CN102433303A - In-vitro culture method for T lymphocytes - Google Patents
In-vitro culture method for T lymphocytes Download PDFInfo
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Abstract
The invention discloses an in-vitro culture method for T lymphocytes, and radically solves the problems of high cost, inconvenience of use and unfavorable medical research and clinical application existing in the conventional in-vitro culture method for the T lymphocytes. The method comprises the following steps of: directionally sorting the T lymphocytes, inducing, differentiating, culturing, amplifying and the like. A special cell factor combination is used for performing induction differentiation on the T lymphocytes; three factors are a CD3 monoclonal antibody, gamma-interferon and interleukin-1 alpha respectively; the optimal concentration ratio of the three factors is 10:1:1; the total final concentration of the three factors in a culture medium is 1,200ng/ml; and the experiment shows that the combination is an extremely effective way of inducing the T lymphocytes to differentiate.
Description
Technical field
The mononuclearcell that the present invention relates to samples such as a kind of handler's of being used for peripheral blood, Cord blood, hydrothorax, ascites becomes cytokine induced kill cell (Cytokine-Induced Killer; Abbreviation CIK) method; Particularly a kind of lymphocytic extracorporeal culturing method of T that is used for; This method can be applied to clinical cell biological treatment; Also can be applied to basic medical research, clinical application research and biotechnology research, especially can be applied to the research of immunity system research and tumor-killing effect.
Background technology
Along with tumour is serious day by day to the healthy threat of human survival; Variation with rapid changepl. never-ending changes and improvements is also taking place in reply tumor treatment pattern; Various novel drugs, new technology, novel method emerge in an endless stream; Wherein the cell biological treatment is the 4th type of tumor therapeuticing method that behind operation, radiation and chemotherapy, develops, and becomes developing direction important in the tumor biotherapy.Cancer is the cell disease that tumour cell is escaped the immunosurveillance system, and the defective of body's immunity is that cancer takes place and development key.The antineoplastic immune of body is mainly the T lymphocyte and mediates.The cell biological treatment technology is fed back to autoimmune cell in the body through external evoked, differentiation, amplification again, walks around in-vivo tumour dysimmunity mechanism, and the immunoreation through excitating organism resists, suppresses and kill cancer cells.Continue after LAK, infiltration tumor lympha cell and differentiation antigen (Cluster of Differentiation is called for short CD) the 3 monoclonal antibody activated killer cell, the knurl effect of killing of CIK comes into one's own day by day.
The CIK cell be 1991 by reported first such as the Schmidts of Stanford Univ USA.They find that under in-vitro simulated human internal environment's condition peripheral blood lymphocyte can and be bred the heterogeneous effector cell who has efficient cytotoxic activity for a group by the directional induction of the various kinds of cell factor.Because this cell mass is expressed CD3, CD8 and CD56 membrane protein molecule simultaneously; So be called nk cell appearance T lymphocyte again; With the restricted advantage of killing knurl of the non-main histocompatibility complex of powerful anti-tumor activity of T lymphocyte and nk cell, bring into play anti-knurl and kill the knurl effect but specificity is gathered in tumor by local.The CIK cell is gone back to the nest in spleen and lymphoglandula in vivo, in liver, distributes at most, and secondly be peripheral blood.CD3 wherein
+, CD8
+, CD56
+Be the main effects cell in the CIK cell mass, in normal people's peripheral blood, only account for lymphocytic 1-5%, and stimulate cultivation after 10-25 days external through the multiple factor, cell quantity can increase more than 1000 times before cultivating.
Compare with other adoptive immunotherapy cells, the CIK cell have propagation fast, kill tumor activity strong, kill that the knurl spectrum is wide, spinoff is little, to advantages such as normal marrow hemopoiesis influence are slight.Tumor cell line to chemotherapy resistance has lethal effect, and the growth of normal hematopoiesis granulocyte colony is not influenced.Through getting a small amount of mononuclearcell of patient, under external specified conditions, prepare a large amount of CIK cells, feed back the directly effective function of tumor of removing of performance of the interior back of body; Especially patient's effect is remarkable behind adversary's postoperative or the chemicotherapy; Residual small metastatic lesion be can eliminate, cancer cells diffusion and recurrence prevented, enhance immunity power; And realized prolongation patient lifetime, improve multiple targets such as patients ' life quality fast.Therefore, CIK is considered to the preferred cell of the antitumor cellular immunization treatment of adopting of a new generation.Tumor biotherapy center applications BMDC (Dendritic Cell is called for short DC) and CIK cell are united the method for feedback at present, are used for disease treatments such as malignant melanoma, kidney, lung cancer, liver cancer, esophagus cancer, cancer of the stomach, bladder cancer.But the method for each laboratory culture CIK at present, materials used, incubation time are all inconsistent, cause culture cycle, quality and the quantity of effective cell uneven, have brought the unsettled risk factors of effect to clinical application.
In addition, in one Chinese patent application number is 200910045790.4 " a kind of CIK cell and preparation method thereof and cell preparation ", a kind of CIK cell and preparation method thereof and cell preparation are disclosed.The inducible factor of the CIK cell of its record is combined as phytohaemagglutinin, anti-CD3 monoclonal antibody and anti-CD28 monoclonal antibody.Employed phytohaemagglutinin is a kind of mitogen in this scheme, can activate small lymphocyte and be converted into lymphoblast, then division growth; Discharge lymphokine; Improve the phagocytic function of scavenger cell, but because its difficult purification, and cost is high; So all the time only in the laboratory as stimulating lymphopoietic reagent, be difficult to be applied to clinical extensive use.In one Chinese patent application number was 200310109565.5 " a kind of CIK cells in vitro cultural method ", a kind of CIK cells in vitro cultural method was disclosed.This cultural method has adopted phytohaemagglutinin and two kinds of mitogen associatings of CD 3-resisting monoclonal antibody activating cells equally; Though can strengthen the stimulus intensity of cell; But this method need be accomplished by bio-reactor; Not only cost is high, uses inconvenience, and is unfavorable for medical research and clinical application.
To sum up, existing CIK cell (T lymphocyte) extracorporeal culturing method is badly in need of improving.
Summary of the invention
The purpose of this invention is to provide the lymphocytic extracorporeal culturing method of a kind of T of being used for, this cultural method can be used for the in-vitro directed sorting of T lymphocyte, induce, breaks up, cultivates, increases.
The technical scheme that the present invention adopted is: this is used for the lymphocytic extracorporeal culturing method of T, and comprise the T lymphocyte is carried out directed sorting, induces, breaks up, cultivates, increases that its technical essential is: the concrete operations step is following:
The first step, the T lymphocyte is carried out directed sorting:
The hemocyte that dilution is good slowly injects on the cell sorting liquid with the ratio of 1:1, and centrifugal treating; With the first layer liquid after the transfer pipet sucking-off sorting is the equal slurry layer of blood plasma or cell tissue, discards, and drawing second layer cell is that ring-type oyster white lymphocyte is in new centrifuge tube; In the cell of new collection, add saline water, abandon supernatant after the centrifugal treating; Repeat to wash twice; Wherein the compound method of cell sorting liquid is: get 9% Ficoll solution 720ml and 33.4% urografic acid methylglucamine salt 30ml thorough mixing, in this mixed solution, add the gelatin of 7.5g again, fully mixing; The specific density of adjusting solution with the Ficoll of the urografic acid methylglucamine salt of high density or dilution is to 1.077g/ml; Put into temperature at last and be 121 ℃ high pressure steam sterilization 30min, room temperature preservation is subsequent use;
Second goes on foot, the T lymphocyte is induced, breaks up, cultivated:
Get and encapsulate one in bottle, the said bottle that encapsulates is the Tissue Culture Flask that has encapsulated CD3 monoclonal antibody, gamma-interferon, interleukin 1 α, three kinds of factors in advance, with saline water or substratum rinse once and discard, adds patient's autologous plasma 2 ~ 5ml; Get the substratum re-suspended cell, behind the mixing its immigration is encapsulated in the bottle; Then use substratum rinse centrifuge tube, rinse liquid is moved into once more encapsulate in the bottle; To encapsulate bottle and put into 5% CO
2, cultivate 24h in 37 ℃ the incubator;
Encapsulate the preparation of bottle: get CD3 monoclonal antibody, gamma-interferon, interleukin 1 α and join in the 40ml phosphate buffered saline buffer; The concentration ratio of CD3 monoclonal antibody, gamma-interferon, three kinds of factors of interleukin 1 α is 10:1:1; Process the mixing solutions that total concn is 1200ng/ml, fully mixing; This mixing solutions is joined in the 600ml culturing bottle, and 4 ℃ left standstill 24 hours at least, processed initial package by bottle; With initial package by bottle under-40 ℃ of conditions freezing 24 hours; Start the compressor of freeze drier, make the temperature in the machine chamber reduce to-40 ℃, will encapsulate a bottle bottleneck and unscrew, put into kiln; The open vacuum pump reaches pressure<15Pa, vacuum-freeze-dry 20 hours; Open CO
2Intake valve cuts out vacuum pump; Treat that pressure closes CO behind 110K
2Intake valve takes out and encapsulates bottle, tightens lid, seals mouth, 4 ℃ of preservations; In the quality guaranteed period of regulation, use;
The 3rd goes on foot, the T lymphocyte is increased:
Draw fresh culture dissolved cell factors A, i.e. the interleukin II of 200,000 units injects and encapsulates bottle, and the concentration of cytokine A this moment in culturing bottle is 0.4 ten thousand units/ml; In 5%CO
2, cultivate 2 ~ 3 days in 37 ℃ of incubators after, in encapsulating bottle, add fresh culture 100ml; Again in 5% CO
2, continue to cultivate in 37 ℃ of incubators 1 ~ 2 day, when cell be paved with basically encapsulate bottle at the bottom of the time, change bag;
Blow down the attached cell that encapsulates in the bottle with transfer pipet; Draw fresh culture dissolved cell factor B with syringe, i.e. the interleukin II of 2,000,000 units adds and encapsulates in the bottle; Add patient's autologous plasma 2 ~ 5ml simultaneously, the cell culture fluid in the culturing bottle is poured in the culture bag; Encapsulate bottle twice with the fresh culture rinse, rinse liquid is poured in the culture bag in the lump, supplemented medium is to 1000ml, and the concentration of cytokine B this moment in culturing bottle is 0.2 ten thousand units/ml; Culture bag is put into 5%CO
2, cultivate 3 ~ 4 days in 37 ℃ of incubators after, add fresh culture 1000ml, continue to put into 5%CO
2, cultivate in 37 ℃ of incubators; After the fluid infusion second day, examine bacterium, prove whether the culture in the culture bag can be used for adoptive therapy or medical research.
Also comprise the inspection bacterium, feed back step:
During said inspection bacterium, take out culture bag, inject two diphase blood culture flasks with syringe respectively by thief hole absorption culture, one bottle is sent with the third party and carries out aseptic detection, and one bottle is used for self check; Place 37 ℃ of constant incubators to cultivate two-way Blood culture bottle; Diphase blood culture flask is cultivated after 3 days if asepsis growth proves that the culture in the culture bag can be used for adoptive therapy or medical research;
During feedback, prepare 8 of centrifuge tubes, culture is poured in the centrifuge tube, the residue culture is put back to incubator and is continued to cultivate; Under 4 ℃ of conditions, the centrifugal treating supernatant discarded is with the saline water re-suspended cell and focus in 2 centrifuge tubes; Under 4 ℃ of conditions, the centrifugal treating supernatant discarded; With the saline water re-suspended cell and focus in 1 centrifuge tube; Under 4 ℃ of conditions, the centrifugal treating supernatant discarded is used the saline water re-suspended cell; Under 4 ℃ of conditions, the centrifugal treating supernatant discarded is used the saline water re-suspended cell; Draw a cell-dispersing agent with syringe and inject new centrifuge tube, this cell-dispersing agent is 10% human serum albumin, then aseptic screen cloth is put on this centrifuge tube; Draw cell suspension, inject the centrifuge tube that sterile filters is housed; Through the big cell mass of filter screen elimination; Fetch one in defeated bag, pour cell into and add saline water subsequent use.
Said cultural method be used for the mononuclearcell of handler's peripheral blood, Cord blood, hydrothorax, ascites in-vitro directed sorting, induce, break up, cultivate, increase.
Advantage and positively effect that the present invention has are: because the present invention has adopted special combination of cytokines to be used for the T lymphocyte is induced differentiation, these three kinds of factors are respectively CD3 monoclonal antibody, gamma-interferon, interleukin 1 α; The optimum concn ratio of these three kinds of factors is 10:1:1, and the total final concentration of these three kinds of factors in substratum is 1200ng/ml, and experiment shows that this combination is the utmost point efficient manner of inducer T lymphocyte differentiation.Simultaneously,, increased area and time that the T lymphocyte contacts with cytokine, made it more acceptant to the cytokine stimulation and induced differentiation rapidly with the cytokine method inducer T lymphocyte differentiation of coated cell culturing bottle in advance.In addition, the present invention has adopted freeze dried method preparation to encapsulate bottle, and this method is very effective for the activity that guarantees cytokine.If this method is applied to industrialization production, both can prolong the quality guaranteed period that encapsulates bottle, reduced requirement again to transport condition, be a kind of excellent way that is applicable to actual production.
Gamma-interferon is by CD4
+Or CD8
+The homodimer gp that cell produces can directly or indirectly be brought into play antitumor action through number of ways, the activity of enhanced natural killer cell, scavenger cell and cytotoxic T cell.In inducing the CIK cell formation process, add gamma-interferon and can reduce the interleukin II consumption; The order of gamma-interferon adding simultaneously is closely related with cytotoxic activity, adds interleukin II behind the adding gamma-interferon earlier and can improve cytotoxic activity.This is can impel interleukin 2 receptor quantity increase on the PMBC because add gamma-interferon earlier, thereby has activated the effector cell effectively.CD 3-resisting monoclonal antibody can promote cell proliferation as a kind of mitotic division promotor.The early 1980s investigators just find anti-cd 3 antibodies or the external activation immobilized of anti-TXi Baoshouti antibody capable simulation physiology part T cell, make it a large amount of propagation, express interleukin 2 receptor, produce the endogenous interleukin II.Anti-CD3 monoclonal antibody has the effect of anti metastasis, and the growth that antibody suppresses malignant tumour possibly be the effect of immunostimulant, and antibody increases antineoplastic specificity T cell quantity, increased activity, and inducing of the endogenous cell factor amplified this effect simultaneously.CD 3-resisting monoclonal antibody not only plays an important role in the CIK cell cultivation process, at raising CIK cell white blood disease and lymphadenomatous killing and wounding is had promoter action equally on the susceptibility.Interleukin 1 is mainly produced by the activated mononuclear macrophage.The anti-tumor in vivo mechanism of action of interleukin 1 is quantity and the function that strengthens the T cell and increase tumor infiltrating lymphocyte.Interleukin 1 can mediate and express interleukin 2 receptor on the PMNC; Unite the cellulotoxic effect that can obviously improve CIK when using with gamma-interferon, CD3 monoclonal antibody, but interleukin 1 is inoperative to the CIK cell amplification.Interleukin II is that (by the polypeptide that 133 amino acid are formed, relative molecular mass is about 15 * 10 for a kind of cytokine of T lymphocytic emiocytosis
3).It is the core substance that human immunity is replied, and has immunostimulant, effect such as antitumor and anti-infective.Treatment of conditions such as kidney, melanoma, lymphoma, lung cancer, cancer of the stomach, mammary cancer, ovarian cancer, intestinal cancer, bladder cancer, tumor of head and neck, white blood disease and cancerous thoracoascites have been used for clinically.Compare with radiotherapy, chemotherapy, interleukin II is slight to normal cellulotoxic side effect, and ability ameliorate tumor patient's pain improves patients ' life quality.White plaque, hepatitis, AIDS, venereal disease, habituation property junkies and other immunologic hypofunctions patient all can use interleukin II in addition.The CIK cell is a group foreign cell crowd of the common inducing culture of the various kinds of cell factor, and the effect of the various kinds of cell factor is each other collaborative, and the propagation of monofactor pairing effect cell and cytotoxic activity do not have effect or less than the combined action of the various kinds of cell factor.Symphyogenetic mechanism is the final common static T cell that activates, and improves cell expressing interleukin 2 receptor and the ability that produces interleukin II, starts the t cell activation reaction that autocrine approach interleukin II relies on.
In addition, the present invention has prepared a kind of liquid of cell sorting efficiently, and the red corpuscle in the enough effective cracking blood of this cell sorting fluid power fully precipitates red blood cell component, separates with treating isolating mononuclearcell.Moreover test kit of the present invention adopts serum free medium, and serum free medium can reach the biologic cleanliness requirement; Definite ingredients, steady quality is convenient to Quality Control; Overcome the shortcoming of people AB serum, can reduce the chance of patient's inadvertent contamination, significant for applying of immunotherapy.Use cultural method according to the invention to handle the T lymphocyte, can gather in the crops the CD3 more than 95%
+Cell, wherein CD3
+CD8
+Cell reaches more than 80%, CD3
+CD4
+Cell reaches more than the 10-20%, CD3
+CD56
+Cell reaches 16-20%.
Description of drawings
Below in conjunction with accompanying drawing the present invention is further described:
Fig. 1 is that human peripheral obtains CIK cell real scene shooting figure under 40 times of mirrors of 4 days of cultivation after cultural method according to the invention is handled;
Fig. 2 is that human peripheral obtains CIK cell real scene shooting figure under 40 times of mirrors of 7 days of cultivation after cultural method according to the invention is handled;
Fig. 3 is that human peripheral obtains CIK cell real scene shooting figure under 100 times of mirrors of 7 days of cultivation after cultural method according to the invention is handled;
Fig. 4 is that human peripheral obtains CIK cell real scene shooting figure under 40 times of mirrors of 9 days of cultivation after cultural method according to the invention is handled;
Fig. 5 is that human peripheral obtains CIK cell real scene shooting figure under 40 times of mirrors of 10 days of cultivation after cultural method according to the invention is handled;
Fig. 6 is that human peripheral obtains CIK cell real scene shooting figure under 400 times of mirrors of 10 days of cultivation after cultural method according to the invention is handled;
Fig. 7 is the CIK cell that people's hydrothorax obtains after cultural method according to the invention is handled, and feeds back the CD45 that through cells were tested by flow cytometry
+Scatter diagram, that iris out among the figure is CD45
+Cell, the white corpuscle in these cell representative samples comprises lymphocyte, scavenger cell etc.;
Fig. 8 is the CIK cell that people's hydrothorax obtains after cultural method according to the invention is handled, and feeds back the CD3 that through cells were tested by flow cytometry
+Scatter diagram, that iris out among the figure is CD3
+Cell, the T lymphocyte in these cell representative samples;
Fig. 9 is the CIK cell that people's hydrothorax obtains after cultural method according to the invention is handled, and feeds back the CD3 that through cells were tested by flow cytometry
+CD8
+Scatter diagram, among the figure according to CD8 antigen with CD3
+Two portions that cell has been divided into, the top is CD3
+CD8
+Two positive cells, representative be cytotoxic T cell; The below is CD3
+CD8
-Single positive cell;
Figure 10 is the CIK cell that people's hydrothorax obtains after cultural method according to the invention is handled, and feeds back the CD4 that through cells were tested by flow cytometry
+CD8
+Scatter diagram, among the figure according to CD4 and CD8 antigen with CD3
+Cell is divided into four parts, and the upper right side is CD4
+CD8
+Two positive cells; The lower right is CD4
-CD8
+Single positive cell, representative be cytotoxic T cell; The lower left is CD4
-CD8
-The jack to jack adapter cell; The upper left side is CD4
+CD8
-Single positive cell, representative be helper T cell.
Embodiment
Describe in detail in conjunction with Fig. 1 ~ 10 with to the present invention.
The lymphocytic extracorporeal culturing method of T according to the invention is mainly used in the in-vitro directed sorting of T lymphocyte, induces, breaks up, cultivation, amplification etc.; The biological sample of its processing comprises human peripheral, Cord blood, hydrothorax, ascites etc.; Can be applied to clinical cell biological treatment; Comprise kidney, malignant melanoma, white blood disease, mammary cancer, the rectum cancer, cancer of the stomach, lung cancer, the esophageal carcinoma, cervical cancer, ovarian cancer, multiple myeloma, malignant lymphoma malignancy diseases such as (non-T cell lymphomas); Also can be applied to basic medical research, clinical application research and biotechnology research, especially can be applied to the research of immunity system research and tumor-killing effect.Concrete cultural method is following:
Embodiment one:
Existing is to handle sample with the human peripheral, uses in the test kit according to the invention the content sorting to induce the CIK cell, and is applied to clinical biotherapy.The concrete operations step is following:
Step 1. blood treatment
1. the 50ml anticoagulation is injected 50ml centrifuge tube, the centrifugal 10min of 1600 ~ 2000 * g under 4 ℃ of conditions.As can not be at once centrifugal, can be placed on 4 ℃ of preservations, but can not be above 4 hours.
2. with transfer pipet upper plasma is moved in the new 50ml centrifuge tube, marked patient information.56 ℃ of deactivation 30min.
3. saline water dilution hemocyte is used the transfer pipet mixing to 50ml.More than be dilution hemocyte process.
4. will dilute good hemocyte slowly injects on the cell sorting liquid of every pipe 7ml with the ratio of 1:1.Note not breaking the interface.
5. under 20 ℃ of conditions, the centrifugal 20min of 2200 ~ 2500 * g.Cell is divided into four layers from top to bottom in the centrifuge tube at this moment.The first layer: the equal slurry layer of blood plasma or cell tissue; The second layer: ring-type oyster white buffy coat; The 3rd layer: hyaline cell sorting liquid layer; The 4th layer: the red corpuscle layer.With transfer pipet sucking-off the first layer liquid, discard; Draw second layer cell in new 50ml centrifuge tube.
6. in the cell of new collection, add saline water to 50ml, under 4 ℃ of conditions, the centrifugal 4 ~ 5min of 1600 ~ 2000 * g.
7. supernatant discarded.Flick at the bottom of the centrifuge tube pipe, break up deposition.
8. repeating step 6 ~ 7 is twice.It more than is the cell sorting process.
9. get coated cell factor 2 and encapsulate bottle more than 4 hours, with saline water or substratum rinse once and discard, add patient's autologous plasma 2 ~ 5ml.
10. get 25ml substratum re-suspended cell, with behind the abundant mixing of transfer pipet its immigration being encapsulated in the bottle.
11., rinse liquid moved into once more encapsulate in the bottle with 25ml substratum rinse centrifuge tube.
Put into 5%CO 12. will encapsulate bottle
2, cultivate in 37 ℃ of incubators.It more than is cell induction differentiation culture process.
Step 2. add cytokine A
1. blood treatment is second day, from above-mentioned incubator, takes out and encapsulates bottle, observation of cell state.
2. draw fresh culture 2ml dissolved cell factors A with the 5ml syringe, injection encapsulates bottle.
3. will encapsulate bottle and put into 5%CO
2, cultivate in 37 ℃ of incubators.
Step 3. fluid infusion
1. above-mentioned cultivation is in the time of 2 ~ 3 days, and is as shown in Figure 1, and the growth sign appears in cell, in encapsulating bottle, adds fresh culture 100ml.
2. will encapsulate bottle and put into 5%CO
2, cultivate in 37 ℃ of incubators.
Step 4. change bag
1. above-mentioned cultivation is in the time of 1 ~ 2 day, like Fig. 2, shown in Figure 3, when cell be paved with basically encapsulate bottle at the bottom of the time, change bag.
2. blow down the attached cell that encapsulates in the bottle with transfer pipet, draw fresh culture 2ml dissolved cell factor B with the 5ml syringe, adding encapsulates in the bottle, adds patient's autologous plasma 2 ~ 5ml simultaneously.
3. get culture bag, marked patient information.
4. take off culture bag pipe cap, 50ml syringe needle and interior bolt.The culture bag fluid inlet is linked to each other with syringe nozzle.
5. the cell culture fluid that will encapsulate in the bottle is poured in the culture bag; Encapsulate bottle twice with the fresh culture rinse, rinse liquid is poured in the culture bag in the lump.
6. in culture bag, add fresh culture to 1000ml.
7. the clamping fluid inlet is removed syringe, builds pipe cap.
8. culture bag is put into 5%CO
2, cultivate in 37 ℃ of incubators.
Step 5. fluid infusion
1. changeed the bag back 3-4 days, like Fig. 4, Fig. 5, shown in Figure 6, cell begins a large amount of agglomerating growths, and can appropriateness rub culture bag this moment, to break up cell mass, cell individual can better be contacted with the growth factor in the substratum.
2. take off culture bag pipe cap, 50ml syringe needle and interior bolt.The culture bag fluid inlet is linked to each other with syringe nozzle.Add fresh culture 1000ml.
3. clamping liquid inlet.Remove syringe, build pipe cap.
4. culture bag is put into 5%CO
2, cultivate in 37 ℃ of incubators.Accomplish cell amplification, behind the bacterium to be checked, subsequent use.
Step 6. the inspection bacterium
1. after the fluid infusion second day, examine bacterium.
2. taking-up culture bag is drawn culture 5ml with the 5ml syringe by thief hole and is injected two diphase blood culture flasks, and one bottle is sent with the third party and carries out aseptic detection, and one bottle is used for self check.Place 37 ℃ of constant incubators to cultivate two-way Blood culture bottle.
3. culture bag is put into 5%CO
2, cultivate in 37 ℃ of incubators.
4. diphase blood culture flask is cultivated after 3 days if asepsis growth proves that the culture in the culture bag can be used for adoptive therapy.
Step 7. feed back
1. the observation of cell growing state is confirmed the feedback time.
2. prepare 8 of 50ml centrifuge tubes, marked patient information.
3. culture is poured in the 50ml centrifuge tube, the residue culture is put back to incubator and is continued to cultivate.
4. under 4 ℃ of conditions, the centrifugal 4 ~ 5min of 1600 ~ 2000 * g.Supernatant discarded is broken up cell at the bottom of flicking the centrifuge tube pipe.
5. with the saline water re-suspended cell and focus in 2 centrifuge tubes.
6. under 4 ℃ of conditions, the centrifugal 4 ~ 5min of 1600 ~ 2000 * g.Supernatant discarded is broken up cell at the bottom of flicking the centrifuge tube pipe.
7. with the saline water re-suspended cell and focus in 1 centrifuge tube.
8. under 4 ℃ of conditions, the centrifugal 4 ~ 5min of 1600 ~ 2000 * g.Supernatant discarded is broken up cell at the bottom of flicking the centrifuge tube pipe.
9. use the saline water re-suspended cell.
10. under 4 ℃ of conditions, the centrifugal 4 ~ 5min of 1600 ~ 2000 * g.Supernatant discarded is broken up cell at the bottom of flicking the centrifuge tube pipe.
11. use the saline water re-suspended cell.
Inject new 50ml centrifuge tube 12. draw a cell-dispersing agent with the 5ml syringe.Aseptic screen cloth is put on the centrifuge tube.
13. use the pipette, extract cell suspension, inject the centrifuge tube that sterile filters is housed.Through the big cell mass of filter screen elimination.
14. fetch one in defeated bag, marked patient information.Cut off the mouth of pipe, connect the 50ml syringe, pour cell into and add saline water to 150ml.
15. clamp the mouth of pipe with mosquito forceps, heat-sealing machine seals.
In operating process, should note following some:
1. when injecting blood on the cellular segregation liquid, should be softly slow, be sure not to break the interface.
2. culturing bottle should make gas communication by peaceful loose bottleneck after putting into incubator.
Every day the observation of cell upgrowth situation, when finding that cell colony appears in cell in the culture bag, soft culture bag is broken up colony.Prevent to cause the necrocytosis of core because of a large amount of gatherings of cell.
4. operating process requires in the local laminar flow Bechtop of ten thousand grades of Clean Operating Labs, to accomplish, to guarantee the safety asepsis of whole process.
5. culturing bottle horizontal positioned as far as possible.
Fetch defeated preceding cell suspension and carry out cell counting, the result shows that the peripheral blood of 50ml is after present method is handled, but harvested cell number about 5 * 10
9~ 5 * 10
11Individual.Get a certain routine sample and carry out the flow cytometer detection through the cell of present method processing results, the result is shown in Fig. 7 ~ 10.LCA CD45 in the cell mass
+Cell account for 68.61% (see figure 7) of whole feedback cells, wherein T lymphocyte surface antigen CD3
+The ratio of cell reaches 99% (see figure 8), cytotoxic T cell surface antigen CD3
+CD8
+Two positive cells account for 68% (see figure 9), helper T cell surface antigen CD3
+CD4
+Two positive cells account for 32% (see figure 10), this shows, the inventive method is cultivated the T lymphocyte and had very high yield and purity.
Embodiment two:
The present invention also can be used for the lymphocytic external evoked amplification of T in the human cord blood.The concrete operations step is following:
Step 1. blood treatment
1. 80ml anti-freezing Cord blood is injected 4 50ml centrifuge tubes respectively, the centrifugal 10min of 1600 ~ 2000 * g under 4 ℃ of conditions.As can not be at once centrifugal, can be placed on 4 ℃ of preservations, but can not be above 4 hours.
2. discard upper plasma.
3. saline water dilutes 4 pipe hemocytes respectively to 50ml, uses the transfer pipet mixing.More than be dilution hemocyte process.
4. will dilute good hemocyte slowly injects on the cell sorting liquid of every pipe 7ml with the ratio of 1:1.Note not breaking the interface.
5. under 20 ℃ of conditions, the centrifugal 20min of 2200 ~ 2500 * g.Draw second layer cell in new 50ml centrifuge tube.
6. in the cell of new collection, add saline water to 50ml, under 4 ℃ of conditions, the centrifugal 4 ~ 5min of 1600 ~ 2000 * g.
7. supernatant discarded.Flick at the bottom of the centrifuge tube pipe, break up deposition.
8. repeating step 6 ~ 7 is twice.It more than is the cell sorting process.
9. get coated cell factor 2 and encapsulate bottle more than 4 hours, once and discard with saline water or substratum rinse.10. get 25ml substratum re-suspended cell, with behind the abundant mixing of transfer pipet its immigration being encapsulated in the bottle.
11., rinse liquid moved into once more encapsulate in the bottle with 25ml substratum rinse centrifuge tube.
Put into 5%CO 12. will encapsulate bottle
2, cultivate in 37 ℃ of incubators.It more than is cell induction differentiation culture process.
Step 2. add cytokine A
1. blood treatment is second day, from above-mentioned incubator, takes out and encapsulates bottle, observation of cell state.
2. draw fresh culture 2ml dissolved cell factors A with the 5ml syringe, injection encapsulates bottle.
3. will encapsulate bottle and put into 5%CO
2, cultivate in 37 ℃ of incubators.
Step 3. fluid infusion
1. above-mentioned cultivation was added fresh culture 100ml in the time of 2 ~ 3 days in encapsulating bottle.
2. will encapsulate bottle and put into 5%CO
2, cultivate in 37 ℃ of incubators.
Step 4. change bag
1. above-mentioned cultivation is in the time of 1 ~ 2 day, when cell be paved with basically encapsulate bottle at the bottom of the time, change bag.
2. blow down the attached cell that encapsulates in the bottle with transfer pipet, draw fresh culture 2ml dissolved cell factor B with the 5ml syringe, adding encapsulates in the bottle.
3. get culture bag, marked patient information.
Subsequent step is with embodiment one.
Embodiment three:
The present invention also can be used for the lymphocytic external evoked amplification of T in people's hydrothorax or the ascites.The concrete operations step is following:
Step 1. get hydrothorax or ascites 1000ml, under 4 ℃ of conditions, the centrifugal 4 ~ 5min of 1600 ~ 2000 * g.
Step 2. supernatant discarded, flick at the bottom of the centrifuge tube pipe, break up deposition.
Step 3. with saline water rinsing cell 3 times.
Step 4. get and encapsulate bottle, once and discard with saline water or substratum rinse.
Step 5. get 25ml substratum re-suspended cell, with behind the abundant mixing of transfer pipet its immigration being encapsulated in the bottle.
Step 6. with 25ml substratum rinse centrifuge tube, rinse liquid moved into once more encapsulate in the bottle.
Step 7. culturing bottle is put into 5%CO
2, cultivate in 37 ℃ of incubators.
Subsequent step is except that no longer adding patient's autologous plasma, and other steps are with embodiment one.
For make cultural method according to the invention can standard, carry out under the standardized equipment state, its agents useful for same and test equipment are introduced:
Comprising: be used for the aseptic centrifuge tube of carrying liquid, 50 * 50ml/; Be used to inhale the aseptic transfer pipet of tapping body, 9 * 10ml/ props up; The cell sorting liquid that is used for sorting cells, 7 * 7ml/ pipe; The bottle that encapsulates that is used for the inducing culture cell, 1 bottle; Being used to cell provides the serum free medium of growth desired nutritional, 2 * 1L/ bottle; Be used to inhale asepsis injector 7 * 5ml/ of tapping body; Be used for asepsis injector, 7 * 50ml/ as funnel switching liquid; Be used to stimulate the cytokine A of T lymphocytic cell division, i.e. interleukin II, 200,000 units/; Be used for aseptic cell culture bags, 1 * 2L/ as the culturing cell container; Be used to stimulate the cytokine B of T lymphocytic cell division, i.e. interleukin II, 2,000,000 units/; Be used to examine the two-way Blood culture bottle of bacterium, 2; Be used to prevent the cell-dispersing agent of cell aggregation, 5 * 2ml/ props up; The sterile filtration net that is used for filtration cell group, 5; The aseptic feedback bag that is used for the splendid attire cell suspension, 5 * 250ml/; Be used to mark the label of feedback information, 5.
The preparation of mentioned reagent and test equipment and the source as follows:
1, encapsulates the preparation of bottle: get CD3 monoclonal antibody, gamma-interferon, interleukin 1 α and join in the 40ml phosphate buffered saline buffer; The optimum concn ratio of CD3 monoclonal antibody, gamma-interferon, three kinds of factors of interleukin 1 α is 10:1:1; Process the mixing solutions that total concn is 1200ng/ml, fully mixing; This mixing solutions is joined in the 600ml culturing bottle, and 4 ℃ left standstill 24 hours at least, processed initial package by bottle; With initial package by bottle under-40 ℃ of conditions freezing 24 hours; Start the compressor of freeze drier, make the temperature in the machine chamber reduce to-40 ℃, will encapsulate a bottle bottleneck and unscrew, put into kiln; The open vacuum pump reaches pressure<15Pa, vacuum-freeze-dry 20 hours; Open CO
2Intake valve cuts out vacuum pump; Treat that pressure closes CO behind 110K
2Intake valve takes out and encapsulates bottle, tightens lid, seals mouth, 4 ℃ of preservations; In the quality guaranteed period of regulation, use.
2, the preparation of cell sorting liquid
Get 9% Ficoll solution 720ml and 33.4% urografic acid methylglucamine salt 30ml thorough mixing, in this mixed solution, add the gelatin of 7.5g again, fully mixing.The specific density of adjusting solution with the Ficoll of the urografic acid methylglucamine salt of high density or dilution is to 1.077g/ml.In 121 ℃ of high pressure steam sterilization 30min, room temperature preservation is subsequent use.
3, centrifuge tube and transfer pipet are all bought in Corning Incorporated.Syringe is all bought the China doctor material ltd in north, Shenyang.
4, phosphate buffered saline buffer is bought Bioisystech Co., Ltd of China fir Golden Bridge in Beijing (2000ml/ bag); The CD3 monoclonal antibody is bought in Japanese transgenic company (5mg/5ml/ props up); Interleukin 1 α buys in U.S. GIBCO company (100 μ g/ prop up); Interleukin II is bought in Beijing Shuanglu Pharmaceutical Co., Ltd. (200,000 units/prop up, 2,000,000 units/prop up); Gamma-interferon is bought in Shandong Quan Gang pharmaceutcal corporation, Ltd (1,000,000 units/prop up); Cell-dispersing agent is a human serum albumin, buys in the gloomy biological products of German Ztel shellfish ltds (10g/50ml/ bottle); Serum free medium is bought in U.S. GIBCO company (1000ml/ bottle).
Claims (3)
1. one kind is used for the lymphocytic extracorporeal culturing method of T, comprises the T lymphocyte is carried out directed sorting, induces, breaks up, cultivates, increases, and it is characterized in that: the concrete operations step is following:
The first step, the T lymphocyte is carried out directed sorting:
The hemocyte that dilution is good slowly injects on the cell sorting liquid with the ratio of 1:1, and centrifugal treating; With the first layer liquid after the transfer pipet sucking-off sorting is the equal slurry layer of blood plasma or cell tissue, discards, and drawing second layer cell is that ring-type oyster white lymphocyte is in new centrifuge tube; In the cell of new collection, add saline water, abandon supernatant after the centrifugal treating; Repeat to wash twice; Wherein the compound method of cell sorting liquid is: get 9% Ficoll solution 720ml and 33.4% urografic acid methylglucamine salt 30ml thorough mixing, in this mixed solution, add the gelatin of 7.5g again, fully mixing; The specific density of adjusting solution with the Ficoll of the urografic acid methylglucamine salt of high density or dilution is to 1.077g/ml; Put into temperature at last and be 121 ℃ high pressure steam sterilization 30min, room temperature preservation is subsequent use;
Second goes on foot, the T lymphocyte is induced, breaks up, cultivated:
Get and encapsulate one in bottle, the said bottle that encapsulates is the Tissue Culture Flask that has encapsulated CD3 monoclonal antibody, gamma-interferon, interleukin 1 α three kinds of factors in advance, with saline water or substratum rinse once and discard, adds patient's autologous plasma 2 ~ 5ml; Get the substratum re-suspended cell, behind the mixing its immigration is encapsulated in the bottle; Then use substratum rinse centrifuge tube, rinse liquid is moved into once more encapsulate in the bottle; To encapsulate bottle and put into 5% CO
2, cultivate 24h in 37 ℃ the incubator;
Encapsulate the preparation of bottle: get CD3 monoclonal antibody, gamma-interferon, interleukin 1 α and join in the 40ml phosphate buffered saline buffer; The concentration ratio of CD3 monoclonal antibody, gamma-interferon, three kinds of factors of interleukin 1 α is 10:1:1; Process the mixing solutions that total concn is 1200ng/ml, fully mixing; This mixing solutions is joined in the 600ml culturing bottle, and 4 ℃ left standstill 24 hours at least, processed initial package by bottle; With initial package by bottle under-40 ℃ of conditions freezing 24 hours; Start the compressor of freeze drier, make the temperature in the machine chamber reduce to-40 ℃, will encapsulate a bottle bottleneck and unscrew, put into kiln; The open vacuum pump reaches pressure<15Pa, vacuum-freeze-dry 20 hours; Open CO
2Intake valve cuts out vacuum pump; Treat that pressure closes CO behind 110K
2Intake valve takes out and encapsulates bottle, tightens lid, seals mouth, 4 ℃ of preservations; In the quality guaranteed period of regulation, use;
The 3rd goes on foot, the T lymphocyte is increased:
Draw fresh culture dissolved cell factors A, i.e. the interleukin II of 200,000 units injects and encapsulates bottle, and the concentration of cytokine A this moment in culturing bottle is 0.4 ten thousand units/ml; In 5%CO
2, cultivate 2 ~ 3 days in 37 ℃ of incubators after, in encapsulating bottle, add fresh culture 100ml; Again in 5% CO
2, continue to cultivate in 37 ℃ of incubators 1 ~ 2 day, when cell be paved with basically encapsulate bottle at the bottom of the time, change bag;
Blow down the attached cell that encapsulates in the bottle with transfer pipet; Draw fresh culture dissolved cell factor B with syringe, i.e. the interleukin II of 2,000,000 units adds and encapsulates in the bottle; Add patient's autologous plasma 2 ~ 5ml simultaneously, the cell culture fluid in the culturing bottle is poured in the culture bag; Encapsulate bottle twice with the fresh culture rinse, rinse liquid is poured in the culture bag in the lump, supplemented medium is to 1000ml, and the concentration of cytokine B this moment in culturing bottle is 0.2 ten thousand units/ml; Culture bag is put into 5%CO
2, cultivate 3 ~ 4 days in 37 ℃ of incubators after, add fresh culture 1000ml, continue to put into 5%CO
2, cultivate in 37 ℃ of incubators; After the fluid infusion second day, examine bacterium, prove whether the culture in the culture bag can be used for adoptive therapy or medical research.
2. the lymphocytic extracorporeal culturing method of T that is used for according to claim 1 is characterized in that: also comprise the inspection bacterium, feed back step:
During said inspection bacterium, take out culture bag, inject two diphase blood culture flasks with syringe respectively by thief hole absorption culture, one bottle is sent with the third party and carries out aseptic detection, and one bottle is used for self check; Place 37 ℃ of constant incubators to cultivate two-way Blood culture bottle; Diphase blood culture flask is cultivated after 3 days if asepsis growth proves that the culture in the culture bag can be used for adoptive therapy or medical research;
During feedback, prepare 8 of centrifuge tubes, culture is poured in the centrifuge tube, the residue culture is put back to incubator and is continued to cultivate; Under 4 ℃ of conditions, the centrifugal treating supernatant discarded is with the saline water re-suspended cell and focus in 2 centrifuge tubes; Under 4 ℃ of conditions, the centrifugal treating supernatant discarded; With the saline water re-suspended cell and focus in 1 centrifuge tube; Under 4 ℃ of conditions, the centrifugal treating supernatant discarded is used the saline water re-suspended cell; Under 4 ℃ of conditions, the centrifugal treating supernatant discarded is used the saline water re-suspended cell; Draw a cell-dispersing agent with syringe and inject new centrifuge tube, this cell-dispersing agent is 10% human serum albumin, then aseptic screen cloth is put on this centrifuge tube; Draw cell suspension, inject the centrifuge tube that sterile filters is housed; Through the big cell mass of filter screen elimination; Fetch one in defeated bag, pour cell into and add saline water subsequent use.
3. the lymphocytic extracorporeal culturing method of T that is used for according to claim 1 and 2 is characterized in that: be used for the mononuclearcell of handler's peripheral blood, Cord blood, hydrothorax, ascites in-vitro directed sorting, induce, break up, cultivate, increase.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201110449100 CN102433303B (en) | 2011-12-29 | 2011-12-29 | In-vitro culture method for T lymphocytes |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
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Cited By (13)
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| CN102988415A (en) * | 2012-08-15 | 2013-03-27 | 王沁怡 | Natural killer cells (NK) prepared by industrializing human allogeneic nucleated cells and injection |
| CN103364545A (en) * | 2013-05-21 | 2013-10-23 | 佛山迪安医学检验所有限公司 | Method for detecting tuberculosis effector T lymphocytes by using enzyme-linked immunospot assay |
| CN103706340A (en) * | 2013-12-20 | 2014-04-09 | 中国科学院苏州生物医学工程技术研究所 | Waterborne adhesive chromatography media and method of using waterborne adhesive chromatography media for detection |
| CN104109653A (en) * | 2014-06-12 | 2014-10-22 | 浙江瑞顺生物技术有限公司 | Method of large-scale amplification of human peripheral blood DNT cell by utilization of animal-serum-free culture system |
| WO2015103793A1 (en) * | 2014-01-13 | 2015-07-16 | 深圳市汉科生物工程有限公司 | Method for preparing and using cell ghost with active factor as synergist of lymphocyte in vitro culture |
| CN106434551A (en) * | 2016-08-31 | 2017-02-22 | 青岛大学 | Culture method for fish lymphocytes |
| CN106754702A (en) * | 2016-12-26 | 2017-05-31 | 华东理工大学 | The method of Cytokine-induced killer cells dynamic suspension culture |
| CN106854644A (en) * | 2017-03-08 | 2017-06-16 | 广州可伴咨询中心（普通合伙） | A kind of in vitro culture kit of CMV CTL cells |
| CN107002018A (en) * | 2014-12-25 | 2017-08-01 | 东洋制罐集团控股株式会社 | The manufacture method of culture vessel and culture vessel |
| CN107090433A (en) * | 2017-04-28 | 2017-08-25 | 中卫华医（北京）医院管理有限公司 | T cell cultural method for oncotherapy |
| CN107164323A (en) * | 2017-07-13 | 2017-09-15 | 山东省齐鲁细胞治疗工程技术有限公司 | A kind of method for the lymphocyte subgroup for obtaining tool High Fragmentation tumour cell ability |
| CN109593713A (en) * | 2018-12-29 | 2019-04-09 | 广州和能生物科技有限公司 | A kind of cultural method of autologous peripheral blood lymphocyte |
| CN112662625A (en) * | 2021-01-18 | 2021-04-16 | 杭州原生生物科技有限公司 | T cell culture medium and method for expanding and culturing T cells by using same |
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| CN102988415A (en) * | 2012-08-15 | 2013-03-27 | 王沁怡 | Natural killer cells (NK) prepared by industrializing human allogeneic nucleated cells and injection |
| CN103364545A (en) * | 2013-05-21 | 2013-10-23 | 佛山迪安医学检验所有限公司 | Method for detecting tuberculosis effector T lymphocytes by using enzyme-linked immunospot assay |
| CN103706340A (en) * | 2013-12-20 | 2014-04-09 | 中国科学院苏州生物医学工程技术研究所 | Waterborne adhesive chromatography media and method of using waterborne adhesive chromatography media for detection |
| WO2015103793A1 (en) * | 2014-01-13 | 2015-07-16 | 深圳市汉科生物工程有限公司 | Method for preparing and using cell ghost with active factor as synergist of lymphocyte in vitro culture |
| CN104109653A (en) * | 2014-06-12 | 2014-10-22 | 浙江瑞顺生物技术有限公司 | Method of large-scale amplification of human peripheral blood DNT cell by utilization of animal-serum-free culture system |
| CN107002018A (en) * | 2014-12-25 | 2017-08-01 | 东洋制罐集团控股株式会社 | The manufacture method of culture vessel and culture vessel |
| CN107002018B (en) * | 2014-12-25 | 2019-12-03 | 东洋制罐集团控股株式会社 | The manufacturing method of culture vessel and culture vessel |
| US10745659B2 (en) | 2014-12-25 | 2020-08-18 | Toyo Seikan Group Holdings, Ltd. | Culture container and method for manufacturing culture container |
| CN106434551A (en) * | 2016-08-31 | 2017-02-22 | 青岛大学 | Culture method for fish lymphocytes |
| CN106754702A (en) * | 2016-12-26 | 2017-05-31 | 华东理工大学 | The method of Cytokine-induced killer cells dynamic suspension culture |
| CN106754702B (en) * | 2016-12-26 | 2020-10-30 | 华东理工大学 | Dynamic suspension culture method for cell factor induced killer cells |
| CN106854644A (en) * | 2017-03-08 | 2017-06-16 | 广州可伴咨询中心（普通合伙） | A kind of in vitro culture kit of CMV CTL cells |
| CN107090433A (en) * | 2017-04-28 | 2017-08-25 | 中卫华医（北京）医院管理有限公司 | T cell cultural method for oncotherapy |
| CN107164323A (en) * | 2017-07-13 | 2017-09-15 | 山东省齐鲁细胞治疗工程技术有限公司 | A kind of method for the lymphocyte subgroup for obtaining tool High Fragmentation tumour cell ability |
| CN109593713A (en) * | 2018-12-29 | 2019-04-09 | 广州和能生物科技有限公司 | A kind of cultural method of autologous peripheral blood lymphocyte |
| CN112662625A (en) * | 2021-01-18 | 2021-04-16 | 杭州原生生物科技有限公司 | T cell culture medium and method for expanding and culturing T cells by using same |
| CN112662625B (en) * | 2021-01-18 | 2023-03-17 | 曹彤 | T cell culture medium and method for expanding and culturing T cells by using same |
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