Mammalian cell culture, basic techniques

Introduction
Terminologies
Types of tissue culture
Applications

Culturing
Sub-culturing
Cryopreservation
Detection of contaminants
In vitro transformation of cells
Cell viability

Rules for working in the Lab
Advantages
Limitations

1. BASIC TECHNIQUES OF MAMMALIAN CELL CULTURE 1 By KAUSHAL KUMAR SAHU Assistant Professor (Ad Hoc) Department of Biotechnology Govt. Digvijay Autonomous P. G. College Raj-Nandgaon ( C. G. )

2. 2 CONTENTS Introduction Terminologies Types of tissue culture Applications Culturing Sub-culturing Cryopreservation Detection of contaminants In vitro transformation of cells Cell viability Rules for working in the Lab Advantages Limitations

3. 3 INTRODUCTION Cell culture can be defined as the process of cultivating cells and tissues outside the body of an organism( in vitro ) in an artificial environment, which stimulates the in vivo conditions such as temperature, nutrition and protection from microorganisms. 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.

4. 4 TERMINOLOGIES 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 primary culture is sub-cultured and they show an ability to continually propagate. Anchorage Dependency : cells grow an monolayers adhreing to the substrate(glass/plastic). Passaging /Sub-culturing : the process of splitting the cells. Finite Cells : when the cells has finite life span. Continues Cell Line : when the cell grow upto infinite life span.

5. 5 TYPES OF TISSUE CULTURE CULTURE MEANING ADVANTAGES DISADVANTAGES cell tissue organ Tissue from an explant is dispersed, mostly enzymatically, into cell suspension which may then be cultured as a monolayer or suspension culture. Scale-up is possible Control of physical condition Homogeneity of sample Cells may lose differentiated characteristics instability Growth of tissue separate from the organism. Facilitated using liquid or semi-solid growth medium such as broth or agar. Some normal functions may be maintained. Better than organ culture for scale-up but not ideal. Original organization of tissue is lost. Entire embryo or organs are excised from body and cultured. Normal physiological functions are maintained. Cells remain fully differentiated. Scale-up is not recommended. Growth is slow.

6. 6 APPLICATIONS Excellent model system for studying The normal phenotype, 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 Virology, Genetic Engineering, Gene therapy Large scale manufacturing of biological compounds like vaccines, insulin, interferon, other therapeutic protein.

7. 7

8. 8 Culturing :- 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 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 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

9. 9 Culturing :-

10. 10 Continues cell lines Cell lines which either occur spontaneously or induced virally or chemically transformed into Continuous cell lines Characteristics of continuous cell lines -smaller, more rounded, less adherent with a higher nucleus /cytoplasm ratio -Fast growth and -reduced serum and anchorage dependence and grow more in suspension conditions -ability to grow up to higher cell density -different in phenotypes from donor tissue -stop expressing tissue specific genes

11. 11 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

12. 12 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

13. 13 Cryopreservation :- Storage at freeze condition.

14. 14 Detection of contaminants :-

15. 15 Invitro transformation of cells :-

16. 16 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

17. 17 Rules for working in the Lab 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

18. 18 ADVANTAGES control of the environment characterization and homogeneity of the sample Economy, scale and mechanization Invitro modeling of Invivio conditions

19. 19 LIMITATIONS To grow cells outside their normal environment, three major controls are involved : - keen observation - providing right kind of physic-chemical environment - nutrients in its simples absorbable form culturing technique needs a great deal of expertise. tissue sample consists a mixture of heterogeneous cell population continually growing cells often show genetic instability differences in behavior of cells cultured or in its natural form should include proper balance of hormones

20. 20 CONCLUSION Although animal tissue culture is benefiting mankind but there are many ethical reasons as well as requirement of expertise in the field and a well established lab is required, it becomes difficult for practical implementation of it with ease.