CN103045530B

CN103045530B - Method for suspension culturing cell

Info

Publication number: CN103045530B
Application number: CN201210542003.9A
Authority: CN
Inventors: 徐明明; 张静; 马景霞
Original assignee: SHANDONG BINZHOU WO HUA BIOTECH ENGINEERING Co Ltd
Current assignee: SHANDONG BINZHOU WO HUA BIOTECH ENGINEERING Co Ltd
Priority date: 2012-12-14
Filing date: 2012-12-14
Publication date: 2015-06-17
Anticipated expiration: 2032-12-14
Also published as: CN103045530A

Abstract

The invention provides a method for suspension culturing cell, comprising the steps of: (1) using the cell fusion technology to fuse an adherence infinite continuous cell line and limit subculture suspension cells to establish a fusion cell line capable of infinite subculturing and suspension culturing; (2) screening the fused cells grown in suspension; and (3) subculturing the fused cells for more than 10 passages to determine the stability of the cell line. The beneficial effects of the method are that: the cell fusion technology is used in the present invention for domesticating the adherent cells into the suspension culture cells, the advantages of the suspension culture is used, the basis for obtaining a desired product from suspension cells is established, the suspension cells domesticated by the method can be cultured by utilizing a common serum-containing cell culture liquid, and the culture cost is greatly reduced compared with a serum-free culture liquid. The method uses the full suspension culture, helps to simplify the production process, shorten the production time, and improve production efficiency, and is easy for expanding culture.

Description

A kind of method of suspended culture cell

Technical field

The present invention relates to field of pharmaceutical biology, be specifically related to a kind of method of suspended culture cell.

The cultural method of cell in vitro comprises adherent culture and suspension culture, and adherent culture (refers to the cultivation that cell attachment carries out at certain solid phase surface; Suspension culture makes cell be in the cultural method of dispersion suspension in nutrient solution all the time by vibration or wheelwork; Wherein suspension culture can be divided into again microcarrier suspension culture and full suspension culture.Compared with adherent culture, suspension culture tool has the following advantages: (1) can expanding production amount continuously; (2) nutritive substance be conducive in cell culture medium fully contacts with gas, and is easy to control culture condition (temperature, pH, oxygen partial pressure and CO ₂deng); (3) culture condition is stablized, and is tending towards homogeneous, is convenient to carry out quantitative examination; (4) be easy to carry out in system airtight continuously, decrease the chance of operation steps and pollution; (5) can cultured continuously for a long time, both can save manpower, and make again cell can continue to maintain logarithmic phase; (6) cell of suspension culture still keeps originally to susceptibility and the biological characteristics of virus.Based on above-mentioned advantage, how to overcome shortcoming when attached cell is cultivated, the advantage making full use of suspension culture becomes the great difficult problem in cell cultures.

Summary of the invention

For above deficiency, the invention provides a kind of method that attached cell carries out suspension culture, attached cell utilizes cell-fusion techniques to carry out suspension culture by present method, takes full advantage of the advantage of suspension culture, make attached cell can carry out simplifying, efficiently, a large amount of cultivation.

The present invention is achieved by the following technical solutions:

A method for suspended culture cell, it comprises the steps:

(1) utilize cell-fusion techniques adherent unlimited continuous cell line and Limited passage suspension cell to be merged, set up and can infinitely to go down to posterity and can the fused cell system of suspension culture;

With the unlimited continuous cell line that square vase adherent culture is recovered from liquid nitrogen, culture condition is: pH7.0 ~ 7.2, temperature 36 ~ 37 DEG C, and substratum is cell culture fluid; Incubation time 48 ~ 72h, cell monolayer grows to 90%, and cell density is 3 ~ 6 × 10 ⁵cells/mL;

Individual layer in square vase is grown to the cell EDTA-trysinization dispersion of 90%, adding 10mL serum-concentration is 15% cell culture fluid, stops digestion and obtains cell suspension, and carry out cell counting;

Get the animal derived lymphocyte equal with above-mentioned passage cell number, after mixing, carry out the centrifugal 10min of 800rpm, obtain cell mixing mud;

By resuspended with 10mL serum-free cell culture medium for above-mentioned cell mixing mud, centrifugal 10min at 800 rpm, repeated washing 1 time, removes remaining serum;

At the bottom of springing centrifuge tube, scattered by the cell mud of centrifugal acquisition, Xiang Guanzhong adds the PEG 1mL that concentration is 50%, at the uniform velocity add, and limit edged rocks gently in 1min; Add 9mL serum-free cell culture medium, mix, the centrifugal 5min of 1000rpm, abandons supernatant; Slowly add 10mL serum-free cell culture medium, re-suspended cell, the centrifugal 5min of 1000rpm, abandons supernatant; Add the cell culture fluid that 5mL serum content is 15%, re-suspended cell, proceed in the disposable Tissue Culture Flask of 5mL, put 37 DEG C of incubators and cultivate; Establish normal lymphocytes to contrast simultaneously;

(2) fused cell of suspension growth is screened;

Cultivate 24h, to blow and beat gently bottom culturing bottle 3 ~ 5 times with suction pipe, nutrient solution is transferred to new cell culture container together with suspension growth cell, 37 DEG C of cultivations; Cultivate 3 ~ 5 days, in culturing process, shake to make a movement culture vessel every 6 ~ 8h, so that the growth of cell, cultured continuously more than 7 days, repeats above-mentioned steps for several times, and removing adherent growth cell, obtains the cell of complete suspension growth.

By suspension cell 37 DEG C of cultured continuously more than 7 days, until remaining lymphocyte or self merge lymphocyte natural apoptosis after, to obtain unlimited passage cell and lymphocytic fused cell.

(3) determination of cell stability;

Cultivate 3 ~ 4 days in above-mentioned steps (2) cell cultivation process, supplemented serum content is the cell culture fluid 1 time of 15%; Cultivate more than 7 days, obtain the unlimited passage cell of complete suspension growth and lymphocytic fused cell, the centrifugal 5min of 1000rpm, removes supernatant, resuspended with fresh medium, is placed in new cell culture container and continues to cultivate, go down to posterity 1 time; Repeat above-mentioned steps, cell is passed more than 10 generations, basis of microscopic observation.

Cell culture temperature in above-mentioned steps (3): 36 ~ 37 DEG C, pH:7.0 ~ 7.4.

The invention has the beneficial effects as follows: the present invention utilizes cell-fusion techniques to be domesticated for by the cell of adherent culture can the cell of suspension culture, take full advantage of the advantage of suspension culture, obtain object product for utilizing suspension cell to lay the foundation, present method tame suspension cell the ordinary cells nutrient solution containing serum can be utilized to cultivate, compared with serum-free medium, greatly reduce toxigenic capacity.Present method adopts full suspension culture, has following advantage compared with microcarrier suspension culture: without the need to the microcarrier of costliness, thus effectively reduce production cost; Can save microcarrier cultivate in additional step as digestion harvested cell etc., thus simplify production process, shorten the production time, enhance productivity, and be easy to carry out enlarged culturing.

Accompanying drawing explanation

Fig. 1 is the situation of basis of microscopic observation culturing cell of the present invention.

In figure: A: cultivate 10 days, fused cell growing state; B: cultivate 10 days, normal chicken embryo splenocyte (lymphocyte) growing state; C: cultivate 17 days, fused cell growing state; D: cultivate 17d, normal chicken embryo splenocyte growing state.

Embodiment

Below in conjunction with the drawings and specific embodiments, the present invention will be further described, so that those skilled in the art better can understand the present invention, but therefore do not limit the present invention.

Embodiment 1

(1) utilize cell-fusion techniques, DF1 and chicken embryo spleen lymphocyte are merged, obtain DF1 and the lymphocytic fusion suspension cell line of chicken embryo.

Get spleen by aseptic for 18 age in days SPF chicken embryos, scissors shreds, and adds 1mL pancreatin, and 37 DEG C of water-bath digestion 10min, repeatedly blow and beat for several times, add 10mL 15% MEM nutrient solution, stop digestion, 4 layers of filtered through gauze.Filtrate is transferred to 15mL centrifuge tube, and carries out cell counting, cell density is about 10 ⁶/ mL;

Individual layer in square vase is grown to the DF1 cell dissociation of 90%, add 10mL 15% MEM and stop digestion, counting cells number;

The DF1 getting equivalent mixes with chicken splenic lymphocyte (is total to about 2 × 10 ⁶individual cell), the centrifugal 10min of 800rpm, removes supernatant, resuspended with serum-free cell culture medium, the centrifugal 10min of 800rpm, then repeated washing 1 time, removes remaining serum;

Springing centrifuge tube gently, the cell mud upper step obtained loosens, and in pipe, slowly add the PEG 1mL that concentration is 50%, and at the uniform velocity add at 1min, and limit edged rocks gently;

Slowly add 9mL serum-free cell culture medium, mixing, the centrifugal 5min of 1000rpm, abandons supernatant; Slowly add 10mL serum-free cell culture medium, re-suspended cell, the centrifugal 5min of 1000rpm, abandons supernatant; Add the cell culture fluid that 5mL serum content is 15%, re-suspended cell, proceed in the disposable Tissue Culture Flask of 5mL, put 37 DEG C of incubators and cultivate.Meanwhile, a part of spleen suspension (about 10 is separately got ⁶individual cell), be placed in 15mL centrifuge tube, the centrifugal 5min of 1000rpm, abandons supernatant, adds the cell culture fluid that 5mL serum content is 15%, re-suspended cell, proceeds in the disposable Tissue Culture Flask of 5mL, puts 37 DEG C of incubators and cultivates, in contrast.

(2) screen the fused cell of suspension growth and determine cell stability;

Cultivate 3d, add the cell culture fluid nutrient solution that 2mL serum content is 15%; Cultivate 1 week, rock nutrient solution gently, and collect nutrient solution, the centrifugal 5min of 1000rpm, removes supernatant, adds fresh medium and cultivates, secondary to going down to posterity 1 time; Repeat above-mentioned steps, cell is passed more than 10 generations, basis of microscopic observation, is shown in Fig. 1;

By above-mentioned steps and observation, can see that within 17 days, normal chicken embryo spleen lymphocyte is basic all dead afterwards in cultivation, and the fused cell of DF1 and chicken embryo spleen lymphocyte grows normally, show the fusion successfully achieving DF1 and chicken embryo spleen lymphocyte, and obtain the fused cell system of Absorbable organic halogens, suspension culture.In Nostoc commune Vanch liquid, lymphocyte does not divide substantially, and life cycle is shorter, and fused cell then can merisis, and shows the characteristic that can infinitely go down to posterity.

Claims

1. a method for suspended culture cell, it comprises the steps:

(1) utilize cell-fusion techniques DF1 and chicken embryo spleen lymphocyte to be merged, obtain DF1 and the lymphocytic fusion suspension cell line of chicken embryo;

Get spleen by aseptic for 18 age in days SPF chicken embryos, scissors shreds, and adds 1mL pancreatin, and 37 DEG C of water-bath digestion 10min, repeatedly blow and beat for several times, add 10mL 15% MEM nutrient solution, stop digestion, 4 layers of filtered through gauze;

Filtrate is transferred to 15mL centrifuge tube, and carries out cell counting, cell density is 10 ⁶/ mL;

The DF1 getting equivalent mixes with chicken splenic lymphocyte, and the centrifugal 10min of 800rpm, removes supernatant, resuspended with serum-free cell culture medium, the centrifugal 10min of 800rpm, then repeated washing 1 time, removes remaining serum;

Slowly add 9mL serum-free cell culture medium, mixing, the centrifugal 5min of 1000rpm, abandons supernatant; Slowly add 10mL serum-free cell culture medium, re-suspended cell, the centrifugal 5min of 1000rpm, abandons supernatant; Add the cell culture fluid that 5mL serum content is 15%, re-suspended cell, proceed in the disposable Tissue Culture Flask of 5mL, put 37 DEG C of incubators and cultivate;

Meanwhile, separately get a part of spleen suspension, be placed in 15mL centrifuge tube, the centrifugal 5min of 1000rpm, abandons supernatant, adds the cell culture fluid that 5mL serum content is 15%, re-suspended cell, proceeds in the disposable Tissue Culture Flask of 5mL, puts 37 DEG C of incubators and cultivates, in contrast;

(2) screen the fused cell of suspension growth and determine cell stability;

Cultivate 3d, add the cell culture fluid nutrient solution that 2mL serum content is 15%; Cultivate 1 week, rock nutrient solution gently, and collect nutrient solution, the centrifugal 5min of 1000rpm, removes supernatant, adds fresh medium and cultivates, and this is for going down to posterity 1 time; Repeat above-mentioned steps, cell is passed more than 10 generations, basis of microscopic observation.