What do cells need to survive?
• • • • • • • Warmth Water Appropriate CO2 /O2 concentration Attachment Food Neighbors (not too many) Sterile environment
All these factors are provided in a healthy body; cells removed from their native environment need to have them artificially supplied.
How to Give Cells What They Need (The Easy Parts)
• Warmth: cell culture incubator (37˚C)
– Substrates need to be stable at this temperature.
• Moisture: Keep cells covered in media; when exchanging liquids, don’t leave the cells uncovered for long.
– Microfluidics: bubble‐free liquid dispensing.
NBTC incubator
• CO2 /O2: 5% CO2 atmosphere maintained in the incubator, cell culture materials cannot be air‐tight (vented, covered caps for flasks; Petri dishes, etc.)
– 3‐D matrices need gas permeability.
How to Give Cells What They Need (Attachment)
Attachment promoting substrates can be obtained by several methods:
– Tissue Culture Polystyrene (TCPS)—read labels on petri dishes and flasks; must be treated for tissue culture. – Hydrophilic substrates improve attachment and spreading
• O2 plasma treatment or chemical surface modification
– Protein‐containing media OR adhesion protein pre‐treatment
• Site‐specific cell attachment via membrane
– Adhesion peptide‐modified surface chemistry
How to Give Cells What They Need (Food)
• Feed cells every 1‐3 days with fresh media • Maintain a consistent media composition—no rapid protocol changes • Cell culture protocols should describe the media components required for particular cell types. These often include:
• • • • • • • • Glucose or other sugars (nutrition) Serum (complex mixture of proteins, salts, hormones, etc) Proteins (nutrition, also via serum) Trace elements (nutrition, also via serum) pH indicator (pink at pH 7.2, yellow under acidic conditions) Antibiotics (often unnecessary) Salts (osmotic pressure/pH balance) Growth factors/hormones (cell‐type dependent)
How to Give Cells What They Need (Some Neighbors)
Under‐confluent(~0 to 40%): For most cell types, this is an abnormal condition. Cells will grow rapidly and move across the surface looking for neighbors in an attempt to “heal”.
Confluence (~50 to 70%): Most cells have one or more neighbors. Growth rate has slowed but cells are ready for passaging.
Over‐confluence (>80%): Cells are packed to closely together or overlapping. Growth/division slows, apoptosis occurs, cells do not replate well.
How to Give Cells What They Need (Sterile Conditions)
• What’s the problem?
– Bacteria, yeast, fungus, viruses, mycoplasma, any unwanted cells.
•
Why?
– – – – Crowd out desirable cells (faster growth rates) Faster consumption of media Creation of waste products that are toxic to cells Infected cells may have different morphology/growth rates/gene expression
• Case study: HeLa
– Infections lead to poor experimental results.
Sterile materials, which are free from cellular contamination, must be used at all times. Sterile techniques must be developed in order to minimize the chances of infection. How are sterile conditions maintained?