1. Tissue Culture (Cell Culture) Toxicology Testing In Vitro

2. Introduction The concept of tissue culture is not new!! (1885)
Toxicological test LD50 methods are necessary to assess the hazard and safety of various substances such as medicine, consumer products and industrial chemicals
In recent years animal or replace animal use

3. Content Def. Historical usage Use of tissue culture
Advantages of tissue culture
Types of tissue culture
Requirements of tissue culture
Work area and Equipments
Cytotoxicological studies

4. Definition
Tissue culture and Plant tissue culture: Is a term used for the process of keeping tissue alive and growing artificially in the lab Using Liquid or Semisolid growth media

5. Tissue culture means :
the ability of tissues to survive and grow outside the body in an artificial environment (by using liquid,
semisolid or solid growth media such as broth or agar.)

6. Historical Usage
Firstly, they chose the frog as a source of tissue because it was a cold-blooded animal, and consequently incubation was not required.
Then at 1885 they used the medullary plate of an embryonic chicken and maintained it in warm saline solution for several days.
In medical science it is carried into warm-blooded animals, where normal and pathological development are close to human using:
Pure strains of rodents.
Embryonated hen’s egg.
In 1902 Leo Loeb placed fragments of the skin (tissue transplantation) from the embryos of guinea pigs in agar and in coagulated serum and inserted them into adult guinea pigs.

7. Uses of tissue culture Used in culturing of specific plants.
Investigation of the normal physiology and biochemistry of the cell (development of nerve cell).
Study the effect of various chemicals or drugs on specific cell types (cytotoxicological- effects)
Study the protective or curative effect of certain substance.
Used for growing viruses and production of anti viral vaccines.
Cancer research
Used medically to generate artificial tissues.(liver, articular cartilage & skin grafting)

8. Advantages of Tissue Culture
Control of the surrounding environment
Sample is Homogenous
Use the specific cells
Cheaper!
Result in short time
Humane!

9. Types 1. Primary cultures:
Secondary cultures (cell line)
1. Primary cultures:
it refers to cultures prepared directly from animals.

10. Organ culture
Slice tissue culture (small pieces of tissue are cultured in enriched media)
Histotypic (histoculture) : (high density slice culture of one cell type)
Primary cell culture (single cells which could be plated on a coated surface)
Reaggregated culture (single cell reaggregate into small balls).

11. Transformed cell line (from tumor cells)
2. Secondary cultures:-
Cells that survive the crisis and continue to grow are referred to cell line.
Continuous cell line (cells can passage indefinitely and express stable phenotype)
Transformed cell line (from tumor cells)
Clonal cell line (cells could be cloned in continuous cell lines to obtain genetically homogenous population)

12. Plating density : number of cells per volume of of tissue culture.
Cells can grow either on suspension or adherent culture.

13. Critical Requirements
Appropriate tissue
A suitable Growth medium
Sterile condition (Aseptic)
Specific medium (Growth regulatory)
Frequent sub culturing

14. Work area and Equipment
Laminar flow hood
CO2 incubator
Inverted microscope
Dissecting microscope
Inverted fluorescence microscope
Liquid nitrogen containers
Vessels (Roller bottles, Screw cap flasks, multiwell plate)
Hot air oven, Autoclave, Water bath, Refrigerator, Deep freezer, Water distiller, Vacuum pump, Pipette, pH meter, syringe filters, Magnetic stir plate…..etc

16. Hood

18. Co2 incubator

21. Inverted Microscope

23. Liquid Nitrogen Container

26. Cryo tubes

27. Roller bottles

28. Screw cap flasks

29. Single use filter

30. Bottle top filter

31. Tissue culture plate (24- well)

32. 6 well
96 well

35. Pipette

36. Toxicological Studies
Morphomatic analysis (size, number)
Estimation of some metabolites or enzymes
Measurement of viability of the cell
Dye uptake (diacetyl fluorescien and fluorescien)
Chromium release from dead cells
Estimation of Cytokines
Detection of DNA change 36

37. Estimation of LC50
Evaluation of the curative and protective effects of substances against toxicants 37

38. A) Hepatic cells