CN103361311B - Method for obtaining leukocytes by using washing disposable leukocyte-removing plastic blood bag - Google Patents
Method for obtaining leukocytes by using washing disposable leukocyte-removing plastic blood bag Download PDFInfo
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Abstract
The invention discloses a method for obtaining leukocytes by using a washing disposable leukocyte-removing plastic blood bag. The method comprises the following steps of: removing substances adhered to a leukocyte filtering device of the disposable leukocyte-removing plastic blood bag and a tee joint above the leukocyte filtering device by washing by a sterilizing washing solution; immersing the leukocyte filtering device and the tee joint into a sterilizing hypertonic solution; washing the leukocyte filtering device treated by the sterilizing hypertonic solution by a sterilizing washing solution, and collecting the leukocytes; re-suspending a leukocyte precipitate by using a sterilizing leukocyte protecting solution, and culturing the leukocytes so that the shapes of the leukocytes are normalized; and separating, and collecting the obtained living leukocytes. The method disclosed by the invention is easy to operate, has low requirement on experimental equipment, ensures high leukocyte collection efficiency and is a simple method for recovering the precious leukocyte resources.
Description
Technical field
The present invention relates to a kind of leukocytic method of obtaining, relate in particular to a kind of utilization and clean disposable use white blood cell removing plastic blood bag and obtain leukocytic method and special solution.
Background technology
Along with the continuous use of the medical procedure such as clinical operation, the probability that makes to transfuse blood also increases greatly, and the usually red cells transfusion of transfusing blood clinically.Blood station separating red corpuscle is used " disposable use white blood cell removing plastic blood bag " conventionally at present, by the leucocyte filtration device in blood bag, filter white corpuscle, reach and collect erythrocytic object, and in this process, retaining in a large amount of white corpuscles in leucocyte filter, blood station has been disposed it as Biohazard Waste.White corpuscle is important immunocyte source, as thrown away as Biohazard Waste, will cause a large amount of wastings of resources.
In recent years cell therapy cancer demonstrates more and more higher using value in curative effect, and wherein immunocyte treatment is main by extracting people's peripheral blood, extracts white corpuscle, enrichment mononuclearcell, by adding cytokine, carry out amplification cultivation, feed back in patient body, reach therapeutic purpose and effect.Along with cellular immunization treatment technology is more and more applied, biotechnology worker constantly explores the method for the more efficient immunocyte of amplification, and the foundation of these methods needs to take white corpuscle as basic experiment support in a large number, and these experiments generally do not need red corpuscle, therefore rationally efficient recovery white corpuscle resource has important meaning.Through retrieval, by reclaiming the white corpuscle in " disposable use white blood cell removing plastic blood bag " filtration unit, by discarded white corpuscle collect, separated, enrichment, the patent and the document that reach leukocytic recovery and maximum using have not been reported.
Summary of the invention
For the deficiencies in the prior art, the problem to be solved in the present invention is to provide a kind of utilization and cleans disposable use white blood cell removing plastic blood bag and obtain leukocytic method and special solution.
Utilization of the present invention is cleaned disposable use white blood cell removing plastic blood bag and is obtained leukocytic method, and step is:
(1) in hundred grades of clean areas, use the leucocyte filtration device on sterile water for irrigation preliminary flushing " disposable use white blood cell removing plastic blood bag ", remove the bloodstain adhering on it; With syringe, draw washing fluid, be inserted in the threeway of blood bag filtration device top, rinse 2-3 time, remove the material adhering on it;
(2) in hundred grades of clean areas, re-use the high sepage washing and filtering of sterilizing device 30s, then with high sepage to the standing processing of its submergence 1-2min;
(3) in hundred grades of clean areas, with sterile water for irrigation, rinse the filtration unit after high sepage is processed, will be transferred in aseptic centrifuge tube containing the leukocytic washing fluid that comes off simultaneously;
(4), with 1600 revs/min of centrifugal 8-15min, abandoning supernatant, collects white corpuscle;
(5), in hundred grades of clean areas, with the resuspended white corpuscle of sterilizing white corpuscle protection liquid, precipitate and cell suspension is transferred in aseptic Tissue Culture Flask, at 37 ℃, 5%CO
2culturing cell 2-3h in incubator, makes white corpuscle recover normal shape;
(6) in hundred grades of clean areas, the leukocyte suspension after cultivating is transferred in aseptic centrifuge tube, with 1000 revs/min of centrifugal 3-5min, abandoning supernatant, precipitation is the survival white corpuscle of results;
Wherein:
Above-mentioned washing fluid formula is: PBS damping fluid, D-Hank ' s liquid, DPBS, the 0.9%NaCl aqueous solution, 1640 culture medium solutions or other lymphocyte serum-free culture based sols, preferably 1640 culture medium solutions;
Above-mentioned high sepage formula is: NaCl15.0g/L, albumin 50.0ml/L, NaHCO
3in right amount; Osmotic pressure is 350～500mOsm/kg, and pH value is 7.35～7.45, uses NaHCO
3regulate pH;
Above-mentioned white corpuscle protection liquid formula is: in every liter of 1640 culture medium solutions, contain albumin 10mg, AB serum 60ml, Regular Insulin 5mg, Sodium Selenite 5mg, hepess20mM, L-glutaminate 1mM, beta-mercaptoethanol 0.1mM; Osmotic pressure is 300mOsm/kg, and pH value is 7.35～7.45.
Utilization of the present invention is cleaned disposable use white blood cell removing plastic blood bag and is obtained the washing fluid that white corpuscle method is used, it is characterized in that: described washing fluid formula is: PBS damping fluid, D-Hank ' s liquid, DPBS, the 0.9%NaCl aqueous solution, 1640 culture medium solutions or other lymphocyte serum-free culture based sols, preferably 1640 culture medium solutions.
Utilization of the present invention is cleaned disposable use white blood cell removing plastic blood bag and is obtained the high sepage that white corpuscle method is used, and it is characterized in that: described high sepage formula is: NaCl15.0g/L, albumin 50.0ml/L, NaHCO
3in right amount; Osmotic pressure is 350～500mOsm/kg, and pH value is 7.35～7.45, uses NaHCO
3regulate pH.
Utilization of the present invention is cleaned disposable use white blood cell removing plastic blood bag and is obtained the white corpuscle protection liquid that white corpuscle method is used, and it is characterized in that: described white corpuscle protection liquid formula is: in every liter of 1640 culture medium solutions, contain albumin 10mg, AB serum 60ml, Regular Insulin 5mg, Sodium Selenite 5mg, hepess20mM, L-glutaminate 1mM, beta-mercaptoethanol 0.1mM; Osmotic pressure is 300mOsm/kg, and pH value is 7.35～7.45.
In leucocyte filtration device in " the disposable use white blood cell removing plastic blood bag " of blood station separating red corpuscle, there is a large amount of white corpuscles to exist, red corpuscle is only used in blood transfusion clinically, be present in white corpuscle in leucocyte filtration device and be just used as Biohazard Waste and dispose, caused leukocytic waste.Utilization disclosed by the invention is cleaned disposable use white blood cell removing plastic blood bag and is obtained white corpuscle method, by the special reagent of special use, change leukocytic shape size, white corpuscle can be split away off by filtration unit, and then can realize the leukocytic object of collecting waste.In the inventive method, separated viable cell adopts machinery to rinse and low-speed centrifugal, simple to operate, and experimental installation requires low, and white corpuscle collection rate is high, for precious white corpuscle resource recycling provides again an easy approach.
Embodiment
Embodiment 1
(1) in hundred grades of clean areas, use the leucocyte filtration device on sterile water for irrigation preliminary flushing " disposable use white blood cell removing plastic blood bag ", remove the bloodstain adhering on it; With syringe, draw washing fluid, be inserted in the threeway of blood bag filtration device top, rinse 2 times, remove the material adhering on it;
(2) in hundred grades of clean areas, re-use the high sepage washing and filtering of sterilizing device 30s, then with high sepage to the standing processing of its submergence 2min;
(3) in hundred grades of clean areas, with sterile water for irrigation, rinse the filtration unit after high sepage is processed, will be transferred in aseptic centrifuge tube containing the leukocytic washing fluid that comes off simultaneously;
(4), with 1600 revs/min of centrifugal 10min, abandoning supernatant, collects white corpuscle;
(5), in hundred grades of clean areas, with the resuspended white corpuscle of sterilizing white corpuscle protection liquid, precipitate and cell suspension is transferred in aseptic Tissue Culture Flask, at 37 ℃, 5%CO
2culturing cell 2h in incubator, makes white corpuscle recover normal shape;
(6) in hundred grades of clean areas, the leukocyte suspension after cultivating is transferred in aseptic centrifuge tube, with 1000 revs/min of centrifugal 5min, abandoning supernatant, precipitation is the survival white corpuscle of results;
Wherein:
Above-mentioned washing fluid formula is: PBS damping fluid; Process for preparation: analytical balance weighs NaCl8.00g, KCl0.20g, Na
2hPO
4h
2o1.56g, KH
2pO
40.20g, is dissolved in 800ml ultrapure water, is then transferred in the volumetric flask of 1L, and adds ultrapure water to 1000ml, mixes.The PBS preparing is put into super clean bench and use 0.22um membrane filtration to 50ml centrifuge tube, sealing, and put in 4 ℃ of refrigerators standby.
Above-mentioned high sepage formula is: NaCl15.0g/L, albumin 50.0ml/L, NaHCO
3in right amount; Osmotic pressure is 350～500mOsm/kg, and pH value is 7.35～7.45, uses NaHCO
3regulate pH; Process for preparation: analytical balance weighs NaCl15.00g and is dissolved in 800ml ultrapure water, then adds 50.0ml albumin, and is transferred in the volumetric flask of 1L, adds ultrapure water to 1000ml, mixes.By the high sepage NaHCO preparing
3regulate pH to 7.35～7.45, then put it in super clean bench and use 0.22um membrane filtration to 50ml centrifuge tube, sealing, and put in 4 ℃ of refrigerators standby.
Above-mentioned white corpuscle protection liquid formula is: in every liter of 1640 culture medium solutions, contain albumin 10mg, AB serum 60ml, Regular Insulin 5mg, Sodium Selenite 5mg, hepess20mM, L-glutaminate 1mM, beta-mercaptoethanol 0.1mM; Osmotic pressure is 300mOsm/kg, and pH value is 7.35～7.45; Process for preparation: according to white corpuscle protection liquid formula, accurately weigh medium additives, prepare 1000ml white corpuscle protection liquid.With NaCl and NaHCO
3regulate substratum osmotic pressure, use NaHCO
3regulate substratum PH.Use 0.22um membrane filtration to 50ml centrifuge tube the substratum that mixes up PH and osmotic pressure, sealing, and put in 4 ℃ of refrigerators standby.
Embodiment 2
(1) in hundred grades of clean areas, use the leucocyte filtration device on sterile water for irrigation preliminary flushing " disposable use white blood cell removing plastic blood bag ", remove the bloodstain adhering on it; With syringe, draw washing fluid, be inserted in the threeway of blood bag filtration device top, rinse 3 times, remove the material adhering on it;
(2) in hundred grades of clean areas, re-use the high sepage washing and filtering of sterilizing device 30s, then with high sepage to the standing processing of its submergence 1min;
(3) in hundred grades of clean areas, with sterile water for irrigation, rinse the filtration unit after high sepage is processed, will be transferred in aseptic centrifuge tube containing the leukocytic washing fluid that comes off simultaneously;
(4), with 1600 revs/min of centrifugal 15min, abandoning supernatant, collects white corpuscle;
(5), in hundred grades of clean areas, with the resuspended white corpuscle of sterilizing white corpuscle protection liquid, precipitate and cell suspension is transferred in aseptic Tissue Culture Flask, at 37 ℃, 5%CO
2culturing cell 3h in incubator, makes white corpuscle recover normal shape;
(6) in hundred grades of clean areas, the leukocyte suspension after cultivating is transferred in aseptic centrifuge tube, with 1000 revs/min of centrifugal 4min, abandoning supernatant, precipitation is the survival white corpuscle of results;
Wherein:
Above-mentioned washing fluid formula is: 1640 culture medium solutions;
Above-mentioned high sepage formula is: NaCl15.0g/L, albumin 50.0ml/L, NaHCO
3in right amount; Osmotic pressure is 350～500mOsm/kg, and pH value is 7.35～7.45, uses NaHCO
3regulate pH;
Above-mentioned white corpuscle protection liquid formula is: in every liter of 1640 culture medium solutions, contain albumin 10mg, AB serum 60ml, Regular Insulin 5mg, Sodium Selenite 5mg, hepess20mM, L-glutaminate 1mM, beta-mercaptoethanol 0.1mM; Osmotic pressure is 300mOsm/kg, and pH value is 7.35～7.45.
Claims (1)
1. utilize the disposable use white blood cell removing plastic blood bag of cleaning to obtain a leukocytic method, step is:
(1) in hundred grades of clean areas, use the leucocyte filtration device on sterile water for irrigation preliminary flushing " disposable use white blood cell removing plastic blood bag ", remove the bloodstain adhering on it; With syringe, draw washing fluid, be inserted in the threeway of blood bag filtration device top, rinse 2-3 time, remove the material adhering on it;
(2) in hundred grades of clean areas, re-use the high sepage washing and filtering of sterilizing device 30s, then with high sepage to the standing processing of its submergence 1-2min;
(3) in hundred grades of clean areas, with sterile water for irrigation, rinse the filtration unit after high sepage is processed, will be transferred in aseptic centrifuge tube containing the leukocytic washing fluid that comes off simultaneously;
(4), with 1600 revs/min of centrifugal 8-15min, abandoning supernatant, collects white corpuscle;
(5), in hundred grades of clean areas, with the resuspended white corpuscle of sterilizing white corpuscle protection liquid, precipitate and cell suspension is transferred in aseptic Tissue Culture Flask, at 37 ℃, 5%CO
2culturing cell 2-3h in incubator, makes white corpuscle recover normal shape;
(6) in hundred grades of clean areas, the leukocyte suspension after cultivating is transferred in aseptic centrifuge tube, with 1000 revs/min of centrifugal 3-5min, abandoning supernatant, precipitation is the survival white corpuscle of results;
Wherein:
Above-mentioned washing fluid formula is: PBS damping fluid, D-Hank ' s liquid, DPBS, the 0.9%NaCl aqueous solution, 1640 culture medium solutions or other lymphocyte serum-free culture based sols;
Above-mentioned high sepage formula is: NaCl 15.0g/L, albumin 50.0ml/L, NaHCO
3in right amount; Osmotic pressure is 350～500 mOsm/kg, and pH value is 7.35～7.45, uses NaHCO
3regulate pH;
It is characterized in that:
The described white corpuscle protection of step (5) liquid formula is: in every liter of 1640 culture medium solutions, contain albumin 10mg, AB serum 60ml, Regular Insulin 5mg, Sodium Selenite 5mg, hepess 20 mM, L-glutaminate 1mM, beta-mercaptoethanol 0.1mM; Osmotic pressure is 300mOsm/kg, and pH value is 7.35～7.45.
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| CN111454902B (en) * | 2020-04-08 | 2022-12-02 | 郭华 | Method and device for separating and enriching white blood cells, stem cells and/or pathogens from blood |
| CN113025571B (en) * | 2021-03-18 | 2022-05-17 | 武汉中生毓晋生物医药有限责任公司 | Treatment method of human peripheral blood T lymphocytes, immunogen preparation and application |
| CN114561352A (en) * | 2022-03-24 | 2022-05-31 | 青岛市中心血站 | Method for separating mononuclear cells from blood donation complete blood collection device |
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| CN103205395A (en) * | 2011-12-05 | 2013-07-17 | 帕尔公司 | Leukocyte purification |
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| JPH07299120A (en) * | 1994-04-28 | 1995-11-14 | Nissho Corp | Blood separation system |
| CN101332296A (en) * | 2008-06-13 | 2008-12-31 | 中国人民解放军第三军医大学第三附属医院 | Dendritic cell vaccine for promoting spinal cord injury function recovery |
| CN102471762A (en) * | 2009-06-30 | 2012-05-23 | 株式会社钟化 | Blood component separation system and separation material |
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