Cell culture refers to growing cells in an artificial environment outside of their natural environment. Common equipment used in cell culture includes cell culture hoods, incubators, microscopes, and storage containers. Specific techniques involved in cell culture include cell isolation, sub-culturing cells when they reach confluence, cryopreservation of cells in liquid nitrogen, and assays to measure cell viability. Common cell viability assays include those based on tetrazolium dye reduction or detection of ATP.

2. Submitted to :- Dr. Ozair Alam
Submitted by:- Shameer
Course :- M .pharm (P.Analysis)
Year & sem :- 1 yr. & 2 sem.
Session :- 2020-2021
Cell Culture

3. Contents:-
• Introduction
• Cell culture equipments
• Cell isolation
• Sub culturing
• Cryogenic preservation
• Cell viability assay
• Flow Cytometry

4. INTRODUCTION
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.
The cells may be removed from the tissue directly and
disaggregated by enzymatic or mechanical means before
cultivation, or they may be derived from a cell line or cell strain
that has already been already established
Cell culture was first successfully undertaken by Ross Harrison
in 1907
Roux in 1885 for the first time maintained embryonic chick
cells in a cell culture

5. CELL CULTURE EQUIPMENT
The specific requirements of a cell culture laboratory depend
mainly on the type of research conducted
• Mammalian cell culture laboratory specialized in cancer
research
• Insect cell culture laboratory that focuses on protein expression
What common do they have is being free from pathogenic
microorganisms(asepsis) and some of the basic equipment that are
generally required in cell culture
Basic Equipment :-
• Cell culture hood (laminar-flow hood or biosafety cabinet)
• Incubator (humid CO2 incubator recommended)
• Water bath
• Centrifuge
• Refrigerator and freezer (–20°C)
• Cell counter (Automated Cell Counter or hemacytometer)
• Inverted microscope
• Liquid nitrogen (N2) freezer or cryostorage container
• Sterilizer (autoclave)

6. Expanded Equipment
• Aspiration pump (peristaltic or vacuum)
• pH meter
• Confocal microscope
• Flow cytometer
Additional Supplies
• Cell culture vessels (flasks, Petri dishes, roller bottles,
multi-well plates)
• Pipettes and pipettors
• Syringes and needles
• Waste containers
• Media, sera, and reagents
• Cells

7. UNDERSTANDING CELL CULTURE LABORATORY
• Aseptic Work Area
The major requirement of a cell culture laboratory is the need to
maintain an aseptic. work area that is restricted to cell culture work
Although a separate tissue culture room is preferred, a designated cell
culture area within a larger laboratory can still be used fort sterile
handling, incubation, and storage of cell cultures, reagents, and media
The simplest and most economical way to provide aseptic conditions is
to use a cell culture hood
• Cell Culture Hood
The cell culture hood provides an aseptic work area while allowing the
containment of infectious splashes or aerosols generated by many
microbiological procedures
On the basis of varying needs of the laboratories they are divided in to:-
• Class I
• Class II
• Class III

8. Air-Flow Characteristics of Cell
Culture Hoods
Cell culture hoods protect the working
environment from dust and other airborne
contaminants by maintaining a constant,
unidirectional flow of HEPA-filtered air over the
work area.
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

9. What should be the layout of culture hood ?
• It should be large enough to be used by a single person at a time,
• It should be design in such a way that it can be cleaned easily inside out
• Must have adequate lighting
• The positioning of every thing should be in such a way that a person can
easily carry out his/her work with out facing any disturbing or uncomfortable
position
Points to be kept in mind
• Keep the work space in the cell culture hood clean & uncluttered
• Keep every thing in direct ,line of sight
• Disinfect each item placed in the cell culture hood by spraying them with 70%
ethanol & whipping clean
The arrangement of items within the cell culture hood usually adheres to the
following right-handed convention, which can be modified to include
additional items used in specific applications.

11. • A wide, clear work space in the center with your cell
culture vessels
• Pipettors in the front right, where it can be reached easily
• Reagents and media in the rear right to allow easy
pipetting
• Tube rack in the rear middle holding additional reagents
• Small container in the rear left to hold liquid waste

12. 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°C4
The material of construction should be
Stainless steel which will allow 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
Types of
incubator
Dry incubator
Humid CO₂
incubator

13. Storage
A cell culture laboratory should
have storage areas for liquids such
as media and reagents, for
chemicals such as drugs and
antibiotics, for consumables such
as disposable pipettes, culture
vessels, and gloves, for glassware
such as media bottles and glass
pipettes, for specialized equipment,
and for tissues and cells
Glassware, plastics, and specialized
equipment can be stored at ambient
temperature on shelves and in
drawers
however, it is important to store
all media, reagents, and
chemicals according to the
instructions on the label.
Depending upon the type of
need we can use Refrigerators,

14. Microscopes
• A simple inverted microscope is
essential so that cultures can be
examined in flasks and dishes.
It is vital to be able to recognize
morphological changes in
cultures since these may be the
first indication of deterioration
of a culture.
Tissue culture ware
• A variety of tissue culture
plasticware is available, the
most common being specially

15. Washing up and sterilizing
facilities
• Glassware, such as pipettes, conical
flasks, beakers (covered with aluminum
foil) are sterilized in a hot air oven at
160 °C for one hour. All other
equipment, such as automatic pipette
tips and bottles (lids loosely attached)
are autoclaved at 121 °C for 20 min.
• Sterilizing indicators such as sterile test
strip are necessary for each sterilizing
batch to ensure that the machine is
operating effectively.
• Autoclave bags are available for loose
items. Aluminum foil also makes good

16. Water purification unit
• A double distilled or reverse
osmosis water supply is essential
for preparation of media, and
rinsing glassware.
• For media preparation water must
be sterilized by autoclaving at 121
°C for 20 min.
• The distilled water must be glass
distilled and stored in glass if it is
to be used for the preparation of
media Storage in plastic may
result in leaching of toxic
substances from the plastic into
the water.

17. Filter sterilization
• Media that cannot be
autoclaved must be sterilized
through a 0.22 μm pore size
membrane filter
• These are obtainable in
various designs to allow a
wide range of volumes to be
filtered
• They can be purchased as
sterile, disposable filters, or
they may be sterilized by
autoclaving in suitable filter
holders.

18. Cell counting facilities
• It is possible to monitor cell
growth by eyes (looking for
confluence), however, more
accurate cell counts are required
for most experimental Purposes.
• The most commonly used device
is the Improved Neubauer
hemocytometer originally
designed for counting blood
cells
Cells/mL culture = Number of viable cells × 104 × dilution factor

19. Cell isolation
• Isolation of one or multiple cell types from a heterogeneous
population is an integral part of modern biological research
and routine clinical diagnosis and Treatment.
• The main principle of separating any cell type from a
population is to utilize one or more properties that are
unique to that cell type

20. Positive selection Negative selection
• Positive selection
involves the
isolation of a target
cell population by
using an antibody
that specifically
binds that
population.
• As an example, a
positive selection kit
for T cells would use
an antibody specific
for the CD3
molecule on T cells
• Negative selection,
however, involves the
depletion of all cell types
except your cell type of
interest. With our T cell
isolation example, our
negative selection kit
would likely involve
antibodies specific for B
cells (CD19), monocytes
(CD14), NK cells (CD56),
and so on. With the
depletion of these cell
types we would only be
left with our cells of
interest, in this case T
cells (CD3).

21. Cellular characteristics as the basis for
cell separation
• Surface charge and adhesion –determines
the attachment of cells to plastic and other
polymer surfaces and can be used to
separate adherent cells from
suspension/free-floating cells Cell size and
density – The physical properties of size
and density are commonly used for the bulk
recovery of cells; either by sedimentation,
filtration or density gradient centrifugation.
• Cell morphology and physiology – on the
basis of shape, histological staining, media
selective growth, redox potential and other
visual and behavioral properties which can
then be harnessed to isolate those cells.
• Surface markers – Specific binding of
surface antigens to either antibodies or

22. Choosing a cell isolation method for an
experiment depends on the following
criteria
• The exploitable characteristics of the
cells
• The amount of stress
mechanical/chemical/physiological –
that the cell type can endure and still
remain viable
• The levels of cell purity and yield
needed
• The acceptable risk of contamination
(zero in case the cells are needed for
culture)
• The type of isolation method – whether
negative or positive
• Any specific requirements of the
downstream applications e.g. cell

23. Different cell Isolation Techniques
Technique Principle Positive/
negative
Purity Yield
Plastic/ adhesion Surface charge and
adhesion
Both Low High
Density gradient
Centrifugation
Cell density Positive Low High
Filtration Cell size Positive Low High
FACS (Fluorescence
assisted cell sorter)
Surface antigen
binding
Positive High low
MACS (Magnetic
assisted cell sorter)
Surface antigen
binding
Both High Medium
Aptamer binding Surface antigen
binding
Positive High Low
Selective
growth/culture
Physiology Negative Medium/
High
Low/Mediu
m
LCM(Laser capture
microdissection)
Morphology Positive High Low
RBC rosetting Size + surface
antigen
Both High Medium
Immuno-LCM Morphology + Positive High Low

24. Sub culturing
• Once the available substrate
surface is covered by cells (a
confluent culture) growth slows
& ceases
• 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 to 80- 90%
confluency in
flask/dishes/plates
• Enzyme such as trypsin,
dipase, collagenase in
combination with EDTA breaks
the cellular glue that attached
the cells to the surface

25. Procedure of subculturingof adherent
cells
• Cells which are anchorage
dependent
• Cells are washed with PBS
(free of ca & mg ) solution.
• Add enough trypsin/EDTA
to cover the monolayer
• Incubate the plate at 37 °C
for 1-2 minutes
• Tap the vessel from the
sides to dislodge the cells
• Add complete medium to
dissociate and dislodge
the cells
• with the help of pipette
which are remained to be
adherent
• Add complete medium

26. Sub culturingprocedure for
suspension cells
• Easier to passage as
no need to detach
them
• As the suspension
cells reach to
confluency
• Asceptically remove
1/3rd of medium
• Replaced with the
same amount of pre-
warmed medium

27. Cryogenic Storage
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
Types of liquid-
nitrogen storage
systems
Vapor phase Liquid phase
Narrow-necked containers have a slower nitrogen evaporation rate and are more
economical, but wide-necked containers allow easier access and have a larger stora
capacity

28. Materials needed for cryopreservation
• Culture vessels containing cultured cells in log
phase of growth
• Complete growth medium
• Cryoprotective agent such as DMSO (use a
bottle set aside for cell culture; open only in a
laminar flow hood)
• Disposable, sterile 15-mL or 50-mL conical
tubes
• Reagents and equipment to determine viable
and total cell counts (e.g., Countess®
Automated Cell Counter, or hemacytometer,
cell counter and Trypan Blue)
• Sterile cryogenic storage vials (i.e., cryovials)
• Controlled rate freezing apparatus or
isopropanol chamber
• Liquid nitrogen storage container

29. Freezing cells for storage
• Remove the growth medium,
wash the cells by PBS and
remove the PBS by aspiration
• Dislodge the cells by
trypsinversene
• Dilute the cells with growth
medium
• Transfer the cell suspension to a
15 ml conical tube, centrifuge at
200g for 5 mts at RT and remove
the growth medium by aspiration
• Resuspend the cells in 1-2ml of
freezing medium
• Transfer the cells to cryovials,
incubate the cryovials at -80 C
overnight
• Next day transfer the cryovials to
Liquid nitrogen

30. Thawing Frozen Cells
• Remove the cryovial containing the
frozen cells from liquid nitrogen
storage and immediately place it into
a 37°C water bath.
• 2) Quickly thaw the cells (< 1 minute)
by gently swirling the vial in the 37°C
water bath until there is just a small
bit of ice left in the vial.
• 3) Transfer the vial it into a laminar
flow hood.
• 4) Transfer the thawed cells dropwise
into the centrifuge tube containing
the desired amount of pre-warmed
complete growth medium appropriate
for your cell line.
• 5) Centrifuge the cell suspension at
approximately 200 × g for 5–10
minutes. The actual centrifugation
speed and duration varies depending
on the cell type.
• 6) After the centrifugation, check the
clarity of supernatant and visibility of

31. Cell viability assay
Cell-based assays are often used for screening
collections of compounds to determine if the test
molecules have effects on cell proliferation or
show direct cytotoxic effects that eventually lead
to cell death. it is important to know how many
viable cells are remaining at the end of the
Experiment
Methods of cell viability assay
• tetrazolium reduction (MTT assay),
• resazurin reduction (alamar blue assay),
• protease markers,
• ATP detection

32. A variety of tetrazolium compounds have been used to
detect viable cells.
The most commonly used compounds include:-
• MTT {3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide}
• MTS {One Solution Cell Proliferation Assay}
• XTT {Cell Proliferation Kit II}
• WST-1. {WST-1 cell proliferation reagent}
These compounds fall into two basic categories:-
MTT which is positively
charged and readily
penetrates
viable eukaryotic cells
those such as MTS, XTT,
and WST-1 which are
negatively charged and
do not readily penetrate
cells.

33. MTT Tetrazolium Assay
The MTT (3-(4,5-dimethylthiazol-2-yl)- 2,5-
diphenyltetrazolium bromide) tetrazolium reduction
assay was the first homogeneous cell viability assay
developed for a 96-well format that was suitable for
high throughput screening (HTS).

34. Reaction involved in MTT assay
• The quantity of formazan (presumably directly proportional to the
number of viable cells) is measured by recording changes in absorbance
at 570 nm using a plate reading spectrophotometer

35. Resazurin reduction assay (alamar blue assay)
• The reduction of resazurin (presumably directly proportional to the
number of viable cells) is measured by recording changes in
absorbance or fluorescence at 570 nm using a plate reading
spectrophotometer

36. Flow Cytometry
Flow cytometry is the measurement of cells in a
flow system, which delivers the cells singly, past a
point of measurement.
In practice the name refers to the instruments in
which light is focused at the point of
measurement.
Typically light scatters at two different angles and
from one to six or more fluorescence will be
measured.
in this process, a sample containing cells or
particles is suspended in a fluid and injected into
the flow cytometer instrument. The sample is
focused to ideally flow one cell at a time through a
laser beam, where the light scattered is
characteristic to the cells and their components.
Cells are often labeled with fluorescent markers so
light is absorbed and then emitted in a band of
wavelengths. Tens of thousands of cells can be
quickly examined and the data gathered are
processed by a computer

37. Principle
• It is the measurement of cellular properties as cells move in a fluid stream
(flow), past a stationary set of detectors
• It is the technique of quantitative single cell analysis
Components of flow cytometer

38. Instrumentation of Flow cytometer
The data representation of flow Cytometry is done either in histogram of in dot plot format

39. Flow cytometry is routinely used in basic research, clinical practice, and
clinical trials.
Uses for flow cytometry include:-
• Cell counting
• Cell sorting
• Determining cell characteristics and function
• Detecting microorganisms
• Biomarker detection
• Protein engineering detection
• Diagnosis of health disorders such as blood cancers

40. References:-
• Google search engine
• Wikipedia
• A handbook of cell culture basics GIBCO Invitrogen
• A presentation on cell culture