Cell Culture in Development of Biological Therapeutics

What is cell culture?

-refers to the removal of cells from an animal or plant in an artificial environment for scientific research

What is cell culture used in?

-drug screening for drug development and large-scale manufacture of biological therapeutics including vaccines

What are the basic equipment needed for cell culture and what do each do?

-laminar flow hood
-incubator
-inverted microscope
-centifuge
-water bath
-fridge and freezer
-liquid nitrogen storage
-hemocytometer
-pipettes
-cell culture vessels
-consumables

What is the laminar flow hood used for?

important to keep the environment ultra-clean with no biological or particulate contamination

What is the incubator used for?

-enables the correct growth conditions by controlling the temp, humidity and CO2 levels

What is the inverted microscope use for?

-able to visualize the cells to monitor cell morphology, count cells and identify contamination

What is the centrifuge used for?

-needed to pellet suspended cells during various culture protocol

What is the water bath used for?

-pre-warms medium before use and thawing frozen cells and reagents

What is the fridge and freezer used for?

-cell culture media need to be stored at 2-8C while most reagents need to be at -5 to -20 C

What is a hemocytometer used for?

-used to count cells

What are pipettes used for?

-dispenses measured volumes of liquids, generally 2uL to 50mL in cell culture

What are cell culture vessels used for?

used for adherent cell attachment

What are consumables used for?

-include various tubes and pipettes that are single use plasticware, which is more cost effective and reduces the risk of contamination

What are some other useful equipment for cell culture?

-colony counter, plate reader, pH meter

What are the 4 basic reagents for cell culture?

-complete medium:
-buffered solution: PBS used for washing cells
-detaching agent:an enzyme used to detach adherent cells from culture vessels for culturing, such as trypsin.
-cryoprotective agent: an agent that reduces the freezing point of media and slow the cooling rate to reduce the risk of ice crystal formation which can damage cells and cause cell death.DMSO is most commonly used.

What are the cell culture conditions needed?

pH:7.0-7.4
osmolarity:280-320 mOsmol/L
CO2: 5-10%
temp:35-37C

What are primary cell cultures?

-cells isolated directly from intact, dissociated or organ fragments and grown in a dish.
-once a primary culture has been sub-cultured for the first time, it is called a cell line.

What are immortalized cell lines?

-cell lines that have no limit on their lifespan

How can normal cells become immortalized?

-through mutations in growth promoting genes
-treatment with chemicals or intro of a tumor causing virus to activate growth-promting genes

How do adherent cells grow?

-in a monolayer attached to the surface of the cell culture plate

What do suspension cells do?

-they remain in suspension and form clumps, especially at high density

What are the four phases of cell culture growth?

1.lag phase: cells are acclimatizing to cell culture conditions and are not dividing
2.log phase: cells are dividing; best phase for cell experimentation and data collection
3.stationary phase: when cells approach overcrowding, cell growth slows
4.decline phase: when the natural process of cell death predominates

What is cell confluency?

-the percentage of the culture vessel surface area that appears covered by a layer of cells when observed by microscopy

What is the criteria used to consider selecting a cell line?

-species
-functional characteristics
-finite or immortalized
-normal or transformed
-growth conditions and characteristics

What is the morphology of cells generally described by?

-fibroblast, epithelial, endothelial, neuronal or lymphoblasts which indicate both cell of origin and physical appearance

How can cell line authentication be achieved?

-by genetic profiling using polymorphic short tandem repeat(STR) loci

What are the four key ingredients to consider when creating medium?

-basal medium: mixture of nutrients and salts
-glutamine: essential amino acid needed for cell growth
-animal serum: provides required growth factors and nutrients for cells
-antibiotics: prevents bacterial growth

What is aseptic technique?

-used for successful cell culture to keep cultures free from both microbial contamination and cellular cross contamination

Where can primary cell isolation be performed?

-tissues, bone marrow, blood, spleen and lymph nodes

How do you perform primary cell isolation?

-cut the isolated tissue into 2-4mm pieces and add to an appropriate buffer on ice and wash 2-3 times.
-dissociation enzymes are added and incubated; cells are dispersed by gently pipetting
-cell suspension is filtered through a fine mesh and washed 2-3 times
-cells are resuspended in medium and seeded

What enzymes are commonly used in tissue dissociation protocols for cell isolation?

-collagenase
-hyaluronidase
-DNase
-elastase
-trypsin

What is subculturing?

-diluting of cells that have reached high confluence to enable continuous culture propagation

When should adherent cells be passaged? Suspension cells?

-80-90% confluent
-when cells start to clump and culture becomes cloudy

Experiments should not be performed on cell lines with…

-very. high passage numbers

What are the cell passage protocol steps for adherent cells?

-media is removed and cells are washed once with PBS
-detaching agent is added (trypsin) and cells are incubated at 37C until fully detached. this can take 1-20 mins. monitor cells under a microscope
-inactivate trypsin by adding medium to cells and spin in a centrifuge to pellet cells
-remove media, gently resuspend cells in fresh media and plate cells in a new culture vessel

What is the cell passage protocol for suspension cells?

-no trypsin is required
-cells are collected and centrifuged to form a pellet, media is removed and cells are resuspended in PBS for a wash step.
-the buffer is removed after another round of centrifugation and cells are resuspended in fresh media and replated

What is cryopreservation?

-process of cooling and storing cells at a very low temp to maintain viability.

How is cryopreservation done?

-washing and pelleting cells, resuspending them in medium with DMSO and transferring the cell suspension to 1 mL sterile cryovials.
-placed in a cryo-freezer and then transferred to liquid nitrogen storage

How do you revive cell lines?

-cells are thawed rapidly in a 37C water bath and transferred to a culture dish with pre-warmed media
-the media dilutes the DMSO so it is no longer toxic and once they are passaged twice, they can be used for experimentation

/What is a major problem in cell culture? Why?

-mycoplasma infection
-it can alter cell behavior, metabolism and have adverse effects on cells
-important to perform mycoplasma detection assays

How is cell counting performed?

-most common method is with a hemocytometer
-position the cover glass over the chamber.
-add 10-20uL of cell suspension into one of the two chambers
-use a microscope and count the cells in each of the four outer squares
-add the counts together and divide by 4

What is cell transfection? What are the most common reagents used?

-refers to the delivery of nucleic acids into cultured cells.
-cationic lipids that form positively charged complexes and allow for interaction of DNA/RNA with the negatively charged cell membrane