Animalcellculturetechniques

ANIMAL CELL CULTURE INTRODUCTION AND TECHNIQUE

1. Animal Cell Culture Techniques & Its Applications 1

2. Introduction Cell culture can be defined as the process of cultivating cells and tissues outside the body of an organism (invitro) in an artificial environment . Cell culture was first successfully undertaken by Ross Harrison in 1907. Roux in 1885 for the first time maintained embryonic chick cells in a cell culture. 2

3. 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 of an organism. 1897 : Loeb demonstrated the survival of cells isolated from blood and connective tissue in serum and plasma. 1907 : Harrison cultivated frog nerve cells in a lymph clot held by the 'hanging drop' method and observed the growth of nerve fibers in vitro for several weeks. He was considered by some as the father of cell culture. 3

4. 1916 : Rous and Jones introduced proteolytic enzyme trypsin for the subculture of adherent cells. 1940s : The use of the antibiotics penicillin and streptomycin in culture medium decreased the problem of contamination in cell culture. 1952 : George 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 (merge) monolayers of cultured cells. 1955 : Eagle studied the nutrient requirements of selected cells in culture and established the first widely used chemically defined medium. 1961 : Hayflick and Moorhead isolated human fibroblasts and showed that they have a finite lifespan in culture. 4

5. Major development’s in cell culture technology 5 First development was the use of antibiotics which inhibits the growth of contaminants. Second was t h e use of t r ypsin t o re m ove adh e rent ce l l s to subculture further from the culture vessel. Third was the use of chemically defined culture medium.

6. Terminologies 6 Primary Cell Culture : When cells are surgically removed from an organism and placed into a suitable culture environment they will attach, divide and grow. Cell Line: When the primary culture is subcultured and they show an ability to continuously propagate. Anchorage dependency: Cells grow as monolayers adhering to the substrate (glass/ plastic ) ( refers to the need for cells to be adhered to or in contact with another layer of cells .) Passaging/ subculturing: The process of splitting the cells. g r ow u p to in f inite Finite cells: When the cells has finite life span. Contin u o u s cell lines: W h en the cells can lifespan.

7. Equipping Tissue Culture Laboratory Laminar cabinet - Vertical LAF are preferable. They are fitted with HEPA filters and UV. Incubation facilities - Temperature of 37°C . 7

8. 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 (light sources and condenser on the top) Tissue culture ware- Culture plastic ware treated by polystyrene ( thermocol ) 8

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10. Tissue Culture Media Cells have complex nutritional requirements that must be met to permit their propagation in vitro. Previously, scientists employed chick embryo extract, plasma, sera, lymph etc., However, they varied in their growth promoting characteristics and thus hampering the reproducibility of the experiments. Today, a number of chemically-defined formulations have been developed that support the growth of a variety of established cell lines 11

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12. Eagle’s basal media Eagles’s Minimum Essential Media (MEM) Dulbecco’s Modified Essential Media (DMEM) Iscove’s Modified Dulbecco’s Medium (IMDM) Roosevelt Park Memorial Institute (RPMI 1640) HAM’s F12 The various nutrients required are: glucose, fats and fatty acids, lipids, phospholipids and sulpholipids ( lipid containing sulphur ) ATP and amino acids Vitamins Minerals Serum: Ser u m ca n pro v ide vari o us growth factors, h o r m o n es, cell ad h esion factor and oth e r fact o rs needed b y t he m ost mammalian cells for their long term growth and metabolism . 13

13. Properties and Special Requirements of Media pH : Optimum pH between 7.2 to 7.4 is generally needed for mammalian cells. Phenol red is used as an internal indicator . (pH indicator) S. f pH Colour of the medium 7.8 Pink / purple 7.6 Pink / purple 7.4 Red 7.0 Orange 5.5 Yellow < 6.5 Lemon Yellow • CO 2 and Bicarbonate: NaHCO 3 and 20 mM HEPE ( maintaining physiological  Ph ). 14

14. Oxygen Cells depend upon glycolysis for the supply of O 2 Selenium controls O 2 diffusion ( Selenium  helps your  body  make special proteins, called antioxidant enzymes) Glutathione acts as free radical scavenger (cleaning) Temperature The optimum temperature of mammal is 37 o C. Change of ± 5°C is acceptable. Humidity For cell growth 100% humidity is essential to reduce evaporation of the media. Antibiotics penicillin (100 U/ml) for bacteria, streptomycin (100 mg/ml) for bacteria, or gentamycin (50mg/ ml) for bacteria, and nystatin (50mg/ml) for fungi and yeast 14

15. 15 Serum Free Media   welldefined  and controlled culture media that has been processed in some form ( e.g.,filtered  to remove inhibitors and/or growth factors).  Serum-free media are not necessarily protein-free or chemically defined; most are optimised for the growth  requirements of cell-specific applications. Serum-free media do not contain or require the addition of serum

16. Types of tissue culture 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 cell line over physical t han in Development of a several generations Scale-up is possible Absolute control of environment Homogeneity of sample L e s s c o m po un d n ee d ed animal models Disadvantages Cells may lose some differentiated characteristics. Hard to maintain Only grow small amount of tissue at high cost Dedifferentiation EXPLANT CULTURE Is the growth of tissues or cells separate from the organism. This is typically facilitated via use of a liquid, semi-solid, or solid growth medium, such as broth or agar. Advantages Some normal functions may be maintained. Better than organ culture for scale-up but not ideal. Disadvantages Original organization of tissue is lost. ORGAN CULTURE The entire embryos or organs are excised from the body and culture 16 • Instability, aneuploidy 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.

17. Suspension culture- the culture of tissue and cells cultured in liquid medium, producing a suspension of single cells and cell clump. In cell  culture  a  monolayer  refers to a layer of cells in which no cell is growing on top of another, but all are growing side by side and often touching each other on the same growth surface.

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19. Primary Cultures 20 When cells are surgically removed from an organism and placed into a suitable culture environment they will attach, divide and grow. Most of the primary culture cells have a finite lifespan of 50- 60 divisions invitro . Primary cells are considered by many researchers to be more physiologically similar to in vivo cells Due to their limited lifespan, one cannot do long-term experiments with these cells

20. Cell lines A cell culture developed from a single cell and therefore consisting of cells with a uniform genetic make-up . Cell culture and cell lines have assumed an important role in studying physiological, pathophysiological and the differentiation processes of specific cells. It allows the examination of stepwise alterations in the structure, biology, and genetic makeup of the cell under controlled environments. This is especially valuable for complex tissues, such as the pancreas, which is composed of various cell types, where  in vivo examination of individual cells is difficult, if not impossible.

21. Tissue explants are excised using sharp scalpel. Mechanical disruption by pestle and mortar. Then filtered using a 0.22µ Filter fitted to a syringe. Enzymatic digestion by Trysin or collagenase Cells are counted on a Haemocytometer. 1-2 × 10^5 cells / mL is seeded in to the media. 5mL of cells is suspended into 25cm^2 flask. The flasks are incubated in a CO 2 incubator . The flasks are observed daily for their normal growth characteristics. Media is changed every 2-3 days until the cells attain 80 % confluent. 20

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23. Isolation of cell lines for in vitro culture Resected Tissue Cell or tissue culture in vitro Primary culture Sub-culture Secondary culture Sub-culture Cell Line Immortalization Successive sub-culture Single cell isolation Clonal cell line Senescence Immortalised cell line Loss of control of cell growth Transformed cell line

24. Isolation of the tissue When work with human or animal tissues, medical ethical rules should be observed Embryonic or fetal animal tissues more than half term also needs guidelines Work with human tissue should be carried out at Containment Level 2 in a Class II biological safety cabinet Sterilization of the site 70% alcohol Remove the tissue aseptically Transfer to lab in dissection BSS(borate buffered saline-pH maintain) or collection medium

25. Isolation of mouse embryo Convenient source of cells for undifferentiated mesenchymal ( multipotent ) cell cultures Mouse embryo fibroblasts are used as feeder layers(a popl of connective tissue) Full term- 19-21 days Optimal age for preparing culture 13 days Since isolation of above half term embryo needs license, 9-10 day embryo is preferable Kill the mouse by cervical dislocation

26. Swabbing with alcohol Tearing the skin to expose the abdominal wall

27. Cut longitudinally along median line of exposed abdomen with sterile scissors

28. Revealing the uterus Dissecting the uterus and transferring to DBSS(dissection balanced salt solution)

29. Dissection of embryos from the uterus by tearing the uterus Take the intact uteri to tissue culture lab and transfer to fresh petri dish

30. Free the embryos from the membrane and placenta, cut the head if necessary

31. Chopping the embryos Primary explants Disaggregation and suspension culture enzymatic mechanical

32. Primary explants Primary culture  refers to the stage of the  culture  after the cells are isolated from the tissue and proliferated under the appropriate conditions until they occupy all of the available substrate (i.e., reach confluence). A fragment of tissue transplanted from its original site and maintained in artificial medium Original method developed by Harrison, Carrel and others A fragment of tissue was embedded in blood plasma Mixed with heterologous serum and embryo extract Placed on a coverslip that was inverted over a concavity slide. The clotted plasma held the tissue in place The explant could be examined with a conventional microscope

33. Cells migrate out from fragments of tissue that is adhered to a suitable substrate The tissue is chopped finely and rinsed Pieces are seeded onto culture flask surfaces Small volume of medium is added with high concentration of serum After the tissue adheres, increase the volume of medium Primary explants

34. Suitable for small amount of tissue Risk of losing cells during mechanical or enzymatic disaggregation Disadvantages Poor adhesiveness of some tissue Only cells capable of migration outgrow Primary explants To promote attachment Place a glass coverslip on top of explant Coating the dish with polylysine , fibronectin or other ECM proteins Purified fibrinogen or thrombin Small amount of medium with high concentration of serum. I ncrease the number of positively charged sites   fibronectin - cell  adhesion molecule

35. Disaggregation using enzymes Trypsin, collagenase , dispase , hyaluronidase Alone or in combinations ( collagenase with dispase , dispase with trypsin ) In combination with EDTA (chelating agent for Ca 2+)(stable-water soluble complex) Crude preparations or purified enzyme preparation Latter(end) generally less toxic and more specific Dispase  is a protease which cleaves fibronectin , collagen IV, and to a lesser extent collagen I . PRONASE- Mix of protease from ECF- denature proteins

36. NOTE The five most common types of collagen are: Type I :  skin ,  organs ,  bone  (main component of the organic part of bone) Type II :  cartilage  (main collagenous component of cartilage) Type III : reticulate (main component of  reticular fibers Type IV : forms basal lamina, the epithelium-secreted layer of the  basement membrane . Type V : cell surfaces,  hair  and  placenta

37. Some of the cell-cell adhesion molecules are calcium dependant. Also integrins which bind to ECM components have calcium ion binding domains. So all these interactions affected by Ca depletion. Proteoglycans need hyaluronidase ( degradation of hyaluronic acid (HA )) and glycoproteins are protease sensitive Enzymes varies in effect Trypsin and pronase gives most complete disaggregation but may damage the cells Collagenase and dispase give incomplete disaggregation but are less harmful Non mammalian enzymes Trypzean , recombinant maize derived trypsin TrypLE , recombinant and microbial in origin. Disaggregation of tissue yields higher number of cells that are more representative of the whole tissue in a shorter time Embryonic tissue disperses more readily and gives more proliferating cells DISPASE- CLEAVES FIBRONECTIN, COLLAGEN IV AND COLLAGEN I

38. Enzymatic disaggregation is mostly used when high recovery of cells is required from a tissue. Disaggregation of embryonic tissues is more efficient with higher yield of cells by use of enzymes. This is due to the presence of less fibrous connective tissue and extracellular matrix. Enzymatic disaggregation can be carried out by using trypsin, collagenase or some other enzymes.

39. Trypsin The term trypsinization is commonly used for disaggregation of tissues by the enzyme, trypsin. Many workers prefer to use crude trypsin rather than pure trypsin for the following reasons: The crude trypsin is more effective due to the presence of other proteases ii. Cells can tolerate crude trypsin better . iii. The residual activity of crude trypsin can be easily neutralized by the serum of the culture media (when serum-free media are used, a trypsin inhibitor can be used for neutralization ). Disaggregation of cells can also be carried out by using pure trypsin which is less toxic and more specific in its action. The desired tissue is chopped to 2-3 mm pieces and then subjected to disaggregation by trypsin. There are two techniques of trypsinization -warm trypsinization and cold trypsinization

40. Warm Trypsin This method is widely used for disaggregation of cells. The chopped tissue is washed with dissection basal salt solution (DBSS), and then transferred to a flask containing warm trypsin (37° C). The contents are stirred, and at an interval of every thirty minutes, the supernatant containing the dissociated cells can be collected. After removal of trypsin, the cells are dispersed in a suitable medium and preserved (by keeping the vial on ice ). The process of addition of fresh trypsin (to the tissue pieces), incubation and collection of dissociated cells (at 30 minutes intervals) is carried out for about 4 hours. The disaggregated cells are pooled, counted, appropriately diluted and then incubated.

41. Trypsinization with cold pre exposure Particularly suitable for small amounts of tissue Cold trypsin method gives a higher yield of viable cells Improved survival after 24 h Preserves more different cell types than warm method No stirring or centrifugation needed Incubation at 4 o C can be left overnight unattended

42. Treatment with collagenase Collagen is the most abundant structural protein in higher animals. It is mainly present in the extra­cellular matrix of connective tissue and muscle. The enzyme collagenase (usually a crude one contaminated with non-specific proteases) can be effectively used for the disaggregation of several tissues (normal or malignant) that may be sensitive to trypsin. Highly purified grades of collagenase have been tried, but they are less effective when compared to crude collagenase.

43. The desired tissue suspended in basal salt solution, containing antibiotics is chopped into pieces. These pieces are washed by settling, and then suspended in a complete medium containing collagenase. After incubating for 1-5 days, the tissue pieces are dispersed by pipetting. The clusters of cells are separated by settling. The epithelial cells and fibroblastic cells can be separated. Collagenase disaggregation has been successfully used for human brain, lung and several other epithelial tissues, besides various human tumors, and other animal tissues. Addition of another enzyme hyaluronidase (acts on carbohydrate residues on cell surfaces) promotes disaggregation. Collagenase in combination with hyaluronidase is found to be very effective for dissociating rat or rabbit liver. This can be done by per-fusing the whole organ in situ. Some workers use collagenase in conjunction with trypsin, a formulation developed in chick serum, for disaggregation of certain tissues.

44. Mechanical disaggregation To avoid the risk of proteolytic damage Methods The cells that spill out when the tissue is carefully sliced or scraped can be collected The dissected tissue can be pressed through a series of sieves(strain solid from liquid) for which the mesh is gradually reduced in size The tissue fragments can be forced through a syringe (with or without a wide-gauge needle) Pipette the tissue repeatedly Due to a risk of proteolytic damage to cells during enzymatic digestion

45. Gives a cell suspension more quickly than does enzymatic digestion Scraping (left over reprocessing) and sieving are the gentlest mechanical methods Mostly suitable only for soft tissues, such as spleen, embryonic liver, embryonic and adult brain, and some human and animal soft tumors The viability of the resulting suspension is lower Suitable when the availability of tissue is not a limitation and the efficiency of the yield is unimportant

46. Separation of viable and non viable cells Adherent primary culture nonviable cells are removed at the first change of medium Primary cultures maintained in suspension Nonviable cells are gradually diluted out when cell proliferation starts Centrifuge the cells on a mixture of Ficoll (hydrophilic polsac dissolved in aq sol) and sodium metrizoate ( radiopaque occurs in Na salt ) viable cells collect at the interface between the medium and the Ficoll / metrizoate the dead cells form a pellet at the bottom of the tube.

47. Cell Suspension Culture Suspension culture  is a type of  culture  in which single  cells  or small aggregates of cells  multiply while  suspended  in agitated liquid medium. It is also referred to as cell culture  or  cell suspension culture . Suspension cells do not attach to the surface of the culture vessels. These cells are also called anchorage independent or non-adherent cells which can be grown floating in the culture medium. Hematopoietic stem cells (derived from blood, spleen and bone marrow) and tumor cells can be grown in suspension. These cells grow much faster which do not require the frequent replacement of the medium and can be easily maintained. These are of homogeneous types and enzyme treatment is not required for the dissociation of cells; similarly these cultures have short lag period.

48. Continuous 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 gets transformed into Continuous cell lines. • Characteristics of continuous cell lines -s m al l e r , m o re rou n de d , less ad h erent with a hig h er n u cleus /cytoplasm ratio -Fast growth and have aneuploid chromosome number -red u ced ser u m and a nc h orage d epe nd ence and grow m o r e in suspension conditions -ability to grow upto higher cell density -different in phenotypes from donar tissue -stop expressing tissue specific markers - Loss of Contact inhibition 22

49. Common cell lines Human cell lines • MCF-7 • HL 60 • HEK-293 • HeLa breast canc Leukemia Human em Henrietta l ac ryonic kidney ks li Primate cell • Vero nes African green M onkey kidney ep helial cells Cos-7 African green monkey kidney cells And others such as CHO from hamster, sf9 & sf21 from insect cells H e la - Epith e l ial 53 MCF-7 breast M R C5 - Fib r o b l a s t HT1080- kidney 3LL - lungs

50. er BAE1-Endothelial b ac SHSY5Y-Neuronal m it

51. Subculturing Incubate the cells. Remove spent media from the culture vessel. Add the pre-warmed dissociation reagent such as trypsin. Gently rock the container to get complete coverage of the cell layer. Incubate the culture vessel at room temperature for approximately 2 minutes. Add equivalent of 2 volumes of pre-warmed complete growth medium. Disperse the medium by pipetting over the cell layer surface several times. Then split the cells into 2 or 3 flasks containing complete media. 55

52. Cryo p reser v ation Next day transfer the cryovials to Liquid nitrogen. Remove the growth medium, wash the cells by PBS and remove the PBS by aspiration. Dissociate the cells by trypsin Dilute the cells with growth medium. Centrifuge at 200g for 5 min at RT and remove the growth medium by aspiration Resuspend the cells in 1-2ml of freezing medium containing DMSO ( dimethyl sulfoxide ) . Transfer the cells to cryovials, incubate the cryovials at -80°C overnight 25

53. 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 (lack cell wall) detected by direct DNA staining with intercalating fluorescent substances e.g. Hoechst 33258 Mycoplasma also detected by enzyme immunoassay by specific antisera or monoclonal abs or by PCR amplification of mycoplasmal RNA The best and the oldest way to eliminate contamination is to discard the infected cell lines directly 26

54. Invitro Transformation of Cells Transformed, Infinite or Established Cells Changed from normal cells to cells with many of the properties of cancer cells Some of these cell lines have actually been derived from tumors or are transformed spontaneously in culture by mutations Chemical or gamma ray treated cells can become infinite with loss of growth factors Viral infection with SV40 T antigen can insert oncogenes and lead to gene alteration No matter how transformation occurred, the result is a cell with altered functional, morphological, and growth characteristics

55. Advantages of Tissue Culture 59 Control of the environment. Characterization and Homogenity of sample. Economy, Scale and Mechanization. Invitro modelling of Invivo Conditions.

56. Limitations 60 To grow cells outside their normal environment, three major controls are involved. Observing s t rict asepsis Providing the right kind of physic-chemical environment Nutrients in its simplest absorbable form Culturing technique needs a great deal of expertise. Tissue samples consists of a mixture of heterogenous cell populations Continuously growing cells often show genetic instability. Differences in the behavior or cells in cultured and in its natural form. Should include proper balance of the hormones.

57. Applications of Cell Culture 61 Excellent model systems for studying: The normal physiology, cell biology and biochemistry of cells The effects of drugs, radiation and toxic compounds on the cells Study mutagenesis and carcinogenesis Used for gene transfer studies. Large scale manufacturing of biological compounds (vaccines, insulin, interferon, other therapeutic protein)

58. P r oducts Mon oclo n al Antibodies (Mab’s) Immuno- biological R egul a t or s Virus v accin e s Hormones Produced by hybridoma cell Used for diagnostic assay systems (determine drugs, toxins & vitamin); therapeutic purposes & biological separations – chromatographic separations to purify protein molecules Interferon – anticancer glycoprotein (secreted animal cell or recombinant bacteria) Lymphokines Interleukines (anticancer agent) Prophylactics (prevent disease) Virus is collected, inactivated and used as vaccine A weakened form will induce a protective response but no disease Large molecules: 50-200 amino acids Produce by hormone- synthesizing organ May also produce by chemical synthesis Example : Erythropoietin (s-kidney)

59. Products Enz ymes Insecticides Whole cells and tissue culture Urokinase, rennin, asparaginase, collaginase, pepsin, trypsin, etc.. Production of some insect viruses that are highly specific and safe to envirionment Artificial organs and semi synthetic bone and dental structure