1. Presenters: Manji M, J. Gerald, S. Jamila. Facilitator: Dr. Russa

2.  Cell culture is the process by which prokaryotic, eukaryotic or plant cells are grown under controlled conditions.  Cells, tissues and organs isolated and maintained in a laboratory setting are said to have been ‘cultured’.

3.  Usually refers to eukaryotes.  For bacteria, refereed to as bacterial culture  Cell culture was first successfully undertaken by Ross Harrison in 1907

4.  1880 - Arnold showed that leucocytes can divide outside the body  1885: Roux maintained embryonic chick cells in saline  1903 - Jolly studied the behavior of animal tissue explants immersed in serum, lymph, or ascites fluid.  1907 - Ross Granville Harrison cultured frog tadpole spinal chord in a lymph drop hanging from a cover slip of a cavity slide.  1913 - Carrel developed a complicated methodology for maintaining cultures free of contamination

5.  1965: Harris & Watkins successfully fused human and mouse cells by virus  1975: Kohler & Milstein produced the first Hybridomas capable of secreting monoclonal antibodies.  1982: Human insulin became the first recombinant protein to be licensed as a therapeutic agent.

6. Glass  most commonly used substrate  easily washed and readily sterilized  optically clear to allow microscopic observation  Glass made of alum-borosilicate (e.g. Pyrex) is preferred to soda-lime glass  Slides, test tubes and flasks, must be washed with non­toxic detergent

7. Plastic  Sterile substrates available are meant for single use.  Non autoclavable.  most commonly used plastic; ◦ polystyrene.; Polyethylene ◦ polycarbonate,; perspex ◦ polyvinyl chloride; teflon ◦ Cellophane; cellulose acetate, etc.

8. Metals  Stainless steel and titanium, being relatively chemically inert  Stainless steel should be acid washed, to remove surface impurities.

9.  The efficiency of the cultured cells through increased cell attachment can be enhanced by treating the surface of the substrate with purified fibronectin or collagen.  Then, the surface of the substrate is treated with a monolayer of special types of cells. This layer is called feeder layer.  The cells used as feeder layer are glial cells, normal foetal intestine, mouse embryo, fibroblasts, etc.

10.  Artificial environment  Appropriate source of energy for the cells which they can easily utilize and compounds which regulate the cell cycle  May or May not contain serum

11.  Classified based on ◦ Source – Natural/ Artificial ◦ Composition – Defined/ Undefined ◦ Consistency – Solid, Semi Solid, Liquid ◦ Serum content – Serum and Serum-free media ◦ Special Need

12. Serum Advantages of serum in culture medium  Serum binds and neutralizes toxins  Serum contains a complete set of essential growth factors, hormones, attachment and spreading factors, binding and transport proteins  It contains the protease inhibitors  It increases the buffering capacity  It provides trace elements.

13. Disadvantages of serum in culture medium  It is not chemically defined and therefore it’s composition varies a lot  It is sometimes source of contamination by viruses, mycoplasma, prions etc.  It increases the difficulties and cost of down stream processing  It is the most expensive component of the culture medium.

14. The Laminar Flow Cabinet  Provides clean air to the working area;  Removes and suspends contaminants introduced  Prevents room air from entering the hood

15. Incubator  Used to keep the cells in a desirable state to allow them to proliferate.  Human cells are placed in incubators with a temperature of 37°C, relative humidity of 95% and 5% carbon dioxide

16. Water bath  To warm or thaw media  Usually takes 10 - 15 minutes for media to warm

17. Vacuum pump  For permanent aspiration of liquids  Use glass pasteur pipettes

18. Dissecting Microscope  Used for dissecting and obtaining target tissues in case for primary culture

19. Inverted Phase Microscope  A phase contrast microscope with objectives below the specimen.  Used for observing the growth status of cultured tissue

20.  Other equipments;  Hot/dry air oven  Autoclave  Refrigerator/deep freezer  Water distiller  Syringe filter  Micropipette  Ph Meter

21. Growth Kinetics

22.  Lag phase: Period of adjustment necessary for growth to commence  Exponential phase: state of balanced growth dividing at constant rate  Stationary phase: Accumulation of waste products, exhaustion of nutrients, pH changes  Death Phase: Population decreases with time due to lack of nutrients and toxic by products

23.  How “covered” the growing surface appears  Optimal confluency for moving cells to a new dish is 70-80% ◦ too low, cells will be in lag phase and won’t proliferate ◦ Too high and cells may undergo unfavorable changes and will be difficult to remove from plate.

24.  When cells contact each other, they cease their growth.  Cells arrest in G0 phase of the cell cycle  Transformed cells will continue to proliferate and pile upon each other

25.  Growth slows and ceases in a highly confluent culture  Cells to be kept in healthy & in growing state have to be sub-cultured or passaged

26.  Cells that attach to surfaces in vivo require a surface to attach to in vitro. ◦ Other cells or specially treated plastic or other biologically active coatings  Blood cells are primary exception.  Transformed cells may not require attachment

27.  Primary Cell Cultures: Cells that are cultured directly from a subject. Have a limited lifespan except those derived from tumors. ◦ After a certain number of population doublings (called the Hayflick limit), cells undergo the process of senescence and stop dividing.  Secondary Culture (Extended culture or multipassage culture): Cells taken from a primary culture and passed or divided in vitro. ◦ Cell Strain

28.  An established or transformed or immortalized cell line has acquired the ability to proliferate indefinitely either through random mutation or deliberate modification, such as artificial expression of the telomerase gene.  Numerous cell lines are well established as representative of particular cell types.

29. 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.

30.  Isolation of cells ◦ isolated from tissues for ex vivo culture ◦ purified from blood or released from soft tissues by enzymatic processes eg using trypsin, collagenases etc ◦ Or by explant culture - pieces of tissue can be placed in growth media, and the cells that grow out are available for culture.

31.  Maintaining cells in culture ◦ Appropriate temperature and gas mixture (typically 37°C, 5% CO 2 ) in a cell incubator ◦ Growth medium – pH, Glucose conc, growth factors, presence of nutrients ◦ Anchorage dependence (Suspension cultures vs Adherent cultures) ◦ Contact Inhibition: When cells contact each other, they cease their growth.

32.  Manipulation ◦ Prevent contamination  Sterile technique  Use of antibiotics ◦ Media Changes  Replenish nutrients  Avoid buildup of toxic by products and dead cells ◦ Passaging/splitting cells  Transfer small no of cells to a new vessel ◦ Transfection  Introduction of foreign DNA to express a protein of interest – Electroporation, Retroviral Infection, Microinjection

33. Two types  Chemical Contaminants ◦ Endotoxins ◦ traces of disinfectants  Biological ◦ microbes (bacteria, viruses) ◦ cross-contamination of cells from other cell lines

36. Cell Line MeaningOrganism Tissue of origin Morpholog y HEK 293 Human embryonic kidney Human Kidney (embryoni c) Epithelium HeLa "Henriett a Lacks" Human Cervical cancer Epithelium HL 60 Human leukemia HumanMyeloblast Blood cells MCF 7 Michigan Cancer Foundation -7 Human Mammary Gland Invasive breast ductal carcinoma

38.  Derived from Henrietta Lacks - 1951  Oldest and Most commonly used cell line  First human cell line to prove successful in vitro, which was a scientific achievement with profound future benefit to medical research  ‘Immortalised Cells’ - can divide an unlimited number of times in a laboratory cell culture plate as long as fundamental cell survival conditions are met – have an active version of telomerase

39.  Chromosome number - Horizontal gene transfer from human papillomavirus 18 (HPV18) to human cervical cells (Chromosome number of 82)  Common contaminant – grows very easily and proliferates very fast, contaminates 10-20% of cell lines

40.  In vitro cultivation of tissues at defined temperature using an incubator & supplemented with a medium containing cell nutrients & growth factors is collectively known as tissue culture

41.  Culture of native tissue that retains most of the in vivo histological features  Parts of an organ or a whole organ can be cultured in vitroin vitro  The media used for a growing organ culture are generally the same as those used for tissue culture

42. The synthetic scaffold of an ear sits bathed in cartilage-producing cells, part of an effort to grow new ears for wounded soldiers

44. CATEGORY ORGAN CULTURE EXPLANT CELL CULTURE Source Embryonic organs Adult tissue fragments Tissue fragments Disaggregated tissue, primary culture, propagated cell line EffortHighModerateLow Characterization Easy, histology CytologyMarkers Biochemical, molecular, immunological, cytological assay HistologyInformativeDifficult Not applicable Biochemical differentiation PossibleHeterogenous Loss, but may be reduced Propagation Not possible Possible from outgrowth Standard procedure Replicate sampling, reproduciblity, homogeneity High intersample variation Low intersample variation QuantitationDifficultDifficult Easy, many techniques available

45.  Model systems for studying  Basic cell biology eg. metabolism and energy production  Interactions between disease causing agents and cells, ie pathophysiology of diseases  Effects of drugs on cells, toxicity testing  Process and triggering of aging

46.  Genetic Engineering  Production of vaccines for polio, measles, mumps, rubella, and chicken pox, rabies, hepatitis B  Production of valuable biological products i.e enzymes, hormones, anticancer agents, antibodies.

47.  Gene therapy  Cells having a functional gene can be replaced to cells which are having non-functional gene

48.  Tissue/Organ culture and engineering  Artificial pancreas – using islets cells to produce and regulate insulin particularly in DM  Artificial bladder – Anton Atala ( Wake Forest University ) has successfully planted artificially grown bladders into seven out of 20 human test subjects.

49.  A beating rat heart has been cultured by Doris Taylor at the University of Minnesota  Artificial kidney has been cultured by H. David Humes at the University of Michigan  A jaw bone has been cultured at Columbia University

50.  lung has been cultured at Yale  Lab-grown cartilage tissue was successfully used to repair knee cartilage  Tissue engineered airway

53. LIMITATIONS Category Examples Necessary Expertise Environmental control Quantity and cost Sterile handling Chemical contamination Microbial Contamination Cross-contamination Workspace Incubation, pH control Containment and disposal of biohazards Capital equipment for scale-up Medium, serum Disposable plastics

54.  www.wikipedia.com, search words: cell culture, tissue culture, organ culture  Essential Cell Biology, An introduction to the molecular biology of the cell. (Alberts, Bray Johnson, Lewis Raff et al)

55.  ATCC - The Global Bioresource Center that focuses in Cell Culture http://www.atcc.org/  European Collection of Cell Cultures (ECACC) that preserves and distributes cell lines http://www.hpacultures.org.uk/collections/ecacc.jsp  Landmarks in Cell Culture http://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=mboc4&part=A1506&ren dertype=table&id=A1516  National Cell Culture Center (NCCC) that produces cell lines for fundamental research http://www.nccc.com/  PubMed- a datatbase that provides research articles related to Cell Culture http://www.ncbi.nlm.nih.gov/sites/entrez

56. Don’t you worry! We will get you a new Brain now that Organs can be cultured.