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What is the purpose of an organ bath?

It's an ideal way to expose isolated tissue to a known drug concentration and measure responses that are produced.


What does the aqueous solution in an organ bath act as?

A physiological fluid. It is usually 20ml in capacity.


What is a typical solution used in an Organ bath?

Krebs solution.
It has glucose, K, Na, Ca, Mg and Cl- which nourishes the tissue.


What is the purpose of an oxygen tube?

Aeration of krebs solution so tissue has oxygen supply.
Usually 95% oxygen, 5% CO2


How is a constant temperature of 37 degrees maintained?

A waterjacket surrounding the organ bath with a constant flow of warm water, warming the krebs.


What things are needed to keep the tissue alive in an organ bath?

krebs solution
oxygen tube
constant temperature (37 degrees)


What does the force transducer do in an organ bath?

It measures the force produced from the contraction of the tissue. It is connected to the tissue hook.


Where are drugs injected?

The organ bath solution (via a pipette).


What is the resting tension in an organ bath?

The tension when tissue is placed in the bath without contracting. If there is no resting tension, no contraction can be recorded (as there will be slack and this may not signal the force transducer).


What are the 3 types of important info you can obtain from a recording of response of isolated tissue?

1. The responses of an isolated tissue to endogenous compounds and to nerve or muscle stimulation may be characterised.

2.The mechanism of action of a new drug may be studied.

3. The potency of a drug may be determined.


Organ baths were widely used in what year?

1960's until the 1990's. Today we look at drug effects on a molecular level, rather than just tissue level.


In an organ bath, do you need to consider ADME processes?



What is good about using mammalian cell cultures?

You can investigate drug receptor interactions and look at the activation of a signal transduction pathways. It can also be used to study the processes that might occur in disease conditions.

Can be used for drug screening and development, testing of toxic drugs, testing of new therapies and large scale manufacturing of biological compounds.

Can also culture for graft/repair purposes.


What are primary cells?

Cells that are used directly from tissue. They are treated with enzymes that break down the extracellular components holding the cells together (dissociation). They then go to tissue growth plate.


What are advantages and limitations of using primary cells?

They are the closest approximation to the 'real thing'.

BUT they are time consuming and numbers produced can be small and they have a finite life span as they become senescent.


What are immortilized cell lines?

Cells that have acquired the ability to proliferate indefinitely.


How can a cell become immortilized?

Spontaneously through random mutation or by deliberate genetic modification. Many are derived from tumor tissue (e.g. HeLa cells from cervical cancer).


What are SHSY-5Y cells?

Immoritlized cells derived from a neuroblastoma.


What is transformation of cells?

The process by which the cells are genetically modified so they acquire the ability to divide indefinitely.


How were HEK293 (human embryonic kidney) cells transformed?

With a specific viral DNA that led to their immortalization.


What are the advantages of transformation?

They are readily available and easy to grow, can proliferate indefinitely leading to a potentially limitless supply of cells for use, results obtained in experiments are quite consistent and highly reproducible.


What are disadvantages of transformation?

They may have only a few of the in-vivo characteristics of the tissue they originated from, their cell properties may change over time or after transformation, or they may mutate into sub-clones with different properties.


What is the 'aseptic' technique?

Reducing the probability of contamination of the culture from microorganisms in the environment.


What are the 4 elements of the aseptic technique?

A sterile work area, good personal hygiene, sterile reagents and media, and sterile handling.


What is MSDS formation?

Material safety data sheets (includes properties of chemical, toxicity, etc).


What is tissue culture media?

A media for mammalian cells with nutrients like inorganic salts, amino acids, carbohydrates, vitamins, minerals, growth factors, hormones, and trace elements necessary to support the growth of the cells in this artificial environment.


What are tissue culture incubators?

Incubators that maintain the physico-chemical environment for the cells. These incubators maintain a stable temperature (37oC) and humidity (98%), and deliver CO2 and oxygen or air to maintain pO2 and pH.


Mammalian cells require what kind of work conditions?



Which hood type is most commonly used?

Class II hood. They don't make much noise.


A tissue culture hood regulates what?



What are laminar flow hoods used for?

Aseptic techniques.


What is confluency?

How much of the cell culture covers the surface of the vessel. Expressed as a percentage.


What are limitations beyond 100% confluency?

Size of cell after division and nutrient supply. The environment becomes more acidic as cells use nutrients.


What indicator is used to monitor cell culture pH?

Phenol red. At neutral, it's red. As it becomes more acidic, it changes to orange/yellow and at this point, cells in culture need to be passaged.