Animal cell culture Lecture 1 Introduction

2. 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. Homogenous population may (clone) or heterogeneous population

3. TISSUE CULTURE In vitro cultivation of organs, tissues & cells at defined temperature using an incubator & supplemented with a medium containing cell nutrients & growth factors is collectively known as tissue culture.

4. TISSUE CULTURE Different types of cell grown in culture includes connective tissue elements such as fibroblasts, skeletal tissue, cardiac, epithelial tissue (liver, breast, skin, kidney) and many different types of tumor cells.

5. HISTORY Roux in 1885 for the first time maintained embryonic chick cells in a cell culture ( use saline). Cell culture was first successfully undertaken by Ross Harrison in 1907 ( lymph) 1911: Lewis made the first liquid media consisted of sea water, serum, embryo extract, salts and peptones. They observed limited monolayer growth.

6. HISTORY 1913: Carrel introduced strict aseptic techniques so that cells could be cultured for long periods. 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.

7. HISTORY 1948: Earle isolated mouse L fibroblasts which formed clones from single cells. 1952: Gey established a continuous cell line from a human cervical carcinoma known as HeLa (Helen Lane) cells. 1955: Eagle studied the nutrient requirements of selected cells in culture and established the first widely used chemically defined medium.

8. HISTORY 1961: Hayflick and Moorhead isolated human fibroblasts (WI-38) and showed that they have a finite lifespan in culture. 1965: Ham introduced the first serum-free medium which was able to support the growth of some cells 1975: Kohler and Milstein produced the first hybridoma capable of secreting a monoclonal antibody.

9. MAJOR 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. Advantages 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. Advantages 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. Advantages To produce artificial tissues ( skin) Use single cell ( impossible in vivo)

13. Disadvantages Cell characteristics can change Cell can adapt to different nutrients If mixed cells cultivated some types will disappear. Activity of enzymes may altered by environment.

14. Animal tissue Cultures Classification according to Source and aim Passages Shape of growth (Culture Morphology) 1-classification a: A- Organ culture B-Tissue culture C-Cell culture

15. Types

16. A- Organ Culture The entire embryos or organs are excised from the body and culture favors the retention of a spherical or three-dimensional 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. Fragments of excised tissue are grown in culture media a fragment of tissue is placed at a glass (or plastic)�liquid interface, where, after attachment, migration is promoted in the plane of the solid substrate Advantages Some normal functions may be maintained. Better than organ culture for scale-up but not ideal. Disadvantages Original organization of tissue is lost. B-Tissue Culture

18. C- Cell Culture Implies that the tissue, or outgrowth from the primary explant, is dispersed (mechanically or enzymatically) into a cell suspension, which may then be cultured as an adherent monolayer on a solid substrate or as a suspension in the culture medium Advantages Development of a cell line over several generations Scale-up is possible Disadvantages Cells may lose some differentiated characteristics.

19. classification b Primary Cultures Derived directly from excised tissue and cultured either as Outgrowth of excised tissue in culture Dissociation into single cells (by enzymatic digestion or mechanical dispersion) Advantages: usually retain many of the differentiated characteristics of the cell in vivo Disadvantages: initially heterogeneous but later become dominated by fibroblasts. the preparation of primary cultures is labor intensive can be maintained in vitro only for a limited period of time.

20. Continuous Cultures derived from subculture (or passage, or transfer) of primary culture Subculture = the process of dispersion and re-culture the cells after they have increased to occupy all of the available substrate in the culture can be serially propagated in culture for several passages There are two types of continuous cultures Cell lines Continuous cell lines

21. Cell lines finite life, senesce after approximately thirty cycles of division usually diploid and maintain some degree of differentiation.

22. Continuous cell lines can be propagated indefinitely generally have this ability because they have been transformed tumor cells. viral oncogenes chemical treatments. the disadvantage of having retained very little of the original in vivo characteristics

23. 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

25. 3- Culture Morphology Suspension (as single cells or small free-floating clumps) or as a monolayer that is attached to the tissue culture flask. The form taken by a cell line reflects the tissue from which it was derived from blood tend to grow in suspension from solid tissue (lungs, kidney) tend to grow as monolayer's. Attached cell lines can be classified as endothelial, epithelial, neuronal or fibroblasts and their morphology reflect the area within the tissue of origin

27. Anchorage dependent or independent Cell lines derived from normal tissues are considered as anchorage-dependent grows only on a suitable substrate. ( Epith, CT) Suspension cells are anchorage-independent e.g. blood cells

28. Adherent cells Cells which are anchorage dependent Add enough trypsin/EDTA to cover the monolayer Incubate the plate at 37 C for 5 mts 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

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

30. confluency Once the available substrate surface is covered by cells (a confluent culture) growth slows & ceases.

31. hep 3B - 70% confluency

32. 100% confluency

33. After 24 h

34. confluency 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

35. Design and Equipment for the Cell Culture Laboratory 

36. Design and Equipment for the Cell Culture Laboratory  1. Laboratory design- in a safe and efficient manner 2. safety cabinets

38. Laminar- flow hood

39. Laminar- flow hood The working environment is protected from dust and contamination by a constant, stable flow of filtered air Two types: horizontal, airflow blow from the side facing you, parallel to the work surface, and is not circulating; - vertical, air blows down from the top of the cabinet onto the work surface and is drawn through the work surface and either recalculated or vented.

40. Laminar- flow hood - The efficiency of the hood depends on a minimum pressure drop across the filter When the filter resistance builds up, the pressure drop increases and the flow rate of air falls. Below 0.4 m/s, the stability of the laminar flow is lost and sterility can not be maintained.

42. Laminar- flow hood Routine maintenance checks of the primary filters are required (every 3-6 months). They might be removed and discarded or washed in soap and water. Every 6 months the main high efficiency particulate air (HEPA) filter above the work surface should be checked for airflow and hole

43. Before use Ultraviolet lights are used to sterilize the air and exposed work surfaces in laminar flow cabinets between use. Detergent 70% alcohol

44. Cell Culture Incubator

45. It requires a controlled atmosphere with high humidity and super controlled of CO2 tension. � The incubator should be large enough, probably 50-200 l have forced air circulation, temperature control + 0.5oC, and a safety thermostat that cuts out if the incubator overhears. �

46. - It should be stainless steel, and easily cleaned. � A double cabinet, one above the other, independently regulated, is preferable to one large cabinet. � Incubators are supplied either with a heated water jacket as a method for distributing the heat evenly around the cabinet or with surface heater elements for heating.

47. Autoclave A simple bench-top autoclave may be sufficient, but a larger model with a timer and a choice of presterilization and poststerilization evacuation will give more capacity and greater flexibility in use

49. Refrigerators and Freezers �4C �-20C �-80C � Liquid N2 tank

50. Inverted microscopy

56. Multiwell plates

63. Cell culture contaminants  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

67. Contamination 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

68. Safety