INSTITUTE: UIPS

DEPARTMENT: PHARMACY
M. Pharmacy-Ist Sem (Pharmacology)
Subject Name and Code: Cellular and Molecular
Pharmacology– 22PHT-624

DISCOVER . LEARN . EMPOWER

Lovedeep Singh, Assistant Professor, UIPS, CU
Course Outcome
CO Number Title Level

CO1 Describe the fundamental knowledge on the structure Remember

and functions of cellular components.
CO2 Describe the molecular pathways affected by drugs. Understand

CO3 Explain the science of genomics Understand

CO4 Illustrate the applicability of molecular pharmacology Understand

and biomarkers in drug discovery process.
CO5 Analyse molecular biology techniques as applicable for Understand
pharmacology.

CO6 Appraise the cell viability assay Understand

Equipment's used in
cell culture lab

Lovedeep Singh, Assistant Professor, UIPS, CU

Basic equipment's used
1. Laminar Flow Hood or Cell Culture Hood Or Biosafety Cabinet:
• It is required for carrying aseptic culturing of cells.
• HEPA filters are provided in the laminar flow to prevent contamination.
2. CO2 Incubator:
• The purpose of incubators is to provide the appropriate environment for cell growth.
3. Hemocytometer and Coulter counter
4. Liquid nitrogen or Cryostorage container.
5. Others such as centrifuge, refrigerators, sterilizers (autoclaves), water bath,
microscope.
6. Expanded Equipment's: Aspiration pump (peristaltic or vacuum), pH meter, Confocal
microscope, Flow cytometer.
Lovedeep Singh, Assistant Professor, UIPS, CU
7. Additional Supplies: Cell culture vessels (e.g., flasks, Petri dishes, roller bottles, multi-
well plates), Pipettes, Syringes, needles, Waste containers, Media, sera, and reagents.

Culture Hood
Three kinds of cell culture hoods, designated as Class I, II and III, have been developed to
meet varying research and clinical needs.
❖ Class I cell culture hoods offer significant levels of protection to laboratory personnel
and to the environment when used with good microbiological techniques, but they do not
provide cultures protection from contamination.
▪ They are similar in design and air flow characteristics to chemical fume hoods.
❖ Class II cell culture hoods are designed for work involving BSL-1, 2, and 3 materials,
and they also provide an aseptic environment necessary for cell culture experiments.
Lovedeep Singh, Assistant Professor, UIPS, CU
▪ A Class II biosafety cabinet should be used for handling potentially hazardous materials
(e.g., primate-derived cultures, virally infected cultures, radioisotopes, carcinogenic or
toxic reagents).
❖ Class III biosafety cabinets are gas-tight, and they provide the highest attainable level of
protection to personnel and the environment.
▪ A Class III biosafety cabinet is required for work involving known human pathogens and
other BSL-4 materials.
Air-Flow Characteristics of Cell Culture Hoods
▪ Cell culture hoods protect the working environment from dust and other airborn
contaminants by maintaining a constant, unidirectional flow of HEPA-filtered air over
the work area.

Lovedeep Singh, Assistant Professor, UIPS, CU

▪ The flow can be horizontal, blowing parallel to the work surface, or it can be vertical,
blowing from the top of the cabinet onto the work surface.
▪ Depending on its design, a horizontal flow hood provides protection to the culture (if the
air flowing towards the user) or to the user (if the air is drawn in through the front of the
cabinet by negative air pressure inside).
▪ Vertical flow hoods, on the other hand, provide significant protection to the user and the
cell culture.
The basic layout of a cell culture hood is shown in next slide

Lovedeep Singh, Assistant Professor, UIPS, CU

The basic layout of a cell culture hood for right-handed workers Left-handed workers may
switch the positions of the items laid out on the work surface

Lovedeep Singh, Assistant Professor, UIPS, CU

Incubator
➢ The purpose of the incubator is to provide the appropriate environment for cell
growth.
➢ The incubator should be large enough for your laboratory needs, have forced air
circulation, and should have temperature control to within ±0.2°C.
➢ Stainless steel incubators allow easy cleaning and provide corrosion protection, especially
if humid air is required for incubation.
➢ Although the requirement for aseptic conditions in a cell culture incubator is not as
stringent as that in a cell culture hood, frequent cleaning of the incubator is essential to
avoid contamination of cell cultures.

Lovedeep Singh, Assistant Professor, UIPS, CU

Types of Incubators
There are two basic types of incubators
➢ Dry incubators are more economical, but require the cell cultures to be incubated
in sealed flasks to prevent evaporation.
▪ Placing a water dish in a dry incubator can provide some humidity, but they do not
allow precise control of atmospheric conditions in the incubator.
➢ Humid CO2 incubators are more expensive, but allow superior control of culture
conditions.
▪ They can be used to incubate cells cultured in Petri dishes or multi-well plates,
which require a controlled atmosphere of high humidity and increased CO2
tension.

Lovedeep Singh, Assistant Professor, UIPS, CU

Refrigerators
▪ For small cell culture laboratories, a domestic refrigerator (preferably one without a
autodefrost freezer) is an adequate and inexpensive piece of equipment for storing
reagents and media at 2–8°C.
▪ For larger laboratories, a cold room restricted to cell culture is more appropriate.
▪ Make sure that the refrigerator or the cold room is cleaned regularly to avoid
contamination.

Freezers
▪ Most cell culture reagents can be stored at –5°C to –20°C; therefore an ultradeep freezer
(i.e., a –80°C freezer) is optional for storing most reagents.
▪ A domestic freezer is a cheaper alternative to a laboratory freezer.

Lovedeep Singh, Assistant Professor, UIPS, CU

▪ While most reagents can withstand temperature oscillations in an autodefrost (i.e., self-
thawing) freezer, some reagents such as antibiotics and enzymes should be stored in a
freezer that does not autodefrost.
Cryogenic Storage
▪ A cryogenic liquid is defined as a liquid with a normal boiling point below -240°F (-
150°C).
▪ Cell lines in continuous culture are likely to suffer from genetic instability as their passage
number increases; therefore, it is essential to prepare working stocks of the cells and
preserve them in cryogenic storage
▪ Do not store cells in –20°C or –80°C freezers, because their viability quickly
decreases when they are stored at these temperatures.
▪ There are two main types of liquid-nitrogen storage systems, vapor phase and liquid
phase, which come as wide-necked or narrow-necked storage containers.
Lovedeep Singh, Assistant Professor, UIPS, CU
▪ Vapor phase systems minimize the risk of explosion with cryostorage tubes, and are
required for storing biohazardous materials.
▪ liquid phase systems usually have longer static holding times, and are therefore more
economical.
▪ Narrow-necked containers have a slower nitrogen evaporation rate and are more
economical, but wide-necked containers allow easier access and have a larger storage
capacity.

Cell Counter
A cell counter is essential for quantitative growth kinetics, and a great advantage when
more than two or three cell lines are cultured in the laboratory
I. Haemocytometer: A predetermined volume of the sample is used to count the number
of cells by examining it under the microscope.
Lovedeep Singh, Assistant Professor, UIPS, CU
▪ The sample is loaded onto the haemocytometer (a slide with grids on it).
▪ The maximum amount of sample volume that can be loaded is 0.1 μL.
▪ To differentiate between viable and non viable cells, dye such as tryptan blue (0.2%) is
used.
▪ The non viable cells are stained blue, while viable cells do not get stained.
▪ This is because viable cells are not permeable, but non-viable cells become permeable
and allow the entry of the dye inside the cell.
▪ On the other hand, using mixture of citric acid (0.1 mol/L) and crystal violet (0.1%), the
nuclei of cells can be stained purple and be counted.

Lovedeep Singh, Assistant Professor, UIPS, CU

Cell counting using hemocytometer, wherein viable cells (nonstained) and non-viable
cells (stained) are counted on a slide using a microscope.
Lovedeep Singh, Assistant Professor, UIPS, CU
II. Coulter Counter
▪ It is an electronic device, which works on the principle of
counting the cells in a pre-determined volume of sample by
allowing it to pass between two electrodes.
▪ By passage of the cells, the flow of current between the
electrodes is impeded, which results in voltage change and
a signal is recorded electronically.
▪ An advantage of method is that it takes very less time to
analyze the whole sample.
▪ However, it is disadvantageous in that it counts all the cells
present in the sample, whether viable or non-viable. Also, to
insure correct results, the sample must be free of any clumps of
cells, otherwise wrong results will be obtained.
Lovedeep Singh, Assistant Professor, UIPS, CU
 Aseptic Technique Checklist
Work Area
1. Is the cell culture hood properly set up?
2. Is the cell culture hood in an area free from drafts and through traffic?
3. Is the work surface uncluttered, and does it contain only items required for your
experiment?
4. Did you wipe the work surface with 70% ethanol before work?
5. Are you routinely cleaning and sterilizing your incubators, refrigerators, freezers, and
other laboratory equipment?
Personal Hygiene
1. Did you wash your hands?
2. Are you wearing personal protective equipment?

Lovedeep Singh, Assistant Professor, UIPS, CU

3. If you have long hair, is it tied in the back?
4. Are you using a pipettor to work with liquids?
Reagents and Media
1. Have you sterilized any reagents, media, and solutions you have prepared in the
laboratory using the appropriate procedure?
2. Did you wipe the outside of the bottles, flasks, and plates with 70% ethanol before
placing them on your work surface?
3. Are all your bottles, flasks, and other containers capped when not in use?
4. Are all your plates stored in sterile resealable bags?
5. Does any of your reagents look cloudy? Contaminated? Do they contain floating
particles?
6. Have foul smell? Unusual color? If yes, did you decontaminated and discarded them?

Lovedeep Singh, Assistant Professor, UIPS, CU

Handling
1. Are you working slowly and deliberately, mindful of aseptic technique?
2. Did you wipe the surfaces of all the items including pipettor, bottles, and flasks with 70%
ethanol before placing them in the cell culture hood?
3. Are placing the caps or covers face down on the work area?
4. Are you using sterile glass pipettes or sterile disposable plastic pipettes to manipulate all
liquids?
5. Are you using a sterile pipette only once to avoid cross contamination?
6. Are you careful not to touch the pipette tip to anything non-sterile, including the outside
edge of the bottle threads?
7. Did you mop up any spillage immediately, and wiped the area with 70% ethanol?

Gurfateh Singh, Professor, UIPS, CU

Contamination of cell cultures
✓ Contamination of cell cultures is easily the most common problem encountered in
cell culture laboratories, sometimes with very serious consequences.
Cell culture contaminants can be divided into two main categories:
❖ Chemical contaminants such as impurities in media, sera, and water, endotoxins,
plasticizers, and detergents.
❖ Biological contaminants such as bacteria, molds, yeasts, viruses, mycoplasma, as well
as cross contamination by other cell lines.
✓ While it is impossible to eliminate contamination entirely, it is possible to reduce its
frequency and seriousness by gaining a thorough understanding of their sources and by
following good aseptic technique.

Lovedeep Singh, Assistant Professor, UIPS, CU

1. Bacterial contamination:
✓ Bacterial contamination is easily detected by visual inspection of the culture within a few
days of it becoming infected; infected cultures usually appear cloudy (i.e., turbid),
sometimes with a thin film on the surface.
✓ Sudden drops in the pH of the culture medium is also frequently encountered.
✓ Under a low-power microscope, the bacteria appear as tiny, moving granules between the
cells, and observation under a high-power microscope can resolve the shapes of
individual bacteria.
2. Yeasts contamination:
✓ Yeasts are unicellular eukaryotic microorganisms in the kingdom of Fungi, ranging in
size from a few micrometers (typically) up to 40 micrometers (rarely).
✓ Like bacterial contamination, cultures contaminated with yeasts become turbid, especially
if the contamination is in an advanced stage.
Lovedeep Singh, Assistant Professor, UIPS, CU
✓ There is very little change in the pH of the culture contaminated by yeasts until the
contamination becomes heavy, at which stage the pH usually increases.
✓ Under microscopy, yeast appear as individual ovoid or spherical particles, that may bud off
smaller particles.
3. Molds contamination
✓ Similar to yeast contamination, the pH of the culture remains stable in the initial stages of
contamination, then rapidly increases as the culture become more heavily infected and
becomes turbid.
✓ Under microscopy, the mycelia usually appear as thin, wisp-like filaments, and sometimes as
denser clumps of spores.
4. Virus contamination
✓ Their extremely small size makes them very difficult to detect in culture, and
to remove them from reagents used in cell culture laboratories.
Lovedeep Singh, Assistant Professor, UIPS, CU
✓ Because most viruses have very stringent requirements for their host, they usually do not
adversely effect cell cultures from species other than their host.
✓ However, using virally infected cell cultures can present a serious health hazard to the
laboratory personnel, especially if human or primate cells are cultured in the laboratory.
✓ Viral infection of cell cultures can be detected by electron microscopy, immunostaining
with a panel of antibodies, ELISA assays, or PCR with appropriate viral primers.
Note:
▪ Antibiotics should not be used routinely in cell culture, because their continuous
use encourages the development of antibiotic resistant strains.
▪ It allows low-level contamination to persist, which can develop into full-scale
contamination once the antibiotic is removed from media, and may hide mycoplasma
infections and other contaminants.

Lovedeep Singh, Assistant Professor, UIPS, CU

 References:
Textbooks

T1 Basic Cell Culture (Practical Approach ) by J. M. Davis (Editor)

T2 Animal Cell Culture: A Practical Approach by John R. Masters (Editor)

T3 Current porotocols in molecular biology vol I to VI edited by Frederick M.Ausuvel etla.

Reference Books

R1 The Cell, A Molecular Approach. Geoffrey M Cooper.

R2 Pharmacogenomics: The Search for Individualized Therapies. Edited by J. Licinio and M –L.Wong

R3 Handbook of Cell Signaling (Second Edition) Edited by Ralph A.et.al

R4 Molecular Pharmacology: From DNA to Drug Discovery. John Dickenson et.al

R5 Basic Cell Culture protocols by Cheril D.Helgason and Cindy L. Miller

 THANK YOU

For queries
Email: lovedeep.e13279@gmail.com

Lovedeep Singh, Assistant Professor, UIPS, CU