SFM Screening Kit Applications Manual
The SFM Screening Kit is intended for researchers seeking to identify
the most appropriate SFM formulation for a specific cell type. While the
literature references SFM formulations which have been used to cultivate
mammalian cells, unless the same strain as that referenced is employed the
best medium may not be known. Further, the SFM formulations used in the past
are not necessarily the best formulations for a given cell type. Therefore,
it is recommended that several media be tested for suitability before selecting
Athena?s SFM Screening Kit contains a 100 ml sample of each of five
serum-free media. The media are provided ready-to-use. The kit includes three
proprietary media along with two general use formulations as follows:
||epithelial cells, human breast explants
||epidermal-like cells, human esophageal epithelial cells, human skin explants, established cancer cells
||immortalized normal prostatic cells, established human prostate cancer cell lines
||1:1 mixture of DMEM and Ham's F12
||Iscove's Modified Dulbecco's Medium
In addition, each kit contains a 25 ml sample of FNC Coating Mix?.
Because SFM?s are low in protein it is important to pre-coat the growth substratum
with proteins as a way of facilitating cell attachment. The FNC Coating Mix? is
specially formulated to provide a superior coating than traditional methods by
employing a mixture of proteins designed to lay down a matrix which induces cell
attachment and proliferation.
There are two approaches for testing the SFMs. The best technique is to
use a serum or medium step-down procedure. In this method, the SFM being testing
is supplemented with serum or the medium currently being used (?base medium?). At
each feeding, the percent of serum or base medium is reduced until the cells are
fully adapted to the SFM formulation. Typically, once the serum concentration is
below 1.0% the suitability of a given SFM over another will become evident. An
alternative method is to subculture the cell line in each of the respective SFM
formulations. The formulation giving the best growth is selected as the most
suitable and the cells are adapted to that medium using the above procedure. While
this is a quick and simple test, not all cell lines will readily adapt to an abrupt
shift in medium composition. Therefore, an incremental adaptation to SFM may be more
Protocol #1: Step-Down Metheod
||Cultivate the cell line to be tested in the base medium employed. If the serum concentration is greater than 5%, reduce the amount of serum to 5%. Once the cells are growing well, proceed to step #2.
||Transfer the cells to SFM supplemented with 2% FBS (or 1:1 mixture of base medium to SFM). When the cells are growing well, proceed to step #3. If the cells do not grow well, increase the serum concentration to 3 or 4% (2:1 mixture of base medium to SFM) and repeat this step.
||Transfer the cells to SFM supplemented with 1% FBS (1:4 mixture of base medium to SFM). Once the cells are growing well with 1% serum, continue to step down the serum concentration to 0.75% (1:6 mixture of base medium to SFM) and then to 0.5% (1:10 mixture of base medium to SFM). Adjust the size of the step down increments as needed to maintain good growth. Once the cells are growing well in 0.5% serum (or 1:10 mixture of base medium to SFM), proceed to step #4. It is during this latter series of step down cultivations where difference between the various media will become evident. Choose the one which provides the best growth for the cell line while accounting for product production where relevant.
||Transfer the cells to SFM without serum or base medium. Passage the cells with a higher inoculum than normal when using SFM. Allow 2-3 feedings before scaling the culture.
For attachment-dependent cells it is strongly recommended that the growth
substratum be pre-coated with FNC Coating Mix? or other suitable protein mixture
before adding the cells. This becomes more important as the serum concentration is
reduced in the medium. It is advisable when performing the step down cultivation,
to continue to grow a portion of the cells in the medium from which they came. This
is to ensure an adequate supply of adapted cells should the cells not grow in the
next step. Do not use cells which have recovered from a severe lag before growing.
This is to avoid selection for good growers which may have picked up a mutant growth
phenotype. The use of conditioned medium may help to improve the step down process
as the cells switch metabolism to accommodate the SFM conditions. However, use this
sparingly. Mixtures containing 10% to 25% conditioned medium added to the supplemented
SFM should suffice. For cell lines designed for producing a native or recombinant
protein, production of the target protein should be monitored during the step down
process to ensure that the cell line maintains good expression levels.
Protocol #2: Rapid Test
Caution: Apply this procedure only for cell lines known to tolerate a range of culture formulations.
||Prepare six cultures of the cell line to be tested.
||Culture the cells to be tested in the base medium until they reach 50% confluence or 50% maximum cell density.
||Remove the medium from the cultures and replace it with each of the five media and the base medium.
||Continue cultivating the cells. Observe the growth over a period of 2-3 days.
||If the cells can tolerate a particular SFM formulation, continued growth without any aberrant morphologies should be observed