Biological safety cabinet. Incubation facilities 37 °C for mammalian cells,. CO. 2. 5% & 95% air at 99% relative humidity. Refrigerators- Liquid media kept at 4 °C,.
Basics of Cell Culture (Tissue culture) By Dr. Aisha M. A. Shahlol PhD.in Medical Microbiology Sabha university
Basics of Cell Culture (Tissue culture)
By Dr. Aisha M. A. Shahlol PhD.in Medical Microbiology Sabha university
Introduction زراعة الخلية هي العملية التي تزرع فيها الخاليا بدائية النواة والخاليا حقيقية النواة أو الخاليا النباتية تحت ظروف محكمة . ولكن في واقع االمر معمليا يشير المصطلح إلى زراعة الخاليا المشتقة من الخاليا الحيوانية
Cell culture was first successfully undertaken by Ross Harrison in 1907
Tissue culture
Types of cells
Primary culture
الخاليا األولية لها عمر محدد
Common cell lines
Culture media
Basic equipments used in cell culture Biological safety cabinet Incubation facilities 37 °C for mammalian cells, CO2 5% & 95% air at 99% relative humidity. Refrigerators- Liquid media kept at 4 °C, enzymes (e.g. trypsin) & serum at -20 °C Microscope- An inverted microscope with 10x to 100x magnification Ordinary microscope Cooling centrifuge Tissue culture ware- Culture plastic ware treated by polystyrene
Why sub culturing.? Once the available substrate surface is covered by cells (a confluent culture). Cells to be kept in healthy & in growing state have to be sub-cultured or passaged It’s the passage of cells when they reach 80 to 90% confluency in flask/dishes/plates Enzyme such as trypsin and collagenase in combination with EDTA breaks the cellular links that attached the cells to the surface
Culturing of cells
Culturing of cells Prepare the cell by enzyme
Deep freezing
Add to 75 cm2 or 175 cm2 flask Incubate the plate at 37 °C for 1-2 min Wash by PBS
Add DMSO (90% culture+10% DMSO)
Add complete medium
Again step No.1 and No.3
Add serum Incubate at 37 °C for 2 day
Tissue culture applications Model systems for
Studying basic cell biology, interactions between disease causing agents and cells, effects of drugs on cells, & nutritional studies
Toxicity testing
Study the effects of new drugs
Cancer research
Study the function of various chemicals, virus & radiation to convert normal cultured cells to cancerous cells
Tissue culture applications
% K.pneumoniae
Tissue culture applications
18
18 16 14 12 10 8 6 4 2 0
16
Ksa KD&Ksp
9 5 2 0
0
0
75%
Low
Moderate
High
Very high
Cytotoxicity levels
FIG.(4) Cytotoxicity levels of K.pneumoniae isolated in this study to vero cells