Basics of Cell Culture (Tissue culture)

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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

Thanks