claim 1 extracts preparation method, it is characterised in that
In step 5, described resuspended step is: precipitates with erythrocyte cracked liquid re-suspended cell successively, splits under room temperature
Solve the erythrocyte 5-15min of residual, after being centrifuged, abandon supernatant；Precipitate with hydroxyethyl starch solution re-suspended cell, room
Stand 20-40min under temperature, take the centrifugal rear supernatant discarded of supernatant liquid；Resuspended carefully by RPMI-1640 culture medium
Born of the same parents are precipitated, and abandon supernatant after being centrifuged, and gained cell precipitation is placental hematopoietic stem cell.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106967675A (en) * | 2017-05-31 | 2017-07-21 | 东莞市保莱生物科技有限公司 | A kind of method of the separation and Extraction candidate stem cell from placenta |
| CN109251893A (en) * | 2018-09-18 | 2019-01-22 | 深圳市宝迪生物工程有限公司 | A kind of preparation method of in vitro placental hematopoietic stem cell |
| CN109337871A (en) * | 2018-11-05 | 2019-02-15 | 潍坊市康华生物技术有限公司 | A kind of placental hematopoietic stem cell preparation method |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106967675A (en) * | 2017-05-31 | 2017-07-21 | 东莞市保莱生物科技有限公司 | A kind of method of the separation and Extraction candidate stem cell from placenta |
| CN109251893A (en) * | 2018-09-18 | 2019-01-22 | 深圳市宝迪生物工程有限公司 | A kind of preparation method of in vitro placental hematopoietic stem cell |
| CN109337871A (en) * | 2018-11-05 | 2019-02-15 | 潍坊市康华生物技术有限公司 | A kind of placental hematopoietic stem cell preparation method |
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