exam 2

medium

singular

media

plural

defined medium

all ingredients are known or defined, i.e., contains no serum

undefined medium

not all ingredients are known, i.e., addition of serum

why do we use cell culture medium

more complete than salt solutions, e.g., HBSS, by providing nutrients

what is media used for

maintenance of cells

growth of cells

replication of cells

inability to culture cells successfully is due to not knowing what?

their nutrient requirements:

• growth factors

• hormones

• energy sources

• amino acids

• fatty acids

constituents of cell culture media

salts

carbs

amino acids

vitamins

hormones

growth factors

antibiotics

water

serum

salts in cell culture media

salts found in balanced salt solutions (BSS)

most media contains EBSS

isotonic

what is the major energy source in cell culture media?

carbs - glucose

amino acids in cell culture media

cells require essential aa’s, along w/ cysteine and tyrosine

vitamins in cell culture media

bring stability to media

fat soluble ones more stable than water soluble ones

growth factors in cell culture media

mitogenic activity

polypeptides that act in paracrine manner, either alone or w/ other GFs and hormones

antibiotics in cell culture media

originally introduced to reduce frequency of contamination

water in cell culture media

need very high-quality water via water purification systems

serum in cell culture media

plasma w/ clotting factors (e.g fibrinogen) removed

contains >300 protein

how much CO2 is EBSS formulated for?

5%

how much CO2 is HBSS formulated for?

0.04%

besides bicarbonate, other buffering agents include what?

phosphate and HEPES to yield pH 7.2 - 7.4

types of salts in cell culture media

Na+

K+

Mg2+

Ca2+

Cl-

SO4-

what does Ca2+ in cell culture media?

cell signaling

exocytosis

adhesion

what do Na+, K+, and Cl- do in cell culture media?

regulate membrane potential

how is glucose converted to energy in cell culture media?

metabolized by glycolysis to pyruvate

aerobic conditions in cell culture

pyruvate enters citric acid cycle

anaerobic conditions in cell culture

pyruvate converts to lactate

why are non-essential amino acids added in cell culture media?

to compensate for cell’s inability to make them

primary energy source for cells in culture

glutamine, and aa’s are the building blocks for proteins

fat soluble vitamins

A, D, E, K

thiamine B1 - water soluble

labile to heat, water, O2

riboflavin B2 - water soluble

labile to sunlight (milk in opaque containers)

pantothenic acid - water soluble

labile to heat

ascorbic acid (vitamin c) - water soluble

labile to heat, O2, UV light

what kind of vitamins does Eagle’s MEM contains?

only water soluble vitamins

B vitamins

choline

folic acid

inositol

nicotinamide

what is in most of the complex media including serum free recipes?

biotin

examples of hormones

growth hormone

insulin

hydrocortisone

triiodothyronine (T3)

others depending on cell type

hormones in cell culture media

often used to replace serum in serum-free media

example of growth factors

fibroblast growth factor (FGF)

epidermal GF (EGF)

transforming GF (TGF)

insulin-like GF (IGF)

insulin

penicillin and streptomycin

penicillin inhibits gram + bacteria

streptomycin inhibits gram - bacteria

typically used in combination

gentacimicin

effective against gram - and gram + bacteria

when are antibiotics not necesary?

using laminar flow hood and having good aseptic technique

what is a risk of using antibiotics in cell culture media?

can hide presence of low levels of contaminants

can encourage growth of antibiotic-resistant organisms

resistivity

expressed in megaohms

used to classify water w/ fewer ions

what kind of water is cell culture media made with?

18 megaohm water

total protein concentration in serum

60-80 mg/mL or 60 g/L

sera used in cell cultures come from

bovine fetus

newborn calf

human

horse, pig, goat

chicken

NuSerum

synthetic serum replacement

more chemically defined

low protein

functions of serum in cell culture

promote attachment and spreading of anchorage-dependent cells

provides source of minerals, lipids, aas, fatty acids, carbs

contains factors that stimulate growth (e.g hormones, GFs)

cellular damage brought about by freezing and thawing is generally caused by what?

water expansion

ice crystals

dehydration

osmotic effects

in a research lab, what is one of the best ways to lyse cells?

subject them to freeze-thaw cycles

in cryosurgery, what is one of the best ways to lyse cells?

freeze tissue to kill it

e.g applying liquid nitrogen to warts

liquid nitrogen temp needed for cryopreservation

-196C

liquid nitrogen (vapor/gas phase) temp needed for cryopreservation

-135C to -190C

or -150C to -190C

water content in cells

cells have a higher water content

what happens when water is cooled below 0C

water content changes

at what temp is water most dense?

4C

it’s still liquid

what happens as water freezes?

become less dense ie. due to air content, ice floats

water molecules crystallizes into open hexagonal form ie. more space than liquid form

structure of liquid water

semi-ordered

structure of frozen water

ordered

water molecules crystallizes into open hexagonal form

what damages cellular membranes and organelles?

ice crystals

intracellular ice

damages intracellular membranes and organelles

can be lethal to cells

extracellular ice

not as harmful as intracellular ice

plasma membrane can prevent penetration of cell by this type of ice

how does recrystallization occur?

during freezing and thawing

large ice crystals increase over small ice crystals, leading to cell damage

when does recrystallization occur?

when cells are warmed from -196C and especially when temps are warmer than -130C

what kind of ice forms first?

extracellular ice forms before intracellular ice

leads to increase in extracellular osmotic concentration

what happens when extracellular ice forms before intracellular ice?

liquid water leaves inside of cells, increasing intracellular osmotic concentration and therefore damaging cells

what is a major cause of cell damage?

osmotic effects (resulting in solute effects)

osmotic effects

as water in a solution freezes, ice crystals form

leads to higher concentrations of solutes and increase osmotic pressure

water that has what in solution freezes at lower temperatures, depending on the concentrations of these substrates?

ions and other substances

as each molecule of pure water freezes, what happens?

the solute concentration that remains increases, resulting in continuous drop in the freezing of that solution

temp. of solution falls below true freezing point of water (supercooling), until ice crystal forms around a “seed”, followed by nucleation to form ice

supercooling

temp. of solution falls below true freezing point of water