protocol 3&4 starred-cell culture/splitting and seeding

basal media

cells grown in this, providing nutrients necessary for cell growth.

basal media nutirents

vitamins, amino acids, fatty acids, lipids, carbohydrates, trace elements

how to determine best media for a given cell line

emperically

our cells will use what media

mixture of DMEM and Ham’s F-12

10% concentration is what

the final serum concentration of our media

serum

added to the base media to provide the desired final concentration

serum adds what

vitamins, hormones, and growth factors

most common used serum

fetal bovine (FBS)

additives helps with what

to help combat potential contamination problems

what additive do we use

penicillin/streptomycin

temperature

cells are grown in incubators that maintain this

what temp do cells want to maintain in incubator

37 celcius

how to get humidity

have flasks with caps loosened or a semipermeable membrane, allowing for gas exchange.

failure to humidify environment

evaporation occurring and would change the concentration of salts

how to maintain appropriate humidity

incubator contains a pan of water

what’s critical for cell survival

maintenance of neutral pH

cells are gown in

5% CO2 atmosphere

where do cells get their source of bicarbonate

media containing sodium bicarbonate

buffer system of CO2

maintains optimal pH in human blood

medias pH indicator

phenol red

phenol red in acidic conditions

will cause the media to turn yellow

phenol red in basic conditions

will cause the media to turn purple/pink

primary cultures

limited number of cell doubling before they become senescent

splitting frequency

depends on the growth rate of cells. CHO cells need this about twice a week

morphology of CHO cells

grow a monolayer, contact inhibited, adhere to flask, have a polygonal shape, and centralized nucleus

types of vessels for cell culture

culture dishes, flasks, multi-well plates, and chamber slides

vessels for culturing adherent cells

manufactured with a special coating on the surface to assist cells in forming an attachment

why do vessels come in different sizes

the surface area where adherent cells grow is important for approximating the number of cells in that vessel

cofluency

percentage of a vessels surface area that is covered with cells

since cell division results in a increase of cells, cultured cells in confined space

must be passaged on a regular basis

how to determine when the cells need to be passaged

based on characteristics of the cell line and assessing the confluence of cells in the vessel

splitting cells at a proper time is

essential for maintaining the quality and phenotype of the culture

cell seeding/spreading

when you need multiple vessels of cells, the cells must be transferred to different vessels for a new culture

using proper technique and a standardized method for cell seeding is crucial, why

for obtaining reproducible experimental results

cell seeding is dependent on 1

confluency required for the experiment

cell seeding is dependent on 2

doubling-time of the particular cell line

cell seeding is dependent on 3

amount of time between seeding and performing the experiment

bicarbonate reaction

CO2+H2O ←——> H2CO3 (carbonic acid) ←——> HCO3-

immortal cells

transformed and grow continuously for years on end