Basic Tissue Culture Techniques

Preface

All human tissue culture work should be carried out wearing gloves and long-sleeved gown in a biosafety cabinet contained in an appropriate area. Gloves and consumable tissue culture products must be disposed in a biohazard bin.

All solutions below are assumed to be sterile unless stated otherwise.

Media

The priniciple media type used in the Boyd Laboratory is RMPI-1640, purchased from CSL in powder form and made up in 20-40 L batches to pH7.0, stored at 4•C up to 2 years. Most major suppliers of culture reagents and media also stock RPMI. Other media types are sometimes used for specific applications for example SFM (Life Tech) for growing hybridomas for antibody purification; DMEM for some mouse lines; Iscoves for chicken cell culture.

RPMI is a useful universal media type since it can be used for human cellular work and is easily adjusted for mouse work by the addition of 2g NaCl/10L (290 mOsm to 325 mOsm)

Media Supplements

Thawing cell lines from liquid nitrogen stocks

Counting cells

Methods include ethidium bromide/acridine orange (EtBr/AcOr -see here for recipe) or Trypan Blue dye inclusion/exclusion, but others are also valid such as the use of a Coulter™ counter or similar, though the latter do not truly count viability.

Ethidium Bromide Acridine Orange Method

The main centre grid is 1mm x 1mm in area and 0.1 mm deep i.e. 10 -4 mL

e.g. Cell count x 2 x dilution (ie x 10 or x 50) x 10 4 = cells / mL

Plating cells

This term refers to decanting an appropriate cell suspension into tissue culture vessels which may be of a tray type of various well numbers, flasks of varying volumes or even petri dishes (where the term orginates)

Freezing cells down

Cells to be frozen are harvested from the culture vessel and centrifuged as before, then resupsended in cold 10% DMSO in FBS(can be kept as a frozen stock in aliquots)

Growth techniques

You must be taught to recognise healthy from non healthly cells by someone experienced and learn when cells should be transferred/passaged.

Adherent vs Suspension culture

Cells that grow in suspension vs those that adhere to the tissue culture vessel are handled differently.

Method

Expansion

Cells resuspend as above may be divided in two for expansion, usually when 70-90% confluency (=100% surface area of vessel). They may be able to take a higher dilution ie 1/10 or more if hardy and in exponential phase. Generally this is done by taking one well of a given size and dividing the cells between 2 wells of the same size and keeping the volume the same eg:

Maintenance / Back Up Culture

To keep stable cells in culture a convenient method is to take the cultures to a 24 well tray and serially dilute the cells across the row ie 1 mL + 1 mL dilution then add 1 mL back to the finished dilutions to make 2 mL final volume in each well. One row is usually enough with most cells to last a maximum of 1 week before repeating the process. Assay the cells regularly to maintain their specificity and do not do this routinely, rather if there is a specific reason. Ideally this should be done with a different media sample e.g. use parallel bottles or make a small aliquot at the start & filter it into a 50 mL tubes or 100 mL bottle.

Use of antibiotics

Antibiotics may be necessary for some cell lines to propagate under selective pressure, however for routine culture of most cell types, the use of antibiotics to prevent infection must be discouraged.

Reasons for not using antibiotics

Disposal of infected media/cells