Meet the Cells – Get your questions answered about some popular cell lines.

There are a number of feeds on the market. Most major suppliers of cell culture media will have some type of feed to go with their protein expression media intended for scale productions.

Generating Vero suspension cells, will be a challenge and there are several aspects which will be very important to consider:

· Media should include fluids which reduce shear forces, if the cells are being grown in a shaking format

· It may be necessary to screen many cells, to find a clone which might have the ability to grow in a suspension format

· It may be necessary to continually passage cells, until the suspension adapated cells finally prevail

· May want to try formulations of media designed for suspension cells

Here is a link with some additional information that may be helpful - http://www.lifetechnologies.com/us/en/home/references/protocols/cell-culture/serum-protocol/adaptation-of-cell-cultures-to-a-serum-free-medium.html

Here is a website with background information on using CRISPR for genome editing.
http://www.lifetechnologies.com/us/en/home/life-science/genome-editing/geneart-crispr/crispr-cas9-based-genome-editing.html .

CRISPR-Cas systems have become powerful tools for genome editing and the system as a good choice for your needs. This website also has a short video that will probably be helpful. The CRISPR Nuclease vector is the system for mammalian cells if you don’t have any promoter constraints.

Your question is a good one and difficult to answer without knowing more about your specific culture conditions. The buildup of lactic acid during fed batch culture could come from several reasons. There is a correlation between lactate amounts and the reduction of culture productivity, so this is a big problem without an easy solution. Some cells are more sensitive than others. Hybridoma culture can be a particular problem since they stop producing antibodies once the lactic acid cut off point is reached. Lactic acid accumulation in media occurs because at low oxygen concentrations glucose is metabolized to pyruvate via glycolysis. If oxygen is available then pyruvate is decarboxylated to form Acetyl CoA in the mitochondria. During anaerobic conditions and especially in the stationary phase when the cell density is high and oxygen low Lactic acid is formed. If you measure the oxygen concentration this may be evident. You might add galactose or another more difficult to metabolize carbon source to reduce lactate or you could limit glucose.

Transitioning your cells from one media to another or from adherent to suspension growth things can get tricky. I assume you are transitioning in a serum containing media like Ham’s F12 for your CHO cells, since serum-free media is generally made for suspension culture. You want to use early passage cells if possible. Seed your cells at a higher density than normal. My suggestion is to use Ham’s F12 without calcium or magnesium and some people reduce the serum from 10% to 5%. Also you might want to add a low concentration of the detergent Pluronic F-68 to reduce shear forces. Use a sterile shaking flask without baffles and shake at about 125 rpm. Good luck. Keep culturing your cells and the viability should increase as selection occurs.

I am assuming you are starting with some fibroblast growth medium like DMEM supplemented with ESC-qualified FBS. If you are using GIBCO Essential 8 medium as your SFM you can transition directly. Essential 8 is designed for feeder free growth. Here is a reference https://www.lifetechnologies.com/us/en/home/references/protocols/cell-culture/stem-cell-protocols/ipsc-protocols/generation-human-induced-pluripotent-stem-cells-fibroblasts.html

If not, There are two ways to do this. Try a sequential adaptation. Assume you are using old media A and want to transition to new media B. Start in media A and know something about the kinetics of growth. Transition to 80%A:20%B grow several passages then move to 60%A:40%B then 40%A:60%B and so on until your cells are in 100new medium B. The other method involves removing 50% of the old media and supplementing with 50% the new media. If you are removing serum you will need to coat the dishes with some attachment matrix like geltrex or vitronectin if you want attachment.