An insight into the tissue culture technique and its implication Dr.Samina Hyder Haq

History of tissue culture 1885: Roux maintained embryonic chick cells in saline 1885: Roux maintained embryonic chick cells in saline 1965:Ham introduced serum free culture medium that support living cells 1965:Ham introduced serum free culture medium that support living cells 1965: Harris & Watkins successfully fused human and mouse cells by virus 1965: Harris & Watkins successfully fused human and mouse cells by virus 1975: Kohler & Milstein produced the first Hybridomas capable of secreting monoclonal antibodies. 1975: Kohler & Milstein produced the first Hybridomas capable of secreting monoclonal antibodies. 1978: Sato developed a serum free medium with a cocktail of hormone and growth factors 1978: Sato developed a serum free medium with a cocktail of hormone and growth factors !982: human insulin is produced !982: human insulin is produced 1985: human growth factors were produced 1985: human growth factors were produced 1986: lymphoblastoid gamaIFNlicences 1986: lymphoblastoid gamaIFNlicences 1987:tissue type plasminogen activator(tPA) became commercially available 1987:tissue type plasminogen activator(tPA) became commercially available 1989:Recombinant erythropoitin in trial 1989:Recombinant erythropoitin in trial 1990:Recombinent products in trial (factorVI.HBsAg, Il2, EGF, mAbs) 1990:Recombinent products in trial (factorVI.HBsAg, Il2, EGF, mAbs)

Plant Tissue culture

Micropropagation

What is tissue culture Organ culture Explant Culture Cell culture

Technique and instrument

Carbon dioxide incubator

Microscope

Tissue culture Ware

Culture Media Sterilization

Cell counting

Changing Medium

Other miscellaneous Equipment Fridge Freezer for storing medium Fridge Freezer for storing medium Liquid nitrogen Container to freeze certain cell lines Liquid nitrogen Container to freeze certain cell lines Incubator for warming up of the medium Incubator for warming up of the medium Bench centrifuges to separate out cell pellet. Bench centrifuges to separate out cell pellet.

Types of cell culture 1: Primary Cell culture 1: Primary Cell culture 2: continuous Cell lines 2: continuous Cell lines

Secondary or continuous Cell Lines Attached Cell Lines NameSpecies and tissue of originMorphology MRC-5 (Prod. No )Human lungFibroblast HELA (Prod. No )Human cervixEpithelial VERO (Prod. No )African Green Monkey KidneyEpithelial NIH 3T3 (Prod. No )Mouse embryoFibroblast L929 (Prod. No )Mouse connective tissueFibroblast CHO (Prod. No )Chinese Hamster OvaryFibroblast BHK-21 (Prod. No )Syrian Hamster KidneyFibroblast HEK 293 (Prod. No )Human KidneyEpithelial HEPG2 (Prod. No )Human LiverEpithelial BAE-1 (Prod. No )Bovine aortaEndothelial

SUSPENSION Cell lines Suspension Cell Lines NameSpecies and tissue of originMorphology NSO (Prod. No )Mouse myelomaLymphoblastoid-like U937 (Prod. No )Human Hystiocytic LymphomaLymphoblastoid Namalwa (Prod. No )Human LymphomaLymphoblastoid HL60 (Prod. No )Human LeukaemiaLymphoblastoid-like WEHI 231 (Prod. No )Mouse B-cell LymphomaLymphoblastoid YAC 1 (Prod. No )Mouse LymphomaLymphoblastoid U 266B1 (Prod. No )Human MyelomaLymphoblastoid SH-SY5Y (Prod. No )Human neuroblastomaNeuroblast

Storing and maintaining Continuous Cell Lines

Advantages of Cell Culture Cell types kept Constant and homogenous. Cell types kept Constant and homogenous. There is direct access to the cells so effect of toxicity of drug and chemicals studied without being lost to other tissues and excreted. There is direct access to the cells so effect of toxicity of drug and chemicals studied without being lost to other tissues and excreted. Replace/ reduce the number of animals.Legal, moral, ethycal isses?(animal rights Group). Replace/ reduce the number of animals.Legal, moral, ethycal isses?(animal rights Group). Control of the environment (pH, osmotic pressure, temperature,oxygen and Carbondioxide tension.) Control of the environment (pH, osmotic pressure, temperature,oxygen and Carbondioxide tension.)

Disadvantages of Cell Culture  Contamination can be Chemical (culture medium) Or Biological (adding antibiotics).  Finding a Happy Environment.  De-differentiation  Origin of Cells  Major differences from in-vivo(loss of ECM)

Implication for Cell Culture 1. Model system (many specialized functions are restored) 1. Model system (many specialized functions are restored) 2.Toxicity Test (viability of cells) 2.Toxicity Test (viability of cells) 3.Cancer research 3.Cancer research Virology Virology Cell Based manufacturing Cell Based manufacturing Genetic councelling Genetic councelling Genetic enginering Genetic enginering Drug screening & development Drug screening & development Gene Therapy Gene Therapy

Production of monoclonal antibodies

Tissue Engineering Carbon nanotube scaffolding Carbon nanotube scaffolding Name of scaffoldig Made up of Nanofiber Like carbon TextilePolyglycolide Gas Foam Foam like structure due to CO2 gas

Growing cells in 3D Forming Tissue in Bioreactor

Application of Monoclonnal Antibodies IMMUNOLOCALIZATION IMMUNOLOCALIZATION IMMUNOBlotting IMMUNOBlotting Cancer Treatment Cancer Treatment

Immunolocalization

Immunoblotting

Cancer Treatment

Stem cell research Embryonic Cell Lines Embryonic Cell Lines Adult Cell Line Adult Cell Line Homeiopoitic Stem Cell Homeiopoitic Stem Cell

Embryonic Cell lines

Placental stem cell Placental stem cells do not possess the antigenic properties, making rejection of the stem cells impossible. It has successfully used in treating various neurological disorders like Parkinson, paralysis and stroke patients.

Haematopoitic stem Cell

Adult Mesenchymal Cell lines

Organ transplant