CN101418283A

CN101418283A - A kind of method of simple high efficiently preparing CIK cell

Info

Publication number: CN101418283A
Application number: CNA2007101338335A
Authority: CN
Inventors: 周曙明; 范云峰
Original assignee: Individual
Current assignee: Individual
Priority date: 2007-10-23
Filing date: 2007-10-23
Publication date: 2009-04-29

Abstract

The invention discloses a kind of method of simple high efficiently preparing CIK cell.The present invention utilizes the white corpuscle in the O type healthy blood donor component blood, under the serum-free condition of suspension culture, stimulates its propagation with multiple factors such as phytohaemagglutinins, can obtain the considerable CIK cell of quantity through cultivating and repeatedly going down to posterity.Operation steps of the present invention is simple, the cells expanded height, and the external knurl of killing is energetic, does not have main histocompatibility restriction, can be used as the ordinary method of CIK cell cultures.

Description

A kind of method of simple high efficiently preparing CIK cell

Technical field

The present invention relates to the method for a kind of simple high efficiently preparing CIK cell in the adoptive immunotherapy.

Background technology

The CIK cell is with CD ₃ ⁺CD ₅₆ ⁺The T cell is main foreign cell group.Researchists such as Yun were with CD in 1989 ₃Monoclonal antibody amplification mouse boosting cell has obtained a cell mass, called after CD ₃Activated killer cell (CD ₃-AK) (Yun YS, HargroveME, Ting CC.In vivo antitumor activity of anti-CD ₃-induced activated killer cells.CancerRes, 1989,49 (17): 4770-4774.).On this working foundation, (Schmidt-Wolf IGH such as Schmidt-Wolf, NegdnRS, Kiem HP, et al.Use of a SCID Mouse/Human lymphoma model to evaluate cytokine-inducedkiller cells with potent antitumor cell activity.J Exp Med, 1991,174-139.) with IFN-γ, IL-2, CD ₃McAb, IL-1 is the cultivator peripheral blood mononuclear cell altogether, has obtained a cell mass that powerful anti-tumor activity is arranged, and is the CIK cell.The CIK cell have rate of propagation fast, kill that the knurl spectrum is wide, killing activity is high, still responsive to the drug-resistant tumor strain, to many advantages such as normal marrow hemopoiesis precursor cell toxicity are little.These advantages have been quickened the research of CIK cell aspect clinical application, have entered clinical II experimental phase phase at China's approved at present.

In the culturing process of traditional C IK cell, IFN-γ, CD ₃McAb, IL-2 are essential cytokines.The CIK cell of present clinical use, its cultural method is by (Schmidt-Wolf IGH that creates such as Schmidt-Wolf, Grimm B, BrandtK, et al.Propagation of large numbers of cells of a human mixed-lineage T-lymphoid/myeloid.Br JHaematol, 1995,90:512-517.), follow following routine.(1) extracts the blood of capacity patient, separate peripheral blood mononuclear cell wherein from body; (3) the washing and precipitating cell is used phosphoric acid buffer more; (2) cultivate with RPMI 1640 perfect mediums that contain 10% allos AB serum; (4) only singlely hatched in advance 24 hours with IFN-γ.

PHA is a kind of nonspecific polyclonal activator as mitogen, and all clones of T lymphocyte populations are activated, and promotes the cell rapid amplifying.The mononuclearcell that activates the bleeding of the umbilicus source is united in propositions such as the Tan Wen of East China University of Science pine with PHA and two kinds of factors of CD3McAb, strengthen cells in vitro amplification ability, be activated the back to these two kinds of factor wash-outs at cell, and be placed in the stirring type bioreactor cultivate obtain the CIK cell (number of patent application: 200310109565.5, title: a kind of CIK cells in vitro cultural method).

The BIOTARGET-1 substratum is a kind of novel serum free medium that BIOLOGICAL INDUSTRIES develops for the cultivation peripheral blood lymphocyte specially, and its prescription is based on the serum free medium (Application No.: 20060073591 of inventions such as Abitorabi; Title: Cell culture media).

The deficiency that existing C IK cell culture processes exists:

In the clinical application at present, conventional CIK cell culture processes is: extract patient's peripheral blood 50～200ml, anticoagulant heparin is through equivalent Ficoll parting liquid (proportion 1.077g/cm ³) density gradient centrifugation is after 30 minutes, absorption mononuclearcell layer transfers to 2 * 10 with the RPMI1640 that contains 10%AB serum ⁶/ ml adds recombinanthumanifn-1000U/ml, places 37 ℃ of 5%CO ₂Incubator in cultivate after 24 hours, add CD ₃McAb 50ng/ml, recombinant human il-2 300U/ml and IL-1 100U/mL continue to cultivate 2～3 days, change fresh medium in later per 2～3 days, and adjusting cell count is 2 * 10 ⁶/ ml adds IL-2 300U/ml simultaneously, after two weeks in batches collecting cell feed back.

In this method a large amount of extract peripheral blood of patients can cause the health of patient's weakness stress, increase the burden of hemopoietic system, can influence the effect of treatment to a certain extent.Contain 10%AB allos serum in the substratum, unknown composition difference between each batch wherein is bigger, can pair cell cultivate and cause unpredictable result, and the pathogenic agent microorganism may import by serum, causes side effect if be used for clinical meeting.Owing to there is not the stimulation in advance of mitogen PHA, the cell growth phase is to slowly.

The method of Tan Wensong etc. is carried out commercialization and is promoted certain degree of difficulty in addition.Its mononuclearcell derives from bleeding of the umbilicus, and the technical requirements that bleeding of the umbilicus is collected is very high, and general section office do not have the special talent.PHA and CD ₃Will be behind two kinds of factor associatings of McAb activating cells by wash-out, the amplification efficiency of cell can descend to some extent after making.In addition, contain 10%AB allos serum in the employed several cell culture mediums of this method, the disadvantageous effect that therefore also exists unknown composition in the serum or pathogenic agent microorganism to bring.

Summary of the invention

The objective of the invention is to make the simple high efficiency of cultivation of CIK cell, easily grasped, satisfy the needs of clinical study and treatment by medical institutions.

The method that the present invention relates to comprises following flow process: getting the whole blood that O type healthy blood donor newly adopts and be rich in leukocytic raffinate after component blood separates, is 1.077g/cm through proportion ³Ficoll liquid density gradient centrifugation (1500rpm 35min), the peripheral blood mononuclear cell of getting interfacial layer, physiological saline washing 2 times is suspended in the BIOTARGET-1 substratum of serum-free, adjusting cell concn is 2 * 10 ⁶/ ml adds PHA 100ng/ml and IFN-γ 1000U/ml, places 37 ℃ of 5%CO ₂Incubator in cultivated 24 hours, add CD ₃McAb 50ng/ml, IL-2 300U/ml continues down to cultivate in the same terms, looks growing state and goes down to posterity and cultivate and change fresh medium, and add IL-2 300U/ml, and from the 14th day per two days one time collecting cell, the whole cycle is about 30 days.

The peripheral blood mononuclear cell that cultivation of the present invention is required derives from the whole blood that blood station O type healthy blood donor newly adopts and be rich in leukocytic raffinate after component blood separates, so material obtains easily.The BIOTARGET-1 nutrient media components of serum-free is clear, avoids the unpredictable result who causes because of composition unknown in the serum.Adopting physiological saline washing and precipitating cell is may be to the deleterious material of body, as phosphoric acid buffer for fear of using.Why stimulating with PHA and IFN-γ pair cell at initial 24 hours, is because PHA can increase amplification efficiency as mitogen, and IFN-γ is that inducing cell has that extremely knurl toxicity is necessary.

Description of drawings

Accompanying drawing can better illustrate meaning of the present invention and effect, rather than limitation of the invention.

Fig. 1 analyzes for the CIK cell purity of cultivating 0 day, 14 days and 28 days.

Fig. 2 is the amplification times of the CIK cell of cultivating 0 day, 14 days and 28 days.

Fig. 3 when cultivating 14 days the CIK cell to the killing activity of human leukemia cell line (K562), lung adenocarcinoma cell line (SPCA-1), cervical cancer cell strain (HeLa).

Embodiment

Embodiment 1

Buy single part of 200ml whole blood from the Red Cross blood station and after component blood separates, be rich in leukocytic raffinate, centrifugal (500rpm10min), abandon supernatant, sedimentation cell physiological saline resuspension, suspension liquid is tiled in Ficoll liquid, density gradient centrifugation (1500rpm 35min), the peripheral blood mononuclear cell of absorption interfacial layer, physiological saline washing 2 times, adjusting cell concn with serum-free BIOTARGET-1 substratum is 2 * 10 ⁶/ ml adds PHA 100ng/ml and IFN-γ 1000U/ml, places 37 ℃ of 5%CO ₂Incubator in cultivated 24 hours, add CD ₃McAb 50ng/ml, IL-2 300U/ml, the same terms continue down to cultivate, and per 2～3 days observation of cell growing ways go down to posterity according to the density of cell growth and to cultivate or with continuing cultivation behind the fresh medium diluting cells that contains IL-2300U/ml.

Be cultured to the 14th day and the 28th day, the antigenic phenotype with the flow cytometry analysis cell identifies main effects cell---CD in the CIK cell ₃ ⁺CD ₅₆ ⁺T cell, purity are respectively 42.38% and 61.72%; And 0 day the time in the peripheral blood mononuclear cell purity of CIK cell be 1.81%.(see figure 1)

When the 14th day and the 28th day, the amplification times of cell is respectively about 140 times and 720 times.(see figure 2)

In the time of 14 days with the CIK cell respectively with human leukemia cell line (K562), lung adenocarcinoma cell line (SPCA-1) and cervical cancer cell strain (HeLa) with the effect target of 10:1 than mixing, mtt assay detects it and kills tumor activity and be respectively 74.2%, 71.7% and 58.3%.(see figure 3).

Claims

1. the method for a simple high efficiently preparing CIK cell is characterized in that, comprises the steps:

1). from the O type healthy blood donor's that newly adopts white corpuscle component blood, separate peripheral blood mononuclear cell, centrifugal, the washing and precipitating cell;

2). with the serum-free BIOTARGET-1 substratum that contains phytohaemagglutinin (PHA) and IFN-(IFN-γ) with cell dilution after cultivation 24 hours, add anti-CD ₃Monoclonal antibody (CD ₃McAb) and interleukin II (IL-2) continue to cultivate 3 days;

3). according to adding the fresh culture that contains IL-2 in the every 2-3 of the upgrowth situation of cell days.

4). the cultivation of repeatedly going down to posterity obtains the CIK cell of high amplification times.

2. method according to claim 1 is characterized in that, peripheral blood mononuclear cell derives from the raffinate (this raffinate be rich in white corpuscle) of whole blood after component blood separates that blood station O type healthy blood donor newly adopts.

3. method according to claim 1 is characterized in that, washing and precipitating cell solutions employed is a medical saline.

4. method according to claim 1 is characterized in that, serum free medium is not for containing the BIOTARGET-1 substratum of various serum.

5. according to claim 1 and the described method of claim 4, it is characterized in that Ficoll liquid separates the peripheral blood mononuclear cell that obtains and is diluted to 2 * 10 with the serum free medium that contains 100ng/ml PHA and 1000U/ml IFN-γ earlier ⁶Cultivated about 24 hours behind individual/ml.

6. according to claim 1 and the described method of claim 5, it is characterized in that culture system is adding CD ₃Need not before McAb and the IL-2 cell that suspends is carried out centrifugation and cleaning.