cell culture

CHO-K1

Chinese Hamster Ovary; immortalized cell line with a 14-17 hour doubling time; adherent cells that attach to the bottom of the plate

Immortalized cell line

Transformed cells that grow indefinitely like cancer; used to study cell behavior and drug responses and to manufacture proteins and antibodies

Primary cell culture

Cells isolated directly from a live organism; have a limited lifespan due to the Hayflick limit

Hayflick limit

The set number of times normal (primary) cells can divide before they senesce; does not apply to immortalized lines

Confluence

The percentage of the dish surface covered by cells; affects growth (contact inhibition) and cell behavior

Contact inhibition

When crowded adherent cells slow or stop dividing due to cell-cell contact signaling; transformed/cancer-like cells often lose this checkpoint

FBS (fetal bovine serum)

Media supplement needed for CHO-K1 growth though the reason is not fully clear; used at 10% and is expensive

CHO-K1 media recipe

Ham's F12 (or DMEM/F12 1:1), 5 mM glutamine, 10% FBS; must be warmed to 37 C

Glutamine in media

Unstable amino acid used as an energy source; added at 5 mM to CHO-K1 media

Phenol red

A pH indicator in media that turns yellow at low pH (acidic conditions)

Antibiotics in this lab's media

None - the lab runs antibiotic-free so aseptic technique is critical

Trypsin

A serine protease found naturally in the small intestine; digests proteins in cell-cell and cell-matrix adhesions to detach adherent cells from plates

EDTA

Chelates calcium and magnesium ions which weakens cell-cell contacts

Trypsin neutralization

Trypsin is stopped with an EQUAL amount of complete Ham F12 media; the serum in complete media inhibits trypsin

Trypsinization protocol steps

Pour out flask media; add 5 mL 37 C trypsin/EDTA; wait 10-15 min under microscope; neutralize with equal Ham F12 complete media; gently triturate; transfer to 15 mL tube; centrifuge 100 x g for 5 min; decant; resuspend in 10 mL complete media; aliquot 1 mL per tube

Trypan blue 0.4%

Viability stain for cell counting; dead cells take up the dye while live cells exclude it giving the percentage live

Countess II

Invitrogen automated cell counter used to count cells and report concentration and percent live

Cell counting mix and load

Mix 10 uL cell suspension with 10 uL trypan blue; load 10 uL into the slide's half-moon sample area by capillary action; let settle 30 seconds; insert until it clicks

Optimal cell count range

1 x 10^5 to 4 x 10^6 cells/mL

NB 8(II) cell culture notebook data

Cell concentration, percentage live, and a drawing of the cells

Plating steps

Label plate (CHO K1 date section group names); 3 mL sterile media in dish; add cells aseptically; triturate gently and add dropwise around the dish; lid on; swirl slowly in all directions without lifting the dish

Incubator conditions

37 C and 5% CO2

Why 5% CO2

CO2 works with bicarbonate buffer in media (CO2 + H2O

Inverted microscope

The objectives are below the stage so you view cells up through the bottom of the dish

BSC alarms and blowers

Both must be ALWAYS ON even when the cabinet is not in use

BSC glass rules

Never open glass above the alarm level; always fully close glass when not in use

BSC cleaning

Clean before AND after use with alcohol (70% ethanol)

BSC UV sterilization

UV lamp before use for 15-20 minutes - no more no less

BSC entry rule

Wipe all bottles gloved hands etc with alcohol before they go into the cabinet; do not remove sterile items stored inside

Nose-scratch rule

Take hands out remove gloves itch get new gloves wipe with alcohol then put hands back in

Air-flow rule

Wait until you hear the air flowing before starting; with air off nothing in the cabinet is considered sterile

Waste rules

Anything that touches cells goes in the ORANGE biohazard bag for sterilization; liquid waste must be treated with BLEACH before pouring down the drain

Blowers-off rule

Tape and close all sterile items (plates tips etc) before the blowers are turned off

Mammalian vs bacterial culture

Forget bacterial culture: mammalian cells grow slowly are fragile and sterile mammalian culture is unforgiving and far more expensive; bacteria can grow anywhere and outcompete everything

Electronics in lab

Contamination bombs - using them during lab automatically disqualifies the extra credit challenge

Monoclonal antibodies

Identical antibodies to one specific epitope of a protein produced by a clone from a single cell; more specific and stronger data but more expensive

Therapeutic proteins made by cell culture

Insulin human growth hormone fertility drugs made by bacteria yeast or cultured mammalian cells

Sterile cell culture plastics

One company's dish can work better than another for a cell line because microscopic surface texture affects ECM-like attachment

Extra credit challenge

1 point for no contamination both partners handling cells proper BSC protocol proper labeling correct incubator placement watching other groups and turning in TA sheets

Extra credit disqualifiers

Contamination causing another group's contamination touching another group's supplies not listening to the TA not cleaning up improper waste disposal using electronics