cell and tissue culture.pptx by taimoor khan

cell and tissue cultureintroduction ppts

1. Cell and Tissue Culture Technology Semester 7 th Taimoor Khan Department of Biotechnology, University of Swabi , KPK, Pakistan

2. Course Objectives To understand the process of tissue culture technology . To study the nutritional and physical requirements of primary cell culture and established cell lines. To use as viable media for the cultivation of viruses; and in diagnosis. To understand the cellular differentiation.

3. Introduction Tissue culture is a general term for the removal of cells, tissues, organs from animals or plants and their subsequent placement into an artificial environment conductive to growth. Cell may be removed from tissue directly Maybe derived from cell lines

4. Terms for understanding Organ Culture: T he culture of whole organ or organ fragments with the intent of studying their continued function is known as organ culture. Cell culture: when the cells are removed from the organ fragment prior to or during cultivation, thus disrupting their normal relationship with neighboring cells.   Suspension culture: Facilitated via use of a liquid, semi-solid, or solid growth medium, such as  broth  or  agar.

5. C ells of interest have been isolated from living tissue. M aintained under carefully controlled conditions i.e , Temp. PH, Humidity. Conditions may vary for different cell types. A rich medium that supplies the essential nutrients (amino acids, carbohydrates, vitamins, minerals), growth factors, hormones, and gases (CO2, O2 ).

6. Cells Isolation Cells may be isolated from a donor organism (primary cells) or an immortalized cell line. Every 30 seconds a patient dies from a disease that could be treated with tissue replacement https://www.ted.com/talks/anthony_atala_growing_new_organs

7. Tissue culture William Roux(1885-1924) – demonstrated that it is possible to maintain living cell outside the body in a saline buffer for few days. Leo loab (1869-1959) – able to culture cells from outside the body Leo loab William Roux

8. History 19th-century  English physiologist Sydney Ringer developed salt solutions containing the chlorides of sodium, potassium, calcium and magnesium suitable for maintaining the beating of an isolated animal heart outside the body. 1885  Wilhelm Roux removed a section of the medullary plate of an embryonic chicken and maintained it in a warm saline solution for several days, establishing the basic principle of tissue culture

9. 1907 to 1910  Ross Granville Harrison , working at Johns Hopkins Medical School, published results of his experiments, establishing the methodology of tissue culture Small pieces of living frog embryonic tissues were isolated and grew outside. 1870-1959

10. Harrison’s Hanging drop technique Well established method for examining living, unstained and small organisms. Traditional procedure employs a glass slide with a concavity. Into which a drop of fluid, containing microorganism, hangs from coverslip. https://embryo.asu.edu/pages/hanging-drop-tissue-culture

11. Dr. Carrel’s Immortal cells Alex carrel discovers cell culture of embryonic and adult tissues of many species. Applied other culture media included: The diluted plasma with varying conc. of salt solution The serum The term tissue culture was defined for first time in 1911. “ A plasmatic medium inoculated with small fragments of living tissues ”

12. D-Flasks Development of first cell culture flask (1923). Culture flask (also called D 3.5 flask) was made of PYREX glass.

13. Evaluation of cell culture technique Introduction to aseptic technique. Use of trypsin solution results in obtaining single cell suspension and cell detachment for subculture. 3% trypsin – used for plasma digestion and did not damage most cells. 5% trypsin was tested, most cells were dead.

14. 1996  T he first use of regenerative tissue was used to replace a small length of urethra , led to the technique of obtaining samples of tissue, growing it outside the body without a scaffold, and reapplying it ( less than 1  cm).

15. Gottlieb Haberlandt first pointed out the possibilities of the culture of isolated tissues, plant tissue culture. He presented his original idea of  totipotentiality  in 1902 , stating that " Theoretically all plant cells are able to give rise to a complete plant ” .

16. Application of cell culture Mass culture of animal cell lines is fundamental to the manufacture of viral  vaccines, Antibodies, peptides   and other products.

17. Applications H uman   stem cells culture -- used to expand the number of cells and differentiate the cells into various somatic cell types for transplantation. Biological products  S ynthetic   hormones M onoclonal antibodies Interleukins   Stem cells are undifferentiated or partially differentiated cells that can change into various types of cells and proliferate indefinitely to produce more of the same stem cell.

18. Types of cell culture

19. Types Primary cell culture – when cells are surgically removed from an organism and are placed into suitable culture medium, they will attach, divide and grow It is further classified based on type of cell; Adherent cell culture 2. Suspension cell culture

20. Adherent cell culture Attachment Fibroblasts/epithelial cells/endothelial) Culture surface are often coated with (collagen, laminin or fibronectin) to enhance cell adherence . Growth conditions Cells cultured in growth medium. As cells proliferate they spread and form a monolayer. Once they reach confluence (covering the surface), the cells may need to be passaged ( subcultured )

21. Sub culturing (passaging) When cells become too confluent, they need to be enzymatically detached (often using trypsin), split into smaller portions and transferred into new culture vessel.

22. Suspension cell culture Those cell which donot require attachment to any surface for proliferation. Instead cells grow freely in medium. i.e commonly used for hematopiotic stem cell (blood cells such as lymphocytes or leukocytes) I mmune cells or transformed cell lines. Hybridomas cells

23. Cont.. Cell growth – agitation or shaking can be applied to keep the cells evenly distributed in medium. Large scale bioreactors can be used for mass production in biotechnology. Advantage: suspension culture can be easily scaled up to large volume making them ideal for industrial processes. i.e vaccine production, antibody production etc.

24. Most of primary cell culture have finite lifespan. Due to limited lifespan one cannot do long term experiments with these cells. Primary cells are considered to be more physiologically similar to in vivo cells.

25. Primary cell Two basic mechanism for obtaining primary culture: Explant culture: small pieces of tissue are attached (using plasma clots or fibrinogen) to a glass or treated plastic culture vessel and immersed in culture medium After few days individual cells will move from the tissue explants out onto a substrate were the begin to grow

26. Enzymatic Dissociation: More widely used Addition of digestive enzymes (proteolytic enzyme or collagenase) to tissues fragments to breakdown bonds holding cell together.

27. 2. Cell lines After the first subculture the primary culture become cell lines. Limited life span As they are passaged, cell with the highest growth rate predominate.

28. 3. Cell strain: A cell strain is the subpopulation of cell lines that has been positively selected from the culture. Cell strain often have undergone additional genetic changes since the initiation of the parent line. Cells have finite lifespan of 40-60 division in vitro. Useful in vaccine production.

29. Heyflick’s phenomenon Cells will continue to grow and divide for number of passages. At certain point they die, even after giving sufficient nutrients. There appears to be a correlation between maximal number of passages and aging. No. of passages decreases when cells are harvested from older individuals.

30. Finite or continuous A cell line or cell strain may be finite or continuous depending upon whether limited culture life span or it is immortal in cells. Finite cell line Limited lifespan Go through limited number of generation Properties Contact inhibition Anchorage dependence Less growth rate Doubling time (24-94hr) Continuous cell line Grow independitely Grow either in monolayer or single layer Properties Absence contact inhibition Absence anchorage dependence High growth rate Doubling time (12-24hrs)

31. Secondary cell culture When primary cell culture occupy all the available substrate ( i.e reach confluence). At this stage cells have to be substituted ( i.e. passaged) by transferring them into a new vessel with fresh growth medium to provide more room for continued growth.

32. Sub culturing or secondary culturing is necessary to periodically provide necessary nutrients and growing space for continuously growing cells lines. Process involve removing growth media, washing plates, dissociating adherent cells enzymatically.

33. There are certain advantages in propagation of cells by suspension culture method. These advantages are:  (a) The process of propagation is much faster., (b) The frequent replacement of the medium is not required., (c) Suspension cultures have a short lag period, (d) treatment with trypsin is not required,  (e) a homogenous suspension of cells is obtained,  (f) the maintenance of suspension cultures is easy and bulk production of the cells is easily achieved., (g) scale-up is also very convenient.

34. The cell lines are known by : a ) A code e.g. NHB for Normal Human Brain. b) A cell line number- This is applicable when several cell lines are derived from the same cell culture source e.g. NHB1, NHB2 . c) Number of population doublings, the cell line has already undergone e.g. NHB2/2 means two doublings.

35. COMMONLY USED CELL LINES cell lines are an invaluable scientific tool. They allow us to dissect the internal workings of tissues in a controlled environment without the ethical implications of working with whole organisms. Starting with the first successful immortal cell line HeLa, the number of available cell lines has since diversified into a plethora(large amt ) of options.

36. HeLa Unlike the other cell lines, HeLa is named after an individual, an American women called Henrietta Lacks. Shortly after establishment of this cell line, HeLa cells were used to proliferate the famous polio vaccine, and they continue to be the most widely used cell line in research labs worldwide. 1952– HeLA cells as growth medium for polio viruses

37. Cont.. According to the British newspaper The Guardian, HeLa “has led to hundreds, if not thousands, of new pieces of knowledge, and helped to shape the way medicine moved in the second half of the 20th century and the first decade of this one” .

38. HEK 293 HEK293 , or human embryonic kidney-derived epithelial cells , are arguably one of the most widely used cell lines in cell biology research. HEK293 is a rapidly dividing, robust line cell with a good reputation for post-translational modification of its heterologously expressed proteins.

39. Chinese hamster ovary cells  Chinese hamster ovary cells ( CHO s) are clearly ovary-derived cells, but this time, we are taking mammalian cells. Similarly to Sf9 cells, they can exist both as adherent or suspension cells in culture. CHO cells are used in various applications such as recombinant protein production and studies of the  epidermal growth factor receptor  .

40. Sf9 insect epithelial cells Derived from the ovaries of the fall armyworm moth ( Spodoptera frugiperda ), these cells are probably related to all insect cell lines in labs worldwide .   Sf9 cells can be cultured as adherent or suspension cells. Most cell lines are adherent cells, which grow only on the surfaces of culture vessels. This limits the amount of cells you can expect to obtain from each culture.

41. Similarly to  E.coli , suspension cells can grow in the entire volume of the medium, thus increasing the amount of cells that can be harvested from a vessel. Because of the high volume: cell number ratio, suspension cultures allow a much more effective use of medium than adherent cultures

42. Basic requirements for tissue culture

43. Media constituents Macronutrients Mineral elements such as; Potassium Calcium Magnesium Nitrogen Phosporous Sulphur Micronutrients Iron Manganese Copper Zinc Boron Molybdenum

44. One type of ion may be contributed by more than one salt in the medium. For example in MS medium, NO3 are contributed by NH4NO3, as well as KNO3 and K+ by KNO3 and KH2PO4.

45. Inorganic Nutrients Inorganic nutrients is usually supplied in the form of ammonium (NH4+) and nitrate (NO3+). Some micro nutrients are essential for growth in culture. Include cofactors for enzymes; Boron (BO33 from H3BO3) Manganese (MN2 from MNSO4).

46. Organic Nutrients To achieve best possible growth of tissues in culture, medium needs to have supplemented with organic nutrients. Sucrose (a source of carbon and energy) Vitamins and aminoacids (Thiamine—Vitamin B1) Nicotinic acid –Vitamin B3) Pyridoxine (Vitamin B6) Glycine (simplest aminoacid )

47. Carbon source Most plant cultures are unable to photosynthesize bcz of the unavailability of chloroplast or sunlight. Results in poor gaseous exchange. Sucrose –Rich carbon sucrose at conc. of 2-5% w/v. Glucose and fructose

48. Plant G rowth Regulators Basal medium –addition of growth regulators to culture medium in order to trigger various types of growth and differentiations. Plants already have growth factors (hormones). Supplement growth factors to achieve best possible growth. Growth regulator required in minute quantity (0.001-10mM).

49. Supply of growth regulators in medium vary accordingly to; Variety of plant used Nature of tissue Stage of culture (initiation of callus, somatic embryogenesis, shoot differentiation or multiplication).

50. Growth Regulators Auxins – involve in development process Gibberellin Cytokinin -- Elongation of stem -- Cell division -- Embryonic differentiation At low level conc. auxin favors root initiation. High conc. induce callus formation

51. Gelling agents Agar (obtained from red algae), especially gelidium amansii Agarose – comprising b-D(1-3) and ,3,6 anhydro -a-L(1-4) galactopyranose molecule, it is obtained by further purifying agar to remove agropectins with its sulphate group.