This preview shows page 1. Sign up to view the full content.
Unformatted text preview: um. Unfortunately, the types and concentration of these factors in serum vary considerably from batch
to batch. This often results in problems controlling growth and function. When growing
normal functional cells, sera are often replaced by specific growth factors. Suspension and microcarrier
cultures can be grown in glass
and plastic spinner vessels. CHO-K1 cells growing on a
microcarrier bead The medium also controls the pH range of the culture and buffers the cells from abrupt
changes in pH. Usually a CO2-bicarbonate based buffer or an organic buffer, such as
HEPES, is used to help keep the medium pH in a range from 7.0 to 7.4 depending on the
type of cell being cultured. When using a CO2-bicarbonate buffer, it is necessary to regulate
the amount of CO2 dissolved in the medium. This is usually done using an incubator with
CO2 controls set to provide an atmosphere with between 2% and 10% CO2 (for Earle’s
salts-based buffers). However, some media use a CO2-bicarbonate buffer (for Hanks’ saltsbased buffers) that requires no additional CO2, but it must be used in a sealed vessel (not
dishes or plates). For additional information on this topic, see the article: The Gaseous
Environment of the Cell in Culture by W.F. McLimans in Growth, Nutrition and Metabolism
of Cells in Culture (1972; Ref. 5).
Finally, the osmolality (osmotic pressure) of the culture medium is important since it helps
regulate the flow of substances in and out of the cell. It is controlled by the addition or subtraction of salt in the culture medium. Evaporation of culture media from open culture vessels (dishes, etc.) will rapidly increase the osmolality resulting in stressed, damaged or dead
cells. For open (not sealed) culture systems, incubators with high humidity levels to reduce
evaporation are essential. For additional information, see article by C. Waymouth: Osmolality
of Mammalian Blood and of Media for Culture of Mammalian Cells (1970; Ref. 6). 5 How to Decide if Cultured Cells Are “Happy”
Evaluating the general health or “happiness” of a culture is usually based on four important
cell characteristics: morphology, growth rate, plating efficiency and expression of special
functions. These same characteristics are also widely used in evaluating experimental results.
The Morphology or cell shape is the easiest to determine but is often the least useful. While
changes in morphology are frequently observed in cultures, it is often difficult to relate
these observations to the condition that caused them. It is also a very difficult characteristic
to quantify or to measure precisely. Examining the morphology of
these stained roller bottles
(containing MRC-5 human
fibroblasts) is a good way to
check if the cells are “happy”.
The bottle on the left was
rotated at too high a speed
resulting in poor attachment
and growth and very
“unhappy” cells. Often, the first sign that something is wrong with a culture occurs when the cells are microscopically examined and poo...
View Full Document
- Fall '11