CN111321119A - Liver cancer cell line suitable for large-scale serum-free adherent culture and establishment method and application thereof - Google Patents
Liver cancer cell line suitable for large-scale serum-free adherent culture and establishment method and application thereof Download PDFInfo
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Abstract
The invention discloses a liver cancer cell line suitable for large-scale serum-free adherent culture and an establishment method and application thereof, wherein serum-free culture medium and microcarrier screening are adopted to obtain a Huh-7 large-scale serum-free adherent culture condition suitable for the liver cancer cell line, and the liver cancer cell line suitable for the large-scale serum-free adherent culture is established through domestication culture. The cell line established by the invention can be used for serum-free preparation of biological products such as vaccines and the like.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a liver cancer cell line suitable for large-scale serum-free adherent culture, and an establishment method and application thereof.
Background
With the rapid development of biomedical technology, cell culture technology is widely applied to the field of biological products. The culture medium used in the cell culture technology is not only a basic substance for supplying nutrition to cells and promoting the growth and proliferation of the cells, but also a living environment for the growth and proliferation of the cultured cells. The culture medium is divided into natural culture medium and synthetic culture medium according to the source, wherein the natural culture medium is most commonly bovine serum. Serum contains multiple active substances necessary for cell growth, such as cell growth factor and adhesion promoting factor.
The most commonly used medium is a basal medium synthesized by adding bovine serum such as M199, DMEM, MEM, or the like. However, due to the complex serum components and the biological risks, serum-free culture has become a trend in the production of biological products. In addition, the existing production process has the problems of low yield, difficult purification and the like.
Disclosure of Invention
The invention aims to: aiming at the defects in the prior art, the liver cancer cell line suitable for large-scale serum-free adherent culture and the establishment method and the application thereof are provided.
The technical scheme adopted by the invention is as follows:
a method for establishing a liver cancer cell line adapting to large-scale serum-free adherent culture comprises the following steps:
recovering the Huh-7 cells and culturing the Huh-7 cells by using a DMEM medium containing 8-12% FBS; after the cells grow full, digesting and passaging by pancreatin, and then culturing by using a DMEM medium containing 3-7% FBS; after the cells grow full, continuing to digest and passage by using pancreatin, and then culturing by using a DMEM medium containing 1-2% FBS; after the cells grow full, continuously digesting and passaging by using pancreatin, and finally culturing by using a CDBHK-21 serum-free culture medium or adding a Cytodex1 microcarrier after resuspending by using the CDBHK-21 serum-free culture medium.
Further, the Huh-7 cells were recovered and cultured in DMEM medium containing 10% FBS; after the cells grow full, digesting and passaging by pancreatin, and then culturing by using a DMEM medium containing 5% FBS; after the cells grow full, continuing to digest and passage with pancreatin, and then culturing with a DMEM medium containing 2% FBS; after the cells grow full, continuously digesting and passaging by using pancreatin, and finally culturing by using a CDBHK-21 serum-free culture medium or adding a Cytodex1 microcarrier after resuspending by using the CDBHK-21 serum-free culture medium.
Further, when subcultured with the CD BHK-21 serum-free medium, the subculture ratio was 1: 2-4.
Further, when subcultured with the CD BHK-21 serum-free medium, the subculture ratio was 1: 3.
the liver cancer cell line established by the method.
The application of the liver cancer cell line suitable for large-scale serum-free adherent culture in expressing foreign protein.
The application of the liver cancer cell line suitable for large-scale serum-free adherent culture in preparation of vaccines.
The application of the liver cancer cell line suitable for large-scale serum-free adherent culture in preparing virus vectors.
In summary, due to the adoption of the technical scheme, the invention has the beneficial effects that:
1. according to the invention, the liver cancer cell line Huh-7 needing serum culture is acclimated into the cell liver cancer cell line suitable for large-scale serum-free adherent growth by screening the CDBHK-21 serum-free culture medium and the Cytodex1 microcarrier, so that the method is suitable for a cell factory culture mode and a bioreactor combined microcarrier culture mode, and has the advantages of simple operation method and good acclimation effect;
2. the method comprises the steps of firstly resuscitating and culturing by using a DMEM medium containing 10% FBS, then culturing by using a DMEM medium containing 5% FBS, then culturing by using a DMEM medium containing 2% FBS, and finally culturing by using a CDBHK-21 serum-free medium or adding Cytodex1 microcarrier after resuspension by using a CDBHK-21 serum-free medium, wherein in the whole domestication culture process, the concentration of serum is gradually reduced, cells are gradually adapted to low serum until the cells grow in the serum-free state, and the cells are prevented from dying due to sudden inadaptation under the serum-free condition; compared with the traditional domestication method and other domestication methods for reducing the gradient, the whole domestication process is smooth, and the method has the advantages that after the cells corresponding to each serum gradient are overgrown, the cells are digested and subcultured by pancreatin, multiple passages are not needed, the whole domestication period is controlled to be completed within 21 days, the domestication period is obviously shortened, the domestication cost is saved, and the subsequent industrial large-scale production is facilitated;
3. the serum-free culture medium used in the invention does not contain animal serum, has low cost and low biological risk, improves the stability of culture, and simplifies the later purification process;
4. the microcarrier used in the invention is combined with serum-free culture medium for culture, so that the application scene of the invention is expanded, the utilization rate of the culture medium is improved through the microcarrier with a large surface area/volume ratio, and a more uniform growth environment is provided;
5. the Huh-7SF cell line has the advantages of good adherence effect, full shape, uniform size, high proliferation speed, high activity and high infection rate, and is suitable for large-scale production.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.
FIG. 1 is a diagram of the cell condition obtained in example 1;
FIG. 2 is a graph of growth of Huh-7SF cells undergoing in vitro proliferation;
FIG. 3 is a graph of the growth curves of Huh-7SF and Huh-7 cells;
FIG. 4 is a graph of the growth of Huh-7SF and Huh-7 cells;
FIG. 5 is a graph comparing the tumorigenicity of Huh-7SF and Huh-7 cells;
FIG. 6 is a graph of mouse tumorigenesis of Huh-7SF cells that were genetically modified (infected with recombinant adenovirus) by irradiation of 200Gy for 2 h;
FIG. 7 is a graph of the tumorigenesis of 2h unirradiated Huh-7SF cell mice genetically modified (recombinant adenovirus infection);
FIG. 8 is a graph showing mouse tumorigenesis of Huh-7SF cells irradiated to 200Gy without gene modification (recombinant adenovirus is not infected);
FIG. 9 is a graph of mouse tumorigenesis of non-genetically modified (recombinant adenovirus not infected) unirradiated Huh-7SF cells;
FIG. 10 is a morphological diagram of Huh-7 cultured in a flask or cell factory with AIM-V medium;
FIG. 11 is a schematic diagram of Huh-7 cultured in a flask or cell factory with CP-SFM medium;
FIG. 12 is a graph showing the morphological results of Huh-7 in a flask or cell factory with CD BHK-21 medium;
FIG. 13 shows the morphology of 24h and 96h Huh-7 cells cultured in VirusPro Medium on microcarriers;
FIG. 14 shows the morphology of 24h and 96h Huh-7 cells on microcarriers cultured in VP-SFM medium;
FIG. 15 is a representation of Huh-7 cells cultured in CD BHK-21 medium for 24h and 96h on microcarriers;
FIG. 16 shows the morphology of 24h and 96h Huh-7 cells cultured in X-VIVO medium on microcarriers;
FIG. 17 is a cell morphology map of Huh-7SF on microcarrier Cytodex 1;
FIG. 18 is a cell morphology map of Huh-7SF on microcarrier Cytodex 3.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the detailed description and specific examples, while indicating the preferred embodiment of the invention, are intended for purposes of illustration only and are not intended to limit the scope of the invention. The components of embodiments of the present invention generally described and illustrated in the figures herein may be arranged and designed in a wide variety of different configurations.
Thus, the following detailed description of the embodiments of the present invention, presented in the figures, is not intended to limit the scope of the invention, as claimed, but is merely representative of selected embodiments of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments of the present invention without making any creative effort, shall fall within the protection scope of the present invention.
It is noted that relational terms such as "first" and "second," and the like, may be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Also, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. Without further limitation, an element defined by the phrase "comprising an … …" does not exclude the presence of other identical elements in a process, method, article, or apparatus that comprises the element.
The features and properties of the present invention are described in further detail below with reference to examples.
Example 1
The liver cancer cell line suitable for large-scale serum-free adherent culture provided by the preferred embodiment of the invention is obtained by the following steps (cell factory culture mode):
recovering the Huh-7 cells and culturing the cells by using a DMEM medium containing 10% FBS; after the cells grow full, digesting and passaging by pancreatin, and then culturing by using a DMEM medium containing 5% FBS; after the cells grow full, digesting and passaging by pancreatin, and then culturing by using a DMEM medium containing 2% FBS; after the cells are full, digesting and passaging by pancreatin according to the passage ratio of 1: 3 subculturing with CD BHK-21 serum-free medium, preserving after continuous passage for 15 times, and naming the cell line as Huh-7SF, wherein the cell condition is shown in figure 1.
Example 2
The liver cancer cell line suitable for large-scale serum-free adherent culture provided by the preferred embodiment of the invention is obtained by the following steps (bioreactor combined with microcarrier culture mode):
recovering the Huh-7 cells and culturing the cells by using a DMEM medium containing 10% FBS; after the cells grow full, digesting and passaging by pancreatin, and then culturing by using a DMEM medium containing 5% FBS; after the cells grow full, digesting and passaging by pancreatin, and then culturing by using a DMEM medium containing 2% FBS; after the cells are full, digesting and passaging by pancreatin according to the passage ratio of 1: 3 resuspending equivalent cells by using a CD BHK-21 serum-free culture medium, and adding a Cytodex1 microcarrier (3g/L) for culture to obtain the cell suspension.
Experimental example 1
In vitro proliferation Using Huh-7SF cells
0.5 x10 inoculations in 6-well plates, respectively5，1.0*105，1.5*105，2.0*105,2.5*105,3*105The growth state and morphology of the cells were observed.
As a result, as shown in FIG. 2, when the amount of the Huh-7SF cell inoculated was small, the growth rate was slow, and the growth became slow, or the cell morphology was likely to change.
Comparison of growth status of Huh-7SF and Huh-7 cells
1. Huh-7SF and Huh-7 with the same cell number were seeded in 6-well plates, and the cell morphology, the cell growth rate, and the cell number were observed.
As shown in FIGS. 3 to 4 and Table 1 below, the cell morphology of Huh-7SF cells did not change much from that of Huh-7SF cells, and the cell proliferation rate was increased.
TABLE 1 cell number comparison Table
Comparison of the tumorigenicity of Huh-7SF and Huh-7 cells
5 nude mice were inoculated with each of Huh-7SF and Huh-7. Inoculating 4-6 weeks female nude mice, each 5x1060.1 ml. Tumor formation was observed.
As shown in FIG. 5, the results of the experiment revealed that the tumorigenicity of Huh-7SF cells was increased.
Experimental example 2
Preparing HBx/IL-12 gene modified (recombinant adenovirus infection) irradiation inactivated Huh-7SF cell vaccine and related experiments:
huh-7SF infection time and irradiation dose screening
1. Time of infection screening
Using the Huh-7SF cells obtained in example 1, subculturing and expanding; Ad-HBx/IL-12 is adopted to infect for 2h, 24h and 48h respectively, and cell proliferation and IL-12 expression are measured by irradiation with 1 piece of copper sheet of 0Gy, 20Gy, 40Gy, 60Gy, 80Gy and 100 Gy. The results were: the expression level of IL-12 after 2h of adenovirus infection is higher than 24h and 48h, and 2h of infection is selected as the optimal infection time.
2. Screening of irradiation dose
Using the Huh-7SF cells obtained in example 1, subculturing and expanding; irradiation with 1 piece of copper sheet, 0Gy, 80Gy, 100Gy, 120Gy, 150Gy, 180Gy, 200Gy irradiation dose was performed to determine whether cells were monocloned and to determine cell survival rate and IL-12 expression. Combining the results, the high irradiation dose, namely irradiation 200Gy, is selected as the optimal irradiation dose.
Experiment of tumorigenesis after Huh-7SF cell infection irradiation
Preparing a nude mouse inoculated with 4 cells, wherein the nude mouse is inoculated with 5x10 cells, and each nude mouse is inoculated with 5x10 cells60.1ml, nude mice were allowed to grow for 20 weeks to observe tumor growth.
As shown in FIGS. 6 to 9, after 200Gy irradiation of 2h of Huh-7SF gene modification (infection), no tumor formation occurs in the nude mice, and the cell vaccine prepared by using the Huh-7SF cell on the surface has good safety.
Experimental example 3
Culturing the recovered Huh-7 cells by using DMEM containing 10% FBS after recovery; after the cells are full, carrying out digestion and passage by using pancreatin, and culturing by using DMEM containing 5% FBS; after the cells are full, carrying out digestion and passage by using pancreatin, and culturing by using DMEM containing 2% FBS; after the cells are full, the cells are subjected to trypsinization and passaged, and the equivalent cells are respectively screened by three serum-free culture media of AIM-V, VP-SFM and CD BHK-21.
As shown in FIGS. 10-12, the cells cultured with AIM-V and VP-SFM had poor adherence, and the cells cultured with CD BHK-21 had good adherence and faster proliferation.
Experimental example 4
Culturing the recovered Huh-7 cells by using DMEM containing 10% FBS after recovery; after the cells are full, carrying out digestion and passage by using pancreatin, and culturing by using DMEM containing 5% FBS; after the cells are full, carrying out digestion and passage by using pancreatin, and culturing by using DMEM containing 2% FBS; after the cells are full, the cells are subjected to trypsinization passage, and the cells are screened and cultured by four serum-free culture mediums of VirusPro, X-VIVO 15, VP-SFM and CD BHK-21 respectively, namely the basic suspension equivalent amount of the cells and cytodex1 microcarrier.
The state of the cells inoculated for 24h and 96h was observed, respectively, and the results are shown in FIGS. 13 to 16 and Table 2 below.
TABLE 2 microcarrier culture table
The results show that the adherence of the cells cultured by X-VIVO 15 and VP-SFM is poor, and the cells are easy to fall off from the microcarrier; the adherence of VirusPro cultured cells is poor until the cells die in the later period; in the culture of CD BHK-21, the cells adhere well and proliferate quickly.
Experimental example 5
Culturing the recovered Huh-7 cells by using DMEM containing 10% FBS after recovery; after the cells are full, carrying out digestion and passage by using pancreatin, and culturing by using DMEM containing 5% FBS; after the cells are full, carrying out digestion and passage by using pancreatin, and culturing by using DMEM containing 2% FBS; after the cells are full, trypsinizing for passage, resuspending equivalent cells by using a CD BHK-21 serum-free culture medium, adding a Cytodex1 microcarrier or a Cytodex3 microcarrier for screening culture, and continuing to culture for 24-48 hours after about 70-80% of cells on the microcarrier are fused and Ad-EGFP recombinant adenovirus is added for infection for 2 hours.
The cell state was observed in white light and fluorescence, respectively, and the results are shown in FIGS. 17-18, where the cells adhered well to the Cytodex1 microcarriers and the infection efficiency was high, while the cells adhered poorly to the Cytodex3 microcarriers and the cells were easy to shed.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.
Claims (8)
1. A method for establishing a liver cancer cell line adapting to large-scale serum-free adherent culture is characterized by comprising the following steps:
recovering the Huh-7 cells and culturing the Huh-7 cells by using a DMEM medium containing 8-12% FBS; after the cells grow full, digesting and passaging by pancreatin, and then culturing by using a DMEM medium containing 3-7% FBS; after the cells grow full, continuing to digest and passage by using pancreatin, and then culturing by using a DMEM medium containing 1-2% FBS; after the cells grow full, continuously digesting and passaging by using pancreatin, and finally culturing by using a CDBHK-21 serum-free culture medium or adding a Cytodex1 microcarrier after resuspending by using the CDBHK-21 serum-free culture medium.
2. The method for establishing the liver cancer cell line adapting to the large-scale serum-free adherent culture according to claim 1, which comprises the following steps:
recovering the Huh-7 cells and culturing the cells by using a DMEM medium containing 10% FBS; after the cells grow full, digesting and passaging by pancreatin, and then culturing by using a DMEM medium containing 5% FBS; after the cells grow full, continuing to digest and passage with pancreatin, and then culturing with a DMEM medium containing 2% FBS; after the cells grow full, continuously digesting and passaging by using pancreatin, and finally culturing by using a CDBHK-21 serum-free culture medium or adding a Cytodex1 microcarrier after resuspending by using the CDBHK-21 serum-free culture medium.
3. The method for establishing a liver cancer cell line adapted to large-scale serum-free adherent culture according to claim 1 or 2, wherein the subculture is performed in a CD BHK-21 serum-free medium at a passage ratio of 1: 2-4.
4. The method for establishing the hepatoma cell line adaptive to large-scale serum-free adherent culture according to claim 3, wherein the subculture is performed by using a CD BHK-21 serum-free medium, wherein the subculture ratio is 1: 3.
5. a liver cancer cell line established by the method of any one of claims 1-4.
6. The use of the hepatoma carcinoma cell line adapted to large-scale serum-free adherent culture of claim 5 for expressing foreign proteins.
7. The use of the hepatoma carcinoma cell line adapted to large-scale serum-free adherent culture according to claim 5 for the preparation of a vaccine.
8. The use of the hepatoma carcinoma cell line adapted to large-scale serum-free adherent culture according to claim 5 for the preparation of viral vectors.
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| US20070172846A1 (en) * | 2005-11-12 | 2007-07-26 | Introgen Therapeutics, Inc. | Methods for the Production and Purification of Adenoviral Vectors |
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| US20150204855A1 (en) * | 2013-02-01 | 2015-07-23 | Human Provincial Tumor Hospital | Human Hepatoma Cell Line HLCZ01 and Uses Thereof |
| CN105132375A (en) * | 2015-09-16 | 2015-12-09 | 广西壮族自治区肿瘤医院 | Serum-free medium for liver cancer stem cells and culture method for serum-free medium |
| CN105861441A (en) * | 2016-04-01 | 2016-08-17 | 中国人民解放军第二军医大学 | Hepatoma cell line STL-C1 derived from human hepatoma a-carcinoma tissue and establishment method thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| US20070172846A1 (en) * | 2005-11-12 | 2007-07-26 | Introgen Therapeutics, Inc. | Methods for the Production and Purification of Adenoviral Vectors |
| CN102604889A (en) * | 2012-03-19 | 2012-07-25 | 中国农业科学院哈尔滨兽医研究所 | HEK (human embryonic kidney) 293 cell line applicable to serum-free culture and application thereof |
| US20150204855A1 (en) * | 2013-02-01 | 2015-07-23 | Human Provincial Tumor Hospital | Human Hepatoma Cell Line HLCZ01 and Uses Thereof |
| CN103352027A (en) * | 2013-05-07 | 2013-10-16 | 中国人民解放军第二军医大学 | Tumor stem cell suspension culture method |
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