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. 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.

8. STEPS OF TISSUE CULTURE Isolation of tissue Disaggregation of cells – initiation of culture Incubation and growth

9. 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

10. 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.

11. TYPES OF PRIMARY CULTURE

12. 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.

13. 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.

14. 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.

15. Enzymatic Disaggregation Warm Trypsin Cold Trypsin Collagenase

16. WarmTrypsin

17. COLD TRYPSIN

18. COLLAGENASE

19. 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

20. 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

21. SEPARATION OF NONVIABLE CELLS  For adherent cultures first change of media.  Gradual dilution of suspension cells when proliferation starts.

22. 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

23. 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.

24. Tissue Primarycell culture Cell line Continuous cell line dispersion Subculture Finite numbers Indefinite numbers Stored Stored

25. 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

26. 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.

27.  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

28.  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

29. 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

30. 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.

31. REFERANCE  Culture of Animal Cells- R. Ian Freshney  Biotechnology- U. Satayanarayan  Cell and tissue culture- john paul fifth edition

Primary and established cell line culture

KAUSHAL SAHU

Primary and established cell line culture