CN113337473A - Serum-free suspension culture domestication method of engineering cell strain FCHO/IL-24 - Google Patents
Serum-free suspension culture domestication method of engineering cell strain FCHO/IL-24 Download PDFInfo
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Abstract
The invention discloses a serum-free suspension culture domestication method of an engineering cell strain FCHO/IL-24, which comprises the following steps: (1) the serum concentration is reduced through gradient, and the domesticated engineering cell strain FCHO/IL-24 can be cultured under the condition of 0.5 percent of serum adherence; (2) directly acclimating the engineering cell strain FCHO/IL-24 obtained in the step (1) from 0.5% serum adherent culture to 0.5% serum suspension culture; (3) and (3) carrying out suspension culture on the engineering cell strain FCHO/IL-24 obtained in the step (2) in a serum-free culture medium. According to the method, the serum concentration is reduced through gradient, the domesticated engineering cell strain FCHO/IL-24 can be cultured on the condition of 0.5% serum adherence, then suspension culture is carried out, and finally the domesticated engineering cell strain FCHO/IL-24 can be subjected to suspension culture in a serum-free culture medium, so that the high cell density can be maintained, rhIL-24 can be stably secreted and expressed, and a foundation is laid for the subsequent optimization of suspension culture conditions and serum-free culture medium components.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a serum-free suspension culture domestication method of an engineering cell strain FCHO/IL-24.
Background
Cancer is a malignant disease that seriously threatens human health and is the first cause of death in the world. Although the medical technology improves the resection rate of solid tumors after surgery and the emerging various chemotherapeutics and targeted drugs improve the five-year survival rate of tumor patients and obviously improve the life quality of the patients, the relapse caused by the characteristics of malignant infiltration and metastasis growth of tumor cells, the drug resistance of the anti-tumor drugs and the damage to normal cells cause that the tumor patients have to endure serious toxic and side effects of the drugs, otherwise, the treatment can only be stopped. The development of a treatment method with low toxicity, strong specificity and good curative effect is urgently needed to effectively control the malignant process of the tumor and search for an anti-tumor medicament which can specifically eliminate malignant tumor cells and has no toxic or side effect on normal cells, and the method is always a research hotspot of tumor biology.
Significantly, Jiang et al, 1995, first reported that melanoma differentiation-associated gene-7/interleukin 24 (mda-7/IL-24) was capable of inhibiting melanoma cell proliferation, encoding a protein consisting of 206 amino acid residues with a molecular weight of approximately 23.8 kDa. Subsequent researches find that the IL-24 has broad-spectrum and specific antitumor activity, can specifically inhibit the growth of various tumors in vitro and in vivo, selectively induces the apoptosis of tumor cells, has almost no influence on normal cells, does not depend on the expression levels of genes such as p53, p21, Rb and the like, and is obviously different from other tumor-inhibiting genes.Meanwhile, IL-24 has the ability of stimulating the anti-tumor immune response of the body, can stimulate the secretion of peripheral blood mononuclear cells to produce inflammatory cytokines such as IL-6, IL-1 beta, IL-12, GM-CSF, IFN-gamma and the like, and can also increase CD3+CD8+The number of T cells can trigger the body to generate immune response for inhibiting tumor cells at the primary part and tumor cells at the metastatic part, which is a characteristic that cancer inhibition genes such as p53 and Rb do not have. Moreover, IL-24 can enhance tumor cell chemoradiotherapy sensitivity, and can produce additive or synergistic antitumor effect when combined with targeted drugs, chemotherapeutic drugs or radiotherapy. Because of the characteristics, the IL-24 is called a magic bullet for treating tumors, and the intensive research on the magic bullet not only has important theoretical significance, but also has important clinical application value.
In the use of IL-24 in clinical tumor therapy studies, IL-24 recombinant adenovirus, "INGN 241", was first developed by Introgen Therapeutics, USA. The I-phase clinical test result published by the company in 2002 shows that the INGN241 has good treatment effect on 28 patients with solid tumors (breast cancer, lung cancer and colon cancer), but the development of adenovirus is limited by defects (such as targeting, effective elimination, biological safety and the like), and no report is available in II-phase clinical tests from 2005, which suggests that the safety problem may be one of the main reasons. Compared with adenovirus, the protein anti-cancer medicine is safer. Compared with the traditional chemical synthesis medicines, the protein medicines become the main varieties of new biotechnological medicines, and the medicines are very close to normal physiological substances in vivo and have the characteristics of high pharmacological activity, small side effect, small dosage, strong biological activity, good curative effect and the like. At present, rhIL-24 is expressed in Escherichia coli, yeast, insect and mammal cells, but the biological activities of genetically engineered proteins obtained in different host cells have significant differences. Escherichia coli is easy to produce endotoxin, foreign proteins cannot be folded correctly, and the foreign proteins cannot be subjected to correct post-translational modification, which is also the reason that the rhIL-24 expressed by the Escherichia coli has extremely low biological activity. Yeast, which can be easily glycosylated but is limited to high mannose type glycosyl modification; insect cells, which are capable of complex glycosylation modification, are also limited to simple mannose-type glycosylation, and there is little literature on rhIL-24 expression using these two systems. The rhIL-24 expressed by Escherichia coli and yeast has the immunoregulation effect, but the dosage is large (the unit is mu g/mL), while the dosage unit of the rhIL-24 secreted and expressed in the supernatant of HEK293 cells is ng/mL; similarly, in the experiment that the rhIL-24 inhibits the proliferation of melanoma cells in vitro, the rhIL-24 expressed by escherichia coli shows activity at the level of mu g/mL, and the rhIL-24 secreted and expressed by mammalian cells has obvious tumor inhibition effect at the level of 5 ng/mL. The rhIL-24 secreted and expressed by the mammalian cells can inhibit the proliferation of various tumor cells, such as esophageal cancer, melanoma, pancreatic cancer, lung cancer, prostatic cancer, cervical cancer, breast cancer and the like. These results indicate that the rhIL-24 secreted and expressed by mammalian cells has high-efficiency and specific antitumor activity.
The expression of rhIL-24 by mammalian cells is mostly realized by constructing a stably expressed HEK293 cell line by using a recombinant plasmid pCEP4-IL-24 or infecting immortalized human normal cells or tumor cells by using recombinant adenovirus Ad-IL-24. A CHO fixed-point integration cell strain FCHO/IL-24 for expressing rhIL-24 is constructed by utilizing a Flp-In system, and the expression level of the rhIL-24 is far higher than that of three randomly integrated HEK293 cell strains (HEK293/pSecTag2A-IL-24, HEK 293/pcDNA3.1myc/His-IL-24 and HEK293/pCEP4-IL-24) under the same cell density and culture time.
Site-directed integration of engineered cell line FCHO/IL-24 was cultured adherently in F12 medium containing 10% serum, resulting in limited levels of secreted expression of rhIL-24. The large-scale culture of animal cells is always a hot spot in the research field of cell culture, and how to improve the expression level of exogenous proteins in mammalian cells is more important. The large-scale production of the bio-pharmaceutical industry depends on the serum-free suspension culture of the engineering cells, the culture mode not only overcomes various defects of animal serum, but also can be used for long-term continuous culture, is easy to expand the production capacity and control the culture conditions, has few operation steps and few pollution opportunities, and is an effective method for improving the protein level of the engineering cell strain expression target. In the research aiming at the expression of rhIL-24 by mammalian cells, no report for further improving the level of the rhIL-24 expressed by an engineering cell strain FCHO/IL-24 exists at present, and no report for serum-free suspension domestication of the cell strain exists.
Disclosure of Invention
In order to solve the problems of the background art, the invention aims to provide a method for the serum-free suspension culture acclimatization of an engineering cell strain FCHO/IL-24. According to the method, the serum concentration is reduced through gradient, the domesticated engineering cell strain FCHO/IL-24 can be cultured on the condition of 0.5% serum adherence, then suspension culture is carried out, and finally the domesticated engineering cell strain FCHO/IL-24 can be subjected to suspension culture in a serum-free culture medium, so that the high cell density can be maintained, rhIL-24 can be stably secreted and expressed, and a foundation is laid for the subsequent optimization of suspension culture conditions and serum-free culture medium components.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows: a serum-free suspension culture domestication method of an engineering cell strain FCHO/IL-24 comprises the following steps:
(1) the serum concentration is reduced through gradient, and the domesticated engineering cell strain FCHO/IL-24 can be cultured under the condition of 0.5 percent of serum adherence;
(2) directly acclimating the engineering cell strain FCHO/IL-24 obtained in the step (1) from 0.5% serum adherent culture to 0.5% serum suspension culture;
(3) and (3) carrying out suspension culture on the engineering cell strain FCHO/IL-24 obtained in the step (2) in a serum-free culture medium.
Further, the basic culture medium for culturing the engineered cell strain FCHO/IL-24 in the step (1) under the 0.5% serum adherent condition is 1640 or DMEM/F12, and is preferably DMEM/F12.
Further, the gradient in step (1) reduces the serum concentration by a gradient from 10%, 5%, 2%, 1% to 0.5% of the serum concentration.
Further, the step (1) is specifically as follows: culturing the FCHO/IL-24 engineering cell strain in F12 culture medium containing 10% serum, carrying out passage at a ratio of 1:3 every 2 days, changing into DMEM/F12 culture medium containing 10% serum during passage, and carrying out continuous passage for 3 times; then reducing the serum concentration to 5%, and continuously carrying out passage for 7-8 times under the condition of 5% serum; reducing the serum to 2%, after passage for 7-8 times under the concentration of 2% serum, reducing the serum to 1%, continuously culturing for 10 times, culturing the cells in DMEM/F12 containing 0.5% serum, and continuously culturing for 12 times; the passage intervals and the passage ratios at different serum concentrations were consistent with those of 10% serum.
Further, the inoculation density of the suspension culture in the step (2) is 5X 105Individual cells/mL.
Further, the rotation speed of the suspension culture in the step (2) was 119 rpm.
Further, the serum-free medium in the step (3) is EdenTM-B300S or CDM4CHO, preferably EdenTM-B300S。
Further, the inoculation density of the suspension culture in the step (3) is 5X 105Individual cells/mL.
Further, the rotation speed of the suspension culture in the step (3) was 119 rpm.
The invention has the beneficial effects that: the serum-free suspension culture of the engineering cells is the first step of large-scale culture by using a fermentation tank, and lays a foundation for the subsequent optimization of suspension culture conditions and serum-free medium components; through the stages of serum-free medium optimization, culture condition optimization and the like, the cell culture density and the expression level of the target protein rhIL-24 can be obviously provided, and the clinical tumor treatment experiment of the rhIL-24 can be promoted effectively.
Drawings
FIG. 1A shows the cell morphology under light microscope on the third day of the passage of FCHO/IL-24 cells cultured in F12 containing 10% serum in example 1 of the present invention; FIG. 1B shows the cell morphology of FCHO/IL-24 cells in example 1 of the present invention, which were subcultured for the third day in three media of 1640, DMEM/F12 and DMEM at different serum concentrations (2%, 5% and 10%); FIG. 1C is a graph showing the relative viability of FCHO/IL-24 cells in different media containing 5% serum, from day 1 to day 5, in example 1 of the present invention; FIG. 1D is a graph showing the relative viability of FCHO/IL-24 cells in different media containing 2% serum for days 1-5 in example 1 of the present invention;
FIG. 2A shows the cell morphology of FCHO/IL-24 cells in example 1 of the present invention, which were cultured in DMEM/F12 and 1640 medium with different serum concentrations (1% and no serum), and passaged for the third day; FIG. 2B is a graph showing the relative viability of FCHO/IL-24 cells in different media containing 1% serum for days 1-5 in example 1 of the present invention; FIG. 2C is a graph showing the relative viability of FCHO/IL-24 cells in different serum-free media from day 1 to day 5 in example 1 of the present invention;
FIG. 3A shows the cell morphology of the engineered cells acclimatized in example 2 of the present invention at different serum concentrations (0.5%, 1%, 2%, 5% and 10%); FIG. 3B is a graph showing the growth of cells at different serum concentrations in example 2 of the present invention; FIG. 3C is the relative cell viability corresponding to FIG. 3B;
FIG. 4A is a graph showing the growth curve of FCHO/IL-24 cells in DMEM/F12 medium containing 0.5% serum, directly transferred from adherent culture to suspension culture in example 3 of the present invention; FIG. 4B is a growth curve of FCHO/IL-24 cells in suspension culture in DMEM/F12 medium containing 0.5% serum for screening suitable cell seeding concentration in example 3 of the present invention; FIG. 4C shows the rhIL-24 protein concentration in the supernatant of FCHO/IL-24 cells cultured in 0.5% serum suspension on day 3 in example 3 of the present invention; FIG. 4D shows the cell specific productivity of FCHO/IL-24 cells cultured in 0.5% serum suspension on day 3 in example 3 of the present invention; FIG. 4E is the variation of rhIL-24 concentration in the supernatant of FCHO/IL-24 cells cultured in suspension in 0.5% serum for 24 days in example 3 of the present invention;
FIG. 5A shows the cell lines of example 4 of the present invention in two serum-free media (Eden)TM-B300S and CDM4 CHO); FIG. 5B shows the rhIL-24 concentration in the cell culture supernatant in example 4 of the present invention; FIG. 5C shows the specific productivity of cell lines in cell culture supernatants of example 4 of the present invention.
Detailed Description
For a better understanding of the present invention, the following examples are given to illustrate the present invention, but the present invention is not limited to the following examples.
The reagents employed in the examples of this application include: cell culture media F12, DMEM/F12, DMEM and 1640, purchased from Gibco; human interleukin-24 (IL-24) ELISA kit (F01531), purchased from shanghai west down biotechnology; serum-free medium CDM4CHO (SH30557) from Hyclone; serum-free medium EdenTMB300S, present by professor ma tan song at the university of east china. The site-directed integration engineering cell Flp-In-CHO/IL-24 (FCHO/IL-24) for stably secreting and expressing rhIL-24 is constructed and stored by a subject group.
Example 1
1) DMEM/F12 and 1640 culture medium is more suitable for 5% and 2% serum adherent culture of the engineered cells FCHO/IL-24
Firstly, 200 microliters of F12 culture medium containing 10% serum is used for inoculating FCHO/IL-24 cells in a 96-well plate, 2000 cells are placed in each well, after the cells are attached and spread, four culture media of F12, 1640, DMEM and DMEM/F12 with serum concentrations of 10%, 5% and 2% respectively are replaced, 10% serum is used as a control group, and the morphology of the cells is observed under a light mirror.
FCHO/IL-24 cells were cultured in F12 containing 10% serum, and cell morphology was observed under a light microscope on the third day of passage, as shown in FIG. 1A (magnification of 100-fold and 200-fold, respectively), and when F12 medium reduced serum to 5% or less, a large number of cells died (results not shown); the cell morphology at the third day of passage in three media, 1640, DMEM and DMEM/F12, at different serum concentrations (2%, 5% and 10%) is shown in FIG. 1B, and it can be seen from FIG. 1B that the cell morphology gradually tends to be round in DMEM medium with less stretching and normal in 1640 and DMEM/F12 as the serum concentration decreases.
The MTT method detects the cell proliferation, 650 FCHO/IL-24 cells are laid in each hole of a 96-hole plate, 200 mu L of culture medium is laid in each hole, and 10 multiple holes are arranged in each serum concentration group (10%, 5%, 2%, 1%, 0.5% and no serum) when the cell proliferation activity is determined; cells are not spread in one circle of the peripheral holes, 200 mu L of culture medium is added into 3 holes to serve as a blank control, and 200 mu L of PBS is added into the other holes; 10% serum was used as a control. One plate was measured every day, 20. mu.L of MTT was added to each well, after incubation for 4h in an incubator, the supernatant in the well plate was carefully aspirated, 150. mu.L of DMSO was added to each well, well dissolved, and the OD490 value was measured with a microplate reader. Subtracting the OD value of a blank well (only culture medium) from the measured OD value, simultaneously removing the maximum value and the minimum value from 10 numerical values, and calculating the average value; and calculating the ratio of the average value of the control group to obtain the relative cell activity. The remaining plates were changed every 3 days and the serum concentration was kept constant. The relative viability of FCHO/IL-24 cells in different media containing 5% and 2% serum from day 1 to day 5 is shown in FIGS. 1C and 1D, respectively. As can be seen from fig. 1C, under 5% serum conditions, the cell viability was above 90% in the first 3 days of DMEM/F12 and 1640 groups, and was above 80% in most relative cell viability until day 5, with 10% serum control each; the relative cell activity of the DMEM group is obviously reduced along with the increase of the culture days on the whole, and is reduced to about 50 percent at least. As can be seen from FIG. 1D, at 2% serum concentration, DMEM/F12 and 1640 groups were stable above 90% for the first 3 days, and then decreased to below 60% at the lowest level; the relative cell viability was below 50% after day 3 in the DMEM group.
The cell state is still good when the DMEM/F12 and 1640 culture medium groups are combined with MTT results and the serum concentration is 2%, which indicates that the two culture media are more suitable for reducing the serum.
2) The DMEM/F12 culture medium is more suitable for FCHO/IL-24 cell 1% serum and serum-free culture
According to the same experimental method, the proliferation of the cell lines in DMEM/F12 and 1640 medium containing 1% serum and serum-free was compared by morphology and cell proliferation activity. As shown in FIG. 2A, the DMEM/F12 group cultured on the third day is full and uniform in color, the 1640 group appears dark, and the difference between the two groups is most obvious particularly in the serum-free condition. The MTT results of FIGS. 2B and 2C show that the DMEM/F12 medium group is relatively more viable than the cells. Although the relative cell viability decreased throughout the process, after day 3, the relative cell viability was higher in the DMEM/F12 group than in the 1640 group (. p < 0.01). By combining the cell morphology and the cell activity, DMEM/F12 is more suitable to be used as a basic culture medium for low-serum domestication of engineering cell strains.
Example 2
By reducing the serum concentration through gradient, the domesticated engineered cells FCHO/IL-24 can grow in a DMEM-F12 culture medium containing 0.5% serum in an adherent way.
Firstly, the engineering cell strain is cultured in F12 culture medium containing 10% serum, the culture medium is replaced by DMEM/F12 culture medium containing 10% serum when in passage, the cell growth rate is not slowed down after 3 times of continuous passage, and the cell strain can still be passaged according to the ratio of 1:3 every 2 days. Secondly, the serum concentration is reduced to 5%, the growth speed of the cells is slightly reduced, and after 8 times of passage, the passage interval and the passage ratio are consistent with 10% of serum, namely, the passage is performed at 1:3 every 2 days. Then, reducing the serum to 2%, ensuring that the adaptive state of the cells is slightly good, but still a plurality of cells are not attached to the wall, washing off floating cells during passage, only retaining adherent cells, and obviously increasing the adherent cells after passage for 5-6 times; after approximately 7-8 passages, the passage interval tended to stabilize at 1:3 passages every 2 days. Finally, with the same passaging procedure, the serum was gradually reduced to 1% and 0.5%, taking about 4 months. Under the condition of 10% serum, the cell is generally passaged once every 2 days at a ratio of 1:3, the cell growth rate is reduced after the serum is reduced, the inoculation density can be improved during the passage, and the passage interval and the passage proportion are ensured as far as possible. In the past, when the cells are passaged for 2-3 passages under the 5% serum condition, the serum concentration is reduced to 2%, but the cells are poorly adapted, a large number of cells die, the turbid phenomenon of a culture medium appears, and the cells are only abandoned until the serum concentration is reduced after the cells are continuously passaged for 7-8 times under the 5% serum condition.
Cells cultured under different serum concentrations were frozen in time, and cell morphology under a light microscope was observed, and cell proliferation activity was analyzed by the MTT method, with the results shown in fig. 3.
As can be seen from FIG. 3A, the cells grow well adherent to the skin under different serum concentrations, and the morphology of the cells is similar to that of the cells under the 10% serum condition after continuous culture and passage for many times. As can be seen from fig. 3B, the growth curves of the cells under the 10% and 5% serum conditions almost agreed, but the growth rate of the cells was significantly affected when the serum concentration was reduced to 2% and below. In particular, the cell number was significantly reduced in the 0.5% serum compared to the 10% serum. Fig. 3C is the relative cell viability corresponding to 3B. The engineered cells were able to grow in DMEM/F12 medium at 0.5% serum by reducing the serum concentration through a gradient.
Example 3
1) Through suspension culture experiments, it is clear that 0.5% serum adherent domesticated FCHO/IL-24 cells can be directly subjected to suspension culture. Will be in DM with 0.5% serumCells cultured adherently in EM/F12 medium were suspension cultured in 125mL shake flasks at shaker rotation speed of 119rpm and placed in a 37 ℃ cell incubator. Three inoculation densities were set, 5X 104、1×105、2×105Individual cells/mL, inoculum volume 20 mL. On the 1 st to 10 th days of inoculation, 0.1mL of shaking culture solution is respectively taken, cells are collected by centrifugation, and the cells are counted after trypan blue staining, and the growth curve of the cells is drawn. FIG. 4A shows that cells can grow stably, with maximum viable cell density for 6, 5, and 4 days at three seeding densities, and with greater seeding densities for a shorter time to reach maximum viable cell density; subsequently, the viable cell density decreased significantly, especially after day 7 of culture. And (4) prompting: FCHO-IL-24 cells can be directly adapted to suspension culture.
2) The selection of cell seeding density was performed on the basis that FCHO/IL-24 cells could be cultured in suspension in DMEM/F12 medium containing 0.5% serum. 6 different inoculation densities (1X 10) were selected6，5×105，2×105，1×105，5×104Individual cells/mL), the conditions of the remaining suspension cultures were unchanged, cells were counted after staining with trypan blue as above, and cell growth curves were plotted for days 1-9. As can be seen from FIG. 4B, the higher the seeding density, the earlier the time until the cells reach the maximum density. At 1X 106Maximum density was reached at day 4 at individual cell/mL density and at 5X 104The maximum density was reached before and after day 5 at individual cells/mL density. To compare the effect of different vaccination densities on protein expression levels, three vaccination densities (1X 10) were selected6，5×105， 2.5×105Individual cells/mL) was collected, culture supernatant on day 3 was collected, and IL-24 concentration in the supernatant was measured by ELISA, and fig. 4C and 4D showed that on day 3 of suspension culture, the seeding density had a significant effect on the level of rhIL-24 secreted and expressed by the cell line, and the higher the seeding density, the higher the final rhIL-24 concentration, but the lower the specific productivity of the cells (the amount of target protein expressed per unit time of a single cell, i.e., the amount of rhIL-24 produced by 1 cell in 1 day). Suggesting that the lack of nutrients in the medium may be a major factor limiting the expression level of single cells. Comprehensive consideration, final selection5×105The subsequent experiments were performed at a seeding density of individual cells/mL. Cells were seeded at the selected density and cultured for 24 days by serial subculture in suspension, and the rhIL-24 concentration in the supernatant stabilized around 3.5ng/mL, FIG. 4E.
Example 4
The optimal cell inoculation density of the FCHO/IL-24 cells in DMEM/F12-0.5% serum suspension culture is 5 x 105On a cell/mL basis, two serum-free media (Eden) were used directlyTM-B300S and CDM4CHO) under constant suspension culture conditions, i.e. 20mL of cells in 125mL shake flasks at a cell density of 5X 105cells/mL at 119rpm were cultured continuously for 10 days. Cell growth curves were plotted by cell counting after trypan blue staining, and rhIL-24 concentration in the culture supernatants was measured by ELISA. As can be seen from FIG. 5A, the cells were able to grow directly in two commercial serum-free media, with no apparent clumping; in EdenTMThe density of the cell strain in-B300S is much higher than that of CDM4CHO, and the maximum viable cell density is 3 times of the seeding density, which is about 1.6X 106cells/mL; maximum density was reached on day 6 and day 5, respectively. Similarly, the engineering cell strain can secrete and express rhIL-24 in two serum-free culture media, the concentration of the rhIL-24 in the supernatant of the first 6 days of culture is increased faster, and the concentration of the rhIL-24 is slowed down after the 6 th day; in EdenTMIn B300S serum-free medium, the cell strain secreted rhIL-24 at a level significantly higher than that of CDM4CHO, FIG. 5B; the specific yields all reached the highest on day 2, fig. 5C. The results show that the cell strain is more suitable for Eden without serum baseTMSuspension culture in-B300S.
Comparative example
Applicants initially performed adherent cultures directly in several commercial serum-free media, with the result that the cells were almost totally dead and completely unable to grow normally. Then, when the conventional medium containing 10% serum was replaced with a commercial serum-free medium in a gradient, the cells were hardly adapted and were unable to proliferate normally.
The above description is only a specific embodiment of the present invention, and not all embodiments, and any equivalent modifications of the technical solutions of the present invention, which are made by those skilled in the art through reading the present specification, are covered by the claims of the present invention.
Claims (9)
1. A serum-free suspension culture domestication method of an engineering cell strain FCHO/IL-24 is characterized by comprising the following steps:
(1) the serum concentration is reduced through gradient, and the domesticated engineering cell strain FCHO/IL-24 can be cultured under the condition of 0.5 percent of serum adherence;
(2) directly acclimating the engineering cell strain FCHO/IL-24 obtained in the step (1) from 0.5% serum adherent culture to 0.5% serum suspension culture;
(3) and (3) carrying out suspension culture on the engineering cell strain FCHO/IL-24 obtained in the step (2) in a serum-free culture medium.
2. The method for the serum-free suspension culture domestication of the engineering cell line FCHO/IL-24 according to claim 1, wherein the basic culture medium for culturing the engineering cell line FCHO/IL-24 in the step (1) under the 0.5% serum-adherent condition is 1640 or DMEM/F12, preferably DMEM/F12.
3. The method for the serum-free suspension culture acclimatization of the engineered cell line FCHO/IL-24 according to claim 2, wherein the gradient-decreasing serum concentration in the step (1) is a gradient-decreasing serum concentration from 10%, 5%, 2%, 1% to 0.5%.
4. The method for the serum-free suspension culture and domestication of the engineered cell line FCHO/IL-24 according to claim 3, wherein the step (1) is specifically as follows: culturing the FCHO/IL-24 engineering cell strain in F12 culture medium containing 10% serum, carrying out passage at a ratio of 1:3 every 2 days, changing into DMEM/F12 culture medium containing 10% serum during passage, and carrying out continuous passage for 3 times; then reducing the serum concentration to 5%, and continuously carrying out passage for 7-8 times under the condition of 5% serum; reducing the serum to 2%, after passage for 7-8 times under the concentration of 2% serum, reducing the serum to 1%, continuously culturing for 10 times, culturing the cells in DMEM/F12 containing 0.5% serum, and continuously culturing for 12 times; the passage intervals and the passage ratios at different serum concentrations were consistent with those of 10% serum.
5. The method for the serum-free suspension culture acclimatization of the engineered cell line FCHO/IL-24 according to claim 1, wherein the seeding density in the suspension culture in the step (2) is 5 x 105Individual cells/mL.
6. The method for acclimatizing the engineered cell line FCHO/IL-24 in serum-free suspension culture according to claim 5, wherein the rotation speed of the suspension culture in the step (2) is 119 rpm.
7. The method for the serum-free suspension culture acclimatization of the engineered cell line FCHO/IL-24 according to claim 1, wherein the serum-free medium in the step (3) is EdenTM-B300S or CDM4CHO, preferably EdenTM-B300S。
8. The method for the serum-free suspension culture acclimatization of the engineered cell line FCHO/IL-24 according to claim 1, wherein the seeding density in the suspension culture in the step (3) is 5 x 105Individual cells/mL.
9. The method for serum-free suspension culture acclimatization of the engineered cell line FCHO/IL-24 according to claim 8, wherein the rotation speed of the suspension culture in the step (3) is 119 rpm.
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| CN101302492A (en) * | 2007-05-08 | 2008-11-12 | 中美奥达生物技术(北京)有限公司 | Method for quickly establishing suspending, high-density engineering cell strain |
| CN106987554A (en) * | 2017-04-17 | 2017-07-28 | 广东顺德工业设计研究院(广东顺德创新设计研究院) | Suspension cell strain and its acclimation method |
| CN111233996A (en) * | 2020-01-20 | 2020-06-05 | 北京交通大学 | Method for promoting engineered cell strain to secrete and express rhIL-24 by using sodium butyrate |
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| CN101302492A (en) * | 2007-05-08 | 2008-11-12 | 中美奥达生物技术(北京)有限公司 | Method for quickly establishing suspending, high-density engineering cell strain |
| CN106987554A (en) * | 2017-04-17 | 2017-07-28 | 广东顺德工业设计研究院(广东顺德创新设计研究院) | Suspension cell strain and its acclimation method |
| CN111233996A (en) * | 2020-01-20 | 2020-06-05 | 北京交通大学 | Method for promoting engineered cell strain to secrete and express rhIL-24 by using sodium butyrate |
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