1. Primary cell culture
Tissue culture

2. Brief theory:
Principles of Biosafety
Introduction to the Primary Cell Culture
Practical:
Protocols
Reagents and Techniques for Cell Culture

3. Why primary?
*The growth of cells taken directly form the living organism (e.g. biopsy material) is also known as primary cell culture.
*The culture consists of mixed population of cell types.
*Frequently, some of the cells may survive without proliferating and will therefore be lost in the increasing population of those which are able to multiply in the conditions supplied in vitro. Many of the cells will only survive for one or a few passages before dying (Terminal differentiation & Senescence cells)
Cells may sometimes be converted to cell lines by passage or induction. These may continue to proliferate for a number of cell generations.
In some instances the primary cells are fused with so-called immortal (cancer) cells to produce a hybridoma line.

4. Certain special considerations when using “human tissue”
1-consent forms and confidentiality
2-biohazard potential , viruses, …. (For example, it needs individual tank)

5. Connective tissue: Sources of Connective tissue:
Forms the dermis of the skin
Capsules and stroma of various organs
Mucous and serous membranes
Cartilage, bone, tendons, ligaments, adipose tissue

6. Connective tissues
made of cells and extracellular fibers
Cells:
Fibroblasts
Lymphocytes
Adipocytes
Monocytes
Neutrophils
Macrophages
Fibers:
Collagen
Reticular
Elastic

7. Collagen:
-major component
-rod like fibers
-protein is triple helical polypeptide
-extensive crosslinking
-resistant to hydrolytic attack by many proteases
-attack with a mix of enzymes:
Collagenase
Hyaluronidase
Pronase
Elastase

8. Epithelial tissue: Sources of Epithelial tissue: Epidermis
Glands of the skin (sweat, sebaceous)
0uter corneal layer
Lining of digestive and reproductive tracts
Endothelium
Parts of liver, pancreas, pituitary, gastric and intestinal glands
Types of intracellular attachment:
Zona occludens
Zona adherens
Macula adherens (desmosomes)
Points:
digestive enzymes can break these (collagenase, trypsin, hyaluronidase)
chelating agents enhance activity - deplete Ca+

9. The things that you should know before starting to work with primary cell culture and suitability of material for doing Primary Culture:
Types of tissue
Species of origin
Age of the individual (animal or human)
Dissociation medium used
Enzymes used
Enzyme concentration and purity
Temperature
Incubation Time
The reason for the culture
Suplements

10. Practical order: 1. Isolation of tissue
2.  Dissection and/or disaggregating
cells migrate out from small tissue chunks in flask
disaggregating either mechanically or enzymatically
create cell suspension
some cells will adhere to substrate in culture
3.  Culture in flasks

11. ISOLATION
Common points of tissue isolation:
1-Remove fat or necrotic tissue should be removed
2-Chop tissue finely with sharp instrument (minimize damage)
3-Remove desegregation enzymes by centrifugation
4-Higher conc. of cell needed for primary isolation than subculturing
5-Use richest medium possible
6-Embryonic tissue is best (disaggregates better, get more viable cells, proliferates better) - ES or AS cell culture is primary cell culture

12. Dissection and/or disaggregating can be done in three methods:
A- Explants
Points:
1-original method developed at turn of the century
2-finely chop tissue to very small parts
3-cells migrate away from pieces onto culture flask
4-useful with small tissue samples (biopsies) 
5-slow process, certain cells migrate faster than others (fibroblasts)
B- Mechanical Disaggregation
Points:
1-vigorous pipetting
2-pressing tissue into a mesh
3-wash cells through sieve
4-faster than enzymes but less yield
5-causes mechanical damage to cells

13. C- Disaggregating by Enzymatic action
Points:
1-need to disrupt cell-cell adhesion proteins 
2-crude preparations of enzymes are often used
3-may contain other proteases as contaminants – (work better)
3-purified mixtures are less damaging to cells though
4-cells can be damaged to point of lysis
Enzymes (Strongest to Weakest)
Trypsinaction
Papain
Elastase
Hyaluronidase
Collagenase Type II
Collagenase Type I
Collagenase Type IV
Collagenase Type III
DNase

14. Removal of Fibroblasts:
-Considered as a contaminating cell
-Proliferate rapidly
-Use monoclonal antibody
-Thy-1 found on human fibroblasts but not on other cells
-Will target fibroblasts for destruction or removal
-Also add supplements to media:
Cholera toxin:  10nM
Cis-hydroxy proline: 10ug/ml

15. Troubleshooting: Yield and Viability Issues
Low Yield and Low Viability
Under or over dissociation
Have cellular damage
Solution:
use less harmful digestive enzyme (trypsin to collagenase)
decrease concentration of enzyme
Low Yield and High Viability
Under dissociation
Solution:
first, increase concentration of enzyme or increase incubation time
next, use other enzyme or combinations
High Yield and Low Viability
Good dissociation
Have cellular damage (enzyme too strong)
Solution:
Reduce enzyme concentration &/or Reduce incubation time &/or Dilute enzyme with BSA or soy proteins
Goal: increase viability w/o changing yield
High Yield and High Viability
Good dissociation
Good yield