CN110129179A - A kind of three joint bag and the method using three joint bag separation umbilical cord blood hematopoietic stem cell - Google Patents
A kind of three joint bag and the method using three joint bag separation umbilical cord blood hematopoietic stem cell Download PDFInfo
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- CN110129179A CN110129179A CN201910457872.3A CN201910457872A CN110129179A CN 110129179 A CN110129179 A CN 110129179A CN 201910457872 A CN201910457872 A CN 201910457872A CN 110129179 A CN110129179 A CN 110129179A
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Abstract
The present invention establishes a kind of three joint bag and the method using three joint bag separation umbilical cord blood hematopoietic stem cell, this method isolates candidate stem cell by the way of three joint bag from Cord blood, the whole operation process of three joint bag separation is completed in the environment of closed sterile soft, both the pollution of operating process can have been reduced, it is low to the sterility requirements of operating environment and personnel simultaneously, convenient for operation；Using automatic plasma separating folder removal blood plasma, speed is fast for separation process, easy to operate, improves operating efficiency.
Description
Technical field
The present invention relates to a kind of three joint bag and the methods of application three joint bag separation umbilical cord blood hematopoietic stem cell, belong to navel
The technical field of extraction with blood candidate stem cell.
Background technique
Candidate stem cell (hemopoietic stem cell) refers to the cell that a group is not yet reached maturity, and is all make
The origin of haemocyte and immunocyte, it can not only be divided into red blood cell, leucocyte and blood platelet, can be with cross-system point
The cell of various histoorgans is turned to, there is self-renewing, Multidirectional Differentiation and goes back to the nest (i.e. directional migration to hematopoietic tissue organ)
Potential, belong to multipotential stem cell.Research finds that candidate stem cell can treat a variety of diseases, with the development of medicine, hematopoiesis
Stem cell transplantation has become the main means of the diseases in the blood system such as radical cure leukaemia at present, brings for mankind's Fighting Disease
New hope.
After Cord blood is delivery of baby, remains in the blood in placenta and umbilical cord, be usually to pass into disuse.But it is close several
10 years the study found that containing can rebuild the candidate stem cell of human body hematopoiesis and immune system in Cord blood, and quantity compared with
It is more.Therefore, Cord blood has become coming for the candidate stem cell of the important sources of candidate stem cell, especially consanguinity-less relation
Source is also very important human biological's resource.Candidate stem cell is isolated from Cord blood at present and there is open behaviour by hand
Make contamination probability height, environmental requirement is high, it is complicated for operation the problems such as；Automatic separation system has simple and convenient, but separation costs are high
The shortcomings that, therefore find that a kind of operating process contamination probability is low, and environmental requirement is low, separation method tool easy to operate, at low cost
It is significant.
Summary of the invention
The present invention is in view of the above-mentioned problems, separate umbilical cord blood hematopoietic to propose a kind of three joint bag and application three joint bag
The method of stem cell.
Specific technical solution is as follows:
A kind of three joint bag, including prepare bag, plasma bags and freeze bag, wherein it prepares and is respectively equipped with the first connection on bag
Pipe, the 4th connecting tube and the 5th connecting tube；
First connecting tube is connected with second pipe and third pipeline respectively, and the first connecting tube is equipped with the first sample tap,
Second connecting tube is connected with blood transfusion puncture outfit, and third connecting tube is connected with plasma bags, and third connecting tube is equipped with second and takes
Sample mouth；
4th connecting tube is connected with bag is frozen；
5th connecting tube is connected with frozen stock solution mouth is added；
Second connecting tube is equipped with first clamp, the 4th connecting tube is equipped with second clamp, the 5th connecting tube is equipped with the
Three clips, third connecting tube are equipped with the 4th clip.
Using the method for above-mentioned three joint bag separation umbilical cord blood hematopoietic stem cell, comprising the following steps:
1) it is put into after sterilizing three joint bag in Biohazard Safety Equipment, empties plasma bags and freezes the air in bag, closed all
Clip；
2) the blood transfusion puncture outfit of three joint bag is penetrated into the turncap open end of blood taking bag and tightened；
3) blood taking bag is lifted, first clamp is unclamped, prepares blood inflow in blood taking bag in bag, is closed after the completion of transfer
First clamp, will prepare bag and is placed on shaking table and shake up 5min；
4) it is mixed after ready hydroxyethyl starch being prepared bag by the first sample tap injection；
5) the preparation bag equipped with Cord blood is clamped with the transfering bag after two inflations and is fixed in Centrifuge Cup, after trim
It is placed in centrifuge and is centrifuged 50g, 7min, 10 DEG C；
6) after being centrifuged, Centrifuge Cup is taken out, then taking-up prepares bag from Centrifuge Cup, clamps first clamp with haemostatic clamp
Neighbouring pipeline hangs upside down and stands 5min；
7) after being layered clearly, first clamp is opened, red blood cell velocity is controlled with haemostatic clamp, makes red blood cell with 3-10ml/
The speed of min flows into blood taking bag, until layering interfaces distance is prepared at bag nozzle 1cm, clamps haemostatic clamp, closes the first folder
Son, removes preparation bag, and heat seal is fallen to connect the pipeline of blood taking bag；
8) blood in preparation bag is mixed, is clamped with the transfering bag after two inflations and prepares bag fixed in Centrifuge Cup,
It is placed in after trim in centrifuge and is centrifuged 747g, 15min, 10 DEG C；
9) after being centrifuged, Centrifuge Cup is taken out, then taking-up prepares bag from Centrifuge Cup, is linked into the automatic plasma separating of 21 ± 1ml
In folder, the 4th clip is opened, presses the switch of automatic plasma separating folder, extra blood plasma can be squeezed at the uniform velocity in plasma bags, arrived automatic
It is automatically stopped after plasma-separating clip setting 21 ± 1ml of volume；
10) the 4th clip is closed, removes preparation bag from automatic plasma separating folder, the blood plasma in pipeline is passed through the 4th clip
It squeezes into plasma bags manually, heat seal plasma bags and prepares pipeline between bag, it is stand-by to cut plasma bags；
11) bag surface is prepared with non-dust cloth wiping, it is ensured that be attached to the karyocyte prepared on bag inner wall and fully enter blood
Liquid, mix deshydremia after from the first sample tap take 1ml blood sample carry out cell number, motility rate, surface marker and colony CFU detection；
12) preparation bag is clamped with biology ice bag, is put into 4-10 DEG C of drug storage box and at least 20min is pre-chilled；
13) open third clip, with micro-injection pump by the frozen stock solution of pre-cooling from adding frozen stock solution mouth addition to prepare in bag,
Third clip is closed after adding；
14) after frozen stock solution adds, second clamp is opened, the mixed liquor prepared in bag is transferred to and is frozen in bag, drains and freezes
Air in bag makes deshydremia mixed liquor is full to freeze a bag pipeline, and heat seal is fallen to freeze the pipeline of bag, will freeze bag and is put into program
Program cooling freezing is carried out in cooling instrument；
15) 16ml blood plasma is taken to carry out aerobic bacteria and anaerobic bacteria detection from the second sample tap of plasma bags.
Further, the additional amount of the hydroxyethyl starch in step 1) are as follows: Cord blood net weight=Cord blood gross weight-sky is adopted
Blood bag weight, Cord blood net volume=Cord blood net weight/1.03, hydroxyethyl starch volume=Cord blood net volume × 0.2.
Further, the preparation bag upside down equipped with Cord blood in step 5) is in Centrifuge Cup.
Further, the preparation bag of step 8), which is just put, is placed in Centrifuge Cup.
Further, in step 13), in preparing bag plus during the entire process of frozen stock solution, bag is prepared all by biological ice bag
It clamps, and is placed on micro computer and adopts and shaken up on fluid controller.
Further, the volume ratio of frozen stock solution addition are as follows: deshydremia: frozen stock solution=4:1, frozen stock solution is by dextran
It is formed with two kinds of reagents of DMSO according to the volume ratio mixed preparing of 1:1.
The invention has the benefit that
1) Cord blood separation overall process all carries out in the environment of closed sterile soft, reduces the probability of sample contamination；
2) removal blood plasma is pressed from both sides with automatic plasma separating, speed is fast, and it is easy to operate, improve efficiency；
3) separation process removes red blood cell using gravity using centrifugation is inverted, convenient at the uniform velocity to control.
Detailed description of the invention
Fig. 1 is three joint bag structural schematic diagram of the present invention；
Fig. 2 is 4 surface marker streaming figure of sample of the present invention；
Fig. 3 is 4 survival rate test result figure of sample of the present invention；
Fig. 4 is 4 granulocytes of sample of the present invention-erythroid cells-macrophage-megakaryocyte Colony forming figure；
Fig. 5 is the quick-fried formula erythroid cells Colony forming figure of sample of the present invention 4；
Fig. 6 is that 4 granulocyte-macrophage colony of sample of the present invention forms figure.
In Fig. 2: All Events is all detection cells randomly selected；P1 is expression CD45 sun in detection cell
The cell of property；P2 is to express the cell of the CD34 positive from P1；P3 is to express the cell of CD45 weakly positive from P2；
P4 derives from P3, expresses the cell of the CD34 positive.
Appended drawing reference:
It prepares bag 1, plasma bags 2, freeze bag 3, the first connecting tube 4, the 4th connecting tube 5, the 5th connecting tube 6, second pipe
7, third pipeline 8, the first sample tap 9, blood transfusion puncture outfit 10, the second sample tap 11, frozen stock solution mouth 12, first clamp 13, second
Clip 14, third clip 15, the 4th clip 16.
Specific embodiment
Clear to be more clear technical solution of the present invention, the present invention is described further with reference to the accompanying drawing,
The technical characteristic of any pair of technical solution of the present invention carries out the scheme that equivalencing is obtained with conventional reasoning and each falls within guarantor of the present invention
Protect range.
Embodiment one
A kind of three joint bag as shown in Figure 1, including prepare bag 1, plasma bags 2 and freeze bag 3, wherein it prepares and is set respectively on bag
There are the first connecting tube 4, the 4th connecting tube 5 and the 5th connecting tube 6；
First connecting tube is connected with second pipe 7 and third pipeline 8 respectively, and the first connecting tube is equipped with the first sampling
Mouth 9, the second connecting tube are connected with blood transfusion puncture outfit 10, and third connecting tube is connected with plasma bags, and third connecting tube is equipped with
Second sample tap 11；
4th connecting tube is connected with bag is frozen；
5th connecting tube is connected with frozen stock solution mouth 12 is added；
Second connecting tube is equipped with first clamp 13, the 4th connecting tube is equipped with second clamp 14, sets in the 5th connecting tube
There are third clip 15, third connecting tube to be equipped with the 4th clip 16.
Embodiment two
The method of the three joint bag separation umbilical cord blood hematopoietic stem cell of Application Example one, comprising the following steps:
1) three joint bag is taken, with being put into Biohazard Safety Equipment behind 75% alcohol disinfecting surface, empties plasma bags and jelly
The air in bag is deposited, all clips are closed, bag, plasma bags is being prepared and is freezing labelled on bag；
2) the blood transfusion puncture outfit needle cap of three joint bag is pulled up, puncture penetrates blood taking bag turncap open end and tightens；
3) blood taking bag is lifted, first clamp is unclamped, prepares blood inflow in blood taking bag in bag, is closed after the completion of transfer
First clamp, will prepare bag and is placed on shaking table and shake up 5min；
4) hydroxyethyl starch volume: hydroxyethyl starch volume=(Cord blood gross weight-sky blood taking bag weight is calculated according to formula
Amount) ÷ 1.03 × 0.2, it is softly mixed after ready hydroxyethyl starch is prepared bag by the first sample tap injection；
5) the preparation bag equipped with Cord blood is clamped with the transfering bag after two inflations, upside down is fixed on centrifugation
In cup, it is placed in after trim in centrifuge and is centrifuged 50g, 7min, 10 DEG C；
6) after being centrifuged, Centrifuge Cup is taken out, then taking-up prepares bag from Centrifuge Cup, clamps first clamp with haemostatic clamp
Neighbouring pipeline, hangs upside down in Biohazard Safety Equipment, stands 5min；
7) after being layered clearly, first clamp is opened, red blood cell velocity is controlled with haemostatic clamp, makes red blood cell with 3-10ml/
The speed of min flows into blood taking bag, until layering interfaces distance is prepared at bag nozzle 1cm, clamps haemostatic clamp, closes the first folder
Son, removes preparation bag, and heat seal is fallen to connect the pipeline of blood taking bag；
8) blood in preparation bag is mixed, clamps preparation bag with the transfering bag after two inflations, stands the fixation of placement
It is placed in into Centrifuge Cup, after trim in centrifuge and is centrifuged 747g, 15min, 10 DEG C；
9) after being centrifuged, Centrifuge Cup is taken out, then taking-up prepares bag from Centrifuge Cup, is linked into the automatic plasma separating of 21 ± 1ml
In folder, the 4th clip is opened, presses the switch of automatic plasma separating folder, extra blood plasma can be squeezed at the uniform velocity in plasma bags, arrived automatic
It is automatically stopped after plasma-separating clip setting 21 ± 1ml of volume；
10) the 4th clip is closed, removes preparation bag from automatic plasma separating folder, the blood plasma in pipeline is passed through the 4th clip
It squeezes into plasma bags manually, heat seal plasma bags and prepares pipeline between bag, it is stand-by to cut plasma bags；
11) three joint bag is put into Biohazard Safety Equipment, prepares bag surface with non-dust cloth wiping, it is ensured that be attached to and prepare in bag
Karyocyte on wall fully enters blood, sterilizes the first sample tap with cotton ball soaked in alcohol, mixes after deshydremia from the first sample tap
1ml blood sample is taken to carry out cell number, motility rate, surface marker and colony CFU detection；The testing result of cell number is 11.8 × 108
(cell number is 13.0 × 10 before separating8), according to rate of recovery calculation: the rate of recovery=(karyocyte total number of cells after separation
Total number of nucleated cell before ÷ is separated) × 100%, the rate of recovery 90.8%；With flow cytomery CD34+ cell, detection knot
Fruit is that the percentage that CD34+ cell occupies nucleus is 0.12%；Candidate stem cell passes through 14 culture, sees under the microscope
Observe the different candidate stem cell Colony formings such as red system, grain system.
12) preparation bag is clamped with biology ice bag, is put into 4-10 DEG C of drug storage box and at least 20min is pre-chilled；
13) two kinds of reagents of dextran and DMSO are put into 4-10 DEG C of medicine according to the ratio mixed preparing frozen stock solution of 1:1
At least 20min is pre-chilled in product storage box；Third clip is opened, according to deshydremia: frozen stock solution=4:1 ratio uses micro-injection
The frozen stock solution of pre-cooling from adding frozen stock solution mouth addition to prepare in bag, during the entire process of adding frozen stock solution, is prepared bag all by biology by pump
Ice bag is clamped, and is placed on micro computer and is adopted and shaken up on fluid controller, and third clip is closed after adding；
14) after frozen stock solution adds, second clamp is opened, the mixed liquor prepared in bag is transferred to and is frozen in bag, drains and freezes
Air in bag makes deshydremia mixed liquor is full to freeze a bag pipeline, and heat seal is fallen to freeze the pipeline of bag, will freeze bag and is put into 25ml
It freezes in can, is then placed in programmed cooling instrument and carries out program cooling freezing；
15) 16ml blood plasma is taken to carry out aerobic bacteria and anaerobic bacteria detection from the second sample tap of plasma bags with 20ml syringe.
Embodiment three
The result for taking the sample of 5 cord blood candidate stem cells to separate through three joint bag is as follows:
By taking sample 4 as an example, attached drawing 2-6 enumerates corresponding testing result figure.
The foregoing is only a preferred embodiment of the present invention, but scope of protection of the present invention is not limited thereto,
In the technical scope disclosed by the present invention, any changes or substitutions that can be easily thought of by anyone skilled in the art,
It should all cover in protection scope of the present invention.
Claims (7)
1. a kind of three joint bag, it is characterized in that, including prepare bag, plasma bags and freeze bag, wherein it prepares and is respectively equipped with first on bag
Connecting tube, the 4th connecting tube and the 5th connecting tube；
First connecting tube is connected with second pipe and third pipeline respectively, the first connecting tube be equipped with the first sample tap, second
Connecting tube is connected with blood transfusion puncture outfit, and third connecting tube is connected with plasma bags, and third connecting tube is equipped with the second sample tap；
4th connecting tube is connected with bag is frozen；
5th connecting tube is connected with frozen stock solution mouth is added；
Second connecting tube is equipped with first clamp, the 4th connecting tube is equipped with second clamp, the 5th connecting tube is pressed from both sides equipped with third
Son, third connecting tube are equipped with the 4th clip.
2. the method for application three joint bag as described in claim 1 separation umbilical cord blood hematopoietic stem cell, it is characterized in that, including it is following
Step:
1) it is put into after sterilizing three joint bag in Biohazard Safety Equipment, empties plasma bags and freezes the air in bag, close all clips；
2) the blood transfusion puncture outfit of three joint bag is penetrated into the turncap open end of blood taking bag and tightened；
3) blood taking bag is lifted, first clamp is unclamped, prepares blood inflow in blood taking bag in bag, the first folder is closed after the completion of transfer
Son, will prepare bag and is placed on shaking table and shake up 5min；
4) it is mixed after ready hydroxyethyl starch being prepared bag by the first sample tap injection；
5) the preparation bag equipped with Cord blood is clamped with the transfering bag after two inflations and is fixed in Centrifuge Cup, is placed in after trim
50g, 7min, 10 DEG C are centrifuged in centrifuge；
6) after being centrifuged, Centrifuge Cup is taken out, then taking-up prepares bag from Centrifuge Cup, clamps first clamp with haemostatic clamp and nearby manages
Road hangs upside down and stands 5min；
7) after being layered clearly, first clamp is opened, red blood cell velocity is controlled with haemostatic clamp, makes red blood cell with 3-10ml/min's
Speed flows into blood taking bag, until layering interfaces distance is prepared at bag nozzle 1cm, clamps haemostatic clamp, closes first clamp, remove
Bag is prepared, heat seal is fallen to connect the pipeline of blood taking bag；
8) blood in preparation bag is mixed, is clamped with the transfering bag after two inflations and prepares bag fixed in Centrifuge Cup, trim
After be placed in centrifuge and be centrifuged 747g, 15min, 10 DEG C；
9) after being centrifuged, Centrifuge Cup is taken out, then taking-up prepares bag from Centrifuge Cup, is linked into the automatic plasma separating folder of 21 ± 1ml,
The 4th clip is opened, the switch of automatic plasma separating folder is pressed, extra blood plasma can be squeezed at the uniform velocity in plasma bags, and automatic plasma separating folder has been arrived
It is automatically stopped after setting 21 ± 1ml of volume；
10) the 4th clip is closed, removes preparation bag from automatic plasma separating folder, the blood plasma in pipeline is squeezed manually by the 4th clip
Enter in plasma bags, heat seal plasma bags and prepare pipeline between bag, it is stand-by to cut plasma bags；
11) bag surface is prepared with non-dust cloth wiping, it is ensured that be attached to the karyocyte prepared on bag inner wall and fully enter blood, mix
1ml blood sample is taken to carry out cell number, motility rate, surface marker and colony CFU detection from the first sample tap after deshydremia；
12) preparation bag is clamped with biology ice bag, is put into 4-10 DEG C of drug storage box and at least 20min is pre-chilled；
13) open third clip, with micro-injection pump by the frozen stock solution of pre-cooling from adding frozen stock solution mouth addition to prepare in bag, after adding
Close third clip；
14) after frozen stock solution adds, second clamp is opened, the mixed liquor prepared in bag is transferred to and is frozen in bag, drains and freezes in bag
Air, make deshydremia mixed liquor is full to freeze a bag pipeline, heat seal is fallen to freeze the pipeline of bag, will freeze bag and is put into programmed cooling instrument
Middle progress program cooling freezing；
15) 16ml blood plasma is taken to carry out aerobic bacteria and anaerobic bacteria detection from the second sample tap of plasma bags.
3. as claimed in claim 2 using the method for three joint bag separation umbilical cord blood hematopoietic stem cell, it is characterized in that in step 1)
Hydroxyethyl starch additional amount are as follows: Cord blood net weight=Cord blood gross weight-sky blood taking bag weight, Cord blood net volume=umbilical cord
Blood net weight/1.03, hydroxyethyl starch volume=Cord blood net volume × 0.2.
4. being equipped with navel in step 5) as claimed in claim 3 using the method for three joint bag separation umbilical cord blood hematopoietic stem cell
Preparation bag upside down with blood is in Centrifuge Cup.
5. the preparation bag of step 8) is just as claimed in claim 4 using the method for three joint bag separation umbilical cord blood hematopoietic stem cell
Storing is placed in Centrifuge Cup.
6. in step 13), being prepared using the method for three joint bag separation umbilical cord blood hematopoietic stem cell as claimed in claim 5
It in bag plus during the entire process of frozen stock solution, prepares bag and is all clamped by biological ice bag, and be placed on micro computer to adopt fluid controller enterprising
Row shakes up.
7. as claimed in claim 6 using the method for three joint bag separation umbilical cord blood hematopoietic stem cell, frozen stock solution adding proportion is
Deshydremia: frozen stock solution=4:1, frozen stock solution are formed by two kinds of reagents of dextran and DMSO according to the ratio mixed preparing of 1:1.
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| CN201910457872.3A CN110129179A (en) | 2019-05-29 | 2019-05-29 | A kind of three joint bag and the method using three joint bag separation umbilical cord blood hematopoietic stem cell |
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| CN201910457872.3A CN110129179A (en) | 2019-05-29 | 2019-05-29 | A kind of three joint bag and the method using three joint bag separation umbilical cord blood hematopoietic stem cell |
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| CN110229788A (en) * | 2019-04-11 | 2019-09-13 | 广州市天河诺亚生物工程有限公司 | A kind of preparation method of cord blood mononuclear cells |
| CN112481098A (en) * | 2020-12-11 | 2021-03-12 | 深圳市北科生物科技有限公司 | Four-in-one bag for separating and storing peripheral blood immune cells and separation method |
| CN115486440A (en) * | 2022-10-20 | 2022-12-20 | 许艳杰 | Integrated into one piece connects cryopreserved bag verifying attachment |
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Application publication date: 20190816 |