Tissue Culture and media preparationPurpose: To deﬁne and understand tissue/cell cultures, the different types, and what is required to maintain them.
Tissue culture: the process ofgrowing and maintaining plant oranimal cells in vitro.
Requirements for culturing cells • Sterile environment. • Nutrients for growth. • Stable pH and temperature.
Basic constituents of media • Inorganic salts • Carbohydrates • Amino Acids and vitamins • Serum
Inorganic salts – primarily help to retain osmoticbalance of the cells.Carbohydrates – are the main source of energy forthe cultured cells. Generally in the form of sugarswith the major sugar used being glucose.Vitamins – generally come from the serum, but manymedia are also enriched with B group vitamins.Serum – Albumins, growth factors and growthinhibitors. Most commonly used and the one that weuse is Bovine Growth Serum (BGS). Advantages of using serum: can be used with awide range of cell types and can bind and neutralizetoxins. Disadvantages: risk of contamination.
albumin: A class of simple, water-soluble proteinsthat can be coagulated by heat and precipitatedby strong acids and are found in egg white, bloodserum, milk, and many other animal and plantjuices and tissues.
Buffering Systems – help to regulate pH conditions.Type 1 “Natural” buffering system balance CO2 gas with the CO3/HCO3 content of the culture medium. This Bicarbonate/CO2 buffering system is what we use in our lab. Advantages: low cost, non-toxic, and provides other chemical beneﬁts to the cells.
Most media include phenol red as a pH indicator so thatpH status in constantly indicated by color change.yellow (acidic) orpurple (alkali),then need to change/replenish the media.
Buffering Systems – help to regulate pH conditions.Type 2 Chemical buffering system using HEPES. Advantages: superior buffering capacity and cultures do not require a controlled, gaseous atmosphere. Disadvantages: expensive and can be toxic to some cell lines.
Media Preparation – Requires “tissue culture pure” water. We use DD water. Media should be ﬁlter sterilized. Addition of Serum and antibiotics sometimes added as well. We add sodium pyruvate (needed for cells to convert glucose to ATP) and Bovine Growth Serum (BGS). No antibiotics. If just RPMI, we add penicillin-streptomycin and gentamicine.
Renourishment – Cells need to be renourished fromtime to time. Color indicator is usually the telltalesign. Some cells need to have renourishment soonerthan others (i.e. WEHI cells). Sometimes cells needmore room to stretch their feet, so they need to besplit.
Aseptic Technique – All manipulations performed under a sterile hood (laminar ﬂow hood). Appropriate clothing (latex gloves, lab coat, etc.) Sterilize area with alcohol before and after manipulations. Instruments purchased sterile or autoclaved.
Primary cultures come directly from excised, normalanimal cells or tissue.Advantages: they retain many of the characteristicsof the cell in vivo.Disadvantages: labor intensive and they can bemaintained in vitro only for a limited period of time.Example: when we harvest and culture splenocytesfrom our mice.
Continuous cultures are made up of a single celltype that can be serially propagated in culture eitherfor a limited # of cell divisions or indeﬁnitely.Generally they are tumor cell lines.Advantage: almost limitless availability.Disadvantage: retain very little of the original in vivocharacteristics of the cell.Example: WEHI cells.
Culture morphology – two forms: Grow in suspension as single cells or small, free- ﬂoating clumps. Grow as a monolayer that attaches to the bottom of the tissue culture ﬂask.*Form reﬂects where the cell was taken from, the tissuefrom which it was derived. Cells from blood generallygrow in suspension and cells from solid tissue grow asmonolayers. The WEHI cells grow as a monolayer.
WEHI cells = FibrosarcomaThey divide indeﬁnitely because they have lost thecontrol mechanism that signals the cells to remain inG1.