CN1715400A - Method for freezing and storaging lymphocyte - Google Patents
Method for freezing and storaging lymphocyte Download PDFInfo
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- CN1715400A CN1715400A CNA200410062235XA CN200410062235A CN1715400A CN 1715400 A CN1715400 A CN 1715400A CN A200410062235X A CNA200410062235X A CN A200410062235XA CN 200410062235 A CN200410062235 A CN 200410062235A CN 1715400 A CN1715400 A CN 1715400A
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- lymphocyte
- cell
- store method
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- blood cell
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Abstract
The cell separating and storing method is to take healthy and active cell out of body and to freeze store after separation, activation and proliferation, so as to provide effective, safe and reliable disease preventing and treating material. Specifically, the lymphocyte freezing and storing method includes the steps of: for the donor to apply, to exam the blood of the donor, to collect venous blood, to separate lymphocyte from venous blood via a density centrifuging process, to activate and culture lymphocyte, to collect active lymphocyte, to freeze and store lymphocyte, and to defreeze before use.
Description
Affiliated technical field
The present invention relates to the method that a kind of lymphocyte separates, preserves.
Background technology
Present cell therapy, biotherapy are with the material of the current point of patient, as cell, tissue etc., even use other people material to treat.This method exists some shortcomings.Such as, current patient is a patient, its physical appearance can be reflected on the material of employed treatment usefulness certainly.Its cell of the patient of cancer might be very low to the killing activity of cancer cells, and the patient of virus infection might be infected by lymphocyte, and elderly patients' body, tissue, cell all might occur wearing out or the like.The cancer patients is after passing through operative treatment or radiation cure, chemotherapy, and body has been very weak, and cell also might be subjected to serious damage quantitatively, qualitatively.If after method treatment, carry out the preparation of biotherapy again with other, may need again to have spent the considerable time, and the opportunity of delay treatment.Utilize other people material can solve material aging, problem such as sick and weak, but might be subjected to the infection of material supplier's disease, more likely heteroplastic transplantation occurs and the rejection reaction that takes place.
If the cell or the tissue that might need people's future,, cell young and vigorous at it or tissue activity are preserved when maximum, and be normally old in needs, suffers from and use in the disease, just can address the above problem well.
Summary of the invention
In order to solve the patient, the patient of cancer, amynologic disease, various infection diseases particularly, in the needs cell therapy, can provide a kind of health or comparatively healthy, bigger active treatment material arranged, present technique provides a kind of method, healthy people or stadium are in the patient at initial stage, before cancer patients's operation, or before the radiotherapy, chemotherapy, take out healthy people or patient's cell external, preserved with suitable mode, be used for the prevention or the treatment of the disease of patient own in the future.
The technical problem that solves:
A kind of fundamentally solution to the problems described above is not arranged at present as yet.The technical program provides possibility for addressing the above problem.By taking the cell of volunteer when healthy or healthier, adopt the method for external activation, propagation, preservation, healthy people's cell can be preserved the time more than 20 years.
Present technique provides a kind of store method of cell, and in optimized technical scheme, the lymphocyte that is used for the people is preserved.At first the volunteer preserves for cell and files an application, applicant's blood test, venous blood collection, in venous blood, pass through the Density Gradient Centrifugation isolated lymphocytes, lymphocytic activation and cultivation, the lymphocytic recovery of sensitization, lymphocytic freezing preservation, the processes such as the recovery of thawing of frozen cell during use.
Present technique provides a kind of store method of cell, in optimized technical scheme, wherein preserves the lymphocyte from healthy people.Be used for the prevention or the treatment of cancer.
Present technique provides a kind of store method of cell, in optimized technical scheme, wherein preserves the lymphocyte from healthy people.Be used for the prevention or the treatment of immunocompromised disease.
Present technique provides a kind of store method of cell, in optimized technical scheme, wherein preserves the lymphocyte from healthy people.Be used for the prevention or the treatment of various infection diseases.
Present technique provides a kind of store method of cell, in optimized technical scheme, wherein preserves the lymphocyte from healthy people.Be used for various hypersensitive preventions or treatment.
Present technique provides a kind of store method of cell, in optimized technical scheme, wherein preserves the lymphocyte from healthy people.Be used for the prevention or the treatment of innate immunity insufficiency disorder.
Present technique provides a kind of store method of cell, in optimized technical scheme, wherein preserves the lymphocyte from healthy people.Be used for the prevention or the treatment of acquired immunity insufficiency disorder.
Present technique provides a kind of store method of cell, in optimized technical scheme, wherein preserves the lymphocyte from healthy people.Be used for the prevention or the treatment of various own immunological diseases.
Present technique provides a kind of store method of cell, in optimized technical scheme, wherein preserves the lymphocyte from healthy people.Starting material as various biotherapies.
Present technique provides a kind of store method of cell, in optimized technical scheme, wherein preserve from cancer patient before the operation or the early stage patient's of the state of an illness cell, or other illness patient's is early stage cell when lymphocyte is comparatively healthy.Starting material as treatment.
For the validity of proof technical solution of the present invention, can test according to following method
Embodiment
1. the volunteer signs blood cell and preserves letter of consent.The cell of stipulating the volunteer in the letter of consent does not have volunteer's agreement to divert.
2. the blood to the volunteer carries out routine examination.For guaranteeing the quality of cell, before cultivating, cellular segregation to carry out conventional inspection to blood.Comprising: hiv virus is checked, the inspection of hepatitis virus, the syphilis inspection, and other might influence the inspection of the project of cell quality and patient health.
3. venous blood takes.With the syringe that is added with heparin, the injection needles of 18G, in volunteer's vein, extract blood 5-100ml with usual method.The amount of heparin is according to how many decisions of getting blood.
4. the processing of blood.After the syringe that blood is housed disinfected in alcohol, place the aseptic technique platform, blood is injected centrifuge tube or proper container gently.The blood of taking is with the physiological saline dilution of equivalent, and is stand-by behind the mixing.
5. the separation of cell.With proportion parting liquid Ficoll (Lymphosepar I) (d=1.01-1.10) 2-20ml add in the centrifuge tube, with the blood that pipette, extract above-mentioned steps 4 was diluted, double-layer is on the proportion parting liquid lightly.Centrifuge tube is put into whizzer gently, under the state of room temperature brake off, the centrifugal 5-30 of 1000-2000rpm minute.
6. the recovery of cell: after whizzer comes to a complete stop, centrifuge tube is taken out lightly, the upper solution sucking-off is discarded, or the serum of preserving separately when making cell cultures is used with suction pipe.The middle layer is recovered in the new centrifuge tube with suction pipe sucking-off gently.
7. cell cleans.In the cell that reclaims, add 10-40ml physiological saline, put into whizzer, under the state of room temperature brake on, the centrifugal 5-30 of 1000-2000rpm minute.After supernatant liquor discards, add 10-40ml physiological saline again, after mixing with bench mixer, once centrifugal again under similarity condition.
8. the allotment of cell: the cell after cleaning suspends with the RPMI-1640 5-50ml of human serum that contains 1-10% and 1000-100000 U/ml IL-2, and is standby.
9. the preparation of culturing bottle: add in the flat culturing bottle with physiological saline or the anti-cd 3 antibodies 1-10ml of PBS (-) dissolved 1-40 μ g/ml, 4 ℃-room temperature left standstill 1-24 hour.Discard behind the solution with physiological saline or PBS (-) solution washing 3 times, standby.
10. the cultivation of cell: the cell solution of step 8 is moved in the culturing bottle that step 9 handled, place 37 ℃ of 5%CO
2Cultivated 1-3 days in the incubator.
11. the preparation of freezing preservation liquid: 5ml DMSO is joined among the RPMI-1640 40ml, adding human serum 1-5ml.
12. preserve the adjustment of cell: after the cell after step 10 cultivated is cleaned, counting.Preserve liquid with the cell freezing of step 11 concentration of cell is adjusted into 1 * 10
7-1 * 10
8, move in the test tube and preserve.Preserve 1-10ml enchylema in every developmental tube.
13. the preservation of cell: the test tube that will fill cell places program control thermos cup, puts into 4 ℃ of refrigerators, after 1-12 hour, changes-80 ℃ of refrigerators over to, after 4-24 hour, puts into liquid nitrogen and preserves.
14. thawing of cell: when needed, the cell of freezing preservation is taken out from liquid nitrogen, place 37 ℃ of water-baths to make it quick-thawing.Cell after thawing adds in the needed nutrient solution and cultivates with after the RPMI-1640 washed twice.
Beneficial effect: present technique can provide for the future of Healthy People the selection of a treatment various diseases, or keeps some ratios for the patient at course of disease initial stage Healthy and safe treatment material.
Claims (10)
1. the store method of a blood cell relates to people's lymphocyte is preserved.Its content comprises that the volunteer is that the cell preservation is filed an application, applicant's blood test, venous blood collection, in venous blood, pass through the Density Gradient Centrifugation isolated lymphocytes, lymphocytic activation and cultivation, the lymphocytic recovery of sensitization, lymphocytic freezing preservation, the processes such as the recovery of thawing of frozen cell during use.
2. the store method of the described blood cell of claim 1 is preserved healthy people's lymphocyte, is used for prevention or treatment cancer.
3. the store method of the described blood cell of claim 1 is preserved healthy people's lymphocyte, is used for prevention or treats various immunocompromised diseases.
4. the store method of the described blood cell of claim 1 is preserved healthy people's lymphocyte, is used for prevention or treats various infection diseases.
5. the store method of the described blood cell of claim 1 is preserved healthy people's lymphocyte, is used for prevention or treats various allergy.
6. the store method of the described blood cell of claim 1 is preserved healthy people's lymphocyte, is used for prevention or treats various innate immunity insufficiency disorders.
7. the store method of the described blood cell of claim 1 is preserved healthy people's lymphocyte, is used for prevention or treatment acquired immunity insufficiency disorder.
8. the store method of the described blood cell of claim 1 is preserved healthy people's lymphocyte, is used for prevention or treats various own immunological diseases.
9. the store method of the described blood cell of claim 1 is preserved healthy people's lymphocyte, is used for the starting material as various biotherapies.
10. the store method of the described blood cell of claim 1 is preserved before the cancer patient operation or the early stage cell of the state of an illness, or the patient's of other illness is early stage, and the cell when lymphocyte is comparatively healthy is used for the starting material as the treatment disease.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA200410062235XA CN1715400A (en) | 2004-07-01 | 2004-07-01 | Method for freezing and storaging lymphocyte |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA200410062235XA CN1715400A (en) | 2004-07-01 | 2004-07-01 | Method for freezing and storaging lymphocyte |
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| Publication Number | Publication Date |
|---|---|
| CN1715400A true CN1715400A (en) | 2006-01-04 |
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Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNA200410062235XA Pending CN1715400A (en) | 2004-07-01 | 2004-07-01 | Method for freezing and storaging lymphocyte |
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| CN (1) | CN1715400A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2015103792A1 (en) * | 2014-01-13 | 2015-07-16 | 深圳市汉科生物工程有限公司 | Method for preserving a synergist of lymphocyte in vitro culture |
| CN105638642A (en) * | 2016-01-27 | 2016-06-08 | 上海润泉生物技术有限公司 | Immune cell cryopreservation solution and application thereof |
-
2004
- 2004-07-01 CN CNA200410062235XA patent/CN1715400A/en active Pending
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2015103792A1 (en) * | 2014-01-13 | 2015-07-16 | 深圳市汉科生物工程有限公司 | Method for preserving a synergist of lymphocyte in vitro culture |
| CN105051183A (en) * | 2014-01-13 | 2015-11-11 | 深圳市汉科生物工程有限公司 | Method for preserving a synergist of lymphocyte in vitro culture |
| CN105051183B (en) * | 2014-01-13 | 2018-04-27 | 张明杰 | The method of preservation culture in vitro of lymphocytes synergist |
| CN105638642A (en) * | 2016-01-27 | 2016-06-08 | 上海润泉生物技术有限公司 | Immune cell cryopreservation solution and application thereof |
| CN105638642B (en) * | 2016-01-27 | 2018-10-19 | 上海润泉生物技术有限公司 | A kind of immunocyte frozen stock solution and its application |
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