TissueCultureMethods2

IV. MAINTENANCE

Cultures should be examined daily, observing the morphology, the color of the medium and the density of the cells. A tissue culture log should be maintained that is separate from your regular laboratory notebook. The log should contain: the name of the cell line, the medium components and any alterations to the standard medium, the dates on which the cells were split and/or fed, a calculation of the doubling time of the culture (this should be done at least once during the semester), and any observations relative to the morphology, etc.

A. Growth pattern. Cells will initially go through a quiescent or lag phase that depends on the cell type, the seeding density, the media components, and previous handling. The cells will then go into exponential growth where they have the highest metabolic activity. The cells will then enter into stationary phase where the number of cells is constant, this is characteristic of a confluent population (where all growth surfaces are covered).

B. Harvesting. Cells are harvested when the cells have reached a population density which suppresses growth. Ideally, cells are harvested when they are in a semi-confluent state and are still in log phase. Cells that are not passaged and are allowed to grow to a confluent state can sometime lag for a long period of time and some may never recover. It is also essential to keep your cells as happy as possible to maximize the efficiency of transformation. Most cells are passaged (or at least fed) three times a week.

1. Suspension culture. Suspension cultures are fed by dilution into fresh medium.

2. Adherent cultures. Adherent cultures that do not need to be divided can simply be fed by removing the old medium and replacing it with fresh medium.

When the cells become semi-confluent, several methods are used to remove the cells from the growing surface so that they can be diluted:

• Mechanical - A rubber spatula can be used to physically remove the cells from the growth surface. This method is quick and easy but is also disruptive to the cells and may result in significant cell death. This method is best when harvesting many different samples of cells for preparing extracts, i.e., when viability is not important.

• Proteolytic enzymes - Trypsin, collagenase, or pronase, usually in combination with EDTA, causes cells to detach from the growth surface. This method is fast and reliable but can damage the cell surface by digesting exposed cell surface proteins. The proteolysis reaction can be quickly terminated by the addition of complete medium containing serum

• EDTA - EDTA alone can also be used to detach cells and seems to be gentler on the cells than trypsin. The standard procedure for detaching adherent cells is as follows:

1. Visually inspect daily

2. Release cells from monolayer surface

C. Media and growth requirements

1. Physiological parameters

• A. temperature - 37C for cells from homeother

• B. pH - 7.2-7.5 and osmolality of medium must be maintained

• C. humidity is required

• D. gas phase - bicarbonate conc. and CO₂ tension in equilibrium

  
  

• E. visible light - can have an adverse effect on cells; light induced production of toxic compounds can occur in some media; cells should be cultured in the dark and exposed to room light as little as possible;

2. Medium requirements: (often empirical)

A. Bulk ions - Na, K, Ca, Mg, Cl, P, Bicarb or CO₂
B. Trace elements - iron, zinc, selenium
C. sugars - glucose is the most common
D. amino acids - 13 essential
E. vitamins - B, etc.
F. choline, inositol
G. 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. 
H. antibiotics - although not required for cell growth, antibiotics are often used to control the growth of bacterial and fungal contaminants.

3. Feeding - 2-3 times/week.

4. Measurement of growth and viability. The viability of cells can be observed visually using an inverted phase contrast microscope. Live cells are phase bright; suspension cells are typically rounded and somewhat symmetrical; adherent cells will form projections when they attach to the growth surface. Viability can also be assessed using the vital dye, trypan blue, which is excluded by live cells but accumulates in dead cells. Cell numbers are determined using a hemocytometer.

V. SAFETY CONSIDERATIONS

	Assume all cultures are hazardous since they may harbor latent viruses or other organisms that are uncharacterized. The following safety precautions should also be observed:
	pipetting: use pipette aids to prevent ingestion and keep aerosols down to a minimum
	no eating, drinking, or smoking
	wash hands after handling cultures and before leaving the lab
	decontaminate work surfaces with disinfectant (before and after)
	autoclave all waste
	use biological safety cabinet (laminar flow hood) when working with hazardous organisms. The cabinet protects worker by preventing airborne cells and viruses released during experimental activity from escaping the cabinet; there is an air barrier at the front opening and exhaust air is filtered with a HEPA filter make sure cabinet is not overloaded and leave exhaust grills in the front and the back clear (helps to maintain a uniform airflow)
	use aseptic technique
	dispose of all liquid waste after each experiment and treat with bleach