CN102660499A - Placental hematopoietic stem cell and preparation method thereof and placental hematopoietic stem cell injection - Google Patents
Placental hematopoietic stem cell and preparation method thereof and placental hematopoietic stem cell injection Download PDFInfo
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Abstract
The invention relates to the field of biology and discloses a placental hematopoietic stem cell and a preparation method thereof and a placental hematopoietic stem cell injection. The preparation method comprises the following steps of: after pretreating a placenta, digesting the treated placenta by using collagenase type IV digestive juice and collagenase type II digestive juice, then washing and filtering and respectively collecting first filtrate and first residue; digesting the first filter residue by using collagenase type I digestive juice and the collagenase type II digestive juice in the first step and then washing, filtering and collecting second filtrate; combining the filtrate from two times, centrifuging and discarding supernate, re-suspending and precipitating; adding a resuspension solution into a lymphocytes separation medium; and carrying out density gradient centrifugation, collecting a buffy coat cell solution on an intermediate layer and centrifuging and discarding supernate to obtain the placental hematopoietic stem cell. According to the placental hematopoietic stem cell and the preparation method thereof disclosed by the invention, collagenase type IV and collagenase type II are combined, collagenase type I and collagenase type II are combined and the hematopoietic stem cell is prepared by fully digesting the placenta according to the characteristics of different collagenases, so that the quantity and the vitality of the prepared hematopoietic stem cell are improved.
Description
Technical field
The present invention relates to biological technical field, relate to a kind of placenta hemopoietic stem cell and preparation method thereof and placenta hemopoietic stem cell injection specifically.
Background technology
Hemopoietic stem cell is meant one type of cell with self and multidirectional differentiation capability.Its fundamental characteristics is to have the self ability; Promptly through after the cell cycle activity; Can produce two with the division before the identical hemopoietic stem cell of character; Have simultaneously multidirectional differentiation capability again, promptly under certain envrionment conditions, hemopoietic stem cell has to the ability that respectively is the hemocyte differentiation.
HSCT is widely used in malignant hematologic disease (like acute leukemia, chronic myelocytic leukemia etc.), non-pernicious intractable hemopathy (like aplastic anemia, myelodysplastic syndrome etc.) heredopathia (innate immunity Que Xian Bing ﹑ thalassemia etc.) and some treatment of solid tumors at present.After HSCT is meant patient carried out total irradiation, chemotherapy and immunosuppression pre-treatment, give patient, make and rebuild normal hematopoiesis and immunologic function with normal donor or from the hemopoietic stem cell intravascular infusion of body.
In general, hemopoietic stem cell is present in three positions, is respectively marrow, peripheral blood and Cord blood, is referred to as marrow hemopoietic stem cells, peripheral blood hematopoietic stem cells and umbilical cord blood hematopoietic stem cell respectively according to its source.Along with medical science and development of biology; Contain a large amount of hemopoietic stem cells in the discovered in recent years placenta; Compare with the hemopoietic stem cell in above-mentioned three kinds of sources; The quantity of contained hemopoietic stem cell is very high in the placenta, and the type of joining of transplanting the placenta hemopoietic stem cell do not need to require very strict, and transplants afterreaction and gentlier and not need adopt medicine.In addition, as placenta hemopoietic stem cell source-placenta, wide material sources often become waste after the woman, and its collection can not cause the sensation of mother and newborn infant's any discomfort or produce any bad influence.Plurality of advantages makes the placenta hemopoietic stem cell be expected to replace marrow hemopoietic stem cells, peripheral blood hematopoietic stem cells and umbilical cord blood hematopoietic stem cell to be used for HSCT.
At present separate the technology of preparation hemopoietic stem cell is that all right ripe, the report that adopts polishing and enzyme process to prepare the placenta hemopoietic stem cell is arranged in the prior art from placenta.But polishing is comparatively coarse, and cell can not separate from tissue fully, and quantity is few, and the abrasive method pair cell has injury, influences vigor.And mostly enzyme process is to adopt single enzyme to carry out the enzymolysis preparation; Wherein the overwhelming majority is a type; Like application number is that 01131190.8 Chinese patent " extracts the novel method that hemopoietic stem cell is used to set up hemopoietic stem cell bank " from placenta tissue; Wherein put down in writing the scheme of utilizing single collagenase to prepare the placenta hemopoietic stem cell, but and unexposed hemopoietic stem cell quantity and vigor.
Summary of the invention
In view of this, the object of the present invention is to provide a kind of placenta hemopoietic stem cell and preparation method thereof and placenta hemopoietic stem cell injection, make said preparation method can improve the quantity and the vigor of prepared placenta hemopoietic stem cell.
To achieve these goals, the present invention provides following technical scheme:
A kind of preparation method of placenta hemopoietic stem cell may further comprise the steps:
Step 1, with after the placenta pre-treatment; With type Digestive system and the digestion of II Collagen Type VI enzymic digestion liquid; Wash then, filter; Collect first filtrating and first filter residue respectively, the type mass percent is 0.02-0.2% in the said type Digestive system, and II Collagen Type VI enzyme mass percent is 0.01-0.1% in the said II Collagen Type VI enzymic digestion liquid;
Step 2, with said first filter residue with the digestion of I Collagen Type VI enzymic digestion liquid and the said II Collagen Type VI of step 1 enzymic digestion liquid; Wash then, filter; Collect second filtrating and second filter residue respectively, I Collagen Type VI enzyme mass percent is 0.02-0.2% in the said I Collagen Type VI enzymic digestion liquid;
Step 3, merging first filtrating and second filtrating and centrifugal; Abandon resuspended deposition behind the supernatant, the resuspended liquid that obtains is added in the human lymphocyte parting liquid, then density gradient centrifugation; The tunica albuginea confluent monolayer cells liquid of one deck and centrifugal in the middle of collecting promptly gets the placenta hemopoietic stem cell after abandoning supernatant.
Wherein, as preferably, the volume ratio of said placenta and type Digestive system is 6:1, and the volume ratio of said placenta and II Collagen Type VI enzymic digestion liquid is 6:1, and the volume ratio of said placenta and I Collagen Type VI enzymic digestion liquid is 6:1.
Type Digestive system according to the invention, I Collagen Type VI enzymic digestion liquid and II Collagen Type VI enzymic digestion liquid are all preferably used DMEM/F12 (gibco) nutrient solution dissolving preparation, other the conventional nutrient solution dissolving preparations of in addition also available this area.
The used placenta of the present invention is provided by certain obstetrics and gynecology hospital, chooses the placenta that transmissible diseases such as hepatitis, syphilis, AIDS detect feminine gender and do not have obstetric complication, and warp gets puerpera's informed consent and signs Informed Consent Form before the art.Prepare aseptic PS liquid storage bottle before the art as transporting bottle, in adorn commercially available cell culture fluid, gather back 4 ℃ of conditions and deliver to preparation place in following 12 hours and separate preparation placenta hemopoietic stem cell.
Prior art adopts single collagenase (mainly being type) to carry out the enzymolysis, digestion placenta usually; And then prepare the placenta hemopoietic stem cell; But single collagenase is the various tissues of enzymolysis, digestion placenta fully; The comparatively small amt of the placenta hemopoietic stem cell of feasible preparation, and vigor is not high yet.Therefore, the present invention adopts the associating of type and II Collagen Type VI enzyme, I Collagen Type VI enzyme and II Collagen Type VI enzyme to unite to carry out the digestion placenta tissue twice, utilize the characteristic of different collagen enzyme, and highly digestion is organized, and discharges more hemopoietic stem cell.
Collagen protein composition in the main hydrolysis reticular tissue of collagenase when the tissue of intending digestion includes more reticular tissue or collagen composition, can adopt collagenase dissociated cell method, and placenta is just belonging to this type tissue.I Collagen Type VI enzyme is used for epithelium, lung, and separating of fat and adrenal tissue cell is used for digestion and organizes the connection portion to make it become individual cells; Type comprises at least 7 kinds of proteolytic enzyme compositions, and it can digest multiple tissue; II Collagen Type VI enzyme is applicable to liver, bone, and Tiroidina, heart separates with salivary organization's cell.Various collagenases are different to the restriction enzyme site of collagen, select the collagenase of appropriate type to join together to digest the placenta tissue effect and can improve.As preferably, said digestion digests 20-60min under 37 ℃ of constant temperatures.
Initial period in preparation method according to the invention; Need carry out pre-treatment to placenta, this measure belongs to known in this field, promptly placenta is carried out disinfection, removes the step of placenta coating and washing; Before the enzymolysis, digestion placenta, those skilled in the art all know these pre-treatment step.In order to improve the effect of enzymolysis, digestion, the present invention can also be cut into 1-2mm with placenta
3Fritter.
In preparation method according to the invention, all washings and resuspended all being preferably are adopted saline water washing and resuspended.In addition, in step 1 and step 2, said filtration all is preferably adopts 50-300 purpose screen filtration.
In order further to improve the quantity of the last placenta hemopoietic stem cell that obtains, as preferably, further comprising the steps of after step 1, before the step 2:
With said first residue washing 1-3 time, the washing fluid of collecting after each washing is incorporated into said first filtrating.
Simultaneously, after step 2, further comprising the steps of before the step 3:
With said second residue washing 1-3 time, the washing fluid of collecting after each washing is incorporated into said second filtrating.
Above-mentioned repeated washing to filter residue can avoid remaining in the loss cell that does not digest on the placenta tissue (being filter residue) to the full extent.
In preparing method's step 3 according to the invention, need utilize the human lymphocyte parting liquid that twice filtrating is separated, the filtrating that preferably is combined with 500-1000g cf-before this is centrifugal, abandons the resuspended deposition of supernatant then.When density gradient centrifugation; Because human lymphocyte parting liquid density is less than red corpuscle, greater than mononuclearcell; The tunica albuginea layer that mediates after centrifugal is exactly the enchylema of mononuclearcell; Wherein include hemopoietic stem cell, the cf-of said density gradient centrifugation is preferably 500-800g, and centrifugation time is preferably 10-20min.
The tunica albuginea confluent monolayer cells liquid one deck that mediates has some lymphocyte separation mediums unavoidably in sepn process, and tunica albuginea confluent monolayer cells liquid thickness comparatively, so needs bigger cf-centrifugal the time.As preferably, centrifugal being specially of tunica albuginea confluent monolayer cells liquid according to the invention:
Said tunica albuginea confluent monolayer cells liquid earlier with the centrifugal 5-10min of the cf-of 400-700g, is abandoned the resuspended deposition of supernatant, follow resuspended liquid with the centrifugal 5-10min of the cf-of 300-500g.
In addition; The present invention also provides a kind of placenta hemopoietic stem cell by preparing method's preparation according to the invention, detects its surface marker CD34 and CD45 through flow cytometer, and the result shows that CD34 is positive; CD45 negative cells content is 1.5-1.8%, shows the hemopoietic stem cell that contains higher amount.
With the simultaneous test of the placenta hemopoietic stem cell of polishing, single collagenase method preparation in; The hemopoietic stem cell vigor of preparation method of the present invention preparation is apparently higher than the vigor of the hemopoietic stem cell of all the other two kinds of methods preparations, and quantitatively also higher at hemopoietic stem cell.
The present invention also provides a kind of placenta hemopoietic stem cell injection; Is that the sero-abluminous saline water of 10-30% is formed by placenta hemopoietic stem cell according to the invention with containing mass percent; Said injection can decide the addition that contains sero-abluminous saline water according to different cell count, makes the product of different size.
Can know by above technical scheme; The present invention adopts type and the associating of II Collagen Type VI enzyme, I Collagen Type VI enzyme and the associating of II Collagen Type VI enzyme; Utilize the characteristic of different collagen enzyme fully to digest placenta and prepare the placenta hemopoietic stem cell, improved the quantity and the vigor of prepared placenta hemopoietic stem cell thus.
Embodiment
The embodiment of the invention discloses a kind of placenta hemopoietic stem cell and preparation method thereof and placenta hemopoietic stem cell injection.Those skilled in the art can use for reference this paper content, suitably improve processing parameter and realize.Special needs to be pointed out is that all similarly replace and change apparent to those skilled in the art, they all are regarded as and are included in the present invention.Product of the present invention and method are described through preferred embodiment; The related personnel obviously can be in not breaking away from content of the present invention, spirit and scope to product as herein described and method is changed or suitably change and combination, realize and use technology of the present invention.
In order further to understand the present invention, a kind of placenta hemopoietic stem cell provided by the invention and preparation method thereof and placenta hemopoietic stem cell injection are elaborated below in conjunction with embodiment.
Embodiment 1: preparation method according to the invention prepares the placenta hemopoietic stem cell
Choose the placenta that transmissible diseases such as hepatitis, syphilis, AIDS detect feminine gender and do not have obstetric complication, warp gets puerpera's informed consent and signs Informed Consent Form before the art.Prepare aseptic PS liquid storage bottle before the art as transporting bottle, in adorn commercially available cell culture fluid, gather back 4 ℃ of conditions and deliver to preparation place in following 12 hours and separate preparation placenta hemopoietic stem cell.
In aseptic super clean bench; Get 1-2ml placenta collection liquid and do aseptic detection; Then the placenta coating is removed the clip placenta tissue; The placenta tissue that shreds is put into beaker wash 1-3 time repeatedly with saline water, flush away remains in the blood on placenta tissue surface, with operating scissors placenta tissue is cut into 1-2mm then
3Size.
The placenta that shreds is transferred in the reagent bottle; Adding mass percent in succession by placenta and collagenase volume ratio 6:1 is that 0.1% type Digestive system and mass percent are 0.05% II Collagen Type VI enzymic digestion liquid; Tighten bottle cap and seal bottleneck, put into 37 ℃ of thermostat water baths and digest 45min with sealing film.
Digest the adding 100-500ml saline water washing that finishes, filter with the 50-300 eye mesh screen; Collect first filtrating and first filter residue; First filter residue is with saline water (each 100-500ml) washing 1-3 time, and the washing fluid of collecting after at every turn washing is incorporated in first filtrating subsequent use.
First filter residue is collected in the reagent bottle; Adding mass percent in succession in the ratio of placenta and collagenase volume ratio 6:1 equally is that 0.1% I Collagen Type VI enzymic digestion liquid and mass percent are 0.05% II Collagen Type VI enzymic digestion liquid; Tighten bottle cap and seal bottleneck, put into 37 ℃ of thermostat water baths and digest 30min with sealing film.
Digest the adding 100-500ml saline water washing that finishes, filter with the 50-300 eye mesh screen; Collect second filtrating and second filter residue; Second filter residue is with saline water (each 100-500ml) washing 1-3 time, and the washing fluid of collecting after at every turn washing is incorporated in second filtrating.
Merge first filtrating and second filtrating, with the centrifugal 10min of 800g cf-, supernatant discarded, collecting cell (deposition part).The cell of collecting is suspended with saline water, resuspended liquid is added in the human lymphocyte parting liquid, 650g density gradient centrifugation 15min, the tunica albuginea confluent monolayer cells liquid of one deck in the middle of collecting.
The tunica albuginea confluent monolayer cells liquid of collecting earlier with the centrifugal 5min of the cf-of 400g, is abandoned the resuspended deposition of supernatant, follow resuspended liquid, promptly get placenta hemopoietic stem cell (deposition part) after abandoning supernatant with the centrifugal 10min of the cf-of 300g.
The cell that obtains is at last detected its surface marker CD34 and CD45 with flow cytometer, and the result shows that CD34 is positive, and CD45 negative cells content is 1.5-1.8%, shows the hemopoietic stem cell that contains higher amount.
Embodiment 2: the contrast of cell quantity and vigor
Use with the placenta of embodiment 1 identical source, volume and prepare the placenta hemopoietic stem cell with existing polishing, single collagenase method respectively; Wherein relate to resuspended, washing, step such as centrifugal local all with embodiment 1 in consistent; In single collagenase method; The amount of the single collagenase digesting liquid that is adopted and collagenase mass percent all with embodiment 1 in the amount of corresponding collagenase digesting liquid identical with the collagenase mass percent; The quantity and the vigor of the placenta hemopoietic stem cell of at last this several method being prepared detect, and the result sees table 1.
Table 1 cell quantity and vigor
Can find out by last table, adopt the quantity of the placenta hemopoietic stem cell of the multiple collagenase associating of the present invention digestion method preparation all to be higher than the cell quantity of other several methods preparations, and on cell viability also than higher.
Embodiment 3: preparation method according to the invention prepares the placenta hemopoietic stem cell
Choose the placenta that transmissible diseases such as hepatitis, syphilis, AIDS detect feminine gender and do not have obstetric complication, warp gets puerpera's informed consent and signs Informed Consent Form before the art.Prepare aseptic PS liquid storage bottle before the art as transporting bottle, in adorn commercially available cell culture fluid, gather back 4 ℃ of conditions and deliver to preparation place in following 12 hours and separate preparation placenta hemopoietic stem cell.
In aseptic super clean bench; Get 1-2ml placenta collection liquid and do aseptic detection; Then the placenta coating is removed the clip placenta tissue; The placenta tissue that shreds is put into beaker wash 1-3 time repeatedly with saline water, flush away remains in the blood on placenta tissue surface, with operating scissors placenta tissue is cut into 1-2mm then
3Size.
The placenta that shreds is transferred in the reagent bottle; Adding mass percent in succession by placenta and collagenase volume ratio 10:1 is that 0.2% type Digestive system and mass percent are 0.1% II Collagen Type VI enzymic digestion liquid; Tighten bottle cap and seal bottleneck, put into 37 ℃ of thermostat water baths and digest 35min with sealing film.
Digest the adding 100-500ml saline water washing that finishes, filter with the 50-300 eye mesh screen; Collect first filtrating and first filter residue; First filter residue is with saline water (each 100-500ml) washing 1-3 time, and the washing fluid of collecting after at every turn washing is incorporated in first filtrating subsequent use.
First filter residue is collected in the reagent bottle; Adding mass percent in succession in the ratio of placenta and collagenase volume ratio 10:1 equally is that 0.2% I Collagen Type VI enzymic digestion liquid and mass percent are 0.1% II Collagen Type VI enzymic digestion liquid; Tighten bottle cap and seal bottleneck, put into 37 ℃ of thermostat water baths and digest 25min with sealing film.
Digest the adding 100-500ml saline water washing that finishes, filter with the 50-300 eye mesh screen; Collect second filtrating and second filter residue; Second filter residue is with saline water (each 100-500ml) washing 1-3 time, and the washing fluid of collecting after at every turn washing is incorporated in second filtrating subsequent use.
Merge first filtrating and second filtrating, with the centrifugal 10min of 1000g cf-, supernatant discarded, collecting cell (deposition part).The cell of collecting is suspended with saline water, resuspended liquid is added in the human lymphocyte parting liquid, 500g density gradient centrifugation 20min, the tunica albuginea confluent monolayer cells liquid of one deck in the middle of collecting.
The tunica albuginea confluent monolayer cells liquid of collecting earlier with the centrifugal 10min of the cf-of 700g, is abandoned the resuspended deposition of supernatant, follow resuspended liquid, promptly get placenta hemopoietic stem cell (deposition part) after abandoning supernatant with the centrifugal 5min of the cf-of 500g.
The cell that obtains is at last detected its surface marker CD34 and CD45 with flow cytometer, and the result shows that CD34 is positive, and CD45 negative cells content is 1.5-1.8%, shows the hemopoietic stem cell that contains higher amount.
Method according to embodiment 2 compares test, and detected result shows that the placenta hemopoietic stem cell quantity of present embodiment preparation method preparation is 10
7The order of magnitude, vigor is higher than 92%, and other preparing methods' cell quantity is 10
6The order of magnitude, cell viability is lower than 85%.
Embodiment 4: preparation method according to the invention prepares the placenta hemopoietic stem cell
Choose the placenta that transmissible diseases such as hepatitis, syphilis, AIDS detect feminine gender and do not have obstetric complication, warp gets puerpera's informed consent and signs Informed Consent Form before the art.Prepare aseptic PS liquid storage bottle before the art as transporting bottle, in adorn commercially available cell culture fluid, gather back 4 ℃ of conditions and deliver to preparation place in following 12 hours and separate preparation placenta hemopoietic stem cell.
In aseptic super clean bench; Get 1-2ml placenta collection liquid and do aseptic detection; Then the placenta coating is removed the clip placenta tissue; The placenta tissue that shreds is put into beaker wash 1-3 time repeatedly with saline water, flush away remains in the blood on placenta tissue surface, with operating scissors placenta tissue is cut into 1-2mm then
3Size.
The placenta that shreds is transferred in the reagent bottle; Adding mass percent in succession by placenta and collagenase volume ratio 3:1 is that 0.02% type Digestive system and mass percent are 0.01% II Collagen Type VI enzymic digestion liquid; Tighten bottle cap and seal bottleneck, put into 37 ℃ of thermostat water baths and digest 60min with sealing film.
Digest the adding 100-500ml saline water washing that finishes, filter with the 50-300 eye mesh screen; Collect first filtrating and first filter residue; First filter residue is with saline water (each 100-500ml) washing 1-3 time, and the washing fluid of collecting after at every turn washing is incorporated in first filtrating subsequent use.
First filter residue is collected in the reagent bottle; Adding mass percent in succession in the ratio of placenta and collagenase volume ratio 3:1 equally is that 0.02% I Collagen Type VI enzymic digestion liquid and mass percent are 0.01% II Collagen Type VI enzymic digestion liquid; Tighten bottle cap and seal bottleneck, put into 37 ℃ of thermostat water baths and digest 40min with sealing film.
Digest the adding 100-500ml saline water washing that finishes, filter with the 50-300 eye mesh screen; Collect second filtrating and second filter residue; Second filter residue is with saline water (each 100-500ml) washing 1-3 time, and the washing fluid of collecting after at every turn washing is incorporated in second filtrating subsequent use.
Merge first filtrating and second filtrating, with the centrifugal 10min of 600g cf-, supernatant discarded, collecting cell (deposition part).The cell of collecting is suspended with saline water, resuspended liquid is added in the human lymphocyte parting liquid, 600g density gradient centrifugation 18min, the tunica albuginea confluent monolayer cells liquid of one deck in the middle of collecting.
The tunica albuginea confluent monolayer cells liquid of collecting earlier with the centrifugal 10min of the cf-of 600g, is abandoned the resuspended deposition of supernatant, follow resuspended liquid, promptly get placenta hemopoietic stem cell (deposition part) after abandoning supernatant with the centrifugal 10min of the cf-of 400g.
The cell that obtains is at last detected its surface marker CD34 and CD45 with flow cytometer, and the result shows that CD34 is positive, and CD45 negative cells content is 1.5-1.8%, shows the hemopoietic stem cell that contains higher amount.
Method according to embodiment 2 compares test, and detected result shows that the placenta hemopoietic stem cell quantity of present embodiment preparation method preparation is 10
7The order of magnitude, vigor is higher than 92%, and other preparing methods' cell quantity is 10
6The order of magnitude, cell viability is lower than 85%.
Embodiment 5: preparation method according to the invention prepares the placenta hemopoietic stem cell
Choose the placenta that transmissible diseases such as hepatitis, syphilis, AIDS detect feminine gender and do not have obstetric complication, warp gets puerpera's informed consent and signs Informed Consent Form before the art.Prepare aseptic PS liquid storage bottle before the art as transporting bottle, in adorn commercially available cell culture fluid, gather back 4 ℃ of conditions and deliver to preparation place in following 12 hours and separate preparation placenta hemopoietic stem cell.
In aseptic super clean bench; Get 1-2ml placenta collection liquid and do aseptic detection; Then the placenta coating is removed the clip placenta tissue; The placenta tissue that shreds is put into beaker wash 1-3 time repeatedly with saline water, flush away remains in the blood on placenta tissue surface, with operating scissors placenta tissue is cut into 1-2mm then
3Size.
The placenta that shreds is transferred in the reagent bottle; Adding mass percent in succession by placenta and collagenase volume ratio 7:1 is that 0.15% type Digestive system and mass percent are 0.07% II Collagen Type VI enzymic digestion liquid; Tighten bottle cap and seal bottleneck, put into 37 ℃ of thermostat water baths and digest 40min with sealing film.
Digest the adding 100-500ml saline water washing that finishes, filter with the 50-300 eye mesh screen; Collect first filtrating and first filter residue; First filter residue is with saline water (each 100-500ml) washing 1-3 time, and the washing fluid of collecting after at every turn washing is incorporated in first filtrating subsequent use.
First filter residue is collected in the reagent bottle; Adding mass percent in succession in the ratio of placenta and collagenase volume ratio 7:1 equally is that 0.15% I Collagen Type VI enzymic digestion liquid and mass percent are 0.07% II Collagen Type VI enzymic digestion liquid; Tighten bottle cap and seal bottleneck, put into 37 ℃ of thermostat water baths and digest 30min with sealing film.
Digest the adding 100-500ml saline water washing that finishes, filter with the 50-300 eye mesh screen; Collect second filtrating and second filter residue; Second filter residue is with saline water (each 100-500ml) washing 1-3 time, and the washing fluid of collecting after at every turn washing is incorporated in second filtrating subsequent use.
Merge first filtrating and second filtrating, with the centrifugal 10min of 500g cf-, supernatant discarded, collecting cell (deposition part).The cell of collecting is suspended with saline water, resuspended liquid is added in the human lymphocyte parting liquid, 700g density gradient centrifugation 14min, the tunica albuginea confluent monolayer cells liquid of one deck in the middle of collecting.
The tunica albuginea confluent monolayer cells liquid of collecting earlier with the centrifugal 10min of the cf-of 550g, is abandoned the resuspended deposition of supernatant, follow resuspended liquid, promptly get placenta hemopoietic stem cell (deposition part) after abandoning supernatant with the centrifugal 10min of the cf-of 400g.
The cell that obtains is at last detected its surface marker CD34 and CD45 with flow cytometer, and the result shows that CD34 is positive, and CD45 negative cells content is 1.5-1.8%, shows the hemopoietic stem cell that contains higher amount.
Method according to embodiment 2 compares test, and detected result shows that the placenta hemopoietic stem cell quantity of present embodiment preparation method preparation is 10
7The order of magnitude, vigor is higher than 92%, and other preparing methods' cell quantity is 10
6The order of magnitude, cell viability is lower than 85%.
Embodiment 6: preparation method according to the invention prepares the placenta hemopoietic stem cell
Choose the placenta that transmissible diseases such as hepatitis, syphilis, AIDS detect feminine gender and do not have obstetric complication, warp gets puerpera's informed consent and signs Informed Consent Form before the art.Prepare aseptic PS liquid storage bottle before the art as transporting bottle, in adorn commercially available cell culture fluid, gather back 4 ℃ of conditions and deliver to preparation place in following 12 hours and separate preparation placenta hemopoietic stem cell.
In aseptic super clean bench; Get 1-2ml placenta collection liquid and do aseptic detection; Then the placenta coating is removed the clip placenta tissue; The placenta tissue that shreds is put into beaker wash 1-3 time repeatedly with saline water, flush away remains in the blood on placenta tissue surface, with operating scissors placenta tissue is cut into 1-2mm then
3Size.
The placenta that shreds is transferred in the reagent bottle; Adding mass percent in succession by placenta and collagenase volume ratio 4:1 is that 0.06% type Digestive system and mass percent are 0.03% II Collagen Type VI enzymic digestion liquid; Tighten bottle cap and seal bottleneck, put into 37 ℃ of thermostat water baths and digest 50min with sealing film.
Digest the adding 100-500ml saline water washing that finishes, filter with the 50-300 eye mesh screen; Collect first filtrating and first filter residue; First filter residue is with saline water (each 100-500ml) washing 1-3 time, and the washing fluid of collecting after at every turn washing is incorporated in first filtrating subsequent use.
First filter residue is collected in the reagent bottle; Adding mass percent in succession in the ratio of placenta and collagenase volume ratio 4:1 equally is that 0.06% I Collagen Type VI enzymic digestion liquid and mass percent are 0.03% II Collagen Type VI enzymic digestion liquid; Tighten bottle cap and seal bottleneck, put into 37 ℃ of thermostat water baths and digest 35min with sealing film.
Digest the adding 100-500ml saline water washing that finishes, filter with the 50-300 eye mesh screen; Collect second filtrating and second filter residue; Second filter residue is with saline water (each 100-500ml) washing 1-3 time, and the washing fluid of collecting after at every turn washing is incorporated in second filtrating subsequent use.
Merge first filtrating and second filtrating, with the centrifugal 10min of 750g cf-, supernatant discarded, collecting cell (deposition part).The cell of collecting is suspended with saline water, resuspended liquid is added in the human lymphocyte parting liquid, 800g density gradient centrifugation 10min, the tunica albuginea confluent monolayer cells liquid of one deck in the middle of collecting.
The tunica albuginea confluent monolayer cells liquid of collecting earlier with the centrifugal 10min of the cf-of 500g, is abandoned the resuspended deposition of supernatant, follow resuspended liquid, promptly get placenta hemopoietic stem cell (deposition part) after abandoning supernatant with the centrifugal 10min of the cf-of 300g.
The cell that obtains is at last detected its surface marker CD34 and CD45 with flow cytometer, and the result shows that CD34 is positive, and CD45 negative cells content is 1.5-1.8%, shows the hemopoietic stem cell that contains higher amount.
Method according to embodiment 2 compares test, and detected result shows that the placenta hemopoietic stem cell quantity of present embodiment preparation method preparation is 10
7The order of magnitude, vigor is higher than 92%, and other preparing methods' cell quantity is 10
6The order of magnitude, cell viability is lower than 85%.
Embodiment 7: the preparation of placenta hemopoietic stem cell injection
With the hemopoietic stem cell of the saline water suspension embodiment that contains 20% (mass percent) rHSA 1 preparation, the adjustment cell count is 4 * 10
7Individual/mL branch installs in the cell storage bag, processes placenta hemopoietic stem cell injection.
Embodiment 8: the preparation of placenta hemopoietic stem cell injection
With the hemopoietic stem cell of the saline water suspension embodiment that contains 30% (mass percent) rHSA 4 preparations, the adjustment cell count is 2 * 10
7Individual/mL branch installs in the cell storage bag, processes placenta hemopoietic stem cell injection.
Embodiment 9: the preparation of placenta hemopoietic stem cell injection
With the hemopoietic stem cell of the saline water suspension embodiment that contains 10% (mass percent) rHSA 6 preparations, the adjustment cell count is 3 * 10
7Individual/mL branch installs in the cell storage bag, processes placenta hemopoietic stem cell injection.
The explanation of above embodiment just is used for helping to understand method of the present invention and core concept thereof.Should be pointed out that for those skilled in the art, under the prerequisite that does not break away from the principle of the invention, can also carry out some improvement and modification to the present invention, these improvement and modification also fall in the protection domain of claim of the present invention.
Claims (10)
1. the preparation method of a placenta hemopoietic stem cell is characterized in that, may further comprise the steps:
Step 1, with after the placenta pre-treatment; With type Digestive system and the digestion of II Collagen Type VI enzymic digestion liquid; Wash then, filter; Collect first filtrating and first filter residue respectively, the type mass percent is 0.02-0.2% in the said type Digestive system, and II Collagen Type VI enzyme mass percent is 0.01-0.1% in the said II Collagen Type VI enzymic digestion liquid;
Step 2, with said first filter residue with the digestion of I Collagen Type VI enzymic digestion liquid and the said II Collagen Type VI of step 1 enzymic digestion liquid; Wash then, filter; Collect second filtrating and second filter residue respectively, I Collagen Type VI enzyme mass percent is 0.02-0.2% in the said I Collagen Type VI enzymic digestion liquid;
Step 3, merging first filtrating and second filtrating and centrifugal; Abandon resuspended deposition behind the supernatant, the resuspended liquid that obtains is added in the human lymphocyte parting liquid, then density gradient centrifugation; The tunica albuginea confluent monolayer cells liquid of one deck and centrifugal in the middle of collecting promptly gets the placenta hemopoietic stem cell after abandoning supernatant.
2. according to the said preparation method of claim 1, it is characterized in that the volume ratio of said placenta and type Digestive system is 6:1.
3. according to the said preparation method of claim 1, it is characterized in that the volume ratio of said placenta and II Collagen Type VI enzymic digestion liquid is 6:1.
4. according to the said preparation method of claim 1, it is characterized in that the volume ratio of said placenta and I Collagen Type VI enzymic digestion liquid is 6:1.
5. according to the said preparation method of claim 1, it is characterized in that, further comprising the steps of after step 1, before the step 2:
With said first residue washing 1-3 time, the washing fluid of collecting after each washing is incorporated into said first filtrating.
6. according to the said preparation method of claim 1, it is characterized in that, further comprising the steps of after step 2, before the step 3:
With said second residue washing 1-3 time, the washing fluid of collecting after each washing is incorporated into said second filtrating.
7. according to the said preparation method of claim 1, it is characterized in that centrifugal being specially of said tunica albuginea confluent monolayer cells liquid:
Said tunica albuginea confluent monolayer cells liquid earlier with the centrifugal 5-10min of the cf-of 400-700g, is abandoned the resuspended deposition of supernatant, follow resuspended liquid with the centrifugal 5-10min of the cf-of 300-500g.
8. according to the said preparation method of claim 1, it is characterized in that the said digestion of step 1 and step 2 is digestion 20-60min under 37 ℃ of constant temperatures.
9. the placenta hemopoietic stem cell of any said preparation method preparation of claim 1-8.
10. a placenta hemopoietic stem cell injection is characterized in that, is that the saline water of 10-30% rHSA is formed by the said placenta hemopoietic stem cell of claim 9 with containing mass percent.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104622901A (en) * | 2015-01-09 | 2015-05-20 | 奥思达干细胞有限公司 | Stem cell preparation for treating acute lymphoid leukemia and preparation method of stem cell preparation |
| CN108148806A (en) * | 2018-02-02 | 2018-06-12 | 中国人民解放军军事科学院军事医学研究院 | A kind of fast separating process of placental hematopoietic stem cell |
| CN108913661A (en) * | 2018-06-22 | 2018-11-30 | 安徽 | A kind of preparation and extracting method of human research's candidate stem cell |
| CN109652372A (en) * | 2019-01-09 | 2019-04-19 | 陕西九州细胞基因工程有限公司 | A kind of quick separating of human placenta source candidate stem cell, preparation method |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1407088A (en) * | 2001-09-06 | 2003-04-02 | 周胜利 | Method for establishing hematopoietic stem cells bank by extracting hematopoietic cells from placenta tissues |
| CN101161249A (en) * | 2006-10-12 | 2008-04-16 | 周胜利 | Method for extracting original mesenchyma and hematopoiesis trunks/ancestral cell from caesarean birth placenta for treating medulla scathe |
-
2012
- 2012-05-23 CN CN201210162406.0A patent/CN102660499B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1407088A (en) * | 2001-09-06 | 2003-04-02 | 周胜利 | Method for establishing hematopoietic stem cells bank by extracting hematopoietic cells from placenta tissues |
| CN101161249A (en) * | 2006-10-12 | 2008-04-16 | 周胜利 | Method for extracting original mesenchyma and hematopoiesis trunks/ancestral cell from caesarean birth placenta for treating medulla scathe |
Non-Patent Citations (2)
| Title |
|---|
| 章涛等: "两种分离人胎盘组织单个核细胞方法的比较", 《遵义医学院学报》 * |
| 章涛等: "人胎盘组织造血干/祖细胞的分离富集", 《中国实验血液学杂志》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104622901A (en) * | 2015-01-09 | 2015-05-20 | 奥思达干细胞有限公司 | Stem cell preparation for treating acute lymphoid leukemia and preparation method of stem cell preparation |
| CN104622901B (en) * | 2015-01-09 | 2019-06-28 | 奥思达干细胞有限公司 | A kind of method that human embryo stem cell for directional is divided into candidate stem cell |
| CN108148806A (en) * | 2018-02-02 | 2018-06-12 | 中国人民解放军军事科学院军事医学研究院 | A kind of fast separating process of placental hematopoietic stem cell |
| CN108913661A (en) * | 2018-06-22 | 2018-11-30 | 安徽 | A kind of preparation and extracting method of human research's candidate stem cell |
| CN109652372A (en) * | 2019-01-09 | 2019-04-19 | 陕西九州细胞基因工程有限公司 | A kind of quick separating of human placenta source candidate stem cell, preparation method |
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