Job Title: Research scientist
IntroductionHave 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.
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.
What are the most costly ingredients in today’s CD media for GMP protein production (mammalian) that make them so expensive (~US$100/L)?
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 a very complicated medium because it must allow for as good or better growth in the absence of proteins and peptides as obtained in a medium supplemented with serum or serum proteins. A typical CD medium will contain everything found in a standard basal medium formulation plus about 25 or more trace elements. Because the concentration of the trace elements is too low to weigh out, stock conntrates (often about 20,000X) must be made. An inorganic substitute for transferrin is also needed as well as inorganic components to reduce apoptosis and free radical production. Each component must be put into solution at the right pH and added to the water in the proper order as to maintain the cells in solution.
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.
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.
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 is phenol-red-free and contains HEPES is destroyed by the light in the hood or laboratory in as little as 2 hours. The light breaks down riboflavin and to a lesser degree folic acid, B12 and folinic acid, trytophan and tyrosine form dimers and the result of these changes is that hydrogen peroxide is generated, which initiates the production of free radicals. Phenol red helps protect the medium by absorbing the same wave lengths that destroy the medium and serum helps protect by breaking down the hydrogen peroxide. HEPES increases sensitivity 3 fold. Taking out the bulb in the refrigerator or protecting the media with black plastic helps. What you describe can also be due to mycoplasma contamination of your cells. Mycoplasma rapidly utilize arginine, induce acid formation and alters metabolic processes. You also need to be sure your water is of high quality. Bacteria in the water system can produce toxins and the water may be contaminated with heavy metals, some of which are very toxic. If your cells are free of mycoplasma and you are sure your water is of good quality, the problems most likely are due to light degradation of your media.
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.
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 MEM and supplement with ITS. You would still need to add the 2% FBS (hopefully qualified for MSC's) and of course B-FGF.
Are there special media requirements in terms of nutrients, supplements, etc. necessary if you are using CHO cells for manufacturing antibodies using perfusion mode?
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 at about 0.5 mM in the medium and with CHO cells the glucose concentration should be monitored and adjusted daily to 1.0 G/L.
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?
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 better off buying a specialty medium rather then making your own. All of the CD formulations are about the same qualitatively, but differ quantitatively. They are also complicated in design requiring extensive labor. A typical CD medium would have about 25 trace elements in it. Whether you use fed-batch or perfusion you need to add back the self-limiting amino acids or feed with complete media. With the NSO hybridomas the self-limiting amino acids are cysteine, leucine, methionine, tyrosine and valine (and of course glutamine). To control ammonia and lactate accumulation you need to try to maintain glutamine at 2 mM (to do the same thing with CHO cells you would maintain glucose at 1.0 gm/L). As far as what hybridoma medium to choose, I have only used Hybridoma SFM and later CD-Hybridoma from GIBCO as that is where I used to work.
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.
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 forward. I have never tried to transfect cells in suspension, but I know that GIBCO has what they are calling 293 Free Style Expression media, adapted cells and a cationic lipid they are calling 293 fectin. Other companies may have equivalent products. My own experience with transfection is limited. I have only worked with attached cells and the cationic lipids Lipofectamine and Lipofectamine 2000.
We are looking to streamline our process development and scale up. Do you think it is possible to use a single media for cloning, seed train expansion and production, with just minor modifications at each step? We are trying to eliminate using different media for each step of the process. Ideally we would just have one media, but if that is not possible then it would be nice to have one base media with adding a few components to each step as necessary
You probably could use one medium if you choose the correct one. For cloning you want to start with as small a volume as possible and the medium should have B12 as one of its vitamins. As cells grow they transfer nutrients and growth factors back and forth across their membrane. If the volume of medium is so great that they can not get back what they need to survive because of dilution, the cells will go into apoptosis. Make sure your incubator is humidified as evaporation concentrates the salts and this is also deadly to the cell. For expansion take advantage of the good things in the conditioned medium and only remove about half of the volume and add back an equal volume of fresh medium until the cells are growing exponentialy. Protect your media from light degradation and control for ammonia production. To do this I would switch from glutamine (2.0 mM) to the dipeptide once the cells are expanding and as the cultures increase in concentration per unit volume, increase the concentration of glutamine dipeptide to 6.0 mM. Also be sure your medium has sodium pyruvate and the so-called non-essential amino acids. If your cells have been grown in the presence of pyruvate and you switch to a medium without pyruvate the cells will go into apoptosis. The other worry is lactate produced from glucose raising the osmolality. I would pick a medium with an osmolality between 270 and 290.
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.