27. Tissue Culture

7/28/2019 27. Tissue Culture

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Tissue Culture

(Kultur Jaringan)

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

SPMK


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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 andpreservation of the architecture and / or

function

It is not widely used due to difficulties inhandling the culture


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Cell Culture :

A method to preserve the growing of cellsin 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


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History

J. F. ENDERS

T.H. WELLER

F.C. ROBINS

The Team succeeded in cultivating the

lansing strain of polymyelitis virus in

various human embryonic tissues


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Purpose :

A. Cultivation of Micro Organisms

B. Cytogenetic analyses.

C. Cellular / Genetic therapy- Pluripotent

cells preservation

Cultivation of BACTERIA :

- Treponema pallidum in Rabbit Testis- Chlamydia in Mouse tissue Culture Cells


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Cultivation of VIRUS :

- Adenovirus :

* He La Cells

* KB Cells

* Human Embryo Kidney Cells

- Herpes Simplex Virus:

* Human Fetal Fibroblasts Cells

* Human Kidney Cells* Rabbit Kidney Cells


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- Vericella Zoster Virus :

* Human Fetal Fibroblast Cells

* Human Thyroid Cells

- Cytomegalovirus (CMV) :* Human Fetal Fibroblast Cells

- Eppstein Barr Virus :* Human Umbilical Cord Leucocytes


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- 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


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- 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


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- Influenza Virus :

* Monkey Kidney Cells

* Human Kidney Cells

- Measles Virus :* Primary Human Fetal Kidney Cells

* Human Amnion Cells

* Monkey Kidney Cells


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- Rabies Virus :

* Human Diploid Fibroblasts Strain WI 38

- Corona Virus :

* Human Embryonic Fibroblast StrainHEL

HEK

WI 38Bradburnes Strain


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- Rubella Virus

* BHK (Baby Hamster kidney Cells)

* Vero Cells (Green Monkey Kidney Cells)

* RK 13 (Rabbit Kidney Cells)

* SIRC


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Cell Lines / Culture of Human Origin

A Variety of Human Cells of Both :

- Malignant

- non Malignant OriginHave been carried in continuous passage of

cell culture


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- 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


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- RD Cell Lines

By Mc Allister et. al in 1969

Human Rhabdomyosarcoma

- Human Diploid Fibroblast Cells

- Human Cord Blood Lymphocyte /Leucocytes Cells


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The Goal of Cultivation in Various Cell

Lines :

A Diagnostic :

- Cellular Degeneration

Cytopathic Effect (CPE)

- Plaque Formation

- Metabolic inhibition

- Hemadsorption


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- Mixed hemadsorption

- Immunofluorescent or

immunoperoxedase staining

- Interference

- Antigen Extraction- Vaccine Development


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Three Different Types of Cell Culture

1. Explant CultureA fragment of tissue or organ is used to

initiate a culture

The tissue is embedded into a plasmaclot to aid in attachment to the culture

Vessel


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2. Monolayer Culture

A Single layer of cells growing on asurface of a glass of plastic culture vessels

Using : - Proteolytic Enzyme

- Chelating Agent


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3. Suspension Culture

Cells multiply while suspended in fluid

medium

- Agitated Method

- Logarithmic Growth Phase

- Highly Reproducible

- Methabolic State

- Gain very large population of cellsHigh Titer Virus Antigen Production

Vaccine Production


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B. For Cytogenetic / Chromosomal Analyses

in Prenasal CaresFetal cells are used to diagnose

constitutional chromosome complement ;

1. Amniotic Fluid Cells (Amniocytes)2. Chorionic Villi (CV)

3. Fetal Blood Lymphocytes


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Other Fetal Tissues :

* Placenta

* Skin

* lung

* Kidney

Are also used for cytogenetic evaluation incase of :

- Therapeutic Termination

- Multiple Miscarriage

- Fetal Demise


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In Neonatal & Adult

The most common tissue used for

constitutional chromosome diagnosis

is peripheral blood

Less Frequently :

Bone Marrow

Skin

And other appropriate tissue


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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


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C .Cellular / Genetic Therapy

Pluripotent cells from Bone Marrow / Stem

Cells could be induced to produce theneeded Cells / Genetic material using for

therapeutic purposes


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All Tissue used for chromosome preparationsand growth in culture

1. Suspension Culture

(Free Floating Cells)

- Lymphocyte & Lymphoblast

- Blood

- Bone Marrow2. Monolayer Culture

- Fibroblast

- Epithelial Like Cells

- From : * Skins* Amniotic Fluid

* Most Other Tissues


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Basic Requirement to Grow Cells in Vitro

1. Culture media & Media Ingredient

2. Culture Wave

3. Proper work environment

4. Tissue to be cultured


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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 Glucoseessential Amino Acids Vitamins


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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


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Supplemet :

- 5 or 20% Bovine SerumFetal Bovine

Calf Serum

- L Glutamine- Essential Amino Acid

- Antibiotics at bacteriostatic level :

Penicillin 100 u

Streptomycin 100 u gr/ml


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Culture ware

FlasksTubes

Pipettes

Mostly Dispossible and Made of Plasticsterilized by radiation


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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


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Specimens / Tissue

Eg :

Blood : - Most Frequently Used in

Cytogenetic Diagnosis- Easy to Obtain

- Tissue of choice for the diagnosisof constitutional kartotype

- Heparinized bloodFetal Blood :

- Prenasal Cytogenetic

- Percutaneus Umbilical Blood Sampling

(Pures)- (Cordoleentesis, Funiculo centesis) USG

Gestation :

- 17 Week Gestation


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Blood Sample

Media & Ingredient

+ Stimulating Factor (Mitogen)PHA

Pokeweed Mitogen

CON A

EBVCulture

Harvest : 45 H 96 H

Chromosomal Studies

Established Relation Between Malignancies andChromosomal Change


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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


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AMNIOTIC FLUID

(Amniocytes Cells)

Appropriate Culture

(Monolayer)

Growth Medium CO2 Incubation

Leave 4 5 Days Change the Medium 7th day Harvest onward

Chromosomal Studies

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27. Tissue Culture

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