ABOUT CULTURING IN BAGS

WHY CULTURE:  Cells are generally for one of two reasons:
1. to expand the number of cells
2. or to produce a soluble substance for harvest.

EXPANSION: For cell expansion, it is desirable at the end of the culture period to be able to harvest the cells. Generally, the supernatant is not as important.  It is important that cells do not adhere to the bag.

PRODUCTION:   When using cells to produce something, it is necessary to harvest the supernatant periodically. Harvesting the cells is not as critical.  When producing a biological, it is essential that the critical biological is not lost to the bag; either adsorbed onto the surface or absorbed into the bag.

BAGS: In both cases culturing in bags permits the contents to be placed into a centrifuge for separation.  It is virtually essential to be able to see through the bag to perform this separation in an efficient manner.

CELLS LINES: The culture process is different for each cell line.  Some cell lines must adhere in order to reproduce, and others like to float in suspension.  Either abode can produce expansion or
production.  The mechanics of culture will depend on the cell line being grown, as well as the purpose for which they are being grown.  All types can be grown in bags.

HOW MANY CELLS CAN GROW IN A BAG: Start with 1,000,000 per milliliter.  Here is why:

IF TOO FEW:  If you start with too low a concentration, there may not be enough cell to cell interaction to support the culture at all and the cells may die.  This is not from loneliness, it is from lack of cytokines.

IF JUST ENOUGH:   If you start with just few, it will take time for the concentration to reach a critical mass for exponential growth or for maximal production.

IF TOO MANY: If you start with too many, exponential growth will not occur, maximal production will not occur.

WHAT MAKES THE DIFFERENCE:   In bag culture, respiration is through the walls of the bags so it is not necessary to agitate, mix, rock, or rotate the bags.  The cells settle to the bottom of the bag.  This means that most respiration will occur through the bottom of the bag, therefore, put the bag on a screen, not on a solid flat surface.  Do not fill more than one centimeter thick, because the additional media above the cells will not improve nutrient or gas diffusion, it is a waste to do so.

CELL CONCENTRATION:    Since there is no agitation, the cells will settle into a layer onto the bottom.  Increasing cell concentration will cause the layer to be thicker.  Increasing the thickness of the media fill will also  cause the layer to be thicker.  The thickness can be expressed as 1,000,000 cells per square centimeter.

VALIDATING:   Start with one million per milliliter.  Your particular cell line may require more or less, you will know in short order.

ADHERENT CELLS:   Cells cannot adhere to FEP.  It will be necessary to provide them with a surface to stick to.  AFC can do that.  It can be a plastic sheet like the floor of a T-flask, or it can be a three dimensional matrix.

SPLITTING AND MEDIA CHANGE: With bags, cells can be pelleted by centrifuging the bag, expressing the supernatant expressed with a plasma expressor.  The culture can be split by mixing the bag and removing a given volume or weight.

DISPOSAL: Final disposal of cultures can be safely done by placing the entire AFC bag into the autoclave and running a standard cycle.  The contents will be contained and sterile.