CELL BIOLOGY

CODE:1101

Dr. Mulumba Pius Edgar

 MODULE ONE
THE CELL

Introduction

• The Cell Theory

• Origin of cells
Methods & Tools for studying cells
• light microscopy
• electron microscope
• methods for isolation of cellular organelles
 WHAT DO YOU KNOW ABOUT CELLS???

• We are made of cells. Cells make up our skin, our organs, and our muscles. The brain,
the seat of our thoughts and desires, is made of cells.
• Our blood vessels teem with cells. Fertilization is no more or less than a joining of two
separate cells to produce a single new cell, which then multiplies to produce the
embryo.
• When we grow from a tiny embryo into a large adult, we do so by adding more and
more cells. When we get sick, it is often because our cells have a problem. And when
we grow old, it is because our cells gradually ware out. After we die and are buried,
soon the only remnants of our existence are bones, teeth, and hair, structures that were
sculpted in life by the ceaseless activity of cells.
• Many medicines work by changing how cells behave, and in recent years cells
themselves are being used as medicines to cure sick people. Because all living things
are made of one or more cells, the origin of life corresponds to the origin of cells.
 DISCOVERY OF CELLS

do we achieve This ???) Through a science called microscopy, the use of a

microscope.
DISCOVERY MICROSCOPES

Zacharias Jansen and the first compound microscope

the 1590's,
Their first microscopes were more of a novelty
than a scientific tool since maximum
magnification was only around 9x and the images
were somewhat blurry
Anton van Leeuwenhoek

AMINALCULES
He became interested in science when, as a Dutch businessman, he began grinding lenses and
building simple microscopes as a hobby.
Each microscope consisted of a flat brass or copper plate in which a small, single glass lens was
mounted. The lens was held up to the eye, and the object to be studied was placed on the head of a
movable pin just on the other side of the lens. Leeuwenhoek made over 400 microscopes, many of
which still exist. The most powerful of these instruments can magnify objects about 275 times.
He was the first person to observe single-celled animals (protozoa) with a microscope and to
describe red blood cells in humans and other animals, as well as sperm cells.
 STUDY OF CELLS

The study of cells started about 330 years ago.

Robert Hooke viewed a thin cutting of cork and discovered empty
spaces contained by walls which he termed cells. he saw only cell walls
because cork cells are dead and without protoplasm
Cont.
Robert Brown discovered the nucleus in plant cells.

In 1831, while investigating the fertilization mechanisms of plants in the Orchidaceae and

Asclepiadaceae families, he noted the existence of a structure within the cells of orchids, as well as

many other plants, that he termed the “nucleus” of the cell.

Matthias Jakob Schleiden: all plant tissues are composed of cells, that an embryonic plant arose

from a single cell. He declared that the cell is the basic building block of all plant matter.

Theodor Schwann reached the same conclusion as Schleiden about animal tissue being composed

of cells, ending speculations that plants and animals were fundamentally different in structure.

discovered pepsin, research on the processes of fermentation, purefaction, and muscular and arterial

contraction
 CELL THEORY:
It wasn ’ t until the 1830s that the widespread importance of cells was realized. In 1838, Matthias

Schleiden, a German lawyer turned botanist, concluded that, despite differences in the structure of

various tissues, plants were made of cells and that the plant embryo arose from a single cell.

In 1839, Theodor Schwann, a German zoologist and colleague of Schleiden ’ s, published a

comprehensive report on the cellular basis of animal life. Schwann concluded that the cells of plants

and animals are similar structures and proposed these two tenets of the cell theory:

1. All organisms are composed of one or more cells.

2. The cell is the structural unit of life.

Schleiden and Schwann ’ s ideas on the origin of cells proved to be less insightful; both agreed that
cells could arise from noncellular materials. Given the prominence that these two scientists held in
the scientific world, it took a number of years before observations by other biologists were accepted
as demonstrating that cells did not arise in this manner any more than organisms arose by
spontaneous generation.
By 1855, Rudolf Virchow, a German pathologist, had made a convincing case for the third tenet of
the cell theory:

3. Cells can arise only by division from a preexisting cell.

 CELL THEORY SUMMARY

1. All living organisms are made of 1 or more cells

2. cell is the basic unit of structure and organization in organisms

3. Cells come from pre-existing cells

REVIEW QUESTIONS
1. When Robert Hooke first described cells, what was he actually looking at?
2. What are the three components of cell theory?

Check Out the Quiz and answer the Questions

 MODULE ONE
METHODS AND TOOLS FOR STUDYING
CELLS

Methods & Tools for studying cells

• light microscopy
• electron microscope
• methods for isolation of cellular organelles
 TYPES OF MICROSCOPY
Modern microscopy involves the use of
- Light microscopes (including fluorescent microscopy),
- Transmission electron microscopes (TEMs), and
- Scanning electron microscopes (SEMs).

When Hooke first looked at cells using a

handmade microscope with a candle as a
light source, he used a light microscope.
A light microscope is a microscope that uses light passing through optical lenses to
magnify objects. For many years this was the only type of microscope used, and it allowed
many advancements to be made.
Light microscopes are still very common and widely used today.
A major advantage of light microscopes is that they are
- relatively inexpensive and simple to use.
- They require little maintenance and very little power to operate.
- Light microscopes have a useful magnifying power up to about 1,000×1,000×.
- In order to distinguish between cellular structures, many scientists will often use dyes,
which give particular target structures a certain color. Light microscopes can also be used
to view living cells and tissues, which can then be used in further research.
Class Activity
Research Functions of Each Part
Light Microscope Images

Gram stain Image by medline Plus Medical

Plant Cells observed under a light microscope

Image credit: Essential Cell Biology 4th Edition

Mode of Action
In a light microscope, the condenser focuses the light beam from the source light bulb
so it passes through the slide on the stage. The objective lens and lenses in the
eyepiece magnify the object. Coarse and fine focus knobs adjust focus, and a camera
port (on some models) allows photos to be taken.
VARIATION OF LIGHT MICROSCOPE
A variation on the light microscope is a fluorescence microscope, which is the use of
specific wavelengths of light to excite dyes or naturally occurring compounds in specimens
in order to view them with a microscope. Fluorescence microscopy often uses ultraviolet
wavelengths for excitation, which necessitates a separate bulb from the visible light bulb. A
filter allows only a small range of wavelengths to pass through, which excite the
fluorescent dye.

Credit: Wiedehopf 20 (left) Howard Vindin (right)

 IMAGES Observed Under a Light Microscope

Many kinds of living materials can be viewed with light microscopy or fluorescence microscopy.
Dyes are added during specimen preparation to increase the visibility of cell structures such as
nuclei, spindle fibers, microtubules, and chromosomes.
Confocal Microscopy
A confocal microscope is a specialized type of fluorescence microscope that builds up an
image by scanning the specimen with a laser beam. The beam is focused onto a single
point at a specific depth in the specimen, and a pinhole aperture in the detector allows
only fluorescence emitted from this same point to be included in the image. Scanning the
beam across the specimen generates a sharp image of the plane of focus—an optical
section. A series of optical sections at different depths allows a three-dimensional image
to be constructed. An intact insect embryo is shown here stained with a fluorescent probe
for actin filaments. (A) Conventional fluorescence microscopy gives a blurry image due to
the presence of fluorescent structures above and below the plane of focus. (B) Confocal microscopy
provides an optical section showing the individual cells clearly.

(Courtesy of Richard Warn and Peter shaw.)

ELECTRON MICROSCOPY

There are two broad categories of EM: transmission EM and scanning EM. First, we discuss the
topic of transmission EM, including the special techniques of cryoelectron microscopy.
Transmission Electron Microscopy
Transmission electron microscopes use electrons in away that is analogous to the way light
microscopes use visible light. The various elements in a transmission electron microscope that
produce, focus, and collect electrons after their passage through the specimen are
all related in function to the corresponding elements in a light microscope (see Fig. 1–B). Rather
than a light source, there is an electron source, and electrons are accelerated toward the anode by a
voltage differential. In an electron microscope, the electrons are focused not by optical lenses of
glass, but instead by magnets.
The electron micrograph below shows a small region of a cell in a piece of
testis. The tissue has been chemically fixed, embedded in plastic, and cut
into very thin sections that have then been stained with salts of uranium
and lead. (Courtesy of Daniel S. Friend.)
TRANSMISSION ELECTRON MICROSCOPY Cont…
SCANNING ELECTRON MICROSCOPE

In the scanning electron microscope (SEM), the specimen, which has been coated with a very thin
film of a heavy metal, is scanned by a beam of electrons brought to a focus on the specimen by
magnetic coils that act as lenses. The quantity of electrons scattered or emitted as the beam bombards
each successive point on the surface of the specimen is measured by the detector, and is
used to control the intensity of successive points in an image built up on a video screen. The
microscope creates striking images of three-dimensional objects with great depth of focus and can
resolve details down to somewhere between 3 nm and 20 nm, depending on the instrument.

Scanning electron micrograph of stereocilia

projecting from a hair cell in the inner ear (left ).
For comparison, the same structure is shown by
light microscopy, at the limit of its resolution
(above). (Courtesy of Richard Jacobs and James
Hudspeth.)
Content Here
ISOLATION OF CELLULAR ORGANELLES

What is cell separation?

Cell separation, also commonly referred to as cell isolation or cell sorting, is a process to

isolate one or more specific cell populations from a heterogeneous mixture of cells. There

are a number of cell separation methods available, each with its own pros and cons.
Why do scientists isolate cells?
Conducting experiments on isolated cells allows scientists to confidently answer specific
research questions by minimizing interference from other cell types within the sample. Isolated
cells have many applications within life science research, allowing scientists to:
• Conduct molecular analysis of a single cell type, including RNA expression and epigenetic
analysis
• Genetically modify and expand a particular cell type of interest for disease modelling or cell
therapy research applications (e.g. T cell therapy research)
• Directly use purified cells for adoptive cell transfer experiments in various animal models
• Increase sensitivity of analytical methods (e.g. cell isolation for HLA analysis, cell isolation
for FISH analysis)
• Study the in vitro effects of drug candidates on specific cell populations (e.g. hematotoxicity
testing)
• Fuse enriched plasma cells with myeloma cells to produce hybridomas
How do scientists prepare samples for cell separation?
There are many different ways to prepare samples for optimal cell isolation. The method you
select depends on your starting sample and may involve removing certain elements from it or
simply creating a single-cell suspension.
Cell separation can be performed on a variety of complex biological samples, including:
•Whole peripheral blood
•Leukapheresis products (e.g. Leukopaks)
•Peripheral blood mononuclear cells (PBMCs)
•Bone marrow
•Cord blood
•Spleen and lymph nodes
•Other tissues (e.g. skin, liver, lung, fat, brain, tumor, etc.)
What cell separation methods and techniques are available?
There are many different ways to isolate cells from complex biological samples. Common
characteristics used to isolate cells include cell size, cell density, cell shape, and surface protein
expression. The most common cell separation techniques include:
• Immunomagnetic cell separation
• Fluorescence-activated cell sorting (FACS)
• Density gradient centrifugation
• Immunodensity cell isolation
• Microfluidic cell sorting
•Adhension
•Sedimentation
There are also less commonly used cell separation methods, including buoyancy-activated cell
sorting, aptamer-based cell isolation, complement depletion, and more.
FURTHER READING
1. Immuno magnetic Cell Separation and Cell Isolation
Magnetic cell separation, also known as immunomagnetic cell separation or magnetic cell
sorting, involves targeting cells for selection or depletion using antibodies or ligands directed
against specific cell surface antigens. Labeled cells are cross-linked to magnetic particles, also
known as magnetic beads, that can be immobilized once an electromagnetic field is applied.
Due to its speed and simplicity, magnetic cell separation is one of the most commonly used
methods by which scientists isolate highly purified populations of specific cell subsets.
Fluorescence-activated Cell Sorting
Fluorescence-activated cell sorting (FACS) is a method that uses flow cytometry and
fluorescent probes to sort heterogeneous mixtures of cells. Fluorophore-tagged antibodies
bind to epitopes on specific antigens on the target cells within a single-cell suspension. After
tagging, the flow cytometer focuses the cell suspension into a uniform stream of single cells.
This stream is then passed through a set of lasers that excites the cell-bound fluorophores,
causing light scattering and fluorescent emissions. Based on the wavelengths produced by
the laser excitation, the resulting photon signals are converted into a proportional number of
electronic pulses that assign a charge to the droplet that is formed around the cell. As each
droplet falls between the deflection plates, its charge causes the droplet to either be deflected
into collection tubes or fall into the waste chamber.
Cont…
Immunomagnetic cell sorting is a much faster and simpler procedure than FACS, and is often
the preferred cell isolation method for common cell types. FACS has several advantages over
immunomagnetic cell sorting including the ability to:
•Sort single cells
•Isolate cells based on intracellular markers (e.g. GFP)
•Isolate cells based on surface marker expression levels
•Sort complex cell types with multiple markers at higher purity
Density Gradient Centrifugation
Density gradient centrifugation relies on the varying densities of cells within a heterogeneous
sample. The sample is layered on top of a density gradient medium before being centrifuged.
During centrifugation, each cell type will sediment to its isopycnic point, which is the place in
the medium gradient where the density of the cells and medium are equal.
Common applications include the fractionation of peripheral blood mononuclear cells,
exclusion of dead cells from a cell culture, and separation of plasma from blood cells.
There are several types of density gradient media, each with unique properties that render them
ideal for different purposes.
The following are examples of the most well-known types:

•Lymphoprep , Lympholyte®, and Ficoll-Paque® are similar media that consist of

saccharides and sodium diatrizoate; they have a density of 1.077 g/mL. These media are
commonly used to isolate mononuclear cells from peripheral blood, cord blood, and bone
marrow.
•Percoll® (density: 1.131 g/mL) consists of colloidal silica particles coated with
polyvinylpyrrolidone (PVP) and is widely used to separate cells, organelles, viruses, and
other subcellular particles.
•OptiPrep is a medium consisting of iodixanol in water that is used to isolate viruses,
organelles, macromolecules, and cells.
Cont…..

Density gradient centrifugation is an inexpensive cell separation technique but has limited
specificity, low purity, and low throughput. In addition, even though it is a common
laboratory technique, density gradient centrifugation can be a slow and laborious process
that is difficult to master. Scientists typically need to carefully layer their sample over the
density gradient medium, centrifuge for 30 minutes without brakes, then carefully harvest
and wash the appropriate layer of cells. Technologies like SepMate make this method
easier and faster. SepMate is a specialized tube that allows users to quickly layer blood
over the density gradient medium, prevents the layers from mixing and facilitates fast and
easy harvesting of the target cells. With SepMate , cells can be obtained in as little as 15
minutes.
Immuno-density Cell Separation
Immuno-density cell separation, also referred to as erythrocyte rosetting, is a negative
selection method that uses a combination of antibody-based labeling and density gradient
centrifugation. With this method, antibodies are added to a whole blood sample, labeling the
unwanted cells and cross-linking them to red blood cells. This results in the formation of
complexes called immunorosettes that are much denser than the mononuclear cells being
isolated. During centrifugation, the unwanted cells pellet with the red blood cells, leaving the
target cells in a layer above the density medium.
Immunodensity cell separation doesn’t require any specialized equipment beyond a centrifuge,
can be easily incorporated into established density gradient centrifugation protocols, and can be
used to isolate specific cell subsets directly from whole blood. However, the technique is
limited to negative selection, relies on the operator’s blood sample layering technique, and
requires a high concentration of red blood cells in the starting sample.
RosetteSep is an example of a commercially available immunodensity cell separation reagent
(Figure 1). RosetteSep can be combined with SepMate PBMC isolation tubes for even
faster and easier immunodensity cell separation.
Sedimentation
Sedimentation works on the basis that gravity will cause larger and denser components to
sediment faster than materials that are smaller and less dense. The largest and densest
components in a sample can be pelleted through an initial low-force centrifugation due to their
high rate of sedimentation. The supernatant can then be spun again. Through successive
centrifugations, components with an increasingly lower rate of sedimentation can be isolated.
Leukocytes are commonly separated from erythrocytes through dextran sedimentation.
HetaSep is an example of an erythrocyte aggregation agent that is used to separate nucleated
cells from red blood cells (RBCs) in whole blood.
Sedimentation is inexpensive but generally results in lower purity than other methods.
Adhesion
The unique adhesion profiles of different cell types can be used to separate target cells from
heterogeneous populations. By choosing suitable growth factors and cell culture plates to
selectively favor or inhibit adhesion, adherent cells can be separated from cells in suspension.
Macrophages are inherently adherent and often isolated from peripheral blood and bone marrow
by adhesion. Mononuclear cells can be cultured with serum and a differentiation cocktail,
promoting the formation of an adherent monolayer of macrophages. After removing the
supernatant containing unwanted cells, the macrophages can be isolated.
Alternatively, cells that naturally grow in suspension or have lost anchorage dependency can be
isolated by culturing the heterogeneous cell population in plates designed for ultra-low
attachment. Without a surface to adhere to, adherent cells will fail to survive and the target cells
will remain in suspension1.
Microfluidic Cell Separation
Microfluidics is an umbrella category of cell separation methods.2 Designed to manipulate
fluids on a microscopic level to facilitate single-cell isolation, microfluidic technologies are
frequently built onto microchips and are commonly known as "lab-on-a-chip" devices. These
devices have several advantages, including the smaller volumes of samples and reagents
required for use. Lab-on-a-chip devices are also portable, making them particularly useful as
field-based diagnostic tools.
Microfluidic methods can be divided into active and passive systems. Active microfluidic
systems involve external forces, whereas passive microfluidics make use of the cell’s density
and mass in combination with gravity. These methods can also be classified by the presence or
absence of cell labeling; although some methods involve labeling cells with antibodies, most
methods are known for being label-free.
Cont….
There are several different microfluidic methods used for cell isolation, including:
• Acoustophoresis
• Aqueous two phase systems
• Biomimetic microfluidics
• Cell affinity chromatography
• Deterministic lateral displacement
• Electrophoretic sorting
• Field flow fractionation
• Gravity and sedimentation
• Magnetophoresis
• Microfiltration
• Optical sorting
OTHER CELL SEPARATION TECHNIQUES
This section summarizes other, less commonly used, cell separation methods.

Aptamer Technology
Aptamers are single-stranded RNA or DNA oligonucleotides that form structures that can bind
to highly specific targets. Through systematic evolution of ligands by exponential enrichment
(SELEX) technology, aptamers can be screened and synthesized to target any cell type. These
aptamers have high affinity and specificity toward their targets, and can be labeled with
fluorochromes or magnetic particles to facilitate cell separation. The main advantage of
aptamers is that they lack immunogenicity.
Fluorophore-labeled aptamers have been used to sort mesenchymal stem cells3 from bone
marrow and RNA aptamers have been used to isolate mouse embryonic stem cells
Buoyancy-Activated Cell Sorting
Buoyancy-Activated cell sorting is a cell separation technique that utilises glass microbubbles
labeled with antibodies specific to the target cells. When mixed into the sample, the
microbubbles bind to the target cells. Due to the augmented buoyancy force, the microbubbles
float to the surface, separating the target cells.

Complement Depletion
The complement depletion method takes advantage of the proteolytic cascade initiated by the
complement system of the immune system. The complement system consists of plasma proteins
that can be activated by pathogens or antibodies. Once activated, the plasma proteins induce the
formation of a membrane-attack complex on a cell, resulting in cell lysis. With specific
monoclonal antibodies, any cell population can be targeted and lysed through the complement
cascade.
Laser Capture Microdissection
Laser capture microdissection (LCM) is a technique that uses a narrow laser beam to cleave
target cells or areas from mostly solid tissue samples. Through microscopic visualization, LCM
can isolate cell populations from heterogeneous mixtures using cell morphology or specific
histological and immunological staining. LCM is particularly useful when working with small
sample sizes.

Immunoguided Laser Capture Microdissection

Immunoguided laser capture microdissection combines immunostaining with laser capture
microdissection (see above). This allows immunophenotypes to be used, in addition to
morphology and tissue location, to identify and isolate target cells from the tissue sample. This
technique employs immunohistochemistry or immunofluorescence to guide the dissection
process for isolating cells expressing a specific molecular marker, and is particularly useful
when histological stains do not recognize certain cell populations.
Limiting Dilution
Limiting dilution involves isolating single cells through the dilution of a cell suspension.
This technique can be carried out with standard pipetting tools and is commonly used to
produce monoclonal cell cultures and single cell cultures for single-cell analysis

Micromanipulation
Micromanipulation, a form of manual cell picking, is a cell isolation technique involving
the use of an inverted microscope and ultra-thin glass capillaries connected to an
aspiration and release unit. The system moves through motorized mechanical stages,
allowing the operator to carefully select a specific cell and apply suction via micropipette
to aspirate and isolate the cell.
END OF MODULE ONE