CN102839153A - Amplifying, freezing and storing and recovering method of activated lymphocyte with CD3+CD8+as major - Google Patents
Amplifying, freezing and storing and recovering method of activated lymphocyte with CD3+CD8+as major Download PDFInfo
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Abstract
The invention discloses culturing, freezing and recovering methods of an activated lymphocyte with CD3+CD8+as major, which can solve problems that a patient needs carrying out blood sampling for many times caused by continuously utilizing the activated lymphocyte with CD3+CD8+as major. The method comprises the following steps of: (1) contacting the extracted lymphocyte of the peripheral blood with IL-2, IL-15, an anti-CD3 antibody and an anti-CD28 antibody, so as to amplify the activated lymphocyte with CD3+CD8+as major; (2) freezing and storing the activated lymphocyte; and (3) recovering the activated lymphocyte. The activated lymphocyte cultured via the method disclosed by the invention has clear components, and comprises few CD4+CD25+Treg cells and more CD8+T lymphocyte; feedback time and frequency of the activated lymphocyte can be adjusted according to other treatments for a patient, such as a radiotherapy or a chemotherapy, so that diseases, such as tumor, infectious diseases and immunodeficiency can be treated well.
Description
Technical field
The present invention relates to the amplification of activated lymphocyte, frozen and method for resuscitation, belong to biological technical field.
Background technology
The immunity system of body has the ability of identification and killing tumor cell, has obtained the confirmation of a large amount of datas.In recent years, the treatment of tumour-specific cellular immunization has shown very infusive curative effect in clinical practice.2002; People such as RosenbergSA have reported the tumour-specific tumor infiltrating lymphocyte that the utilizes external a large amount of amplifications feedback method of adopting at the Science magazine; They provide the tumor infiltrating lymphocyte (TILs) that patient self dislikes black cell-specific lethal effect at in-vitro screening, and a large amount of amplifications back feeds back to be given from the body patient.In this method; Combined action through external IL-2 and anti-cd 3 antibodies; Quantity is fed back in the patient body who removes the lymphocyte chemotherapy up to the TILs of kinds of tumors antigen-specific identification 1011, the tumour cell high special, and these cells are main with the T cell of CD3+CD8+CD56-.In the 13 routine patients that receive treatment, 6 routine patients are alleviated (CR, tumour completely dissolve) fully, and 4 routine patients are partly alleviated (PR, tumour disappears more than 50%, no New Development tumour).Tangible tumour all takes place and dwindles and/or disappear in this 10 routine patient.In the long-term clinical practice of Dr.Rosenberg, they find, the tumor infiltrating lymphocyte that is used to come from tumour carries out the treatment of malignant melanoma in late period, its efficient reaching more than 50%.The study group of Greenberg and Yee C has in the treatment that the CD8+T cell clone of tumour-specific kill capability carries out in utilization and finds; Repeatedly use this type cell, unite and use chemotherapeutic or do not use chemotherapeutic to carry out malignant melanoma patient's in late period treatment, can make part patient tumour long-term stability, obviously prolong patient's lifetime.
Yet, in these specific cellular immunity therapeutic processes, exist the technical barrier that specific cell is cultivated amplification.At first, for tumor infiltrating lymphocyte, (1) needs to solve the external former foster technical barrier of being commissioned to train of tumor tissues, to identify the tumor infiltrating lymphocyte that is obtained autologous tumor is had kill capability; (2) tumour that is taken place for the key organ of life need identify that the specificity infiltrating T lymphocyte discerns whether for organizing dependency antigen, to avoid the generation of autoimmune disease.What is called is organized dependency antigen, refers to high expression level in tumour, low tumour antigen of expressing in normal corresponding histoorgan.When the immunotherapy melanoma; Leukodermic spinoff may take place; And to like life-critical organs such as liver, brains; Because the issuable autoimmune disease of specific killing cell of this type antigen induction, the application that makes tumor infiltrating lymphocyte carry out specific active immunotherapy is restricted.Secondly, for using antigen and BMDC (DC) to carry out the lymphocyte clone of external specific for tumour antigen, (1) need obtain a large amount of PMNCs to obtain DC and feeder cell through leukophresis; (2) each purpose antigen must carry out exo-antigen stimulates, and obtaining tumour-specific T cell clone, technical sophistication, length consuming time, cell consumption are big, have limited the acquisition of multiple T cells with antigenic specificity.In addition, activated lymphocytes needs repeatedly infusion just can reach better therapeutic effect, and most medical institutions utilize blood cell separator to gather mononuclearcell, and this acquisition method costs an arm and a leg, and repeatedly gathers the misery of bringing psychology and health to the patient.If the mononuclearcell of once gathering can be frozen, when patient treatment needed, recovering and inducing was activated lymphocytes, just for the patient has practiced thrift expense, had alleviated misery.
The lymphocytic method of traditional amplification in vitro; Be as the lymphocyte activator agent with CD3 antibody; And adopt heavy dose of IL-2, though obtain a large amount of activated T cells so in a short time, be easy to cause the necrocytosis of activation inductive; Finally be difficult to obtain the cytotoxic T lymphocyte of sufficient amount, and contain a large amount of CD4+CD25+Treg cell (seeing embodiment 14).
Low dose of anti-CD3 monoclonal antibody can activation immobilized T cell; Induce it to produce the effect of killing tumor cells; This lethal effect had both comprised the direct killing to tumour cell; Generation kills and wounds indirectly to tumour cell to comprise the relevant cytokine of secretion again, also can be through inducing tumor cell generation apoptosis to its generation effect.Add the apoptosis pathway that can activate tumour cell behind the anti-CD3 monoclonal antibody on the other hand, cause the quantity of apoptotic cell to increase, and this apoptotic effect mainly produces through the Fas approach.
But do not produce killing and wounding and inducing action of straight line increase heavy dose of the adding when CD3 monoclonal antibody is cultivated; Occur the phenomenon that apoptosis quantity descends on the contrary, even occur the effect of T cell inhibiting, the quantity of necrosis and apoptotic cell all descends; This possibly be because the limited amount of TXi Baoshouti (TCR); Under excessive anti-CD3 monoclonal antibody effect, not only can not activated T cell, on the contrary can the factor receptor surface be closed and can't activation.
Summary of the invention
Low in order to have solved existing activated lymphocyte preparation (mainly being meant the CIK cell) purity, the shortcoming that proliferative ability is low.And the solution patient needs the repeatedly problem of blood sampling because repeatedly use cell.The present invention joins and uses co-cultivation such as anti-CD3 monoclonal antibody, anti-CD28 monoclonal antibody, IL-2, IL-15, removes the CD4+CD25+Treg cell that in culturing process, produces through immunomagnetic bead technique in addition, makes that cultured cells kind purity is higher.The method of the frozen and recovery of the present invention can solve the patient and be to use continuously activatory CD3+CD8+ to need the problem of repeatedly taking a blood sample for main lymphocyte.
We are in the clinical observation of using the activated lymphocytes treatment that the present invention cultivated, in several patients, observe tumour long-term stability, combined with chemotherapy tumour phenomenon such as obviously dwindle.This phenomenon, to treat observed effect similar with tumour-specific cellular immunization.Simultaneously, CD8+T lymphocyte per-cent is high more in the activated lymphocytes colony, and result of treatment is good more.Therefore, we suspect in these " non-specific immunity cells ", possibly have tumour-specific CD8+ cytotoxic T lymphocyte, have brought into play its antitumous effect.The T lymphocyte comprises many different antigenic specific cells that are directed against in the human body, and these cells are through after cultivating, and they can have identification and fragmentation effect widely to different tumour antigens or virus antigen in theory.
The CD28 molecule is claimed TP44 again, is a kind of 202 amino acid whose I type transmembrane glycoproteins that comprise.Can provide the T cell activation required costimulatory signal, promote T cell activation propagation.Utilize the part of CD28 monoclonal antibody, activate the TCR signal pathway, can strengthen the proliferation activity of CD3AK cell effectively, and delay the apoptosis of CD3AK cell with the CD3 monoclonal antibody as CD28.CD28 stimulates altogether makes the peripheral blood lymphocyte activation significantly promote cytokine to produce; The collaborative CD3 monoclonal antibody of CD28 monoclonal antibody can obviously improve IFN γ in the peripheral blood lymphocyte supernatant, IL-2, IL-4 level, and the CD28 monoclonal antibody can suppress the mRNA degraded of IFN γ, IL-2, tumour necrosis factor (TNF) etc.
IL-2 is main important ESC by activated T emiocytosis.It is that purifying comes out from the lymphocytic culture supernatant of mitogenstimulated at first, is a kind of important cytokine of regulating the T lymphocyte responses.After the antigen activation, can promote the increment of T cell, but in the later stage, along with the rising gradually with the concentration of IL-2 that increases of activating T cell quantity, the T cell just get into soon the Fas/FasL mediation activation inductive necrocytosis (AICD)., therefore simple use IL-2 can not be the amplification of T cell in the external long period
IL-15 also is a kind of important ESC, and it mainly by monocyte and BMDC expression-secretion, can promote the NK cell, the propagation and the differentiation of T cell and B cell.IL-15 is the propagation by the T clone that can stimulate IL-2 to rely on, and this multiplication effect can not be blocked by anti-IL-2 antibody.Therefore IL-15 has similar biological effect with IL-2, and promptly both can both promote the differentiation increment of T cell, yet; Different with IL-2 is after the IL-15 receptors bind, can stablize the rc chain; And raise anti-apoptotic genes expression bcl-2, thereby performance there are anti-AICD and the effect that prolongs the cell survival time.
The CD4+CD25+Treg cell is that nature produces and ripe in unique cell mass of thymus gland, accounts for CD4+T cell 5% one 10%.It can initiatively suppress the activation of autoreactive T cell through cells contacting dependent mechanism or SC factor dependent mechanism, keeps the autoimmunization tolerance, prevents the generation of autoimmune disease.
The mainly performance negative regulation effect in body immune system of CD4+CD25+Treg cell; Can suppress inappropriate immunoreation; Can limit scope, degree and the action time of immunne response again, the propagation of pairing effect cell, immunocompetent performance play restraining effect.Inhibition shows that the Treg cell can suppress many activity of immune cells, propagation and function, like CD4+Th cell, CD8+ cytotoxic T cell, the restricted NKT cell of CDld, Monocytes, initial/memory B cell and BMDC (DCs) etc.Token stimulus through the TcR mediation can be the polyclonal stimulation that anti-CD3 monoclonal antibody produces; It also can be the antigen-specific sexual stimulus; Be non-antigen-specific in case be activated this effect, and this inhibitive ability of immunity do not have the restricted propagation that can suppress homotype of the same race or allotype T cell of MHC.
Tumour patient immunologic hypofunction in addition; Total lymphocyte reduces in the peripheral blood; Each lymphocyte subgroup ratio is disorderly, and has numerous immunosuppressive factors in the serum, and these factors are unfavorable for the amplification of peripheral blood activated lymphocyte; Particularly after radiotherapy, chemotherapy, very difficult assurance provides the cell of sufficient amount.If can utilize the frozen technology of low temperature to preserve PMNC, to recover when needed and induce to the CIK cell of cytotoxic activity is arranged, this problem just has been readily solved.
It is the amplification method of master's activated lymphocyte with CD3+CD8+ that first aspect of the present invention relates to a kind of
(a) isolated mononuclearcell in the peripheral blood blood sample is cultivated in the substratum that contains lymphocyte activator agent and cytokine IL-2; Said lymphocyte activator agent is anti-cd 3 antibodies and anti-CD28 antibody, and the final concentration of anti-cd 3 antibodies is 0.1-100ng/ml; The final concentration of anti-CD28 antibody is 0.1-50ng/ml; Said IL-2 final concentration is 100U/ml to 5000U/ml;
(b) remove the CD4+CD25+Treg cell with cultivating the lymphocyte that obtains in (a) step with mini MACS magnetic bead sorting;
(c) cultivate in the substratum that contains cytokine IL-2 and IL-15 cultivating the lymphocyte that obtains in (b) step; Said IL-2 final concentration is 50-500U/ml; The final concentration of IL-15 is 10-500ng/ml;
(d) collect the lymphocyte that obtains in (c) step and be used for feedback or frozen.
Below the present invention is specified
In the method for the invention, the final concentration of wherein said step (a) anti-cd 3 antibodies is preferably 1-75ng/ml, and the final concentration of anti-CD28 antibody is preferably 2-30ng/ml.
In the method for the present invention, the lymphocyte activator agent of using in the step (a) can also contain other activator, for example PHA and/or IFN-r.The final concentration of PHA in the preferred step (a) can be 1-100ng/ml for example, and the final concentration of IFN-r can be 100-2000U/ml for example.
In the method for the present invention,, cultivate so in substratum, add the IL-2 of high density for obtaining to have in a large number the lymphocyte of multiplication capacity at short notice.The final concentration of preferred IL-2 is 2000-4000U/ml in the step (a).
In the method for the present invention, make that for removing the CD4+CD25+Treg cell that in culturing process, transforms propagation cultured cells CD8+T cell purity is higher, in step (b), remove the CD4+CD25+Treg cell with the indirect immunomagnetic beads key player on a team of mini MACS.
In the method for the present invention, the final concentration of preferred IL-2 can be for example 200-500U/ml in the step (c), and the final concentration of preferred IL-15 can be for example 200-400ng/ml.
In the method for the present invention, step (c) can also contain other similar functions cytokines for example IL-7 and/or IL-12 and/or IL-21.The final concentration of preferred IL-7 is that the final concentration of 10-200ng/ml and/or IL-12 is that the final concentration of 5-100ng/ml and/or IL-21 is 1-200ng/ml.The final concentration of preferred IL-7 is that the final concentration of 50-100ng/ml and/or IL-12 is that the final concentration of 20-50ng/ml and/or IL-21 is 10-100ng/ml.
In the method for the invention, it is 3-4 days that wherein said step (a) begins to finish required time to step (b), and cell cultures is 6-8 days as being used for clinical feedback required time in the step (c), as to be used for the cell cryopreservation required time be 1-2 days.
Cultured cells of the present invention is mainly the CD3+CD8+T cell, and the CD3+CD8+T cell content can be greater than 90%.
Second aspect, the invention provides a kind of activatory is main lymphocytic frozen method with CD3+CD8+:
(e) getting what be grown in logarithmic phase is that main activated lymphocyte is collected with CD3+CD8+, and collection method is: the speed with 300-2000r/min is centrifugal, 10 minutes, obtains sedimentation cell;
It is 0.1-5 * 10 that the cell adding frozen storing liquid that (g) will in (e) step, obtain makes cell concn
7/ ml; Cell suspension is changed in the frozen pipe;
(h) cell that obtains in (g) is put in the programmed cooling box at subzero 20 ℃ placed 1-5 hour, change in-80 ℃ of refrigerators again and placed 16-48 hour, it is frozen to change the liquid nitrogen container midium or long term over to;
In the method for the invention; Logarithmic phase described in the wherein said step (e) is meant when TLC is 2-100 a times of lymphoblast sum; Preferably; Be that the CD3+CD8+ TLC is original separation obtains in the step (a) the lymphocytic 2-100 of CD3+CD8+ times, preferred, be that the CD3+CD8+ TLC is original separation obtains in the step (a) the lymphocytic 5-20 of CD3+CD8+ a times;
In the method for the invention, in the wherein said step (e) be that main lymphocytic collection is following: get cell and be put in the centrifuge tube, with the centrifugal speed of 400-600r/min centrifugal 10 minutes, remove supernatant with CD3+CD8+;
In the method for the invention; Used frozen storing liquid is that foetal calf serum and DMSO prepare according to the volume ratio of 9:1 in the wherein said step (g); Also can be substratum and the DMSO volume ratio preparation according to 9:1, the mixing liquid and the DMSO that also can be foetal calf serum and substratum prepare according to the volume ratio of 9:1.
In the method for the invention, wherein said step (g) was preferably placed 2-3 hour at subzero 20 ℃, changeed in-80 ℃ of refrigerators again and placed 20-24 hour.
The third aspect, the invention provides a kind of is main lymphocytic recovery of frozen autoactivation and recovery back cultural method with CD3+CD8+:
(i) take out frozen cell in (h) step, melt 37 ℃ of water-bath middling speeds;
(j) cell that obtains in (i) is changed over to rapidly in 37 ℃ of preheating fresh cultures, change in the Tissue Culture Plate then and cultivate;
(k) draw the supernatant of the substratum of institute's culturing cell in (j), and add the fresh culture that contains cytokine IL-2 again; The final concentration of said IL-2 is 1000-5000U/ml;
After treating that (l) cell covers with the hole in (k), be transferred in the culturing bottle that contains the lymphocyte activator agent, and add the fresh culture that contains cytokine IL-15; The final concentration 200-500ng/ml of said IL-15; Contained lymphocyte activator agent is anti-cd 3 antibodies and anti-CD28 antibody, and the final concentration of anti-cd 3 antibodies is 1-100ng/ml, and the final concentration of anti-CD28 antibody is 2-200ng/ml;
Treat (m) in (l) that cell quantity increases 2-10 times the time, adds the fresh culture that contains cytokine IL-2 and IL-15 and carries out enlarged culturing; The final concentration of the factor-containing IL-2 of institute is 50-500U/ml, and the final concentration of IL-15 is 50-500ng/ml;
(n) collect amplifying activated lymphocyte in (m).
In the method for the invention, the final concentration of preferred IL-2 can be for example 1000-2000U/ml in the wherein said step (k).
In the method for the invention, the final concentration of preferred IL-15 can be for example 200-500ng/ml in the wherein said step (l).
In the method for the invention, can for the final concentration of anti-cd 3 antibodies 50-100ng/ml preferably in the wherein said step (l), the concentration of anti-CD28 antibody is 20-80ng/ml.
In the method for the invention, the multiple of the preferred cell quantity amplification of wherein said step (m) is 2-5 times.
In the method for the invention, the final concentration of preferred IL-2 is 50-200U/ml in the wherein said step (m), and the final concentration of IL-15 is 20-100ng/ml.
In the method for the invention, the one the second, substratum described in the third aspect is RPMI-1640 substratum or the similar substratum of other effects, the similar substratum of wherein said other effects is DMEM substratum and/or AIM-V substratum.And, the first, the second, in the method for the third aspect, wherein said substratum can be with a kind of substratum, also can be in RPMI-1640, DMEM, the AIM-V substratum two or more.In addition; In lymphocytic culturing process, can also in substratum, add serum and blood plasma; The source of serum or blood plasma can also can be (the deriving from the lymphocyte difference) of allosome from body (it is identical with lymphocyte to originate) but consider that from security standpoint preferential, selection is from the serum or the blood plasma in body source; More preferably, the selection finished product commercialization serum free medium AIM-V for example that can add serum or blood plasma.
The invention has the beneficial effects as follows:
Existing activated lymphocyte preparation (mainly being meant the CIK cell) purity is low; Proliferative ability is low; A large amount of CD4+CD25+Treg cells are contained in the inside; The CD4+CD25+Treg cell can be secreted a large amount of immunosuppressive factors, can not bring into play too big effect after receiving the influence of immunosuppressive factor after making cell feed back in the body.And because most patient need do chemotherapy, make patient body's endolymph cytoactive reduce, this has just caused existing activated lymphocyte preparation (mainly being meant the CIK cell) to be difficult to blood sampling operation continuously, has influenced the effect that patient uses activated lymphocyte.
The activated lymphocyte composition that the present invention cultivated is clear; The CD4+CD25+Treg cell content is few; And contain a large amount of CD8+T lymphocytes; We are in the clinical observation of using the activated lymphocytes treatment that the present invention cultivated, and CD8+T lymphocyte per-cent is high more in the activated lymphocytes colony, and result of treatment is good more.
The present invention frozen and the recovery method; Can solve the patient and need the repeatedly problem of blood sampling for main lymphocyte for using activatory CD3+CD8+ continuously; Thereby can adjust the feedback time and the number of times of activated lymphocyte according to patient's other treatment such as radiotherapy chemotherapy; With better treatment tumour, diseases such as infection and immunodeficiency symptoms.
Description of drawings
Fig. 1 is not frozen lymphocyte form;
Fig. 2 is the cellular form of frozen back recovery;
Fig. 3 is not frozen lymphocytic cell phenotype;
Fig. 4 is the lymphocytic cell phenotype that frozen recovery is later cultivated.
Embodiment
1. lymphocytic activation
Get patient's peripheral blood 50ml, divide in the centrifuge tube of packing into, with the centrifugal 10min of 1600r/min; Remove upper plasma, add 30ml saline water, mixing; The every 30ml of hemocyte that dilution is good carefully joins in the upper strata of 15mlFicoll centrifugal then 2000r/min, 20min.Behind centrifugal the finishing, leukocytic cream in the middle of getting, with saline water repetitive scrubbing 3 times, in the AIM-V substratum of 37 ℃ of preheatings of adding, making cell concn is 2 * 10
6/ ml adds anti-cd 3 antibodies, and anti-CD28 antibody and IL-2 make that the concentration of anti-CD3 is 60ng/ml, and the concentration of anti-CD28 antibody is 15ng/ml, and the concentration of IL-2 is 4000U/ml.Then culturing bottle is put into CO2gas incubator, 37 ℃, CO
2Concentration is 5%.From the beginning in second day of cultivating, carry out the observation of cell upgrowth situation with microscope.
2.CD4+CD25+Treg the removal of cell
About cell cultures the 3rd day, the observation of cell upgrowth situation is if the cell well-grown then carries out this step operation.Cell suspension is adjusted to suitable cell concn, add PE-labeled antihumanCD25,4 ℃ are taken out after hatching 30min; With the special-purpose PBS centrifuge washing of MACS 3 times; Add the Anti-PE magnetic bead again, put 4 ℃ equally and hatch 30min, cross the MACS post; The resulting CD4+CD25+Treg cell of positive sorting in the MACS post, what elute is to remove the resulting activated lymphocyte of Treg cell.
3. lymphocytic amplification
The lymphocyte of removing the Treg cell is resuspended with the AIM-V substratum, make that cell concn is 2 * 10
6/ ml adds IL-2 and IL-15 and make that the concentration of IL-2 is 200U/ml, and the concentration of IL-15 is 300ng/ml.Use the microscope observing cell upgrowth situation continuously,, then add according to cell concn and add corresponding substratum and corresponding cytokine, make cell concn maintain 2 * 10 if upgrowth situation is good
6/ ml, the concentration that cytokine concentrations also maintains IL-2 is 200U/ml, and the concentration of IL-15 is 300ng/ml.
4. the collection of cell
If cell is used for frozen, then in cell cultures about the 5th day, the CD3+CD8+ TLC begins to collect when being lymphocytic 20 times of the CD3+CD8+ that obtains of original separation.If be used for feeding back, then collect about the 10th day in cell cultures.Collection process is following:
With cell transfer in centrifuge tube, 1600r/min, centrifugal 10 minutes, cell was used the saline water washed twice, if frozen then add corresponding frozen storing liquid; If feed back, then be resuspended in 5% the BSA saline water.
5. the detection of cell phenotype
With the saline water suspension PBMC or the activated lymphocyte that contain 1% human serum albumin, ice bath 10 minutes.Twice of PBS washed cell (300g, 4 ℃ of centrifugal 5min sedimentation cell agglomerates) with precooling.Dye the damping fluid re-suspended cell to whole cell density to 2 * 107cells/ml with precooling.Every pipe adds 50 μ l cell suspensions.Every pipe adds an amount of (various antibody optimum dose) antibody; Comprise FITC mark anti-cd 3 antibodies; PE mark anti-CD 25 antibody, PE-Cy5 mark anti-CD 4 antibodies, the anti-CD8 antibody of APC mark; The anti-CD16 antibody of PE mark, the anti-CD56 antibody of APC mark comprise the negative tube that is used to compensate the monochromatic tube of setting and is added with various homotype contrasts.The ice bath lucifuge is hatched 20min.Every pipe adds 1ml PBS, 300g, and 4 ℃ are centrifugal 5 minutes.The careful suction abandoned supernatant or the supernatant that inclines.The repeated washing process once.Vibration suspension cell deposition.With 0.5 milliliter of PBS re-suspended cell.Can not detect immediately like cell, staining cell must be used 4 ℃ of 4% Paraformaldehyde 96s fixing 30 minutes, washed behind the re-suspended cell 4 ℃ with the dyeing damping fluid and kept in Dark Place.Last machine testing.
Detected result comprises CD4, CD8 ratio and the two ratio, CD3+CD4+CD25+ cell subsets proportion, the detection (see figure 3) of CD3+CD16+CD56+ and CD3-CD16+CD56+ cell subsets.
Wherein in just beginning isolating PMNC, the T cell accounts for 40-70%, and through after the present invention's cultivation, the T cell accounts for more than 95% of cell mass, and wherein the ratio of CD3+CD8+T cell is more than 90%.
6. activated lymphocyte is frozen
Digestion attached cell or collection suspension cell are collected into cell suspension in the centrifuge tube centrifugal 5～10 minutes of 1000rpm; Abandon supernatant; With a certain amount of frozen storing liquid (frozen storing liquid is that foetal calf serum and DMSO prepare according to the ratio of 9:1) that cell is resuspended, counting, adjustment cell density to 10
7About individual/ml, the cell suspension branch is filled in the frozen pipe of 2ml, every pipe 1～1.5ml, frozen mouth of pipe envelope is labelled, carry out record, press the follow procedure cooling: 20 ℃ (1～2 hour) →-80 ℃ of (16～18 hours or overnight) → liquid nitrogen (LN2).
7. the recovery of activated lymphocyte
From liquid nitrogen container, take out frozen pipe; Put into 37 ℃ of water-baths immediately; The frozen pipe of light rolling melts liquid as early as possible, should in 1 minute, melt frozen pipe of cotton ball soaked in alcohol wiping with 75% and pipe lid usually; The cell suspension that will thaw moves in a certain amount of AIM-V substratum that does not add cytokine, and cell concn is diluted to 1.0 * 10
6/ ml, and change in 24 well culture plates and cultivate, spend the night.
8. the continuation of recovery back cell is cultivated
Second day, from every hole, take out general supernatant, and the fresh culture that contains 2000U/ml IL-2 of additional respective amount; After the empty inner cell of treating Tissue Culture Plate covers with, cell changed in the culturing bottle cultivate, add the AIM-V substratum of respective amount; And add the IL-15 of respective amount, and anti-CD3 monoclonal antibody and anti-CD28 monoclonal antibody, make that the concentration of IL-15 is 200ng/ml; The concentration of anti-CD3 monoclonal antibody is 80ng/ml, and the concentration of anti-CD28 monoclonal antibody is 40ng/ml.
When treating above-mentioned cell amplification, add fresh AIM-V substratum and carry out enlarged culturing, make cell concn 3 * 10 to its 3 times
6/ ml, and add respective fine intracellular cytokine IL-2 and IL-15, the concentration that makes IL-2 is at 100U/ml, and the concentration of IL-15 is at 50ng/ml.
9. the results of culturing cell after recovering
Cell cultures is carried out the collection of activated lymphocyte about the 11st day.Collection process is following: with cell transfer in centrifuge tube, 1600r/min, centrifugal 10 minutes, cell was used the saline water washed twice, was resuspended in 5% the BSA saline water.
10. the lymphocyte phenotype of recovery back cultivation detects
With the saline water suspension activated lymphocyte that contains 1% human serum albumin, ice bath 10 minutes.Twice of PBS washed cell (300g, 4 ℃ of centrifugal 5min sedimentation cell agglomerates) with precooling.Dye the damping fluid re-suspended cell to whole cell density to 2 * 107cells/ml with precooling.Every pipe adds 50 μ l cell suspensions.Every pipe adds an amount of (various antibody optimum dose) antibody; Comprise FITC mark anti-cd 3 antibodies; PE mark anti-CD 25 antibody, PE-Cy5 mark anti-CD 4 antibodies, the anti-CD8 antibody of APC mark; The anti-CD16 antibody of PE mark, the anti-CD56 antibody of APC mark comprise the negative tube that is used to compensate the monochromatic tube of setting and is added with various homotype contrasts.The ice bath lucifuge is hatched 20min.Every pipe adds 1ml PBS, 300g, and 4 ℃ are centrifugal 5 minutes.The careful suction abandoned supernatant or the supernatant that inclines.The repeated washing process once.Vibration suspension cell deposition.With 0.5 milliliter of PBS re-suspended cell.Can not detect immediately like cell, staining cell must be used 4 ℃ of 4% Paraformaldehyde 96s fixing 30 minutes, washed behind the re-suspended cell 4 ℃ with the dyeing damping fluid and kept in Dark Place.Last machine testing.
Detected result comprises CD4, CD8 ratio and the two ratio, CD3+CD4+CD25+ cell subsets proportion, the detection (see figure 4) of CD3+CD16+CD56+ and CD3-CD16+CD56+ cell subsets.
11. behind the cryopreservation resuscitation with the comparison of not frozen activated lymphocyte form
Inverted microscope is observed down: PMNC is suspension growth, and size is even, and refractivity is consistent, and activated lymphocyte obviously increases through cultivating the rear section cell, the visible cell colony, and karyon density is strengthened, and volume increases, and after birth is smooth, does not see projection.Volume is obviously greater than common lymphocyte.Mononuclearcell inductive activated lymphocyte after fresh and frozen does not have significant difference on form.
Cell after the recovery is atypia, and cell obviously increases, visible a lot of cell colonies not of uniform size, and karyon density is strengthened, and volume increases cell state and well (sees Fig. 1, Fig. 2)
12. frozen back recovery and not the in-vitro multiplication situation and the phenotype of frozen activated lymphocyte
In the cell cultivation process, sampling every day counting, it is 2.5X10 that the result is presented at first culturing cell quantity
7When freeze-stored cell was not cultivated the 10th day the 11st day left and right sides of cell cultures with frozen back recovery during the left and right sides, cell quantity all can reach 3X10
9About, the ratio of CD3+CD8+T cell is all more than 90% in the cell phenotype, and the ratio of CD4+CD25+T cell is all below 5%
13. the comparison of recovery of frozen back and the external killing activity of not frozen activated lymphocyte
Reagent: 1. the tetramethyl-diazo salt (MTT, 10mg/ml); 2. acidifying Virahol: the Virahol that contains 0.04mol/L HCl; 3. 96 porocyte culture plates.
Tumor cell line: the HepG2 of logarithmic phase growth
Working method:
Frozen and not frozen activated lymphocyte density to 1.5 * 10 of RPMI-1640 substratum adjustment with the 10%FBS that contains IL-2
6/ ml; The incomplete work of cell warp in the vegetative period nutrient solution of taking the logarithm washs, and tire is expected blue dyeing counting, and is subsequent use with RPMI-1640 nutrient solution adjustment cell density to the 2 * 106/ml that contains 10%FBS.
Frozen and not frozen activated lymphocyte and different target cell strains are 5:1 by imitating target, 10:1, and the ratio of 20:1 is got 100 μ l respectively and is inoculated in co-cultivation in the flat culture plate in 96 holes, and three multiple holes are done in each test.If different concns effector cell 200 μ l/ hole single culture are measured effector cell's absorbancy.Simultaneously the target cell strain is adjusted to 3 * 104～1 * 105/ml with the RPMI-1640 nutrient solution that contains 10%FBS; Get 100 μ l cell suspensions and 100 μ l nutrient solutions are inoculated in 96 well culture plates; Each concentration inoculation 3 multiple hole is to measure the absorbancy of different concns tumour cell; The Tissue Culture Plate that above-mentioned inoculation is good is put 37 ℃, and 5%CO2 is hatched 20h; Before stopping cultivation, every hole adds MTT 10 μ l, cultivates 4h behind the mixing again; Supernatant is abandoned in suction, and every hole adds solvent (acidifying Virahol) 100 μ l, and the Dai Jia Za product that vibrates slightly fully dissolves; On ELIASA, measure OD570-620nm; Cytotoxic percentage calculates by following formula:
ODE+T=effector cell hole OD value+target cell hole OD value
The OD value of ODE=respective concentration individual effect cell
The OD value of the independent target cell of ODT=respective concentration
Behind the cryopreservation resuscitation cell and not the comparison of freeze-stored cell killing activity it is as shown in the table, explain that the cell killing activity behind the cryopreservation resuscitation does not descend.
The external knurl relatively (%) of killing of table 1 activated lymphocyte
Different groups same effect target than the time to the cytotoxic activity there was no significant difference of hela (P>0.05)
14. cultural method of the present invention and existing cultural method are cultivated the comparison of activated lymphocyte
5 liver cancer patient peripheral bloods of aseptic collection 50ml collects white corpuscle; White corpuscle is isolated mononuclearcell with lymphocyte separation medium (proportion is 1.077 ± 0.002) after the saline water dilution, wash 2 times, respectively is divided into and equates two parts, and a with the inventive method cultivation, portion is cultivated with existing method.
Existing method is cultivated:
With RPMI 1640 complete culture solutions (including 10% autologous plasma of deactivation) re-suspended cell, the adjustment cell concn is 2X10
6/ ml.Added IFN-on the 0th day
"(1000U/ml), going into incubator hatches and adds IL-2 (1500U/ml), anti-CD3 monoclonal antibody (100ng/ml) after 24 hours again and continue to cultivate.Add fresh RPMI 1640 complete culture solutions (including 10% autologous plasma of deactivation) according to cell concn in every 3-4 days, and add IL-2 to 1000U/ml.To the 7th, 14, collecting cell carried out related detection in 21,28 days.
The present invention cultivates:
Cultivate according to the inventive method, to the 7th, 14, collecting cell carried out related detection in 21,28 days.
Table 2 the inventive method and existing method cells expanded are relatively
| 7d | 14d | 21d | 28d | |
| Existing method | 6.28±2.76 | 67.79±10.53 | 98.37±12.64 | 121.54±11.82 |
| The present invention | 11.73±1.88 * | 150.32±15.37 * | 285.91±17.26 * | 427.65±13.58 * |
*Compare P with existing method cultured cells<0.05
Be its peak when existing cultural method is generally cultivated the 14d left and right sides, it is dead easily to continue culturing cell, and the present invention can cultivate the 28th day, and cell is still in quick growth.
Table 3 the inventive method and existing method are cultivated the comparison of back cell phenotype CD4+CD25+
*Compare P with existing method cultured cells<0.05
The shared ratio of CD4+CD25+ is very high always in the existing cultural method, when especially cultivating 28d.And the shared ratio of CD4+CD25+ is very low among the present invention.
15. real case
Colorectal carcinoma liver shifts the patient, the woman, and 59 years old, blood picture was lower always.Regimen is that chemotherapy adds the activated lymphocyte adoptive therapy that the present invention cultivates, and chemotherapy regimen is RP-54780 scheme: Oxaliplatin120mg/m
2D1,5-Fu400mg/m
2CIV24h D1-5, CF200mg/m
2D1-5 behind chemotherapy 1 end cycle the 3rd day, feeds back the activated lymphocyte (4x10 that the present invention cultivates
9), intravenous drip drips off within an hour, feeds back 1 time in each 1 day; Descend 1 cycle chemotherapy at a distance from 1 week after feeding back 3 times, carry out 4 most of disappearances of cycle MET altogether, patient condition is good; It is normal that blood picture recovers, and the appetite symptom such as lose weight that descends all disappears, and it is also normal to sleep.
Claims (10)
1. one kind is the amplification method of master's activated lymphocyte with CD3+CD8+, it is characterized in that,
(a) isolated mononuclearcell in the peripheral blood blood sample is cultivated in the substratum that contains lymphocyte activator agent and cytokine IL-2; Said lymphocyte activator agent is anti-cd 3 antibodies and anti-CD28 antibody, and the final concentration of anti-cd 3 antibodies is 0.1-100ng/ml; The final concentration of anti-CD28 antibody is 0.1-50ng/ml; Said IL-2 final concentration is 100U/ml to 5000U/ml;
(b) remove the CD4+CD25+Treg cell with cultivating the lymphocyte that obtains in (a) step with mini MACS magnetic bead sorting;
(c) cultivate in the substratum that contains cytokine IL-2 and IL-15 cultivating the lymphocyte that obtains in (b) step; Said IL-2 final concentration is 50-500U/ml, and the final concentration of IL-15 is 10-500ng/ml;
(d) collect the lymphocyte that obtains in (c) step and be used for feedback or frozen.
2. amplification method as claimed in claim 1 is characterized in that, the final concentration of said step (a) anti-cd 3 antibodies is 1-75ng/ml, and the final concentration of anti-CD28 antibody is 2-30ng/ml; The concentration of IL-2 is 2000-4000U/ml.
3. cultural method as claimed in claim 1 is characterized in that, the final concentration of IL-2 is 200-500U/ml in the step (c), and the final concentration of IL-15 is 200-400ng/ml.
4. amplification method as claimed in claim 1 is characterized in that, the lymphocyte activator agent of using in the said step (a) also contains activator PHA and/or IFN-r; The final concentration of said PHA is 1-100ng/ml, and the final concentration of IFN-r is 100-2000U/ml.
5. amplification method as claimed in claim 1 is characterized in that, said step (c) also contains one or more among cytokine IL-7, IL-12, the IL-21; The final concentration of said IL-7 is 10-200ng/ml, and the final concentration of IL-12 is 5-100ng/ml, and the final concentration of IL-21 is 1-200ng/ml.
6. amplification method as claimed in claim 1 is characterized in that, said substratum is RPMI-1640 substratum, DMEM substratum or AIM-V substratum.
Among the claim 1-6 any one what increased is the frozen method of main activated lymphocyte with CD3+CD8+, it is characterized in that,
(e) the weighting profit require 1-6 what be grown in logarithmic phase is that main activated lymphocyte is collected with CD3+CD8+, collection method is: the speed with 300-2000r/min is centrifugal, obtains sedimentation cell;
It is 0.1 * 10 that the cell adding frozen storing liquid that (g) will in (e) step, obtain makes cell concn
7-5 * 10
7/ ml; The cell suspension of preparation is changed in the frozen pipe;
(h) the frozen pipe that obtains in (g) step is put in the programmed cooling box at subzero 20 ℃ placed 1-5 hour, change in-80 ℃ of refrigerators again and placed 16-48 hour, it is frozen to change the liquid nitrogen container midium or long term over to.
8. frozen method as claimed in claim 7 is characterized in that, logarithmic phase is meant that TLC is 2-100 a times that original separation obtains TLC in the said step (e); In the said step (e) be main lymphocytic being collected as: get cell and be put in the centrifuge tube, with the centrifugal speed of 400-600r/min centrifugal 10 minutes, remove supernatant with CD3+CD8+; Used frozen storing liquid is that foetal calf serum and DMSO prepare according to the volume ratio of 9:1 in the said step (g); Perhaps substratum and DMSO are according to the volume ratio preparation of 9:1; The mixing liquid and the DMSO that perhaps are foetal calf serum and substratum prepare according to the volume ratio of 9:1, and said substratum is RPMI-1640 substratum, DMEM substratum or AIM-V substratum; Said step (g) is to place 2-3 hour at subzero 20 ℃, changes in-80 ℃ of refrigerators again and places 20-24 hour.
Claim 7 or 8 frozen be the method for resuscitation of main activated lymphocyte with CD3+CD8+, it is characterized in that,
(i) take out frozen cell in (h) steps of claim 7 or 8, melt 37 ℃ of water-bath middling speeds;
(j) cell that obtains in (i) is changed over to rapidly in 37 ℃ of preheating fresh cultures, change in the Tissue Culture Plate then and cultivate;
(k) draw the supernatant of the substratum of institute's culturing cell in (j), and add the fresh culture that contains cytokine IL-2 again; The final concentration of said IL-2 is 1000-5000U/ml;
After treating that (l) cell covers with the hole in (k), be transferred in the culturing bottle that contains the lymphocyte activator agent, and add the fresh culture that contains cytokine IL-15; The final concentration 200-500ng/ml of said IL-15; Contained lymphocyte activator agent is anti-cd 3 antibodies and anti-CD28 antibody, and the final concentration of anti-cd 3 antibodies is 1-100ng/ml, and the final concentration of anti-CD28 antibody is 2-200ng/ml;
Treat (m) in (l) that cell quantity increases 2-10 times the time, adds the fresh culture that contains cytokine IL-2 and IL-15 and carries out enlarged culturing; The final concentration of the factor-containing IL-2 of institute is 50-500U/ml, and the final concentration of IL-15 is 50-500ng/ml;
(n) collect amplifying activated lymphocyte in (l).
10. method for resuscitation as claimed in claim 9 is characterized in that, the final concentration of IL-2 is 1000-2000U/ml in the said step (k); The final concentration of IL-15 in the said step (l) is 200-500ng/ml, and the final concentration of anti-cd 3 antibodies is 50-100ng/ml, and the final concentration of anti-CD28 antibody is 20-80ng/ml; The multiple of said step (m) cell quantity amplification is 2-5 times; The final concentration of IL-2 is 50-200U/ml in the said step (m), and the final concentration of IL-15 is 20-100ng/ml; Said substratum is RPMI-1640 substratum, DMEM substratum or AIM-V substratum.
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