Stable Cell Line Services

Biologics International Corp (BIC) provides stable cell line generation services, including stable cell line for research assay, CHO stable cell line for protein production, CRISPR gene knockout cell line service. We can simplify your cell line development process and save your research and development time.

Why Develop Stable Cell Lines

Stable cell lines can grow continuously over a long period of time without significant change in the expression level of the target gene. It is widely used in functional studies of genes, drug screening and production of proteins, for both therapeutic and non-therapeutic purposes.

• Functional studies: the expression level of a gene in certain cells can be manipulated to study its functions.
• Drug screening: stable cell lines are very important for drug screening in a combinatorial chemical library.
• Proteins for research: proteins for research purposes can be produced by transient transfection of HEK293 cells. However, this method is not very cost effective in producing recombinant proteins in large quantity. For some highly demanded proteins, a stable cell line is more desirable as production host.
• Proteins/antibodies for pharmaceutical: stable cell lines are essential for FDA approval and useful for preparing recombinant proteins, recombinant antibodies and vaccines for therapeutic or preventive purposes.

How to Develop Stable Cell Lines

• Cell preparation

We are familiar with various cell cultivation approaches. We have successfully built a variety of cell lines, such as CHO, and cells will be prepared two weeks in advance to ensure a highly active state during transfection. At the same time, we also accept cells provided by our customers.

• Determination of antibiotic screening concentration

Determination of the optimal concentration of antibiotic for screening is a key step. We have decades of experiences and are familiar with the best screening concentration of different types of antibiotics. We can thereby sufficiently save the experimental time.

• Plasmid construction and cell transfection
• Screening, detection and amplification of positive clones

The transfected cells are plated onto a 96-well plate using a limited dilution method and the clones are screened using a specific screening medium. Mark up the single clones/cell pools and check the ELISA titers from the supernatants. Screen and scale up the positive clones.

• Pressure selection

To improve the protein expression level of cell lines, a pressure selection is required to carry out on the construction of cell lines for protein production. Clones with higher expression are selected and transferred to the 6-well plate and amplified for pressure selection. The concentration of pressure should be gradually increased for multi-round selections.

• Subcloning and single clone screening

Select the clone/pool with highest expression, and perform monoclonal screening by limited dilution. After 3-4 weeks, we will mark up the single clones and check the ELISA titers from the supernatants, and scale-up the positive single cell clones.

• Quality control of cell lines
• Batch culture: Cells of the same density were inoculated into flasks and counted on a daily basis. Cell growth and protein expression curves are determined using ELISA.
• Stability test: Cells of the same density were inoculated into flasks and sampled every 48 hours for counting. Then cells were continuously inoculated with the same initial density and the expression stability is determined by ELISA. The stability test goes for 10-50 generations.
• Mycoplasma test: Hoechst staining and PCR (Nested-PCR)
• Identify and freeze stable producers: Upon the results of growth and production profile, we will select the top 5-10 clones to be freezed.

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