In the name of God

1. In the name of God

2. Summer School Influenza Unit, Pasteur Institute of Iran summer 2012

3. Cell Culture

4. Introduction Cell culture is the process by which prokaryotic, eukaryotic or plant cells are grown under controlled conditions. But in practice it refers to the culturing of cells derived from animal cells. Cell culture was first successfully undertaken by Ross Harrison in 1907

5. Historical events in the development of cell culture 1878: Claude Bernard proposed that physiological systems of an organism can be maintained in a living system after the death . 1880: Roux maintained embryonic chick cells in a saline culture. 1911: Lewis made the first liquid media consisted of sea water, serum, embryo extract, salts and peptones. They observed limited monolayer growth.

6. Contd.. 1916: Rous and Jones introduced proteolytic enzyme trypsin for the subculture of adherent cells. 1923: Carrel and Baker developed 'Carrel' or T-flask as the first specifically designed cell culture vessel. 1940s: The use of the antibiotics penicillin and streptomycin in culture medium decreased the problem of contamination in cell culture. 1952: Gey established a continuous cell line from a human cervical carcinoma known as HeLa (Helen Lane) cells. Dulbecco developed plaque assay for animal viruses using confluent monolayers of cultured cells.

7. Aseptic techniques Carrel Flask History

8. Contd.. 1955: Eagle studied the nutrient requirements of selected cells in culture and established the first widely used chemically defined medium. 1965: Ham introduced the first serum-free medium which was able to support the growth of some cells. 1978: Sato established the basis for the development of serum-free media from cocktails of hormones and growth factors.

9. Major development’s in cell culture technology First development was the use of antibiotics which inhibits the growth of contaminants. Second was the use of trypsin to remove adherent cells to subculture further from the culture vessel. Third was the use of chemically defined culture medium.

10. Why is cell culture used for? Areas where cell culture technology is currently playing a major role. Model systems for Studying basic cell biology, interactions between disease causing agents and cells, effects of drugs on cells, process and triggering of aging & nutritional studies. Toxicity testing , Study the effects of new drugs. Cancer research, Study the function of various chemicals, virus & radiation to convert normal cultured cells to cancerous cells.

11. Contd…. Virology Cultivation of virus for vaccine production, also used to study there infectious cycle. Genetic Engineering Production of commercial proteins, large scale production of viruses for use in vaccine production e.g. polio, rabies, chicken pox, hepatitis B & measles Gene therapy Cells having a functional gene can be replaced to cells which are having non-functional gene

12. Terminology Primary Cell Culture Derived from an explant, directly from the animal Usually only survive for a finite period of time Involves enzymatic and/or mechanical disruption of the tissue and some selection steps to isolate the cells of interest from a heterogeneous population Clone A population derived from a single cell Sub-culture Transplantation of cells from one vessel to another Established or Continuous Cell Lines A primary culture that has become immortal due to some transformation Most commonly tumour derived, or transformed with a virus such as Epstein-Barr

13. Primary culture Cells when surgically or enzymatically removed from an organism and placed in suitable culture environment will attach and grow are called as primary culture Primary cells have a finite life span Primary culture contains a very heterogeneous population of cells Sub culturing of primary cells leads to the generation of cell lines Cell lines have limited life span, they passage several times before they become senescent Cells such as macrophages and neurons do not divide in vitro so can be used as primary cultures Lineage of cells originating from the primary culture is called a cell strain

14. Continous cell lines Most cell lines grow for a limited number of generations after which they ceases Cell lines which either occur spontaneously or induced virally or chemically transformed into Continous cell lines Characteristics of continous cell lines -smaller, more rounded, less adherent with a higher nucleus /cytoplasm ratio -Fast growth and have aneuploid chromosome number -reduced serum and anchorage dependence and grow more in suspension conditions -ability to grow upto higher cell density -different in phenotypes from donar tissue

15. Types of cells On the basis of morphology (shape & appearance) or on their functional characteristics. They are divided into three. Epithelial like-attached to a substrate and appears flattened and polygonal in shape Lymphoblast like- cells do not attach remain in suspension with a spherical shape Fibroblast like- cells attached to an substrate appears elongated and bipolar

16. Common cell lines Human cell lines -MCF-7 breast cancer HL 60 Leukemia HEK-293 Human embryonic kidney HeLa Henrietta lacks Primate cell lines Vero African green monkey kidney epithelial cells Cos-7 African green monkey kidney cells And others such as CHO from hamster, sf9 & sf21 from insect cells

17. Monkey Kidney cells Polio vaccine first product primary monkey kidney cells human diploid lung fibroblast

18. Fibroblast Baby hamster kidney Epithelial Chinese Hamster Ovary Epithelial Epithelial Canine Kidney Human Keratonocyt Fibroblast Monkey Kidney Fibroblast Mouse fibroblast Common cell lines BHK CHO PER-C6 MDCK Vero 3T3

19. HeLa Cells • Classic example of an immortalized cell line. – These are human epithelial cells from a fatal cervical carcinoma transformed by human papillomavirus 18 (HPV18).

20. HeLa Cells • Adherent cells which maintain contact inhibition in vitro: – As they spread out across the culture flask, when two adjacent cells touch, this signals them to stop growing. • Loss of contact inhibition is a classic sign of oncogenic cells: – Cells which form tumors in experimental animals. – Such cells not only form a monolayer in culture but also pile up on top of one another. • HeLa cells are not oncogenic in animals, but they may become so if further transformed by a virus oncogene.

21. Number of Cell Divisions • Growing cells in culture. – Place cells in a culture dish. – Give them nutrients, growth factors, keep them free from bacterial. – Cells will grow to cover the surface of the dish. – Can take cells out of this culture and start a new culture. • Splitting cells from one dish to another is a passage.

22. Number of Cell Divisions • This ability to split cells and have them continue to divide is not without limits however. • Normal cells have a limit to the number of times which they can be passed in culture. • This number does vary from cell type to cell type, but commonly the limit is between 50 and 100 passages.

23. Contact Inhibition • The phenomenon observed in normal animal cells that causes them to stop dividing when they come into contact with one another. • Cells in a culture flask with the appropriate nutrients and the cells grow and divide. • Continues until the cells are covering the entire surface. • At that point they stop dividing. • These cells can be triggered to begin dividing again by giving them more room. • The cells now being in an environment where they are not in contact with one another begin to divide again.

24. Contact Inhibition • Cancer cells do not display contact inhibition. • Put them in a culture dish, they will grow to create a single layer of cells • Then they will continue to grow multiple layers and create piles of cells.

25. GROWTH CYCLE IN ATTACHEMENTCULTURE • Eukaryotic cells in attachment culture have a characteristic growth cycle similar to bacteria. • The growth cycle is typically divided into three phases. – Lag Phase – Log Phase – Plateau Phase

26. Lag Phase • This is the time following subculture and reseeding during which there is little evidence of an increase in cell number. • It is a period of adaptation during which the cell replaces elements of the glycocalyx lost during trypsinization, attaches to the substrate, and spreads out. • During spreading the cytoskeleton reappears and its reappearance is probably an integral part of the spreading process.

27. Log Phase • This is the period of exponential increase in cell number following the lag period and terminating one or two doublings after confluence is reached. • The length of the log phase depends on the seeding density, the growth rate of the cells, and the density at which cell proliferation is inhibited by density. • In the log phase the growth fraction is high (usually 90%-100%) and the culture is in its most reproducible form. It is the optimal time for sampling since the population is at its most uniform and viability is high. • The cells are, however, randomly distributed in the cell cycle and, for some purposes, may need to be synchronized.

28. Plateau Phase • Toward the end of the log phase, the culture becomes confluent – All the available growth surface is occupied and all the cells are in contact with surrounding cells. • Following confluence the growth rate of the culture is reduced, and in some cases, cell proliferation ceases almost completely after one or two further population doublings. • At this stage, the culture enters the plateau (or stationary) phase, and the growth fraction falls to between 0 and10%. • There may be a relative increase in the synthesis of specialized versus structural proteins and the constitution and charge of the cell surface may be changed.

29. Culture media Choice of media depends on the type of cell being cultured Commonly used Medium are GMEM, EMEM,DMEM etc. Media is supplemented with antibiotics viz. penicillin, streptomycin etc. Prepared media is filtered and incubated at 4 C

30. Culture Media • Ions – Na, K, Ca, Mg, Cl, P, Bicarbonate • Trace elements – iron, zinc, selenium • Sugars – glucose is the most common • Amino acids – 13 essential • Vitamins • Serum – Contains a large number of growth promoting activities such as buffering toxic nutrients by binding them, neutralizes trypsin and other proteases – Contains peptide hormones or hormone-like growth factors that promote healthy growth. • Antibiotics - although not required for cell growth, antibiotics are often used to control the growth of bacterial and fungal contaminants.

31. Serum • Serum/protein free media • Growth factors • Lipid concentrate • Extracts Yeast extract • Insulin • Bovine Serum Albumin ( transport/detoxification)

32. Basic equipments used in cell culture Laminar cabinet-Vertical are preferable Incubation facilities- Temperature of 25-30 C for insect & 37 C for mammalian cells, co2 2-5% & 95% air at 99% relative humidity. To prevent cell death incubators set to cut out at approx. 38.5 C Refrigerators- Liquid media kept at 4 C, enzymes (e.g. trypsin) & media components (e.g. glutamine & serum) at -20 C Microscope- An inverted microscope with 10x to 100x magnification Tissue culture ware- Culture plastic ware treated by polystyrene

33. Equipment Class II Cabinets These cabinets are designed to give operator protection as well as a sterile environment The air is directed downwards from the top of the cabinet to the base, when working in these cabinets it is important not to pas non-sterile objects over sterile ones

34. Equipment Centrifuges There are centrifuges in each cell culture area which are refrigerated 100 x g is hard enough to sediment cells, higher g forces may damage cells Incubators The incubators run at 37C and 5% Carbon Dioxide to keep the medium at the correct pH They all have meters on them to register temperature and gas level There are alarms to indicate when these deviate from set parameters Keep the door open for as short a time as possible

35. Why sub culturing? Once the available substrate surface is covered by cells (a confluent culture) growth slows & ceases. Cells to be kept in healthy & in growing state have to be sub-cultured or passaged It’s the passage of cells when they reach to 80-90% confluency in flask/dishes/plates Enzyme such as trypsin, dipase, collagenase in combination with EDTA breaks the cellular glue that attached the cells to the surface

36. Culturing of cells Cells are cultured as anchorage dependent or independent Cell lines derived from normal tissues are considered as anchorage-dependent grows only on a suitable substrate e.g. tissue cells Suspension cells are anchorage-independent e.g. blood cells Transformed cell lines either grows as monolayer or as suspension

37. Adherent cells Cells which are anchorage dependent Cells are washed with PBS (free of ca & mg ) solution. Add enough trypsin /EDTA to cover the monolayer Incubate the plate at 37 C for 1-2 min. Tap the vessel from the sides to dislodge the cells Add complete medium to dissociate and dislodge the cells with the help of pipette which are remained to be adherent Add complete medium depends on the subculture requirement either to 75 cm or 175 cm flask

38. Suspension cells Easier to passage as no need to detach them As the suspension cells reach to confluency Asceptically remove 1/3rd of medium Replaced with the same amount of pre-warmed medium

39. Culturing Animal Tissue- the Steps Animal tissue is obtained either from a particular specimen, or from a ‘tissue bank’ of cryo-preserved (cryo = frozen at very low temperatures in a special medium) Establishment of the tissue is accomplished in the required medium under aseptic conditions Culture vessels and medium for animal cell culture

40. Working with cryopreserved cells Vial from liquid nitrogen is placed into 37 C water bath, agitate vial continuously until medium is thawed Centrifuge the vial for 10 mts at 1000 rpm at RT, wipe top of vial with 70% ethanol and discard the supernatant Resuspend the cell pellet in 1 ml of complete medium with 20% FBS and transfer to properly labeled culture plate containing the appropriate amount of medium Check the cultures after 24 hrs to ensure that they are attached to the plate Change medium as the colour changes, use 20% FBS until the cells are established

41. Freezing cells for storage Remove the growth medium, wash the cells by PBS and remove the PBS by aspiration Dislodge the cells by trypsin-versene Dilute the cells with growth medium Transfer the cell suspension to a 15 ml conical tube, centrifuge at 200g for 5 min at RT and remove the growth medium by aspiration Resuspend the cells in 1-2ml of freezing medium Transfer the cells to cryovials, incubate the cryovials at -80 C overnight Next day transfer the cryovials to Liquid nitrogen

42. Cell viability Cell viability is determined by staining the cells with trypan blue As trypan blue dye is permeable to non-viable cells or death cells whereas it is impermeable to this dye Stain the cells with trypan dye and load to haemocytometer and calculate % of viable cells - % of viable cells= Nu. of unstained cells x 100 total nu. of cells

43. Cell toxicity Cytotoxicity causes inhibition of cell growth Observed effect on the morphological alteration in the cell layer or cell shape Characteristics of abnormal morphology is the giant cells, multinucleated cells, a granular bumpy appearance, vacuoles in the cytoplasm or nucleus Cytotoxicity is determined by substituting materials such as medium, serum, supplements flasks etc.

44. Contaminant’s of cell culture Cell culture contaminants of two types Chemical-difficult to detect caused by endotoxins, plasticizers, metal ions or traces of disinfectants that are invisible Biological-cause visible effects on the culture they are mycoplasma, yeast, bacteria or fungus or also from cross-contamination of cells from other cell lines

45. Cell Culture Enemies Micro-organisms grow ~10-50 times faster than mammalian cells, which take ~8-16 hours to divide. They are more tolerant to variations in temperature, pH and nutrient supply than cells. Cells are most vulnerable to contamination when our aseptic technique is bad and the culture becomes infected with bugs. This can lead to the development of antibiotic resistant micro-organisms.

46. Cell Culture Enemies Cells are more susceptible to infection at certain times When they have been stressed after recovery from liquid nitrogen Primary cells are often generated by enzymatic disruption and selection procedures Cultures prepared from live animals will often be accompanied by micro-organisms Splitting cells at too high a dilution can allow micro-organisms to dominate the culture Cells release Autocrine growth factors which condition the medium and favour cell growth

47. Cell Culture Enemies IF YOU ARE IN DOUBT ABOUT THE CONDITION OF YOUR CELLS, ASK FOR ADVICE NEVER USE CONTAMINATED CELLS. THEY MAY NOT REACT IN THE SAME WAY AS UNCONTAMINATED CELLS POOR ASEPTIC TECHNIQUE IS THE MAJOR CAUSE OF INFECTIONS

48. Effects of Biological Contamination’s They competes for nutrients with host cells Secreted acidic or alkaline by-products ceses the growth of the host cells Degraded arginine & purine inhibits the synthesis of histone and nucleic acid They also produces H2O2 which is directly toxic to cells

49. Detection of contaminants In general indicators of contamination are turbid culture media, change in growth rates, abnormally high pH, poor attachment, multi-nucleated cells, graining cellular appearance, vacuolization, inclusion bodies and cell lysis Yeast, bacteria & fungi usually shows visible effect on the culture (changes in medium turbidity or pH) Mycoplasma detected by direct DNA staining with intercalating fluorescent substances e.g. Mycoplasma also detected by enzyme immunoassay by specific antisera or monoclonal abs or by PCR amplification of mycoplasmal RNA

50. Basic aseptic conditions If working on the bench use a Bunsen flame to heat the air surrounding the Bunsen Swab all bottle tops & necks with 70% ethanol Flame all bottle necks & pipette by passing very quickly through the hottest part of the flame Avoiding placing caps & pipettes down on the bench; practice holding bottle tops with the little finger Work either left to right or vice versa, so that all material goes to one side, once finished Clean up spills immediately & always leave the work place neat & tidy