Preparation of cell line II_animal cell culture.pptx

Preparation of cell line II_animal cell culture

1. Chapter 6 Preparation of Cell Line Initiation of a Primary Culture

2. Steps in Initiation of a Primary Culture Acquisition of the sample Isolation of the tissue Dissection and/or disaggregation Culture after seeding

3. Techniques for Tissue Disaggregation for Primary Culture Primary explant technique Mechanical disaggregation Enzymatic disaggregation

4. A. Primary Explant Technique The primary explant technique was the original method developed by Harrison [1907], Carrel [1912], and others for initiating a tissue culture. Outline Embed a fragment of tissue in blood plasma or lymph Mix with heterogenous serum and embryo extract Place on a coverslip that is inverted over a concavity slide Transfer for secondary explant culture Subculture the outgrowth to initiate cell line

5. Primary Explant Technique Primary explant culture from mouse squamous skin carcinoma; explant and early stage of outgrowth about 3 days after explantation Outgrowth after removal of explant, about 7 days after explantation . 10× objective

6. Primary Explant Technique Advantage: Particularly useful for small amount of tissue (e.g. skin biopsies) for which there is a risk of loosing cells during mechanical or enzymatic disaggregation Limitation: Poor adhesiveness of some tissue Cells in the outgrowth could be highly selective Relatively slow process

7. B. Mechanical Disaggregation Spillage: collecting the cells that spill out when the tissue is carefully sliced and the slices scraped 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) Pipetting: repeated pipetting of suspension containing tissue fragments

8. Mechanical Disaggregation. (a) Scraping or ‘‘spillage .’’ Cutting action, or abrasion of cut surface, releases cells. (b) Sieving. Forcing tissue through sieve with syringe piston. ( c) Syringing. Drawing tissue into syringe through wide-bore needle or canula and expressing. (d) Trituration by pipette. Pipetting tissue fragments up and down through wide-bore pipette.

9. Mechanical Disaggregation Soft tissues (e.g. spleen, embryonic liver, embryonic and adult brain, soft tumor etc) are suitable for mechanical disaggregation Advantage: Fast, simple Limitation: lower viability of cells, less efficient compared to enzymatic disaggregation

10. C. Enzymatic Disaggregation Sensitivity to proteolytic enzymes Fibronectin Laminin Proteoglycans Thus trypsin digestion would produce cells by tissue disaggregation Sensitive to metal ion (Ca 2+ ) chelation Cadherin Integrin Selectin Trypsin + EDTA therefore improves the yield The purer the trypsin , the less toxic and more predictable it becomes Due to the presence of other proteases in crude trypsin , it becomes more efficient but toxicity is higher

11. Collagen Elastin Fibronectin Laminin

12. Enzymatic Disaggregation Warm trypsin method Cold trypsin method Other enzymatic methods

13. a) Warm Trypsin Method It is important to minimize the exposure of cells to active trypsin in order to preserve maximum cellular viability The tissue is chopped and stirred in trypsin for a few hours at 37°C. The dissociated cells are collected every half hour, centrifuged, and pooled in medium containing serum.

14. a) Warm Trypsin Method The warm trypsin technique is useful for the disaggregation of large amounts of tissue in a relatively short time, particularly for chopped whole mouse embryos or chick embryos. It does not work as well with adult tissue, in which there is a lot of fibrous connective tissue. Mechanical agitation can be damaging to some of the more sensitive cell types, such as epithelium. If reaggregation is found after centrifugation and resuspension , incubate in DNase , 10 to 20 μg / mL , for 10 to 20 min, and recentrifuge .

15. b) Cold Trypsin Method A simple method of minimizing damage to the cells during exposure is to soak the tissue in trypsin at 4◦C for 6 to 18 h to allow penetration of the enzyme with little tryptic activity. Following this procedure, the tissue will only require 20 to 30 min at 37◦C for disaggregation.

16. c) Other Enzymatic Methods Trypsin in combination with- Collagenase Elastase Pronase Hyaluronidase DNase

17. Separation of Viable and Non-viable Cells When tissue is disaggregated and seeded into primary culture, only a proportion of the cells are capable of surviving and generating a primary culture. If it is important to separate the necrotic and apoptotic cells. In case of adherent cells, nonviable cells are removed at the first change of medium. With primary cultures maintained in suspension, nonviable cells are gradually diluted out when cell proliferation starts. If necessary, however, nonviable cells may be removed from the primary disaggregate by centrifuging the cells on a mixture of Ficoll and sodium metrizoate .

18. Ficoll -sodium metrizoate Density Gradient Centrifugation Culture medium Viable cells Ficoll -sodium metrizoate Dead cells

19. Factors to be considered during 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. The concentration of cells in the primary culture should be much higher than that normally used for subculture because the proportion of cells from the tissue that survives in primary culture may be quite low. A rich medium, such as Ham’s F12, is preferable to a simple medium, such as Eagle’s MEM, and if serum is required, fetal bovine often gives better survival than does calf or horse. Isolation of specific cell types will probably require selective serum-free media. Embryonic tissue disaggregates more readily, yields more viable cells, and proliferates more rapidly in primary culture than does adult tissue.