Tissue Culture 01 at Laser Institute for Research and Applications, Beni-Suef University

LASER APPLICATIONS IN BIOLOGY &
MEDICINE LABORATORY.
LAB 01, 31- 3 -2019
By: Esraa Ahmed Mohamed Hassan
Teaching Assistant at
Laser Institute for Research and Applications (LIRA).
Biological Laser Applications Department.
Beni-Suef University.

Lab. Supervisors:
1- Prof. Dr. Tarek Ali, Professor of Laser Physics.
Laser Institute for Research and Applications, Beni-Suef
University.
2- Dr. Ahmed Osama El-Gendy, Associated professor,
Microbiology & Immunology Department. Faculty of
Pharmacy, Beni-Suef University.

Esraa Ahmed, Biological Laser Applications Department.
2 Courses: (Biological & Medical)
Course name: "Laser Laboratory in Biological Applications and
Infection control" (BL503).
Credit Hours: 2 (100 degree).
*************************
Course name: "Laser Laboratory in Medical Applications 1"
(ML505).
Credit Hours: 3 (150 degree).

Grading: Biology Diploma
Attendance 10 Project
Handouts 10
Presentation 10
Quizzes 10x2 Final Exam
Practical 20
Test 30
Attendance 10 Project
Handouts 20
Presentation 10
Quizzes 20x2 Final Exam
Practical 30
Test 40
Grading: Medical Diploma

Topics:
1. Basic tissue culture techniques, Equipment, aseptic techniques,
culturing, sub-culturing, trypsinization, and scaling up.
2. Quantification, Growth curve, Trypan Blue staining, confluence, cell
viability and cyto-toxicity.
3. Maintenance and cryo-preservation of cells, Freezing and thawing of
cells.
4. Contamination, Infection control & Safety measurements.
5. Biological Aspects: Advanced Tissue culture Topics.
6. Medical aspects: Different fiber optics, different delivery systems,
endoscopy and surgical microscopy and colposcopy, Laser cutting of
tissue (including continuous wave, pulsed and plasma-mediated
ablation).

Practical:
1.Tissue Culturing.
2.Sub-culturing and Scaling up.
3.Quantifications.
4.Examination with ELIZA & IFM.

Reports:
1. Media formulation.
2. Cell separation, Characterization and Transformation.
3. Contamination, Mycoplasma detection and Infection control.
*************************
1. Medical laser equipment (each on your specialization).
2. Setting and adjusting the parameters of laser for different
medical applications.
3. Laser tissue coagulation, welding and soldering.
Biology
Medical

What is Cell Culture
1

What is Cell Culture?
Cell culture refers to: “The removal of cells from an animal or plant
and their subsequent growth in a favorable artificial environment”.
Applications

Source of Cells:
Cell culture refers to: “The removal of cells from an animal or plant
and their subsequent growth in a favorable artificial environment”.
*************************
The cells may be removed from:
➢ The tissue directly and disaggregated by enzymatic or mechanical
means before cultivation. Or,
➢ Derived from a cell line or cell strain that has already been
established.

2 Categories of Cell Culture:
Primary
Cells
Established
Cell Lines
➢ Obtained directly from Donor
Tissue.
➢ Only able to divide a finite number
of times due to telomere loss.
➢ Cells eventually reach senescence.
➢ Grow indefinitely.
➢ Useful for long-term
research.

Main Growth Conditions
Monolayer
(Adherent Culture)
Free Floating
(Suspension Culture)
Knowing the growth prosperities help use the appropriate
Cultureware
Flasks
Plates
Roller Bottles

Culture Environment:
➢Physiological E.
➢ Hormones and nutrient concentrations.
➢Physico-chemical E.
➢ Temperature.
➢ pH.
➢ Osmotic pressure.
➢ O2 and CO2 tension.

Culture environment:
Factors affecting the culture environment:
1- Culture Media & Sera:
The most important component of the culture environment, because it
provides the necessary nutrients, growth factors, and hormones for cell
growth, as well as regulating the pH and the osmotic pressure of the
culture.
The 3 classes of media are:
Basal media, reduced-serum media, and serum-free media.
which differ in their requirement for supplementation with serum.

Serum:
1. A source of: Growth and adhesion factors, Hormones, Lipids, Minerals.
2. Regulates cell membrane permeability.
3. Serves as a carrier for lipids, enzymes, micronutrients, and trace
elements into the cell.
Disadvantages:
➢ High cost.
➢ Problems with standardization, specificity, variability.
➢ Unwanted effects such as: stimulation or inhibition of growth and/or cellular
function on certain cell cultures.
If the serum is not obtained from reputable source, contamination can
also pose a serious threat to successful cell culture experiments.

Types of media:
1- Basal Media
The majority of cell lines grow well in basal media, which contain amino acids, vitamins, inorganic
salts, and a carbon source such as glucose, but these basal media formulations must be further
supplemented with serum.
2- Reduced-Serum Media
Another strategy to reduce the undesired effects of serum in cell culture experiments is to use
reduced-serum media.
Reduced-serum media are basal media formulations enriched with nutrients and animal-derived
factors, which reduce the amount of serum that is needed.
3- Serum-Free Media
Replacing the serum with appropriate nutritional and hormonal formulations.
One of the major advantages of using serum-free media is the ability to make
the medium selective for specific cell types by choosing the appropriate
combination of growth factors.

The MIMIC
To keep the cells alive we need to keep the conditions as close to
the physiological conditions as possible
Culture Media
Amino Acids
Inorganic Salts
Vitamins
Macromolecules
Lipids
Growth Factors
Fetal Bovine
Serum
Buffering system
& pH indicator

2- Temperature:
The optimal temperature for cell culture depends on:
✓ The body temperature of the host from which the cells were isolated.
✓ The anatomical variation in temperature (e.g., temperature of the skin may be
lower than the temperature of skeletal muscle).
Optimal Temperatures for Various Cell Lines
Most human and mammalian cell lines are maintained at 36°C to 37°C.
Insect cells are cultured at 27°C for optimal growth.
Avian cell lines require 38.5°C for maximum growth.
Cell lines derived from cold-blooded animals range between 15°C and 26°C.

4- co2 levels
➢ Achieved by: including an organic (e.g., HEPES) or CO2-bicarbonate based buffer.
➢ the pH of the medium is dependent on the delicate balance of dissolved carbon
dioxide (CO2) and bicarbonate (HCO3–), changes in the atmospheric CO2 can alter
the pH of the medium.
➢ most researchers usually use 5 – 7% CO2 in air.
3- pH levels
Most normal mammalian cell lines grow well at pH 7.4.
• Some transformed cell lines have been shown to grow better at slightly more acidic
environments (pH 7.0 – 7.4).
• Some normal fibroblast cell lines prefer slightly more basic environments (pH 7.4 –
7.7).
• Insect cell lines such as Sf9 and Sf21 grow optimally at pH 6.2.

The Growth continues in;
Maintain:
Temperature.
Humidity.
CO2 level: 5-10%
CO2 Incubator

Contamination:
Get to know your Enemy

1- Bacterial Contamination:
Macroscopic detection
Culture media contaminated with bacteria (left) appear
turbid and yellow whereas non-contaminated media
(right) appear clear and red.
➢ Increased turbidity of culture
medium, medium appears
cloudy.
➢ If medium contains phenol red
as pH indicator, a rapid color
change to yellow indicates a
sudden decrease in pH.

1- Bacterial Contamination:
Microscopic detection
Bacteria are much smaller than eukaryotic cells. They appear as dark rod-like
structures, spheres or spiral structures under the microscope, and they may exist as
single cells, in pairs, chains, or clusters.
Common shapes of bacteria: rod (bacillus), spherical
(coccus), and spiral (spirilla).Bacterial contamination

Mycoplasma-positive cell cultures show no visible changes to the media.
Mycoplasma are only about 0.1 - 0.3
µm in diameter, therefore detection via
brightfield microscopy is not possible.
2- Mycoplasma Contamination:
DAPI staining of Mycoplasma-infected cells

Scanning electron micrograph of mycoplasma attached
to the membranes of human fibroblasts. (David M
Phillips/ Science Source/ Getty Images)

3- Fungal Contamination:
Fungal colonies floating on the medium surface.
Fungi and mold can appear as small isolated
colonies of grey, white or greenish color,
floating at the surface of the medium.
Sometimes fungal contaminations will cause
a pH increase of the medium, resulting in
phenol-red containing media to appear pink.

3- Fungal Contamination:
Fungal contamination
It is usually the fungal spores which get
into a culture vessel and then begin to
sprout and form the typical fiber like
hyphae.
Fungi growing on the bottom of the
dish/flask are more easily detected in the
microscope than those floating on the
surface.

4- Yeast Contamination:
➢ Increase in turbidity of the medium, medium becomes cloudy.
➢ Initial stages of yeast contamination are difficult to detect macroscopically, as the
pH changes only slightly, initiating little or no color change in medium containing
phenol red as pH indicator.
Yeast contamination
They can exist as single cells or
in in the form of chains or
branches.

5- Eukaryotic Contamination:
Sometimes the shape or
dimension of the cell could
provide a hint.
• one cell line is contaminated with another cell
• line. There is no visible sign of a mixed culture.
• Pay close attention if the growth rate of your cell line begins to differ from
previous measurements/observations.
Eukaryotic contamination

The Culturing
Should be in Sterile Environment
to avoid:
Microbial Contamination
Bacteria
Fungi
Mycoplasma
Laminar
Air
Flow

Laminar Air Flow

Tissue Culture 01 at Laser Institute for Research and Applications, Beni-Suef University

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