CN102847145A

CN102847145A - Method for preparing dendritic cell vaccine

Info

Publication number: CN102847145A
Application number: CN2012103700726A
Authority: CN
Inventors: 储以微; 郑秀娟; 张丹
Original assignee: Fudan University
Current assignee: Fudan University
Priority date: 2012-09-27
Filing date: 2012-09-27
Publication date: 2013-01-02

Abstract

The invention relates to the technical field of biology, in particular to a method for preparing dendritic cell vaccine. The method is characterized by comprising the following preparation steps: (1) taking autologous blood of a patient, and separating red blood cells, mononuclear cells and plasma from separating medium of human lymphocyte; (2) adjusting concentration of the mononuclear cells; (3) adding the solution into a six-pore plate and performing adherence for 16 hours; (4) sucking and discharging non-adherent cells on the upper layer of each pore of the six-pore plate; (5) adding 3ml of dendritic cell (DC) culture medium, placing the solution in a carbon dioxide cultivating box with the concentration of carbon dioxide of 5% at the temperature of 37 DEG C to perform cultivation for 2-3 days; (6) performing half-quantity liquid changing; (7) enabling the concentration of the autologous tumor antigen in each pore of the six-pore plate to be 20 mu g/ml by loading the autologous tumor antigen in the sixth day; and (8) detecting the quantity and maturity of the matured DC cells after 24 hours. Compared with the prior art, the number of the matured DC cells at least reaches 1*107, and the maturity is larger than 85%.

Description

A kind of dendritic cell vaccine preparation method

Technical field

The present invention relates to biological technical field, specifically a kind of dendritic cell vaccine preparation method.

Background technology

DC is called dendritic cell, it is the important immunocyte of body, have a wide range of applications in the cellular immunization treatment, under special tumor antigen stimulated, ripe dendritic cell can be expressed cytokine profiles and chemotactic factor, as, MHC II molecule HLA-DR, costimulatory molecules CD80, the cytokine profiles such as CD86, effectively inducing antigen-specific and nonspecific immune response reaction, the enhancing human body immunity function.But DC ratio in human peripheral is very low, therefore improves amplification times, increases ripe dendritic cell number, therapeutic effect is played a part crucial.

What dendritic cell was done now is awfully hot, and routine techniques is to use GM-CSF+IL4, and the amplification times of this method is not high, and Maturity is not high, and general ripe dendritic cell number can only reach 2 * 10 ⁶, ripe dendritic cell purity is 60%, referring to Fig. 1.Have adopt to add that Flt3 increases yet, but wherein also can add chemicals, and amplification times and Maturity are not high yet.

Summary of the invention

The objective of the invention is to overcome the deficiencies in the prior art, a kind of preparation method of the dendritic cells in treatment vaccine of using for oncotherapy is provided, the amplification of Effective Raise dendritic cell, make immature dendritic cell amplification times raising under condition of in vitro culture among the human PBMC, improve Maturity, and do not add chemicals, high purity.

For achieving the above object, design a kind of dendritic cell vaccine preparation method, it is characterized in that adopting being prepared as follows step: (1) gets the 17ml sufferer from body blood, adopts the human lymphocyte separating medium to isolate erythrocyte, monokaryon lymphocyte, blood plasma; (2) adjusting monokaryon lymphocyte concentration with the GT-T551 serum-free medium is 2 * 10 ⁶Individual/ml; (3) the monokaryon lymphocyte that will adjust concentration adds in 6 orifice plates adherent 16 hours with the 3ml/ hole; (4) inhale upper strata attached cell not in each hole abandon 6 orifice plates, the lower floor in each hole namely stays the immature DC cell; (5) add respectively DC culture medium 3ml in each hole of 6 orifice plates again, place in the CO2 gas incubator, gas concentration lwevel is 5%, and temperature is 37 ℃, cultivates 2～3 days; (6) inhale half 1.5ml of DC culture medium in each hole of abandoning 6 orifice plates, replenish again DC culture medium 1.5ml in each hole, carry out half amount and change liquid; (7) adding the 6th day that the DC culture medium begins to calculate for the first time, load autologous tumor antigen, making the concentration of the autologous tumor antigen in each holes of 6 orifice plates is 20 μ g/ml; Detect quantity and the Maturity of ripe DC cell after (8) 24 hours; Described DC culture medium by isolated autologous plasma in the above-mentioned steps (1), GM-CSF, IL-4, Flt3, and the GT-T551 serum-free medium formed, wherein autologous plasma accounts for 1% of whole DC culture volume, the concentration that GM-CSF accounts for whole DC culture medium is 50ng/ml, the concentration that IL-4 accounts for whole DC culture medium is 100ng/ml, the concentration that Flt3 accounts for whole DC culture medium is 100ng/ml, 96% of the whole DC culture volume of GT-T551 serum-free culture fiduciary point.

The present invention compares with prior art, and isolated autologous plasma recycling replaces with stripped people AB serum with human plasma; Add Flt3 in this DC culture medium, can strengthen stimulates the dendritic cell amplification times, and ripe dendritic cell number reaches 1 * 10 at least ⁷, the Maturity of mature dendritic cell＞85%; Solve in the dendritic cell preparation process cell quantity few, bred slow problem; With self special tumor stem cell lysate, as tumor antigen, the specific killing power of cell is stronger; And raw material does not add extra chemicals, high purity.

Description of drawings

Fig. 1 is the purity figure of the dendritic cell of the maturation prepared of original method.

Fig. 2 is the purity figure of the dendritic cell of the maturation that adopts the present invention and prepare.

The specific embodiment

Below in conjunction with example the present invention is further described.

(1) get the 18ml sufferer from body blood, get wherein the 1ml sufferer and do fluidic cell from body blood and detect, be used for the treatment of the evaluation of front immune indexes, the 17ml sufferer adopts the human lymphocyte separating medium to isolate erythrocyte, monokaryon lymphocyte, blood plasma from body blood in addition;

(2) adjusting monokaryon lymphocyte concentration with the GT-T551 serum-free medium is 2 * 10 ⁶Individual/ml;

(3) the monokaryon lymphocyte that will adjust concentration adds in 6 orifice plates adherent 16 hours with the 3ml/ hole;

(4) the upper strata non-adherent cell is abandoned in suction in each hole of 6 orifice plates, namely stays the adherent immature DC cell of lower floor in each hole;

(5) in each hole of 6 orifice plates, add respectively again DC culture medium 3ml, place in the CO2 gas incubator, wherein CO ²Concentration is 5%, and temperature is 37 ℃, cultivates 2～3 days;

(6) inhale DC culture medium 1.5ml in each hole abandon 6 orifice plates, the DC culture medium of getting in addition again 1.5ml is placed in each hole;

(7) to add the 6th day that the DC culture medium begins to calculate for the first time, load sufferer autologous tumor antigen makes the DC cell maturation and is prepared into autologous tumor antigenic specificity DC vaccine, and wherein the concentration of the autologous tumor antigen in each hole of 6 orifice plates is 20 μ g/ml;

Detect quantity and the Maturity of ripe DC cell after (8) 24 hours.

Described DC culture medium is comprised of isolated autologous plasma, GM-CSF, IL-4, Flt3, GT-T551 serum-free medium in the above-mentioned steps (1), wherein to account for the volume of whole DC culture medium be 1% to autologous plasma, the concentration that GM-CSF accounts for whole DC culture medium is 50ng/ml, the concentration that IL-4 accounts for whole DC culture medium is 100ng/ml, the concentration that Flt3 accounts for whole DC culture medium is 100ng/ml, and the volume of the whole DC culture medium of GT-T551 serum-free culture fiduciary point is 96%.

Many with respect to original technology amount for taking blood, the situation that amplification times is few, the preparation method of this new tumor dendritic cells in treatment vaccine provided by the invention, be used for prevention and the treatment of tumor, have that amount for taking blood is few, preparation is easy, cell proliferation quantity is many, good effect, characteristics that specific killing power is strong, wherein ripe dendritic cell number reaches 1 * 10 at least ⁷, the Maturity of mature dendritic cell＞85% is referring to Fig. 2.

Claims

1. dendritic cell vaccine preparation method, it is characterized in that adopting being prepared as follows step: (1) gets the 17ml sufferer from body blood, adopts the human lymphocyte separating medium to isolate erythrocyte, monokaryon lymphocyte, blood plasma; (2) adjusting monokaryon lymphocyte concentration with the GT-T551 serum-free medium is 2 * 10 ⁶Individual/ml; (3) the monokaryon lymphocyte that will adjust concentration adds in 6 orifice plates adherent 16 hours with the 3ml/ hole; (4) inhale upper strata attached cell not in each hole abandon 6 orifice plates, the lower floor in each hole namely stays the immature DC cell; (5) add respectively DC culture medium 3ml in each hole of 6 orifice plates again, place in the CO2 gas incubator, gas concentration lwevel is 5%, and temperature is 37 ℃, cultivates 2～3 days; (6) inhale half 1.5ml of DC culture medium in each hole of abandoning 6 orifice plates, replenish again DC culture medium 1.5ml in each hole, carry out half amount and change liquid; (7) adding the 6th day that the DC culture medium begins to calculate for the first time, load autologous tumor antigen, making the concentration of the autologous tumor antigen in each holes of 6 orifice plates is 20 μ g/ml; Detect quantity and the Maturity of ripe DC cell after (8) 24 hours; Described DC culture medium by isolated autologous plasma in the above-mentioned steps (1), GM-CSF, IL-4, Flt3, and the GT-T551 serum-free medium formed, wherein autologous plasma accounts for 1% of whole DC culture volume, the concentration that GM-CSF accounts for whole DC culture medium is 50ng/ml, the concentration that IL-4 accounts for whole DC culture medium is 100ng/ml, the concentration that Flt3 accounts for whole DC culture medium is 100ng/ml, 96% of the whole DC culture volume of GT-T551 serum-free culture fiduciary point.