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Cell culture and maintenance-“Concepts of in-vitro screening”

Submitted To- Presented By-Mrs. Jyothi Y. Bhavya RewariAsst. Proff., Dept. of Pharmacology M.Pharm, Ist year, Pharmacology Krupanidhi College of Pharmacy, Bangalore

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Contents

Introduction Other techniques The “Three Rs“ Cell cultures Computer simulation Conclusion

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Introduction

Most scientists and governments say they agree that animal testing should cause as little suffering as possible, and that alternatives to animal testing need to be developed

Two major alternatives to in vivo animal testing are in vitro cell culture techniques and in silico computer simulation

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Other techniques-

Biotechnology methods

Tissue culture technique

Fibroblast culture

Tissue slices

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Some claim they are not true alternatives since simulations use data from prior animal experiments and cultured cells often require animal derived products

Others say that they cannot replace animals completely as they are unlikely to ever provide enough information about the complex interactions of living systems

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Other alternatives involve the use of humans for skin irritancy tests and donated human blood for pyrogenicity studies

Another alternative is so-called microdosing, in which the basic behaviour of drugs is assessed using human volunteers receiving doses well below those expected to produce whole-body effects

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The “Three Rs", may be the guiding principles for the use of

animals in research

Replacement refers to the preferred use of non-animal methods over animal methods whenever it is possible to achieve the same scientific aim

Reduction refers to methods that enable researchers to obtain comparable levels of information from fewer animals, or to obtain more information from the same number of animals

Refinement refers to methods that alleviate or minimize potential pain, suffering or distress, and enhance animal welfare for the animals still used

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The concept of the “Three Rs”, replace, reduce and refine; was put forward by Russell and Burch in their 1959 ‘The Principles of Human Experimental Technique’

Refinement: The use of analgesics and analgesia, the use of remote telemetry to increase the quality and quantity of data gathered and human endpoints for the animals are examples of refinements

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Reduction: Reduction in numbers of animals used in toxicology testing

Replacement: Alternatives such as in vitro, cell culture, tissue culture, models, simulations, etc. are examples of replacement

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Cell cultures

Cell culture is currently the most successful, and promising, alternative to animal use

For example, cultured cells have been developed to create monoclonal antibodies, prior to this production required animals to undergo a procedure likely to cause pain and distress

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Skin corrosion

Human skin equivalent tests can be used to replace animal-based corrosive studies

Two products, EpiDerm and EpiSkin are derived from human skin cells which have been cultured to produce a model of human skin

These methods are currently accepted replacements in Canada and the European Union

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Skin absorption

Several tissue culture methods which measure the rate of chemical absorption by the skin have been approved by the Organization for Economic Cooperation and Development (OECD), although they have not yet been approved as a replacement in the US

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Phototoxicity

Phototoxicity is a rash, swelling or inflammation, like a severe sunburn, caused by exposure to light following exposure to a chemical

The 3T3 Neutral Red Uptake (NRU) Phototoxicity Test, approved by the Organization for Economic Cooperation and Development (OECD), detects the viability of 3T3 cells after exposure to a chemical in the presence or absence of light

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Human-based

Skin irritation A skinpatch test has been designed and is

used in Canada to measure development of rashes, inflammation, swelling or abnormal tissue growth on human volunteers

Pyrogenicity Pyrogens are most often pharmaceutical

products or intravenous drugs that may cause inflammation or fever when they interact with immune system cells. This interaction can be quickly and accurately tested in vitro using donated human blood.

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Modular immune in vitro construct The MIMIC or modular immune in

vitro construct uses human cells to create a model of the human immune system on which the efficacy of new vaccines and other compounds may be tested, replacing some steps of the vaccine development process that would otherwise be performed on animals

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Computer simulation

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Examples of computer simulations available include models of diabetes, asthma and drug absorption

Computer operated mannequins, also known as crash test dummies, complete with internal sensors and video, have replaced live animal trauma testing for automobile crash testing

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The first of these was “Sierra Sam” built in 1949 by Alderson Research Labs (ARL) Sierra Engineering

Prior to this, live pigs were used as test subjects for crash testing

Computer models have been constructed to model human metabolism, to study plaque build-up and cardiovascular risk and to evaluate toxicity of drugs

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Conclusion