Primary cell culture

Tissue culture

Brief theory:

Principles of Biosafety Introduction to the Primary Cell Culture

Practical:

Protocols Reagents and Techniques for Cell Culture

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.

Certain special considerations when using “human tissue”

1-consent forms and confidentiality 2-biohazard potential , viruses, …. (For example, it needs individual tank)

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

Connective tissue:

Connective tissuesmade of cells and extracellular fibers

Cells:Fibroblasts Lymphocytes Adipocytes Monocytes Neutrophils Macrophages

Fibers:CollagenReticularElastic

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

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+

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 cultureSuplements

Practical order:

1.  Isolation of tissue

2.  Dissection and/or disaggregatingcells 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

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

Dissection and/or disaggregating can be done in three methods:

A- ExplantsPoints: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 DisaggregationPoints: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

C- Disaggregating by Enzymatic actionPoints: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)

TrypsinactionPapainElastaseHyaluronidaseCollagenase Type IICollagenase Type ICollagenase Type IVCollagenase Type IIIDNase

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

High Yield and High ViabilityGood dissociation Good yield

Troubleshooting: Yield and Viability Issues

Low Yield and Low ViabilityUnder 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 ViabilityGood 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