Hello Claire Mc Cord

1) Is it possible to cultivate adherent cell line (CHO and HEK ) into non-adherent cell lines, and if it's possible how can it be done.

There are several steps involved in transforming adherent cell line(CHO and HEK) into non-adherent cell line. You may follow the protocol provided below.

1. Detach the adherent cells from the substratum using detaching agent, such as trypsin.

2. After the trypsinization process, gently pipette the cells to obtain a single-cell suspension.

3. To this single cell suspension, add the culture medium containing serum and additives like 0.3% (w/v) Poloxamer 188, which helps prevent cell clumping in suspension.

4. Transfer the cell suspension to a shake flask and incubate under standard conditions with constant, gentle agitation (for instance, 125 rpm).

5. Monitor the culture daily for growth and cell viability.

6. You have now to adapt your cells to lower serum and serum-free media. Once the cells are growing well in suspension with serum, begin to reduce the serum concentration gradually over multiple passages.

7. A common starting point would be 75% serum-containing to 25% serum free medium. Then move to 50:50, and later to 25:75, before finally moving to 100% serum-free medium.

8. If cells struggle to adapt at a particular serum-to-serum-free medium ratio, go back and maintain them in the previous, less stressful ratio for a few more passages and later try the new ratio again.

9. Observe for normal doubling times and viability (typically >85-90%) at each serum concentration before proceeding to the next lower level. A successful adaptation is indicated by a high viability (often > 90%) and a stable, comparable generation (doubling) time to the original adherent cells. Please note that serum proteins bind to antibiotics. When switching to serum-free medium, you may need to reduce the antibiotic concentration to prevent toxicity to the cells.

10. To confirm that the cells are now growing in suspension, you need to verify that they no longer require a surface to grow and that they maintain high viability and growth rates in the final serum-free suspension medium.

To summarize: To convert adherent cells (CHO and HEK) to suspension cells, gradually acclimate them to a serum-containing, then serum-free, suspension medium over several passages, increasing the agitation and decreasing the serum concentration with each split, until they no longer require surface adhesion to grow.

2) I also wanted to know, as we do have enough room to buy a second incubator, which shaking plate can be used in a CO2 incubator.

For use in a CO2 incubator, you will need a specialized CO2-resistant shaking plate. The warm, humid, and slightly acidic environment inside a CO2 incubator is a harsh condition that standard laboratory shakers are not equipped to handle. The moisture and CO2 can cause corrosion and damage to their exposed internal components, leading to premature failure and potential contamination of your cell cultures. The specialized CO2-resistant shaking plate are standalone units with protected electronics and drives designed to withstand the high humidity, CO2, and acidic conditions found in a CO2 incubator. Companies like Thermo Fisher Scientific, Eppendorf, and Crystal Technology offer such specialized orbital shakers. Provided below is the required information.

https://www.thermofisher.com/in/en/home/life-science/lab-equipment/lab-shakers/orbital-shakers/co2-resistant-shakers.html

https://crystalindustries.com/products/benchtop-equipment/mixers/3d-nutating-mixersbenchtop-equipment/shakers-3d-nutating-mixersbenchtop-equipment/orbital-shaker-designed-for-co2-incubators/

Best,