Best Practices For Cell Culture Media Design And Processes

Introduction

Have you had the problem that a new cell line in your laboratory is not growing to your expectations or cells that had been growing well in your medium all of a sudden start looking sick and are going into apoptosis?

The classical cell culture medium consists of amino acids, vitamins and a source of energy, such as glucose, in a buffered salt solution. These formulations require further supplementation with a protein source such as serum. The classical media formulations were designed using cancer-derived cell lines and can be very sub-optimal for the growth of specialized cells, such as stem cell, recombinant cells and differentiated cells. In the absence of serum and serum proteins it becomes essential that you control for the osmolality, ammonia and the production of free radicals. As important as using the right medium is the proper handling of the both the media and the cells and together can be the difference between a successful or failed experiment.

This Ask the Expert Session is hosted by Paul J. Price, Ph.D., Media Design Consultant. Dr. Price has been a research scientist for over 50 years. Positions he has held include Branch Chief in the Center for Infectious Diseases at the CDC, and founder and Executive Vice-President of Hycor Biomedical.

If you have had a problem with an experiment or cells, take this opportunity to prevent either from crashing. Dr. Price has over 50 years of experience in cell culture and media design and has had the opportunity to make all of the mistakes and find ways to correct them.

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Question 1

We are trying to transition vero cells from serum containing to a commercially available serum-free media for vero, but are having trouble with the cells making the transition. Which steps would you recommend that we take to help the transition for the cells?

0 0 0 Total: 0 Cells, including VERO, work hard to make an environment for themselves allowing good viability and growth. When we discard the so-called spent or conditioned media, we are also discarding the growth and other factors the cells have produced for their survival. With primary neurons if you discard all of the … Continued

Question 2

What are the most costly ingredients in today’s CD media for GMP protein production (mammalian) that make them so expensive (~US$100/L)?

0 0 0 Total: 0 It not the cost of the ingredients that dictate the product cost but the labor involved. A few of the ingredients in a CD formulation are costly (i.e. monothioglycerol) but the concentrations in the 1X formula are so low as to not be a significant factor. A CD medium is … Continued

Question 5

Yes we have. Cell is sensitive to the water, media lot and some contamination which is hard to identified. Sometimes we can’t find the reason which cause the bad growth and crashing.

0 0 0 Total: 0 Several different or combined problems may be causing your cultures to crash. A common cause of cells crashing is the destruction of the medium you are using by light. Both incadescent and fluorescent light can destroy the medium. The time it takes is dependent on the medium. A medium that … Continued

Question 6

If you are trying to remove or reduce serum in mesenchymal cells, what are some key components – supplements, growth factors, etc. that you need to include in your classic media.

0 0 0 Total: 0 To reduce serum in a classical medium you need to pick the right medium. GIBCO has OptiMEM 1 and several Advanced formulations such as Advanced DMEM:F12. You should be able to reduce the serum in these media to 2%. If you prefer to use a published claasical medium consider alpha … Continued

Question 7

Are there special media requirements in terms of nutrients, supplements, etc. necessary if you are using CHO cells for manufacturing antibodies using perfusion mode?

0 0 0 Total: 0 You need to be aware that different nutrients are used at different rates and what is overly utilized is cell specific. With CHO cells glutamic acid, aspartic acid and cystene are self limiting and need to be part of a perfusion medium. Glutamine or glutamine dipeptide needs to be added … Continued

Question 8

What do you recommend for an good productive hybridoma media, but also cost effective. Would you make your own using classical or buy off the shelf?

0 0 0 Total: 0 For best results you probably want to grow your cells in suspension and in a CD-medium. The various CD media are cell type specific as different cells utilize the amino acids at different rates. You also have to decide if you are going with fed-batch or perfusion. You would be … Continued

Question 9

What kind of media do you recommend for transient transfection of HEK293 cells? Are there any key components to include or look for in media. Our cells aren’t doing well right now in our current media.

0 0 0 Total: 0 The medium will differ as will the transfection protocol dependent on if you are growing and transfecting adherent cells are cells in suspension. The life Technologies web-site has protocols on it for transfecting either type of cell. I just looked at a couple of them and they were pretty straight … Continued

Question 11

We are having trouble with our VCC going down to half as we are moving adherent CHO cells to suspension. Any ideas on how media could help with the transition. We are using an off the shelf CHO media right now.

0 0 0 Total: 0 Going from adherent culture to suspension can be tricky, especially when going from a protein containing to a chemically defined (CD) formulation. The following procedure should work for you. Grow your cells in the medium that they have been adapted to and collect the supernatant(call it CM for conditioned medium) … Continued