As one of the most effective eukaryotic expression systems, suspension culture and transfection of HEK293 cells is an important means for transient expression of recombinant antibodies and other recombinant proteins. In the past 10 years, the HEK293 expression system has successfully achieved the expression of more than 6,000 recombinant proteins (including antibodies), and the amount of HEK293 transient transfection serum-free medium has exceeded 200,000 liters. With the rapid development of structural biology and biomacromolecule drug research in recent years, the demand for HEK293 transient expression system is also increasing. Today, we have compiled some experiences of HEK293 suspension culture and transfection to share with you.
1.Selection of cell lines
The common cell lines of HEK293 cells are HEK293F, HEK293E, HEK293T, HEK293FT, HEK293H and HEK293SHEK293F is the most commonly used cell line type for suspension culture and transient transfection of the protein of interest.
2.Choice of medium
HEK293 cells are generally used for suspension cultureSerum-free mediumPerform, because no serum is added during use, the cost is relatively low, and experimental production batch variations caused by sero-derived contamination and instability of serum ** and batch can be avoided.
3.Equipment conditions required for cultivation
HEK293 cell suspension culture can be divided into two methods: shake flask culture and bioreactor culture. The equipment required for shake flask culture includes: biosafety cabinet, thermostatic water bath, centrifuge, cell counter, CO2, thermostatic shaker, -80 freezer, liquid nitrogen tank, cryobox, cryovial, triangular (or Schott bottle).
4.Cultivation conditions
When HEK293 cells are kept in suspension, the CO2 constant temperature shaker conditions are: 365-37, 5%-8% CO2, the speed of the shaker will vary greatly according to the different wheelbase of the shaker and the container used, which can be adjusted according to the actual use, the recommended speed of the 35 mm wheelbase shaker is 150-175 rpm, and the recommended speed of the 50 mm wheelbase shaker is 100-110 rpm. The culture conditions of the bioreactor are: 365-37, 40% dissolved oxygen, pH: 71. Speed: 80 100 rpm.
5.Acclimation of cells
During the suspension culture of HEK293 cells, if the type or brand of medium used needs to be changed, the medium adaptation is generally required. The methods of domestication are divided into two types: direct domestication and indirect domestication. Direct acclimation is to dilute well-growing cells in the logarithmic growth phase directly into a new medium for normal culture, that is, the acclimation medium used for direct acclimation is the medium used for substitution. Indirect acclimation is similar to direct acclimation, but the acclimation medium used is a mixed medium mixed with the original medium and the alternative medium in different proportions, and the mixing ratio of the original medium and the substitute medium is 75:25, 50:50, 25:75, 10:90, and 0:100 from high to low. During the acclimation process, the mixed medium with a high proportion of the original medium was first cultured, and then the proportion of the original medium was gradually reduced until 100% of the culture was used with the alternative medium. Direct acclimation is generally recommended first, and indirect acclimation is not successful when direct acclimation is unsuccessful.
6.Resuscitation of cells.
The initial density of cells should be slightly higher than that of normal passages, and the medium used for recovery should preferably be the medium used for expansion culture prior to cell cryopreservation. During resuscitation, the cryopreservation tube was first placed in a 37 water bath and gently shaken to thaw the cells (the time was controlled within 1 minute), after the cells were thawed, they were transferred to a triangular flask or centrifuge tube with a pipette, and 37 pre-warmed medium (added drop by drop at the beginning, and then the addition speed can be increased), shaken well, and placed in a CO2 constant temperature shaker for culture. Due to the toxicity of DMSO in cryopreservation solution to cells, some media require centrifugation to remove DMSO from cryopreservation during thawing, while others do not.
7.Cryopreservation of cells
HEK293 cells must be selected for cryopreserved cells that are in the logarithmic growth phase (typically day 3 after passage) and have a cell viability of >90%. Depending on the brand of serum-free medium used, the preparation of cryopreservation solution also varies, and there are generally two common forms of preparation: 10% DMSO + 90% fresh serum-free medium; 7.5% dmso + 46.25% fresh serum-free medium + 4625% conditioned serum-free medium (and centrifuged supernatant of cell culture).
8.Transient transfection of cells
PEI transfection is widely used for transfection of HEK293 suspension cells. Although PEI has a certain toxicity to cells, it can basically meet the efficiency requirements of transfection, and the cost of transfection is low.