PA1- Topic 6 Part 1/2/3

the functions / importance of BSS

maintain physiological pH (sodium and bicarbonate supply)

maintain osmotic pressure of cells

maintain the membrane potential (providing sodium, potassium, calcium ions outside cell membrane)

aids in cell attachment (adhesion molecules need magnesium and calcium)

acts as cofactors in enzyme reactions

how proteins and peptides function

common examples of proteins

prevent toxicity, increase viscosity, promote cell attachment and growth, carriers of fatty acids, minerals and hormones

alpha-globulin, albumin, transferrin, fibronectin

amino acids (non-essential and essential)

most animal cells have a requirement of essential amino acids that are not synthesized in the body.

non-essential amino acids are added as well

to compensate the particular cell type’s incapacity to make them

or they are made but lost by leakage in the medium

vitamins

vitamins especially B group are required for cell growth and multiplication

many vitamins are precursors for the metabolic cofactors

how/ why/ what function of phenol red

the color change of phenol red

bacterial contamination and phenol red

is added as a pH indicator of the medium and accounts for the color of the medium

is useful because it is particularly sensitive to slight pH changes around the growth optimum of cells.

7- orange

6.5 yellow

overnight change in color of the culture from red to yellow shows bacterial contamination

what is the purpose of adding antibiotics

which two practices make use of antibiotics unnecessary

the disadvantages of antibiotics

reduce the frequency of contamination

the laminar-flow hood and strict aseptic technique

encourage the development of antibiotic-resistant organisms

encourage poor aseptic technique

hid the presence of low level, cryptic contaminants

hide mycoplasma infections

anti-metabolic effects that can cross react with mammalian cells

since basal media alone is not enough for the long term growth, serum must be added to be a complete medium.

how Attachment factors

supplement of major serum protein (fibronectin) can enhance the attachment of anchiorage-dependent cells. collagen and laminin also plays a role.

cell produce glycoprotein (integrin) that help them bind to fibronectin for adhesion

cell attachment activates cell surface receptors to activate signaling cascade to influence survival, differentiation and proliferation of the cells.

functions of albumin

family of soluble carrier protein, molecular taxi for a range of small hydrophobic molecules esp lipids.

detoxifying role by binding toxic metal ions and other inhibitory factors (absorptive capacity of albumin)

protect cell against damage by shear forces that may occur in stirred or pumped culture systems and cryopreservation.

7 major functions of serum

provide attachment factors

provide growth factors

hormones

nutrients for metabolism

carrier proteins such as albumin and transferrin

protease inhibitors

detoxifying enzymes

substrate ; substance which cells grow on/ in

types of substrate - glass

advantage and disadvantage

optically clear

easily washed without losing its growth-supporting properties

can be sterilized by autoclave or boiling

-usually need to be washed with strong alkali like NaOH or caustic detergents

-so it must be neutralized with acid wash before autoclave sterilization

plastic substrate

its structure ? advantage ? how can plastic polystyrene is able to support cell adhesion?

single use polystyrene flask provide simple, reproducible substrate for culture.

it has good optical quality

since it is a flat growth surface - provide uniform and reproducible culture

plastic substrate is treated to support cell adhesion

because polystyrene is homophobic - which is not suitable surface for cell growth

treated by gamma-irradiation, chemically or with an electric ion discharge to produce a charged surface which is hydrophilic

special matrix-coated surfaces

how is it suitable to be a growth surface?

how it is made, conditioned?

fibronectin, laminin and collagen are major matrix products produced by cells

they promote adhesion, to allow the attachment and spreading that are necessary for cell proliferation

promote the cell-matrix interaction which may be cell type-specific.

for example, chondronectin enhances chondrocyte adherence.

substrate can be conditioned

by applying spent medium

adding purified fibronectin, laminin or collagen to the medium

apply gelatin coating for culture of muscle and endothelial cells

monolayer feeder cells

why is it needed for?

what does it consist?

cultures of some fastidious cells, at low cell density, require support from living cells

irradiated adherent growth-arrested cells so that these cells cannot divide. however, they are viable and bioactive so that they can support the growth of other cells which are feeding on them.

makes the surface suitable for attachment for other cells due to the conditioning of substrate by cell products

also help to enhance cell growth due to supplementation of the medium by leakage or secretion from the layer feeder cells

constituents of gas phase

why the depth of medium is important to consider ?

ambient oxygen with controlled 5.0% of carbodioxide

because cell rely mainly on the dissolved oxygen for respiration which can be toxic due to the elevation in level of free radicals

and depth of culture medium can influence the rate of oxygen diffusion to the cells

so must keep the depth of medium within the range of 2-5mm (0.2-0.5 ml/cm^2)in static culture.

optimum ph color - red

above red is purple and pink

below are yellow and orange

what control ph of the cultures

why is buffer system is required in a basal medium ?

advantages/ disadvantages of CO2-bicarbonate buffer

buffer system

cells produce CO2 and lactic acid from glucose metabolism

open dishes where the evolution of CO2 causes the pH to drop

buffer system in media can compensate the effects caused by acidic waste products to maintain optimal pH range of 7.4 for as long as possible. or to slow down changes in pH

advantages - low toxicity, nutritional benefit to the culture

disadvantages- culture may still undergo pH changes during cell growth

weak buffer with poor buffering capacity, lower pKa

sensitive to temperature and light and changes in CO2%

types of contamination

what are the deleterious effects of these contaminants to the cell culture?

the sources of contamination

how to monitor the contamination

how to determine the microbial contamination/ characteristics features

mycoplasma contamination

how to minimize contaminations

what to do when contamination occurs

bacteria

fungi

mycoplasma

they will flourish in the cell culture environment and rapidly overgrow and in many cases, kill the animal cells by release of toxins, depletion of nutrient medium or depressing the medium pH

techniques such as pipetting, dispensing, manipulations

hygiene like work surface, operator hair, hands, breath and clothing etc

materials and reagents - solutions, glassware and screw taps, instruments and pipettes, culture flasks and media bottles

equipment and facilities -room air, laminar-flow hoods, incubators, refrigerators, infestations

imported biological materials - tissues samples and cell lines

daily inspection of ongoing cultures

-check by eye and with a microscope (100-400x) at each handling of a culture.

-check for mycoplasma every month

-if the culture becomes cloudy overnight and changes from red to yellow, its likely to be contaminated (pH change, color change, cloudy media)

-sudden change in pH

(medium color- from red to yellow)

-cloudy media

-under low microscopy (100x)

if its bacterial, spaces between cells will appear granular or you may see very small black dots. there may be mobility of the bacteria.

fungi, thin filamentous mycelia and sometimes denser clumps of spores

they are not visible by naked eye other than through signs of deterioration in culture

tested by;

fluorescent staining

PCR

ELISA

Immunostaining

Microbiological assays

checking cultures daily by eye and a phase contrast microscope

reagents are checked for sterility before use

ensuring that media are not shared between different users and cell lines

ensuring that the standard of sterile technique is kept high at all times

record the nature of contamination and its specificity

discard all contaminated cultures, including media, flask, pipettes and disposable articles that has been used in conjunction with the cultures

swab the laminar flow and work surface with 70-75% alcohol

review all sterilization procedures in the laboratory

how to ensure sterility of media?

there are two methods of sterilization such as filtration and autoclaving

filter-sterilize heat-liable and non-autoclavable media

sterilize heat-stable reagents by autoclaving

ensure that bottles used to contain medium is sterile

parafilm the bottles individually after medium aliquoting

perform a simple sterility test

(add 6ml of medium to the flask and incubator for 2 days in 37 degree celsius to se if there any contamination)

when should the subculture be started ?

other criteria for subculture

why should we do a subculture ?

all of the available substrata is occupied (confluence)

or when the cell concentration (cells/ml of medium) exceeds the capacity of the medium

in any of the above 2 situations, cell growth ceases or is greatly reduced.

time since last subculture - routine subculture, cell concentration should be determined that allows for the cells to be subcultured after 7 days, with the medium being changed after 3-4 days.

change the type of culture vessel or medium, to increase the stock

trypsinization - function of trypsin

how it is done ? what are things to take note ?

trypsin-EDTA

what is the goal of trypsinization and when should the process stop?

and how to stop?

proteolytic enzyme, break down the proteins that bind the cells to the culture surface

trypsin is added to washed cells in the culture flask for 5-10 minutes to dislodge the cells from the culture surface - do not put the cells into the trypsin for a long time, the cells will be damaged by the trypsin- so bang the edge of the flask with hand

detachments of cells by trypsin is more efficient if magnesium and calcium ions are removed from the medium

combination with EDTA- EDTA is a chelating agent that may be used alone for removal of weakly adherent cells.

the goal is to dislodge the cells from the substrata without damaging them. hence when majority of them are rounded and floating, stop the process.

the action of trypsin is stopped by the addition of serum-supplemented medium.

the excess protein present in the medium serves to reduce the trypsin activity

if the medium is serum-free, use a soybean trypsin inhibitor to stop the action of trypsin.

how to subculture the suspension culture ?

advantages of suspension cultures

scale-up is easier

trypsin treatment is not required

whole process is quicker and less traumatic for the cells

how cryopreservation is done?

why the cells need to be treated with cryoprotectant ?

1.check culture for

2.grow that culture up to late log phase, trypsinize and count

3.resuspend at approximately 10^6-10^7 cells/ml in culture medium containing serum and a cryoprotectant like dimethyl sulfoxide (DMSO) or glycerol at a final conc of 5-10%

cells must be treated with a cryoprotectant DMSO or glycerol to survive freezing and thawing

4.dispense cell suspensions into 1-or2-ml prelabeled cryovials and seal

if the vial is not perfectly sealed, it may inspire liquid nitrogen (during freezing and storage) in the liquid phase of the nitrogen freezer and will subsequently explode violently on thawing or it may become infected.

what is DMSO and why is it and glycerol are needed to add in cryopreservation ?

dimethyl-sulphoxide

to survive freezing and thawing

what are cryoprotectants and what is their mechanism

efflux of water is the key to the cryopreservation process

DMSO or glycerol added to cell resuspension medium (5-10% (v/v)) acts to permeabilize plasma membrane and allow water to flow out of the cells as cooling occurs

cryoprotectants depress the freezing point to -5C. When at -5C to -15C, the cells are sufficiently dehydrated and ice crystals will form in the medium but not inside the cells.

why the cryovials are needed to be perfectly sealed?

because, if the via is not perfectly sealed, it may inspire liquid nitrogen during freezing and storage in the liquid phase of the nitrogen freezer, will subsequently explode (violently) on thawing or it may become infected.

what step is to ensure the vial will cool at -1C/min ?

why it is important to cool at -1C/min

wrap the cryovial in paper towel and submerge it inside the ice box. this is then being transferred to -20C freezer, and subsequently, the cryovial is placed at -70C or -90C in a regular deep freezer.

minimizes or eliminates formation of intra-cellular ice crystals due to gradual cooling.

when the vials have reached -70C, what is the next step?

transfer to liquid nitrogen freezer. This must be done quickly, as the cells will deteriorate rapidly if the temperature rises above -50C.

when the vials are safely located in the liquid nitrogen freezer, make sure to record the: freezing index which shows what is in each part of the freezer.

Thawing is like warming the cells

what things to take not when adding the medium into the cell suspension ?

partially thawed * dilution*

1.check the index

2.label your culture flask

then retrieve the vials from the liquid nitrogen freezzer

place it in water at 37C with a lid (close the lid immediately)

when thawed, swab the vial with 70% ethanol and open it in the laminar flow hood.

transfer the contents into the culture flask

add medium slowly to the cell suspension: 10ml over about 2 min added dropwise at the start and then a little faster, gradually diluting the cells and preservative. (DMSO)

This is particularly important with DMSO, where sudden dilution cause severe osmotic damage and reduce survival by half.

factors affecting cell viability - in cryopreservation and thawing

1. rate of cooling (freezing)

2. warming rate

3. storage temperature

4. cryovials

5. cell no for optimal survival

6. conditions of original cell cultures

what is good about using the DMSO in cryopreservation ?

cryoprotectants generally

to permeabilize the cell membrane and allow the water out of the cell as cooling occurs

to protect the cells from rupture by the formation of ice crystals.

cryoprotectants depress the freezing point to -5C. When at -5C to -15C, the cells are sufficiently dehydrated and ice crystals will form in the medium but not inside the cells.

why put cryovials in 37 C water bath for 1-5mins

reduce the exposure to potenially damaging ice crystals when the vial is partially thawed

what are conditions of original cell cultures for cryopreservation and thawing

healthy with a viability >90%

no microbial contamination

in log phase of growth

finite cell lines should be grown less than the 5th population doubling for freezing

how the student should prepare the BSC and arrange materials in order to avoid contamination during cell culture procedures.

*tutorial bad technique

swab the hood

swab the materials before placing them in the BSC

arrange in clean, working, contaminated area

minimize clutter

position items without blocking the air vents to prevent obstruction to the airflow in the BSC

how to prevent cross-contamination

when does it occur

never work on more than one cell line at a time

never use the same pipette for difference cell lines

never put a pipette back into medium after you have used it to pipette cells.

one cell type may be accidentally introduced into culture flasks of another cell type. occurs when more than 1 cell line is worked on at the same time.

how to ensure the sterility when performing cell culture ?

sterilization of reagents and media

working in laminar flow hood

minimize cluttering

sterile handling

sterile handling includes ?

swabbing -

capping

handling of bottles and flasks

flaming

3 types of cell viability assays

dyes reduced by cellular enzymes

mitochondrial membrane potential dependent dyes

ATP and ADP assays

how many types of dye reduction assays

how it is done

tetrazolium and resazurin assays

they incubate a reagent with the cell sample

and the compounds in the reagent form a dye in a metabolically active environment, and the resulting color change can be quantified to indicate the extent of cell viability.

-measure ABS

MTT Assay ?

MTT is converted into formazan via electron transfer reaction with substrates in the cell such as NADH and NADPH

the formazan crystals are insoluble, and form as precipitate inside the cells and culture medium

the crystals must be solubilized for the color change to be detected via spectrophotometer.

DMSO and SDS solutions are added to dissolve crystals

MTT is light-sensitive, must be kept and used in the dark.

advantages / disadvantages of MTT Assay

advantages - inexpensive, easy to use and adaptable to large scale screening

can be used with all cell types

disadvantages- toxicity, affected by culture conditions, susceptible to chemical interference, less sensitive than fluorescent and luminescent cell viability assays.

MTS, XTT, WST-1 assays

advantages;

disadvantages;

intermediate electron acceptor reagent is added. it enters into the cell and it itself reduced, leaves the cell where it can then transfer the electrons into the tetrazolium salt. the salt is then converted into the soluble formazan product. since the product is soluble, multiple readings can be taken from the same plate across a time-course.

advantages;

easy to use

No DMSO dissolution required

more sensitive and accurate than MTT

disadvantages;

intermediate reagent which is required(may be toxic)

resazurin assays

advantages

disadvantages

resazurin assays use electron transfer to convert one compound into another.

resazurin forms a dark-blue solutionn when dissolved in physiological buffers, which is converted to resorufin.

resorufin is a pink, fluorescent product.

resazurin can penetrate cells, meaning that an intermediate electron acceptor is not required, although inclusion can speed up signal generation.

advantages;

simple, safe and rapid

relatively inexpensive

increased sensitivity over tetrazolium assays

disadvantages;

risk of fluorescence interference from other compounds

light sensitive

positive controls

negative controls

qualitative

semi-quantitative

quantitative

to ensure that the testing procedure results when the expected outcome is present.

to confirm that the experimental setup can detect positive results and that all reagents and instruments are functioning correctly and as intended.

to ensure that no change is observed when a change is not expected

to confirm that any positive result in the experiment is truly due to the test condition and not due to external factors.