Lecture Animal Biotechnology Haji Akbar

Establishment of cell culture: Many types of animal cells can grow in vitro such as tumour cells, neuroblastoma cells etc. Based on experiment continuous (immortal) cell line can be developed from cultured tissue. It is not necessary each cell that place on medium start growth.

Basis on growth responses culture cells are divided into three types: Precursor or master cell or stem cells, undifferentiated but committed cells and mature differentiated cells Cell cultures are at a stage of equilibrium of these types of cells which may be shifted by manipulating growth conditions.

e.g. in the presence of low serum, suitable hormones, cell matrix interaction and high cell density, cell differentiation is promoted where as in the presence of high serum suitable growth factors and low cell density cell proliferation is promoted. In addition the type cells growing in culture determined by their representative sources from where these have been derived.

e.g. high Stem cell will be found in culture driven from embryos b.c they are capable of frequent cell division as compared to adult cell culture. And cells from stress conditions such as muscles fibroblast etc will be committed precursor. (C.P cells have limited life)

i. Evolution of cell line: Primary culture sub culture in special growth medium under control condition. Some cells attached and proliferate to yield single cell line. The suspension should be diluted with fresh medium.

ii. Primary Cell culture: After disaggregation culturing primary cell culture. Refers to cultures prepared directly from the tissue directly taken from animals. Proteolytic enzymes (trypsin and Collagenase) are commonly used to break the protentious material between cells. Animal growing cell type: Anchorage-dependent (adherent cells) commonly obtained from organ fix at place e.g. Kidney cell, liver etc. (not mobile) Anchorage-independent (suspension culture) Grow in liquid medium and don’t attached to surface (e.g. suspension culture of All blood cells)

Primary culture becomes cell line only after first subculture. A cell line is a permanently established cell culture that will proliferate indefinitely given appropriate fresh medium and space. Subculture is needed when nutrient present in medium for cell growth diminish.

Secondary cell culture and cell line: Primary cell culture can be passaged to form secondary cell culture. Primary Cell culture cannot viable for a long time because the cell utilize all nutrients of the medium. Therefore sub culturing is needed. During repeated sub culturing and selection the cell line gets evolved and properly established consisting of proliferating cells.

Thus the unaltered form of cells line is called continues cell line which propagated in logarithmic ways. Any change in continues cell line may discontinue the increase in cell number. Brought about by chemical, spontaneous mutation or viruses (EBV). The phenomenon of alteration in continuous cell line is called “in vitro transformation”. Cell line consists of several similar and dissimilar lineages. A defined cell lineage having specific properties is called “Cell strain”. On basis of life span: Continues (immortal i.e. which will not die) Finite (limited up to generation)

Nomenclature of cell line: Represented in abbreviated form either derived from the source of Cells, name of institute or association with virus e.g. EB, Epstein Barr; WI, Wistar Institute) Numbered If more than one cell line (e.g. EB1, EB2) Furthermore, a cell line is given the number of population doubling e.g. EB1/1.

Types of cell line: Different types of cell line produced from different tissues or organs. Broadly they are grouped into: Finite cell line and continuous cell line. Finite cell line: Grow through a limited number of cell generations and have limited life. Grow slowly and form monolayer. Doubling time ranges from 24 to 96 hours. Anchorage dependent, contact inhibition and density limitation. Continuous cell lines: Obtained from either from transformed cell line in vitro or cancerous cells. Divides rapidly. Generation time hrs. No or reduced density limitation.

The transformed cells in vitro bears the following differences. Enhanced growth and proliferation due to rapid growth rate. Existence of alter ploidy i.e. aneuploidy or heteroploidy due to alter chromosomes number. Different cell shape and organization of microfilaments. Ability to translocate, Different energy metabolism No contact inhibition & no anchorage dependence And Different growth factors requirements and responses to regulator molecules.

Factors affecting subculture in vitro: Several factors that influence differentiation and proliferation of cells when subculture in vitro as below; Mammalian cells need attachment to a solid surface so their maximum numbers are limited by the surface area available. Serum is added in medium (mixture of several kinds of growth promoting and growth inhibitory factors). Various additives are used to protect the cell from agitation or aeration damage. e.g. cellulose and starch derivatives, polyvinyl pyrrolidones, Methyl celluloses, Dextrans Polyvinyl Glycol (PEG), Polyvinyl Alcohols (PVA) etc Optimum pH of the medium should maintained b/w 7.0 to7.6. Animal cells are very sensitive to temperature therefore thermostat should be used to maintained at 37C.

Somatic cell fusion: Fusion of two different cells to produce a hybrid cell. Application in biotechnology: Study control gene expression and differentiation. Gene mapping Malignancy Viral replication Antibodies production via hybridoma technology

Continue!!! Macrophages fuse around the foreign body or bacterial cell in the tissues. Bones cells also known to undergo somatic fusion. In culture cells are induce to fuse by some viruses e.g. Sendai virus. This virus induces first to form heterokaryon and during mitosis chromosomes of heterokeryon are brought two poles and which latter on to form hybrid. Some chemicals such as polyethylene glycol also induce somatic cell fusion. Taxonomically different animals can fuse and form hybrids. This suggests that there is no compatibility b/w membranes, nuclei, organelles of two different groups of animal cells.

Organ culture: Means the maintenance of a part of tissue or part of organ or whole organ in vitro. or The culture of complete living organs (explants) of animals and plants outside the body in a suitable culture medium. Animal organs must be small enough to allow the nutrients in the culture medium to penetrate all the cells. It is easier to culture embryonic organ than the adult animals.(O 2 95% required).

Different methods are required for culturing of embryonic and adult organs. Embryonic organ can be culture by any of the following methods: Organ culture on plasma clots: Prepared by mixing 5 drops of embryo extract with 15 drops of plasma in a watch glass on a cotton wool pad (put in Petri dish). Moistened the cotton to decrease evaporation.

Organ culture on agar medium: Solidify culture medium with agar is also used for organ culture. Adults fail to survive whereas embryonic organ grow well. The media consist of ingredients: Agar (1%basal salt solution) chick embryo extracts and horse serum in the ratio of 7:3:3

Organ culture in liquid medium: Consist of all the ingredients except agar. Perforated metal gauze or cellulose acetate or a raft of lens paper is used to provide support. Whole embryo culture: Chick embryo: eggs incubation at 38C for hrs. sterilization (70% ethanol) Blastoderm placed on watch glass containing medium incubate at 37.5C for further developments

Growth phase of Cells in culture * Lag phase : adapt to new environment; repair cell membrane damage * Log phase : exponential growth: % of cells are dividing * Plateau or Stationary phase : cell growth ~ 0-10% : Contact inhibition of movement : Density limitation of growth

Advantage of using cell culture Can be observed microscopically Genetic homogeneity Environment (pH, Temp., osmotic pressure, O 2 and CO 2 tension) Rapid Requires less amount of material

Primary cell culture and Establishment cell line Preparation of cell suspension from intact tissue Preparation of cell suspension from intact tissue 1. Single cell preparation : use mechanical, Chemical, and/or enzymatic method 2. Disaggregate or dissociate cell : cutting, homogenizing, rotary shaker, vortex, pipette, teasing

Preparation of cell suspension from intact tissue (cont.) Preparation of cell suspension from intact tissue (cont.) ** Enzymes ** Trypsin (crude) : from cattle and pig’s pancrease : contain Chymotrypsin, elastase, ribonuclease, deoxyribonuclease and amylase

Collagenase Collagenase : for connective tissue Pronase Pronase : for fibroblast Elastase Elastase : for fibroblast protein Deoxyribonuclease : for DNA ** Enzymes **

Preparation of cell suspension from intact tissue (cont.) Preparation of cell suspension from intact tissue (cont.) Source of tissue : Young animals e.g. kidney (Monkey, Dog, Rabbit), Chick embryo : Old animal tissue contains a large amount of connective tissue

Preparation of cell suspension from intact tissue (cont.) Preparation of cell suspension from intact tissue (cont.) 1. Removal of tissue and place in Isotonic (or growth medium with antibiotic) 2. Trim off unwanted parts and cut into smaller pieces3. Dissociate cells using enzyme 4. Remove large debris and wash cells

Preparation of cell suspension from intact tissue (cont.) Preparation of cell suspension from intact tissue (cont.) 5. Suspend cells in growth medium accordingly 6. Pipette into culture vessel and incubate

Primary cell culture Cell line subculture Subline or cell strain cloningPhysical separation

Establishment of Suspension culture Establishment of Suspension culture : Heteroploid cell line ; suspension culture 1. Resuspend trypsinized cells to 5 x 10 5 cells/ml 2. Continous stirring 3. Cellular product be removed and replenished with fresh medium

Contamination fungal contamination Bacterial contamination Mycoplasma contamination Viral contamination Other cell line

Sources of contamination Original tissue : primate virus, mycoplasma Biological: Serum Laboratory personnel : from body, aerosols Laboratory environment : culture vessel cap : humidified Incubator : Water bath : Insect

Other Parameters Incubator : humidified Incubation temperature pH and Buffer Gas phase Osmolarity and water grade

Cell Storage *** Prevent genetic drift *** : Freezing Medium * Serum (~ 20-90%) * Culture medium * Cryoprotective agent : ~ 5-10% (DMSO, Glycerol)

Cell Storage : Temp. decline rate 1-10 o C/min * (-20 o C ) Freezer * (-70 o C ) Freeze : 6M-2 yr * liquid nitrogen: Years : cell concentration ~ 5 x x 10 7 cells/ml : % cell viability is decrease 2-3%/yr

** Slow Freeze : Quick Thaw ** Cell Thawing and culture 1. Quick thaw in 37 o C water bath 2. Pipette to culture vessel 3. Slowly growth medium adding 4. Incubate for overnight 5. Refresh with new growth medium

Fibroblast-liked cell Epithelium-liked cell

Monolayer cell culture