Post on 03-Apr-2018
transcript
7/28/2019 27. Tissue Culture
1/36
Tissue Culture
(Kultur Jaringan)
Dr. H. A. Aziz Djamal M.Sc, DTM & H,
SPMK
7/28/2019 27. Tissue Culture
2/36
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
7/28/2019 27. Tissue Culture
3/36
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
7/28/2019 27. Tissue Culture
4/36
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
7/28/2019 27. Tissue Culture
5/36
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
7/28/2019 27. Tissue Culture
6/36
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
7/28/2019 27. Tissue Culture
7/36
- Vericella Zoster Virus :
* Human Fetal Fibroblast Cells
* Human Thyroid Cells
- Cytomegalovirus (CMV) :* Human Fetal Fibroblast Cells
- Eppstein Barr Virus :* Human Umbilical Cord Leucocytes
7/28/2019 27. Tissue Culture
8/36
- 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
7/28/2019 27. Tissue Culture
9/36
- 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
7/28/2019 27. Tissue Culture
10/36
- Influenza Virus :
* Monkey Kidney Cells
* Human Kidney Cells
- Measles Virus :* Primary Human Fetal Kidney Cells
* Human Amnion Cells
* Monkey Kidney Cells
7/28/2019 27. Tissue Culture
11/36
- Rabies Virus :
* Human Diploid Fibroblasts Strain WI 38
- Corona Virus :
* Human Embryonic Fibroblast StrainHEL
HEK
WI 38Bradburnes Strain
7/28/2019 27. Tissue Culture
12/36
- Rubella Virus
* BHK (Baby Hamster kidney Cells)
* Vero Cells (Green Monkey Kidney Cells)
* RK 13 (Rabbit Kidney Cells)
* SIRC
7/28/2019 27. Tissue Culture
13/36
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
7/28/2019 27. Tissue Culture
14/36
- 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
7/28/2019 27. Tissue Culture
15/36
- RD Cell Lines
By Mc Allister et. al in 1969
Human Rhabdomyosarcoma
- Human Diploid Fibroblast Cells
- Human Cord Blood Lymphocyte /Leucocytes Cells
7/28/2019 27. Tissue Culture
16/36
The Goal of Cultivation in Various Cell
Lines :
A Diagnostic :
- Cellular Degeneration
Cytopathic Effect (CPE)
- Plaque Formation
- Metabolic inhibition
- Hemadsorption
7/28/2019 27. Tissue Culture
17/36
- Mixed hemadsorption
- Immunofluorescent or
immunoperoxedase staining
- Interference
- Antigen Extraction- Vaccine Development
7/28/2019 27. Tissue Culture
18/36
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
7/28/2019 27. Tissue Culture
19/36
2. Monolayer Culture
A Single layer of cells growing on asurface of a glass of plastic culture vessels
Using : - Proteolytic Enzyme
- Chelating Agent
7/28/2019 27. Tissue Culture
20/36
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
7/28/2019 27. Tissue Culture
21/36
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
7/28/2019 27. Tissue Culture
22/36
Other Fetal Tissues :
* Placenta
* Skin
* lung
* Kidney
Are also used for cytogenetic evaluation incase of :
- Therapeutic Termination
- Multiple Miscarriage
- Fetal Demise
7/28/2019 27. Tissue Culture
23/36
In Neonatal & Adult
The most common tissue used for
constitutional chromosome diagnosis
is peripheral blood
Less Frequently :
Bone Marrow
Skin
And other appropriate tissue
7/28/2019 27. Tissue Culture
24/36
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
7/28/2019 27. Tissue Culture
25/36
C .Cellular / Genetic Therapy
Pluripotent cells from Bone Marrow / Stem
Cells could be induced to produce theneeded Cells / Genetic material using for
therapeutic purposes
7/28/2019 27. Tissue Culture
26/36
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
7/28/2019 27. Tissue Culture
27/36
Basic Requirement to Grow Cells in Vitro
1. Culture media & Media Ingredient
2. Culture Wave
3. Proper work environment
4. Tissue to be cultured
7/28/2019 27. Tissue Culture
28/36
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
7/28/2019 27. Tissue Culture
29/36
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
7/28/2019 27. Tissue Culture
30/36
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
7/28/2019 27. Tissue Culture
31/36
Culture ware
FlasksTubes
Pipettes
Mostly Dispossible and Made of Plasticsterilized by radiation
7/28/2019 27. Tissue Culture
32/36
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
7/28/2019 27. Tissue Culture
33/36
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
7/28/2019 27. Tissue Culture
34/36
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
7/28/2019 27. Tissue Culture
35/36
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
7/28/2019 27. Tissue Culture
36/36
AMNIOTIC FLUID
(Amniocytes Cells)
Appropriate Culture
(Monolayer)
Growth Medium CO2 Incubation
Leave 4 5 Days Change the Medium 7th day Harvest onward
Chromosomal Studies