Introduction
Primary Culture
Steps of Primary Culture
Isolation Of Tissue
Dissection And Disaggregation
Types Of Primary Culture
Primary Explants Culture
Enzymatic Disaggregation
Mechanical Disaggregation
Cell Line( Finite & Continuous)
Naming A Cell Line
Choosing A Cell Line
Maintenance Of Cell Line
Conclusion
Reference
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Primary and established cell line culture
1. PRIMARY AND ESTABLISHED
CELL LINE CULTURE
By
KAUSHAL KUMAR SAHU
Assistant Professor (Ad Hoc)
Department of Biotechnology
Govt. Digvijay Autonomous P. G. College
Raj-Nandgaon ( C. G. )
2. SYNOPSIS
Introduction
Primary Culture
Steps of Primary Culture
Isolation Of Tissue
Dissection And Disaggregation
Types Of Primary Culture
Primary Explants Culture
Enzymatic Disaggregation
Mechanical Disaggregation
Cell Line( Finite & Continuous)
Naming A Cell Line
Choosing A Cell Line
Maintenance Of Cell Line
Conclusion
Reference
3. ANIMAL TISSUE CULTURE
Animal cell culture basically involves the in vitro maintenance
and propagation of animal cells in a suitable nutrient media.
Thus culturing is a process of growing cells artificially.
Types of animal tissue culture
1.organ culture 2.primary explants culture
3.cell culture
4. ORGAN CULTURE
The entire embryos or organs are excised from the body and culture
Advantages
Normal physiological functions are maintained.
Cells remain fully differentiated.
Disadvantages
Scale-up is not recommended.
Growth is slow.
Fresh explantation is required for every experiment.
5. EXPLANTS CULTURE
Fragments of excised tissue are grown in culture media
Advantages
Some normal functions may be maintained.
Better than organ culture for scale-up but not ideal.
Disadvantages
Original organization of tissue is lost.
6. CELL CULTURE
Tissue from an explant is dispersed, mostly enzymatically, into a
cell suspension which may then be cultured as a monolayer or
suspension culture.
Advantages
Development of a cell line over several generations
Scale-up is possible
Disadvantages
Cells may lose some differentiated characteristics.
7.
8. PRIMERY TISSUE CULTURE
A primary culture refers to the starting culture of cells, tissue or
organs, taken directly from an organism.
Thus the primary culture is initial culture before first subculture.
9. STEPS OF TISSUE CULTURE
Isolation of tissue
Disaggregation of cells –
initiation of culture
Incubation and growth
10. ISOLATION OF TISSUE
Make sure our work is within rules.
Work safely, especially with human tissue.
If we isolate our cells far from culture place (as it is in our case)
keep it on ice (4oC) for up to 72 hours
11. DISSECTION AND DISAGGREGATION
Fat and necrotic tissues are best removed during dissection.
The tissue should be chopped finely with sharp scalpels to cause
minimum damage.
Enzymes used for disaggregation should be removed subsequently
by gentle centrifugation.
13. PRIMARY EXPLANT CULTURE
The primary explant technique was the original method developed
by Harrison [1907], Carrel [1912].
As originally performed, a fragment of tissue was embedded in
blood plasma or lymph, mixed with heterologous serum and
embryo extract, and placed on a cover slip that was inverted over a
concavity slide.
14.
15. ENZYMATIC DISAGGREGATION
Cell–cell adhesion in tissues is mediated by a variety of homotypic
interacting glycopeptides (cell adhesion molecules, or CAMs).
(Cadherins, Integrins, Fibronectin & laminin)
Enzyme are neutralized or inactivated or inhibited after the used.
Increasing the purity of an enzyme will give better control and less
toxicity with increased specificity but may result in less
disaggregation potency.
16. ENZYMES USED IN ENZYMATIC DISAGGREGATION
Enzymes
Trypsin
Collagenase II
Elastase
Hyaluronidase
Dnase
Pronase (bacterial protease)
Usually a combination of enzymes
Crude preparations are usually more efficient.
21. spillage: collecting the cells that spill
out when the tissue is carefully sliced
and the slices scraped [Lasfargues,
1973].
sieving: pressing the dissected tissue
through a series of sieves for which
the mesh is gradually reduced in size.
syringing: forcing
the tissue fragments through a syringe
(with or without a
wide-gauge needle) [Zaroff et al.,
1961]
MECHANICAL DISAGGREGATION
22. INCUBATION AND GROWTH
Appropriate medium supplemented with growth factors.
Some cells require special adhesion surfaces (cover tissue
culture dish with extracellular matrix proteins or synthetic
attachment molecules).
Challenges
– Removal of dead cells
– Separation of cell types
23. SEPARATION OF NONVIABLE CELLS
For adherent cultures first change of media.
Gradual dilution of suspension cells when proliferation starts.
24. SEPARATION OF VIABLE CELL
Selective media
Difference in the speed of attachment
Use of enzymes
Collagenase does not easily disperse epithelial cells but works
well on stroma
25. CELL LINE
The term cell line refers to the propagation of culture after the first
subculture. In other world once the primary culture is subculture is
becomes cell line.
Finite cell line
Limited culture life span (20-100 time division)
Human cell (50-100).
Grow slowly and form monolayer
Doubling time ranges from 24 to 96 hours.
Continuous cell line
Transformed immortal and tumorigenic
Divides rapidly. Generation time 12-24 hrs.
27. NAMING A CELL LINE
New cell lines are given a code or designation
Eg.
NHB = Normal Human Brain
NHB1 = Normal Human Brain cell line no 1
NHB2-1= Normal Human Brain cell line no 2 clone no 1
28. SELECTION OF CELL LINE
Finite or continuous
A continuous cell line generally is easier to maintain, grows
faster, clones more easily, produces a higher cell yield per flask,
and is more readily adapted to serum-free medium.
Normal or Transformed
The researcher should decide whether the cell line should be
malignantly transformed or not.
29. Growth characteristics
Population doubling time
Plating efficiency
Growth fraction
Availability
If the researcher uses a finite cell line he or she should make sure
that there is enough stocks available
If the researcher uses a continuous cell line he or she should
make sure that authenticated stocks are available
30. Validation
The researcher has to make sure that the selected cell line is not
a result of cross-contaminations.
Phenotypic expression
The researcher has to make sure that the selected cell line is
made to express the right characteristics
Stability
The researcher has to make sure that the selected cell line is
stable.cell line is
31. MAINTENANCE OF CELL CULTURE
Cell morphology
Replacement of media
Cell concentration
A decrease in pH (fall 0.1/day =no harm)
(fall 0.4/day = harmful)
Cell type
Morphological changes
32. CONCLUSION
Animal cell culture became a common laboratory technique
in the mid-1900s but the concept of maintaining live cell lines
(a population of cells derived from a single cell and
containing the same genetic makeup) separated from their
original tissue source was discovered in the 19th century.
33. REFERANCE
Culture of Animal Cells- R. Ian Freshney
Biotechnology- U. Satayanarayan
Cell and tissue culture- john paul fifth edition