Tissue Culture (Kultur Jaringan)

Dr. H. A. Aziz Djamal M.Sc, DTM & H, SPMK

Definition : Tissue or Organ Culture : A method to maintain or to preserve the growth tissues, organ primordia, or whole or parts of an organ in vitro in a way which allows differentation and preservation of the architecture and / or function It is not widely used due to difficulties in handling the culture

 Cell Culture : A method to preserve the growing of cells in vitro underconditions where the cells are no longer organized into tissues. It is the type of culture generally employed in several types of diagnostic procedure.
Cell Culture Tissue Culture

 History J. F. ENDERS T.H. WELLER F.C. ROBINS The Team succeeded in cultivating the lansing strain of polymyclitis virus in various human embryonic tissues

 Purpose : A. Cultivation of Micro Organisms BACTERIA : - Treponema Pallidum in Rabbit Testis - Chlomydia in Mouse tissue Culture Cells

VIRUS : - Adenovirus : * Hela Cells * KB Cells * Human Embryo Kidney Cells - Herpes Simplex Virus: * Human Fetal Fibioblasts Cells * Human Kidney Cells * Rabbit Kidney Cells

- Vericella Zoster Virus : * Human Fetal Fibroblast Cells * Human Thyroid Cells
- Cytomegalovirus (CMV) : * Human Fetal Fibroblast Cells - Eppstein Barr Virus : * Human Umbilical Cord Leucocytes

- Entero Virus : * Monkey Kidney Cells * Human Embryonic Kidney Cells * Diploid Strain of Human Fibroblast Cells - Rhino Virus : * Human Embryonic Kidney Cells (HEK) * Diploid Strain of Human Embryonic Lung ( HEL) Cells

- Arbovirus : * Green Monkey Kidney Cells * Hamster Kidney Cells * Chick Embryo Fibroblast Cells Most Convenient : Continuous Cells Lines Derived from monkey kidney * Vero Cells * LLC MK2 cells

- Influenza Virus : * Monkey Kidney Cells * Human Kidney Cells

- Measles Virus : * Primary Human Fetal Kidney Cells * Human Amnion Cells * Monkey Kidney Cells

- Rabies Virus : * Human Diploid Fibroblasts Strain WI 38 - Corona Virus : * Human Embryonic Fibroblast Strain HEL HEK WI 38 Bradburnes Strain

- Rubella Virus * BHK (Baby Hamster kidney Cells) * Vero Cells (Green Monkey Kidney Cells) * RK 13 (Rabbit Kidney Cells) * SIRC

 Cell Lines / Culture of Human Origin

A Variety of Human Cells of Both : - Malignant - non Malignant Origin Have been carried in continuous passage of cell culture

- Hela Cell Lines Established in 1951 Gey et. al Cervical Carcinoma - KB Line by Eagle et. al in 1956 Human carcinoma cells (nasopharynx) - HEp 2 Human Epithelial Line by Toolan in 1954 (Neoplasma) - Epithel Cell of Human Amniotic Membrane (FL Strain) by Fogh and Lund

- RD Cell Lines By Mc Allister et. al in 1969 Human Rhabdomynosarcoma

- Human Diploid Fibroblast Cells

- Human Cord Blood Lymphocyte / Leucocytes Cells

The Goal of Cultivation in Various Cell Lines : 1. Diagnostic : - Cellular Degeneration Cytopathic Effect (CPE) - Plaque Formation - Metabolic inhibition - Hemadsorption

- Mixed hemadsorption - Immunofluorescent or immunoperoxedase staining - Interference - Antigen Extraction - Vaccine Development

 Three Different Types of Cell Culture 1. Explant Culture A fragment of tissue or organ is used to initiate a culture The tissue is embedded into a plasma clot to aid in attachment to the culture Vessel

2. Monolayer Culture A Single layer of cells growing on a sirface of a glass of plastic culture vessels Using : - Proteolytic Enzyme - Chelating Agent

3. Suspension Culture Cells Multiply White suspended in fluid medium - Agitated Method - Logarithmic Growth Phase - Highly Reproducible - Methabolic State - Gain very large population of cells High Titer Virus Antigen Production Vaccine Production

B. For Cytogenetic / Chromosomal Analyses in Prenasal Cares Fetal cells are used to diagnose constitutional chromosome complement ; 1. Amniotic Fluid Cells (Amniocytes) 2. Chorionic Villi (CV) 3. Fetal Blood Lymphocytes

 Other Fetal Tissues : * Placenta * Skin * lung * Kidney Are also used for cytogenetic evaluation in case of : - Therapeutic Termination - Multiple Miscarriage - Fetal Demise

 In Neonatal & Adult The most common tissue used for constitutional chromosome diagnosis is peripheral blood Less Frequently : Bone Marrow Skin And other appropriate tissue

 For Acquired Chromosome Abnormalities - Frequently Associated with Neoplasma * The involved tissue in malignancy process is used for cytogenetic evaluation * Bone marrow * Pleural Fluid * Spinal Fluid * Lymphnode * Other Solid Tumor

 All Tissue used for chromosome preparations and growth in culture 1. Suspension Culture (Free Floating Cells) - Lymphocyte & Lymphoblast - Blood - Bone Marrow 2. Monolagen Culture - Fibroblast - Epithelial Like Cells - From : * Skins * Amniotic Fluid * Most Other Tissues

 1. 2. 3. 4.

Basic Requirement to Grow Cells in Vitro Culture media & Media Ingredient Culture Wave Proper work environment Tissue to be cultured

 Culture Media It is range from Simple Buffer salt solution to very complex media : PBS Hanks Solution Isotonic Condition Earles Base Content : Basal Salt Solution Glucose essential Amino Acids Vitamins

 Some of the most community used cultural media in cytogenetic laboratories : - MEM - RPMI 1640 - Mc Coy 5 A - TC 199 - Ham F 10 Growth Factors & Hormons

 Supplemet : - 5 or 20% Bovine Serum Fetal Bovine Calf Serum - L Glutamine - Essential Amino Acid - Antibiotics at bacteriostatic level : Penicillin 100 u Streptomycin 100 u gr/ml

 Culture ware Flasks Tubes Pipettes Mostly Dispossible and Made of Plastic sterilized by radiation

 Environment 1. Working Area : * Separate Sterile & Dust Free Space lominar Flow Hoods : - UV light - Filtered Air Circulation - Alcohol / Stable Antiseptic - Touch o matic type gas bunner 2. Human Element : - Least / Minimal - Gloves

 Specimens / Tissue Eg : Blood : - Most Frequently Used in Cytogenetic Diagnosis - Easy to Obtain - Tissue of choice for the diagnosis of constitutional kartotype - Heparinized blood Fetal Blood : - Prenasal Cytogenetic - Percutaneus Umbilical Blood Sampling (Pures) - (Cordoleentesis, Funiculo centesis) USG Gestation : - 17 Week Gestation

Blood Sample Media & Ingredient + Stimulating Factor (Mitogen) PHA Pokeweed Mitogen CON A EBV Culture Harvest : 45 H 96 H Chromosomal Studies Established Relation Between Malignancies and Chromosomal Change

AMNIOTIC FLUID * The most common source of fetal cells for prenatal diagnosis * The fluid drown from amniotic sac 20 22 g Dispossible Needle & USG (Amniocentesis) * 2 nd Trisemester, 15 16 Week, 20 30 ml * Early Amniocentesis ( 12 15 Week ) 1 ml fluid / week gestation

AMNIOTIC FLUID (Amniocytes Cells) Appropriate Culture (Monolayer) Growth Medium CO2 Incubation Leave 4 5 Days Change the Medium 7th day Harvest onward Chromosomal Studies