Growing Cells in Culture

Part 1: Terminology

Cell CultureCell CultureThe maintenance of cells outside of the living animal (in vitro) for easier experimental manipulation and regulation of controls.

• Pros– Use of animals reduced– In vitro models allow for control of the

extracellular environment– Able to monitor various secretions without

interference from other biological molecules that occurs in vivo

• Cons– Removal of cells from their in vivo environment

means removing the cells, hormones, support structures and various other chemicals that the cells interact with in vivo.

– It is nearly impossible to recreate the in vivo environment.

Classification of Cell Cultures

• Primary Culture– Cells taken directly from a tissue to a

dish– Cells taken from a primary culture and

passed or divided in vitro.– These cells have a limited number of

divisions or passages. After the limit, they will undergo apoptosis.• Apoptosis is programmed cell death

Primary culture from Poeciliopsis lucida (the desert topminnow)

Making a Primary Culture

• Cell Line – Cells that have undergone a mutation and

won’t undergo apoptosis after a limited number of passages. They will grow indefinitely.. Good model system?

• Transformed cell line – A cell line that has been transformed by a

tumor inducing virus or chemical. Can cause tumors if injected into animal. Good model?

• Hybrid cell line (hybridoma)– Two cell types fused together with

characteristics of each. Good model?

Cell Lines

Our Cell Lines

• MCF-7 –estrogen receptors expressed , alpha > beta

• MDA-MB-231- beta estrogen receptors expressed• Breast Cancer• ATCC• http://www.atcc.org/

Growing Cells in Culture

Part 2: Understanding Cell Behavior

Confluency• How “covered” the growing

surface appears• This is usually a guess• Optimal confluency for moving

cells to a new dish is 70-80%– Too low, cells will be in lag

phase and won’t proliferate. Sense nearest neighbor.

– Too high and cells will stop dividing or pile on one another in tumor like formation.

Contact Inhibition

• When cells contact each

other, they cease their

growth.

• Cells arrest in G0 phase

of the cell cycle

• Transformed cells may

continue to proliferate

and pile upon each other

• May overgrow and die

Anchorage Dependence

• Cells that attach to surfaces in vivo require a surface to attach to in vitro.– Other cells (feeder layer) or specially treated

plastic or other biologically active coatings– Some may grow in suspension as well as

spheroids (unusual)

Passage number

• The number of times the cells have been

removed (or “split”) from the plate and re-

plated.

• Always write this on your plate or flask as

“P#” for the passage number.

Growing Cells in Culture

Part 3: Solutions used in cell culture

Cell Culture Media

• A. Bulk ions - Na, K, Ca, Mg, Cl, P, Bicarb or CO2

B. Trace elements - iron, zinc, seleniumC. Sugars - glucose is the most commonD. Amino acids - 13 essentialE. Vitamins F. Choline, inositol (cell structure and membrane integrity)G. Serum H. Antibiotics - although not required for cell growth,

antibiotics are often used to control the growth of bacterial

and fungal contaminants.

Fetal Bovine Serum

• Contains a large number of growth promoting activities such as buffering toxic nutrients by binding them, neutralizes trypsin and other proteases, has undefined effects on the interaction between cells and substrate, and contains peptide hormones or hormone-like growth factors that promote healthy growth.

Trypsin EDTA• An enzyme used to detach the cells from

a culture dish.• Trypsin cleaves peptide bonds (LYS or

ARG) in fibronectin of the extracellular matrix.

• EDTA chelates calcium ions in the media that would normally inhibit trypsin.

• Trypsin will self digest and become ineffective if left in water bath more than 20 minutes.

• Trypsinizing cells too long will reduce cell viability

Trypan Blue-ViCell

• An exclusion dye

• Living cells cannot take up the dye and will appear bright and refractile.

• Dead cells with broken membranes will absorb the dye and appear blue.

• Usually add 200 l of trypan blue to 200 l of cell suspension in eppendorf tube

70% Ethanol

• Decontamination of work surfaces and incubator interiors.

• 100% ethanol may be used as a solvent as may methanol and DMSO.

• How do you make 70% ethanol?

Bleach

• Used to destroy any remaining cells in dishes and tubes before they are tossed in the trash can (10% bleach).

• Add enough to change media to clear, – wait 5 minutes, – rinse solution down sink– throw away the dish/flask/plate in the trash

can.

Growing Cells in CultureGrowing Cells in Culture

Part 4 : Equipment

CO2 incubator

• Maintains CO2 level (5-10%), humidity and temperature (37o C) to simulate in vivo conditions.

• Humidity helps maintain pH

Inverted Phase Microscope

• A phase contrast microscope with objectives below the specimen.

• A phase plate with an annulus will aid in exploiting differences in refractive indices in different areas of the cells and surrounding areas, creating contrast

A comparison

Phase contrast microscopy Light microscopyCan be used on living cells requires stain, thus killing cells

Biological Safety Cabinet

• The primary purpose of a BSC is to serve as the primary means to protect the laboratory worker and the surrounding environment from pathogens. All exhaust air is HEPA-filtered as it exits the biosafety cabinet, removing harmful bacteria and viruses. We are biosafety level 2 but only work within biosafety level 1.

Centrifuge

• Puts an object in rotation around a fixed axis, applying a force perpendicular to the axis.

Microplate Reader

• Designed to detect biological, chemical or physical events of samples in microtiter plates.

• Common detection modes for microplate assays are absorbance, fluorescence intensity, luminescence.

Basic cell culture instructions

Aseptic Technique

• For best results in tissue culture, we want to work to keep microbial (bacteria, yeast and molds) contamination to a minimum. To do this, there are certain things you must be aware of and guidelines to follow.

• Work in a culture hood set-aside for tissue culture purposes. Most have filtered air that blows across the surface to keep microbes from settling in the hood. Turn off the UV/antimicrobial light and turn on the hood 30 minutes prior to entering the hood.

• Wash hands with soap and water before beginning the procedure and rewash if you touch anything that is not sterile or within the hood. Wear gloves!!

• Spray down your gloves, work surface, and anything that will go into the hood with 70% ethanol. Rewipe at intervals if you are working for a long time in the hood. This will reduce the numbers of bacteria and mold considerably.

• Do not breathe directly into your cultures, bottles of media, etc. This also means to keep talking to a minimum. No singing or chewing gum.

• Work as quickly as you can within limits of your coordination. Also, keep bottles and flasks closed when you are not working with them. Avoid passing your arm or hand over an open bottle.

• Use only sterilized pipets, plates, flasks and bottles in the hood for procedures.

• Take special precautions with the sterile pipets. Remove them from the package just before use. Make certain to set up the numbers on the pipet so that they face you. Never mouth-pipet, use the pipetting aid. Change pipets for each manipulation. If the tip of the pipet touches something outside of the flask or bottle, replace with a new one. Never use a pipet twice.

Basic Cell Culture Procedure for Anchorage Dependent Cells

• View cells using inverted phase microscope• Aseptically aspirate media• Rinse residual media with 2.5mL trypsin, remove• Add 2.5mL Trypsin to cells • Incubate cells at 37° C for 3 minutes• Agitate and view on phase microscope for

floating cells, strike flask if still adherent• Resuspend cells with minimal fresh media• Take sample and count cells (VICell)• Calculate how many cells are needed to add to

new plate or flask

Remember

• Some volumes don’t need to be exact in cell culture

• Rinsing volume of trypsin (as long as it fits in the dish and is sufficient to rinse the serum).

• Volume of trypsin as long a bottom of plate or flask can be covered.

• Volume of media used to resuspend your cells. The same number of cells will be there despite the volume of media used.

– Too little resuspension media will result in very high cell count and would require more dilution (and higher dilution factor). The volume needed to seed your next plate would then be very small, maybe too small to work with.

– Too much media would result in low cell count/ml and you may need a large volume to add to your new plate.

Troubleshooting Low Hemacytometer Counts

Trypsinization not complete

• Trypsin is ineffective – too cold, be sure to warm sufficiently– self digested or expired check date, don't

warm too long– too much serum left on plate -rinse

plate thoroughly with trypsin

Trypsinization technique

• Trypsin doesn't coat plate, completely add full 2.5 mL’s, lay flask down for 3 minutes at 37ºC,

• not left long enough in incubator depends on cell line HACAT can go 8 minutes

• flask may need to be tapped or slapped to facilitate cell removal(this varies by cell line)

Six Essential Calculations

Hemacytometer

• Specialized chamber with etched grid used to count the number of cells in a sample.

• use of trypan blue allows differentiation between living and dead cells

Using the Hemacytometer

• Remove the hemacytometer and coverslip (carefully) from EtOH and dry thoroughly with a kimwipe.

• Center coverslip on hemacytometer

• Barely fill the grid under the coverslip via the divet with your cell suspension.

• Count cells in ten squares (5 on each side) by following diagram at station.

Looking at the grid

under the phase

contrast microscope

How the cells will appear• Bright refractile “spheres” are

living cells,

• Blue cells about the same size as the other cells are dead.

• Keep a differential count of blue vs. clear for viability determination.

• Sometimes there will be serum debris, and this will look red or blue and stringy or gloppy--don’t count it!

These are blood cells,You will not have this many

Count 10 squaresAny 10 will do but we will follow convention

Watch for stringy, reddishmaterial—those aren’t cells!

serum

Top group

Count cells thattouch top and left lines

DO NOTCount cells thattouch bottom and right lines

Bottom Group

Calculate your cells/ml

• Calculate the number of total cells in one ml of your suspension.Total cells counted x (dilution factor) x (10,000)

number of squares

• Here, dilution factor is 2 and # of squares is 10

(our example 62/10 x2 x104 =1.24 x 105)

Determine your percent viability

• Viability is a measure how many of your cells survived your cell culture technique.

# of viable (living) cells x 100

total number of cells counted

Our example 54/62 x 100 =87.09%

Calculate total # of cells in original suspension

Number of cells per ml x total mls of original suspension

Let’s assume 10ml original suspension1.24 x105 x 10 =1.24 x 106 cell total

Total # of viable cells available in original suspension

Total number of cells in original suspension x % viability1.24x106 x 87% =1.08x 106 viable cells in the original suspension

Determine the number of cells you need to add to your flask

• You want the cells to grow happily without overcrowding (or being too sparse) before the next time you come into class.

• Using the calculation on the next slide, figure out the number of cells needed for the size of vessel being used

• You need to take into account:– length of time cells are to be grown.– the size of the cells (not directly in the formula)

– their doubling time

An ExerciseAn Exercise• You will be using a T-25 flask and using cells

that have a doubling time of 18 hours

• X is the number of cells you want by the time you return to passage them (right column of table, next slide)

• X0 is the number of cells that were seeded (we want to solve for this right now)

• t is the time since plating (hours until the next passaging)

• td is the doubling time of the cell line.

dt

t

eXX*)2ln(

0

Vessel 3T3-L1 final count

18 hour doubling rate

3.5cm or 6 well plate 1x106

6cm dish or T25 flask 2 x106

10cm dish 5 x 106

Determine how many mls of cell suspension much to add to your

flask

# of cells needed

cells/ml

Determine total # mls fresh media you will need to add to dish or

flask• Use table in VISTA to see how many mls

will fit in your flask (or we will tell you).

Volume flask will hold – mls suspension to you plan to add

Growing Cells in Culture

Part 5: The protocol

Observing cells in culture

• Check color of media– Healthy growth usually leaves media

slightly orange– Too yellow means bacterial growth– Too purple means low carbon dioxide, cells

dead

• Observe cells under phase microscope– Spread out or rounded?– How confluent?

What to do with growing cells

• If they are at least 70-80% confluent

• Subculture them – Also called passing or

splitting

• Remove media, remove cells, resuspend and transfer some to a new plate

• If they are not very confluent

• Lift and replace onto same plate– Culture more than 4 days old

for our cells– Remove old media, lift cells

from plate and resuspend in fresh media on same plate

• “Feed” them– Culture less than 4 days old– Remove old media and replace

with fresh, warm media

Brief subculturing preview

• Remove media, lift cells from plate

• Resuspend cells in fresh media

• Count cells and determine viability

• Seed new plates with appropriate # of cells and volume of media

Some volumes that do not need to be Some volumes that do not need to be exact—but follow our exact—but follow our

recommendations until you are recommendations until you are comfortablecomfortable

• Rinsing volume of PBS

• Volume of trypsin EDTA

• Volume of media to resuspend cells– Record how much

• Volume of cells removed for counting

• Exact # of cells to be plated

You will need to return to take care of your cells

• Thursday or Friday is an in between point before next week.

• First time through may require up to an hour

• If one member cannot make the return time, that person should work in hood tonight.

• Choose times that will be consistent each week