CN102321583A - Method for separating umbilical cord blood hematopoietic stem cell by gradient centrifugation - Google Patents
Method for separating umbilical cord blood hematopoietic stem cell by gradient centrifugation Download PDFInfo
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Abstract
The invention discloses a method for separating umbilical cord blood hematopoietic stem cells by gradient centrifugation; the centrifugation adopts primary centrifugation which comprises two steps, wherein the set centrifugation conditions of the first step comprise a temperature of 5-15 DEG C, centrifugal force of 25-80 g, and centrifugation time of 5-10 minutes; the set centrifugation conditions of the second step comprise a temperature of 5-15 DEG C, centrifugal force of 400-1000 g, and centrifugation time of 10-20 minutes. The invention adopts a gradient centrifugation method for separating umbilical cord blood hematopoietic stem cells, and reduces the danger caused by stem cell transfer into a transfer bag during secondary centrifugation.
Description
Technical field
The present invention relates to a kind of method of separating umbilical cord blood hematopoietic stem cell, especially a kind of method through ladder spinning umbilical cord blood hematopoietic stem cell.
Background technology
The bleeding of the umbilicus stripping technique is to set up unbilical blood bank and the key link of carrying out umbilical cord blood transplantation research, and the effective active that it is directly connected to the stem cell recovery after the frozen final volume of bleeding of the umbilicus sample, hematopoietic stem separation rate, the cell cryopreservation recovery, cell is kept and the success or failure of next step research.The isolating purpose of bleeding of the umbilicus mainly is red corpuscle, thrombocyte and the part blood plasma of removing in the bleeding of the umbilicus; The nucleated cell (NC) of bleeding of the umbilicus ancestral cells is rich in collection; Through separation bleeding of the umbilicus is concentrated and purifying, improve the concentration of bleeding of the umbilicus ancestral cells, with the needs that satisfy research and preserve.
The bleeding of the umbilicus stripping technique comprises that hydroxyethylamyle (HES) precipitator method, Percoll solution density gradient centrifugation, monoclonal antibody add flow cytometry analysis method, immunological magnetic bead sorting method or chloride leach method etc. at present.Denning-Kendall etc. to the separating effect of different bleeding of the umbilicus separation methods such as lymphocyte liquid (Ficoll) separation, density gradient centrifugation separation, NH4Cl dissolution method, methylcellulose gum method, gelatin partition method, hydroxyethylamyle (HES) settling process carry out detail analysis and relatively after think; The separating effect of 3% gelatin sedimentation method is best; The recovery of its CD34+ cell and colony (CFU-C) recovery is the highest, reaches 86% and 92% respectively.Dissolution method is taken second place, but its final volume is big thereby actual application value is little.Ficoll and Percoll density gradient centrifugation, though use more extensive, its to technology have relatively high expectations and also expense more expensive relatively.And Ficoll method single separating umbilical blood amount is less, and when separating blood volume sample how, the centrifuge tube number of use is many, and workload is big, is easy to pollute, therefore the separation of suitable bleeding of the umbilicus in enormous quantities.
Summary of the invention
Technical problem to be solved by this invention is, a kind of once centrifugal and method of passing through ladder spinning umbilical cord blood hematopoietic stem cell that need not shift is provided.
In order to solve the problems of the technologies described above; The technical scheme that the present invention adopts is: a kind of method through ladder spinning umbilical cord blood hematopoietic stem cell; Said centrifugal employing is once centrifugal, carries out in two steps, and the first step centrifugal condition is set at 5-15 ℃, cf-25-80g, centrifugal 5-10 minute; The second step centrifugal condition is set at 5-15 ℃, cf-400-1000g, centrifugal 10-20 minute.
Particularly, comprise the steps:
(1) Cord blood in the collection bag is fully shaken up, sterilization adds hydroxyethylamyle (HES) in proportion, the blood bag is placed on ASIC micro computer adopts on the fluid controller, fully mixing;
(2) fill around the Cord blood bag with airbag, make the Cord blood bag be in plumbness, place the Centrifuge Cup trim, carry out gradient centrifugation, the first step centrifugal condition is set at 5-15 ℃, 25-80g, centrifugal 5-10 minute; The second step centrifugal condition is set at 5-15 ℃, 400-1000g, centrifugal 10-20 minute;
(3) behind centrifugal the finishing, the Cord blood in the collection bag is divided into three layers, and the upper strata is a blood plasma, and the middle level is a umbilical cord blood hematopoietic stem cell, and lower floor is a red corpuscle, respectively the blood plasma on upper strata and the red corpuscle of lower floor is emitted, and is the umbilical cord blood hematopoietic stem cell of enrichment in the collection bag.
Preferably, the first step centrifugal condition is set at 10 ℃, 50g, centrifugal 7 minutes; The second step centrifugal condition is set at 747g, and 10 ℃, centrifugal 15 minutes.
The invention has the beneficial effects as follows: adopt gradient centrifugation to separate umbilical cord blood hematopoietic stem cell, reduced stem cell and when secondary centrifuging, need transfer to the risk of pollution that brings in the transfering bag.
Embodiment
Below in conjunction with embodiment the present invention is done further explain:
The method of the present invention through ladder spinning umbilical cord blood hematopoietic stem cell, said centrifugal employing is once centrifugal, carries out in two steps, and the first step centrifugal condition is set at 5-15 ℃, cf-25-80g, centrifugal 5-10 minute; The second step centrifugal condition is set at 5-15 ℃, cf-400-1000g, centrifugal 10-20 minute.
Particularly, comprise the steps:
(1) Cord blood in the collection bag is fully shaken up, sterilization adds hydroxyethylamyle (HES) in proportion, the blood bag is placed on ASIC micro computer adopts on the fluid controller, fully mixing;
(2) fill around the Cord blood bag with airbag, make the Cord blood bag be in plumbness, place the Centrifuge Cup trim, carry out gradient centrifugation, the first step centrifugal condition is set at 5-15 ℃, 25-80g, centrifugal 5-10 minute; The second step centrifugal condition is set at 5-15 ℃, 400-1000g, centrifugal 10-20 minute;
(3) behind centrifugal the finishing, the Cord blood in the collection bag is divided into three layers, and the upper strata is a blood plasma, and the middle level is a umbilical cord blood hematopoietic stem cell, and lower floor is a red corpuscle, respectively the blood plasma on upper strata and the red corpuscle of lower floor is emitted, and is the umbilical cord blood hematopoietic stem cell of enrichment in the collection bag.
Preferably, the first step centrifugal condition is set at 10 ℃, 50g, centrifugal 7 minutes; The second step centrifugal condition is set at 747g, and 10 ℃, centrifugal 15 minutes.
Be elaborated in the face of the present invention down:
1. the Cord blood in the collection bag is fully shaken up.With iodine disinfection collection bag sampling point 1 time, 75% alcohol disinfecting 2 times.Cut off the sterilization position with sterile scissors, take a blood sample and originally carry out cell counting and the detection of nucleated cell motility rate.
2. add HES (hydroxyethylamyle) in proportion.Formula is: HES volume/Cord blood weight (containing antithrombotics)=1/5 promptly adds quantity=Cord blood weight (containing antithrombotics) * 0.2 (ml) of HES.
3. the blood bag is placed on ASIC micro computer and adopts on the fluid controller, fully mixing is 10 minutes.
4 use gradient centrifugation to prepare nucleated cell
4.1 around airbag filling Cord blood bag, make the Cord blood bag be in plumbness, place Centrifuge Cup.
4.2 trim, carry out gradient centrifugation.The first step centrifugal condition is set at 10 ℃, 50g (g is a cf-unit), centrifugal 7 minutes; The second step centrifugal condition is set at 747g, and 10 ℃, centrifugal 15 minutes.
5. behind centrifugal the finishing, carefully the Cord blood bag after centrifugal vertically is placed on the blood plasma plasma-separating clip, the pipeline of blood bag lower end is passed the opening of blood plasma-separating clip bottom.
6. clamp the collection bag lower channel with mosquito forceps.With iodine disinfection collection bag sampling point once, twice of 75% alcohol disinfecting.Cut off the sterilization position with sterile scissors, connect transfering bag, unclamp mosquito forceps and emit red corpuscle.When treating the red corpuscle interface, clamp mosquito forceps according to collection bag bottom 1cm.Erythrocytic weight emitted in record.The heat seal pipeline.
7. clamp collection bag top pipeline with mosquito forceps, with iodine disinfection collection bag sampling point once, twice of 75% alcohol disinfecting.Cut off the sterilization position with sterile scissors, connect transfering bag, unclamp mosquito forceps and emit blood plasma, when residue blood volume is to 33ml in the collection bag, clamp mosquito forceps.
8. unclamp the blood plasma folder, take off collection bag, the blood plasma in the pipeline is refluxed.Be the umbilical cord blood hematopoietic stem cell of enrichment in the collection bag.
9. the Cord blood in the abundant mixing collection bag, draw samples branch are done the nucleated cell counting, tongue expects that indigo plant refuses to dye, CD34
+Detections such as cell detection, stem/progenitor cells cultivation.
In sum, content of the present invention is not confined in the above embodiments, and the knowledgeable people in the same area can propose other embodiment easily within technical director's thought of the present invention, but this embodiment is included within the scope of the present invention.
Claims (3)
1. the method through ladder spinning umbilical cord blood hematopoietic stem cell is characterized in that said centrifugal employing is once centrifugal, carries out in two steps, and the first step centrifugal condition is set at 5-15 ℃, cf-25-80g, centrifugal 5-10 minute; The second step centrifugal condition is set at 5-15 ℃, cf-400-1000g, centrifugal 10-20 minute.
2. the method through ladder spinning umbilical cord blood hematopoietic stem cell according to claim 1 is characterized in that, comprises the steps:
(1) Cord blood in the collection bag is fully shaken up, sterilization adds hydroxyethylamyle in proportion, the blood bag is placed on ASIC micro computer adopts on the fluid controller, fully mixing;
(2) fill around the Cord blood bag with airbag, make the Cord blood bag be in plumbness, place the Centrifuge Cup trim, carry out gradient centrifugation, the first step centrifugal condition is set at 5-15 ℃, 25-80g, centrifugal 5-10 minute; The second step centrifugal condition is set at 5-15 ℃, 400-1000g, centrifugal 10-20 minute;
(3) behind centrifugal the finishing, the Cord blood in the collection bag is divided into three layers, and the upper strata is a blood plasma, and the middle level is a umbilical cord blood hematopoietic stem cell, and lower floor is a red corpuscle, respectively the blood plasma on upper strata and the red corpuscle of lower floor is emitted, and is the umbilical cord blood hematopoietic stem cell of enrichment in the collection bag.
3. the method through ladder spinning umbilical cord blood hematopoietic stem cell according to claim 2 is characterized in that, the first step centrifugal condition is set at 10 ℃, 50g, centrifugal 7 minutes; The second step centrifugal condition is set at 747g, and 10 ℃, centrifugal 15 minutes.
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104762262A (en) * | 2015-03-26 | 2015-07-08 | 湖北省生命源干细胞有限公司 | Volume centrifugal separation method of umbilical cord blood stem cells |
| WO2016086476A1 (en) * | 2014-12-01 | 2016-06-09 | 四川新生命干细胞科技股份有限公司 | Method for separating nucleated cell from umbilical blood |
| CN110325280A (en) * | 2017-01-25 | 2019-10-11 | 烟台澳斯邦生物工程有限公司 | The device and method of automation sample treatment for diagnostic purposes |
| WO2020019195A1 (en) * | 2018-07-25 | 2020-01-30 | Yantai Ausbio Laboratories Co., Ltd. | Microbead-based separating gel |
| CN110839612A (en) * | 2019-10-10 | 2020-02-28 | 湖南源品细胞生物科技有限公司 | Cord blood hematopoietic stem cell preserving fluid and preparation method thereof |
| CN111518763A (en) * | 2020-06-04 | 2020-08-11 | 广州同康生物科技有限公司 | Method for separating high-purity umbilical cord stem cells |
| CN111961646A (en) * | 2020-09-01 | 2020-11-20 | 山东省齐鲁干细胞工程有限公司 | Umbilical cord blood stem cell enrichment method without adding settling agent |
| US20220176274A1 (en) * | 2020-12-09 | 2022-06-09 | Hunan Yuanpin Cell Technology Co. Ltd | Method of separating hematopoietic stem cells from umbilical cord blood |
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| CN1615437A (en) * | 2001-12-11 | 2005-05-11 | 株式会社奈特克 | Blood cell separation system |
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Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2016086476A1 (en) * | 2014-12-01 | 2016-06-09 | 四川新生命干细胞科技股份有限公司 | Method for separating nucleated cell from umbilical blood |
| CN104762262A (en) * | 2015-03-26 | 2015-07-08 | 湖北省生命源干细胞有限公司 | Volume centrifugal separation method of umbilical cord blood stem cells |
| CN110325280A (en) * | 2017-01-25 | 2019-10-11 | 烟台澳斯邦生物工程有限公司 | The device and method of automation sample treatment for diagnostic purposes |
| CN110325280B (en) * | 2017-01-25 | 2022-04-05 | 烟台澳斯邦生物工程有限公司 | Apparatus and method for automated sample processing for diagnostic purposes |
| US11883817B2 (en) | 2017-01-25 | 2024-01-30 | Yantai Ausbio Laboratories Co., Ltd. | Equipment and methods for automated sample processing for diagnostic purposes |
| WO2020019195A1 (en) * | 2018-07-25 | 2020-01-30 | Yantai Ausbio Laboratories Co., Ltd. | Microbead-based separating gel |
| CN110839612A (en) * | 2019-10-10 | 2020-02-28 | 湖南源品细胞生物科技有限公司 | Cord blood hematopoietic stem cell preserving fluid and preparation method thereof |
| CN110839612B (en) * | 2019-10-10 | 2021-02-02 | 湖南源品细胞生物科技有限公司 | Cord blood hematopoietic stem cell preserving fluid and preparation method thereof |
| CN111518763A (en) * | 2020-06-04 | 2020-08-11 | 广州同康生物科技有限公司 | Method for separating high-purity umbilical cord stem cells |
| CN111961646A (en) * | 2020-09-01 | 2020-11-20 | 山东省齐鲁干细胞工程有限公司 | Umbilical cord blood stem cell enrichment method without adding settling agent |
| US20220176274A1 (en) * | 2020-12-09 | 2022-06-09 | Hunan Yuanpin Cell Technology Co. Ltd | Method of separating hematopoietic stem cells from umbilical cord blood |
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