Cell Culture

Finite cell lines are established by mechanical or enzymatic disruption of tissues followed by a transfer of the released cells to a growth medium 

• Have fixed lifetimes that typically die out after about 40-50 divisions 

• Anchorage dependent -> they won’t grow and divide unless they can attach to a solid substratum and form a continuous monolayer 

• Exhibit density dependent growth inhibition or contact inhibition (growth can stop due to crowding) 

Established or continuous cell lines can continue to divide as long as they have access to food and room to grow 

• Either derive from tumor tissue or have been genetically modified 

• In procedure used mouse fibroblast line called NIH/3T3 cells 

• Continuous cells but exhibit contact inhibition 

Dulbecco’s Modified Eagle’s Medium (DMEM) -> common growth medium used for cell culture 

• Extremely complex mixture consisting of glucose, vitamins, and amino acids (play nutrional roles) 

• Various antibiotics provide some protection against contamination by bacteria and fungi 

• Glutamine plays a key role in promoting cell adhesion to solid surfaces  

• High levels of fetal bovine serum which promotes cell growth, division, and adhesion 

• Bicarbonate is included at high levels to establish a normal pH of about 7.4 

• PH indicator phenol red 

• At pH 7.4 color is red/orange 

• At high pH color is cherry-red 

• At low pH color is yellow 

HEPES -> second buffer within cell growth media, provides extra protection for when a culture must be removed from the incubator (protection is limited) 

Incubators for cell culture replicate normal growth conditions within the mammalian body by providing temperatures of 37C and high CO2 levels (5-10%) 

Unlike typical biochemical reaction mixtures, the buffering of cell medium is provided by the balance between alkalinity of the bicarbonate ion and the acidity of dissolved CO2 

Cell cultures can be grown in polystyrene T-flasks and multiple-well plates 

• T-flasks are incubated on their sides, cells adhere to the lower surface  

• Can come in multiple sizes (T-25, T-75, T-150) the numbers indicate the approximate area of growth surface in square centimeters 

• Can hold working volumes of 5, 15, 30 mL  

• Multiple-well plates can come in 24 and 96-well 

• Each hold about 2 and 0.3 mL  

Confluency -> proportion of cell medium covered by adherent cells, establishment of a monolayer 

Most cell cultures need to be detached from the substratum before being subcultured 

• Typically done using enzyme trypsin which cleaves linkages between the cell and the substratum 

• EDTA is also present and chelates calcium which is needed for some of the attachment proteins 

• Trypsinization does pose the risk of damaging the cell surface 

Splitting cultures -> resuspending near confluent cultures and dividing them into two flasks 

• A split that divides into two cultures will grow to confluency faster than a split grown into four 

Thinning cultures -> discarding half or more of the suspended cells, retaining the remainder of the original flash and adding fresh medium 

Yellowing cell medium can be a sign that cells have reached a high density and need to be split 

If the medium is cherry-red it could indicate that the cap is screwed on too tightly  

Bacterial contamination can also be indicated by yellow coloring, fungal contamination turns it red 

Inverted microscope -> special microscope that allows for clear viewing of cultured cells by placing the light source and condenser above and the magnifying lens beneath so one can view the cells from underneath 

• Usually equipped with phase contrast optics because cells absorb very little light and are barely perceptible, phase contrast depends on how the light is slowed down and bent when passing through material 

Laminar flow cabinet -> air enters through a HEPA filter to remove small particles like bacteria, air flows towards the user, downdraft system sucks air in that might enter/leave the cabinet and filters it 

Dulbecco’s Phosphate Buffered Saline (DPBS) -> washes away any free calcium and any serum that remains from the medium 

Under a microscope long, flat cells are those still attached to the substratum and round, balled up cells are those that free 

Hemocytometer -> cell counting chamber; thick microscope slide that has a 1/10mm gap that separates the slide from the coverslip 

• 9 major squares each 1mm on a side 

• Counting the cells in one of the large corner squares = sample of cells contained in 0.1 uL