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A Review of Best Practices For Cell Culture Media Design And Processes
Last week, we finished our Ask the Expert discussion on best practices for cell culture media design and processes. Several questions were asked about media design for specific cell lines including hybridoma, CHO, HEK293, vero and mesenchymal. Other topics included reducing/removing serum in media formulations, media considerations for perfusion mode, transitioning adherent CHO cells to suspension, transient transfection, contamination issues, bulk prepared liquid vs. powdered for in-house preparation, CD media components and tips for streamlining process development and scale up.
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 was 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. As a Research Fellow at Life Technologies, he helped design much of the specialized media used around the world for the culture (growth) of neurons, stem cells and other mammalian cells, including those used in Bio-Production. He is currently a consultant to several human health companies wanting to develop products in both the areas of Regenerative Medicine and Bio-Production. In June of this year he was awarded a Distinguished Lifetime Achievement Award by the Society for In Vitro Biology for his role in moving research forward in the production of products from cells (Biotechnology) and the use of cells as products (Regenerative Medicine).
Session question topics included:
- Media for specific cell lines – CHO, hybridoma, HEK293, vero, and mesenchymal
- Reducing/removing serum from culture
- Media formulations for perfusion mode
- Transitioning adherent CHO to suspension culture
- Tips for streamlining process development and scale up
- Transient transfection media
- Contamination challenges
- Bulk prepared liquid vs. powdered for in-house preparation
- CD media components
I have selected a few of the submitted questions and answers to include below. For a full list of questions and answers, please see Ask the Expert discussion on best practices for cell culture media design and processes.
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.
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) 2 to 3 days after plating. The cells should be in log phase growth at this time. Plate the cells as you normally do but now the medium should be a 50:50 mixture of CD (assuming this is the medium you want to adapt the cells to. If not add whatever medium you want to use for suspension culture and for this protocol we will still call it CD) and CM. When the cells are growing well (in phase growth which should be 3-4 days), collect the CM and mix it 50:50 with the fresh CD. Continue this until the suspension cells are growing well. If in the beginning they slow down, go back to saved CM from the previous plating. The CM contains lots of growth and viability enhancing factors that the cells produce for their own survival. Once the cells are growing well as suspension culures, you can use only your selected CD medium.
A question regarding culture media sold as bulk pre-prepared liquid vs. powders for in-house preparation of media. Which is better performance-wise, liquid media from the manufacturer or liquids prepared from powders in-house? Everyone I’ve asked has noted that fresh liquid media from the manufacturer will inherently provide better cell culture performance vs. media prepared in-house from the ‘same’ comparably-fresh powder. Some report seeing >10% increase in yields using bulk liquid media vs. powder. Everyone presumes that the powder grinding process can only result in some loss of activity, if only from the heat involved; and that the culture media companies can simply make better, more consistent, finished liquid culture media than can beed in-house from powders, with the manufacturer simply knowing its products better, likely having heavier-duty and proprietary mixing technology/methods, etc.
The reason 1X liquid media from a manufacturer is often better then medium made in your lab from a DPM is the difference in the quality of water. The better commercial media companies monitor endotoxin and heavy metal contaminents in their water and have processes in place to control them. Water grows bacteria rapidly and unless you control growth you will have endotoxin produced by the gram negative organisms. Many systems have a bacterial filter at the end of the line and the pressure of the water passing through will rupture the gram-negative bacteria and release endotoxin. It is a good practice to change this filter once a month. It is also true that the use of ceramic grinding stones produces a lot of heat. This is why the most heat-labile components are added last during a DPM run, so that they are exposed to the heat the least amount of time. Like with a liquid medium, the timing of addition of the media components is a science in itself. If made correctly and you have high quality water, the DPM should be fine as the components in a standard formulation are usually there in excess. There are ways to make a DPM which allows for the a fine powder to be sprayed through an orifice so rapidly as to result in the components seeing minimal heat (jet milling). Some manufacturers have switched to this method while others still use ball milling or use one or the other dependent on the size of the run. A good manufacturer also protects the liquid medium from light degradation during both manufacturing and storage.
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?
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 spent medium they will go into apoptosis. The best procedure for neurons and many of the stem cells is to remove only about half the volume of medium at each feed and replace with an equal volume of fresh media. With hemoatopioetic stem cells I plate the cells in a minimum amount of medium and just add fresh media at days 3 and 6 and harvest on day 7. If you ever have a cell that is growing poorly, try retaining half the conditioned medium and adding an equal volume of fresh at each feeduntil they are growing well, instead of completely changing the medium. This same conditioned medium can also be used to adapt a cell to growth in a new medium. Save the conditioned medium from cells in log phase growth (day 2 or 3) prior to transfer and at P1 plate them in 50% the medium you wish to adapt them to and 50% of the conditioned medium. At the next passage instead of discarding the spent or conditioned medium collect it prior to trypsinization and plate the new cells in 50% of this medium and 50% of the medium you want to adapt them to. Continue doing this until the cells are growing well and then just use the selected medium. This usually takes about 5 subcultures. If the cultures slow down at let’s say P4, plate the cells in the conditioned medium from P3 with 50% of the desired medium and continue.
Please visit next week’s Ask the Expert session – “Fluorescence detection in living cells and ways to improve your image,” hosted by Timothy Fawcett, Ph.D, Director of the BioTechnical Institute of Maryland (BTI) a non-profit institute located in Baltimore, Maryland.