UNIT 2

Cell culture

Stem Cells
Stem cells are unique cells with the ability to develop into various cell types in the body
during early life and growth. They function as an internal repair system in many tissues,
capable of dividing indefinitely to replenish other cells for the lifespan of the person or
animal. When stem cells divide, the resulting cells can either remain as stem cells or
differentiate into specialized cells such as muscle cells, red blood cells, or brain cells.
Progenitor cell
A progenitor cell is a biological cell that can differentiate into a specific cell type. While both
progenitor cells and stem cells share the ability to differentiate, progenitor cells are more
specified than stem cells. Progenitor cells can only differentiate into their designated "target"
cell type and can divide only a limited number of times, unlike stem cells which can replicate
indefinitely. The precise definition of progenitor cells is still debated and evolving.

Stem Cells Progenitor Cells

1. Can differentiate into many different cell 1. Can only differentiate into a specific cell
types (pluripotent) or a limited range of cell type (unipotent or oligopotent)
types (multipotent)
2. Less specified compared to progenitor cells. 2.More specified than stem cells

3.Can replicate indefinitely. 3.Can divide only a limited number of times

4.Found in early development (Embryonic stage) 4.Typically found in specific tissues and
and various tissues in adults. stages of development.

5.Function of stem cells is the Development of 5.Function of progenitor cell is to Replenish

tissues and organs, repair of damaged tissues specific cell types in adult tissues

Cell-Cell Interactions
Cell-cell interactions refer to direct interactions between the surfaces of adjacent cells. These
interactions are essential for various biological processes in multicellular organisms,
including development, tissue organization, and immune responses. Cell-cell interactions
enable cells to communicate with each other, allowing them to respond to changes in their
environment and coordinate their activities. These interactions can be stable, such as those
formed by cell junctions that facilitate strong adhesion and communication between cells
within tissues.
Tight Junctions
1. Tight junctions are multi-protein complexes that hold cells together and prevent the
movement of water and water-soluble molecules between cells.
2. In epithelial cells, they separate the extracellular fluid around the apical and
basolateral membranes, forming a continuous band just below the apical surface.
3. Tight junctions align on adjacent cells to seal different tissues and body cavities; the
apical surface of gastrointestinal epithelial cells acts as a selective permeable barrier
separating the external environment from the body.
4. The permeability of tight junctions depends on factors such as protein makeup, tissue
type, and cell signaling.
5. Tight junctions are composed of various proteins, with four main transmembrane
proteins: occludin, claudin, junctional adhesion molecules (JAMs), and tricellulins.
6. The extracellular domains form the tight junction barrier through homophilic and
heterophilic interactions with adjacent cells, while cytoplasmic domains anchor them
to the cell cytoskeleton.

Gap junctions
1. Gap junctions are the main site of cell-cell signalling or communication that allow
small molecules to diffuse between adjacent cells.
2. In vertebrates, gap junctions are composed of transmembrane proteins called
connexins.
3. They form hexagonal pores or channels through which ions, sugars,
and other small molecules can pass.
4. Each pore is made of 12 connexin molecules; 6 form a hemichannel on one cell
membrane and interact with a hemichannel on an adjacent cell membrane.
5. The permeability of these junctions is regulated by many factors
including pH and Ca2+ concentration.
Anchoring junctions
1. Of the three types of anchoring junctions, only adherens junctions and desmosomes
are involved in cell-cell interactions.
2. Both are found in many types of cells.
3. Adjacent epithelial cells are connected by adherens junctions on their lateral
membranes, located just below tight junctions.
4. Adherens junctions give shape and tension to cells and tissues and are also sites of
cell-cell signaling.
5. Adherens junctions are made of cell adhesion molecules from the cadherin family,
with over 100 types corresponding to different cells and tissues.
6. The most common cadherins are E-, N-, and P-cadherins, with E-cadherin being the
most abundant in epithelial cells.
Sterilization:

Sterilization is the process of removing, killing, or deactivating all forms of life and
biological agents on a specific surface, object, or fluid. This can be achieved through
heat, chemicals, irradiation, high pressure, and filtration. Sterilization eliminates all
forms of microorganisms and biological agents. After sterilization, an object is
considered sterile or aseptic.it is used in various fields the medicine, research, and
food processing to prevent contamination.

Methods of Sterilization

Heat

Heat sterilization is a method of eliminating all forms of microbial life, including

bacteria, viruses, fungi, and spores, by exposing them to high temperatures. This
process is widely used in medical, laboratory, and food industries to ensure the
sterility of instruments, equipment, and products.

Types of Heat Sterilization:

1. Steam sterilization
Steam sterilization also known as moist heat sterilization, is a method that uses
steam under pressure to kill microorganisms. It is performed using an
autoclave at high temperature for a specific time. This process is faster than
dry heat sterilization. Typical cycles take 3-30 minutes at 121-134°C.The
steam condenses on the surfaces of objects being sterilized, transferring heat
and destroying bacteria, viruses, and fungi.

2. Dry heat sterilization

Dry heat sterilization, the earliest method, is slower than moist heat
sterilization and gradually kills microorganisms with prolonged exposure to
lethal temperatures. Forced hot air can accelerate heat transfer, reducing the
temperature and time needed for sterility. Standard settings are 160 °C for two
hours or a rapid method of 190 °C for 6-12 minutes, depending on item
wrapping. This method is suitable for powders and heat-stable items, avoiding
issues like rusting in steel objects.

Chemical Sterilization
Chemical sterilization is used for heat-sensitive materials, employing gases or liquids
to avoid heat damage. It requires compatibility between the sterilant and materials and
ensuring full surface contact. However, these chemicals can be harmful to humans,
necessitating careful handling and residue removal.
 Types of chemical Sterilization
1. Ethylene oxide
Ethylene oxide (EO, EtO) gas is widely used for sterilization due to its
compatibility with diverse materials, including heat-sensitive items,
making it the predominant method for sterilizing over 50% of disposable
medical devices and 70% of total sterilizations.

2. Hydrogen peroxide
Hydrogen peroxide, in liquid and vapor form (VHP), is a potent oxidizing
agent used for sterilizing heat-sensitive medical tools like endoscopes. It
offers rapid cycles, though limitations in material compatibility and
penetration exist, posing health risks such as skin irritation and respiratory
hazards.

3. Glutaraldehyde and formaldehyde

Glutaraldehyde and formaldehyde are liquid sterilizing agents requiring
long immersion times, up to 22 hours for spores, and are volatile and toxic.
Glutaraldehyde has a short shelf-life and is costly, while formaldehyde,
also used as a gas, is less expensive but highly volatile and used in vaccine
sterilization.

4. Peracetic acid
Peracetic acid (0.2%), FDA-approved for sterilizing medical devices like
endoscopes, is a disinfectant produced from acetic acid and hydrogen
peroxide. It's environmentally friendly, widely used in sanitizers, and
increasingly employed globally for surface fumigation to combat COVID-
19 and other diseases.

Radiation sterilization
Radiation sterilization is a method that uses electromagnetic radiation (such as UV
light, X-rays, and gamma rays) or subatomic particles (such as electron beams) to
eliminate all forms of microorganisms from materials, including bacteria, viruses,
fungi, and spores. This technique can involve ionizing radiation, which has
enough energy to ionize atoms and molecules, or non-ionizing radiation, which is
less energetic.

Types of Radiation Sterilization:

1. Non-ionizing radiation sterilization

Non-ionizing radiation sterilization uses lower-energy electromagnetic
waves, such as ultraviolet (UV) light, to disinfect surfaces and transparent
objects by damaging the DNA of microorganisms. It is effective for surface
sterilization but limited by its inability to penetrate shaded areas or dirt.
 2. Ionizing radiation sterilization
Ionizing radiation sterilization uses high-energy radiation, such as gamma
rays, X-rays, or electron beams, to eliminate microorganisms by damaging
their DNA. This method is effective for sterilizing medical devices,
pharmaceuticals, and food products.

Autoclave
An autoclave is a machine that uses high temperatures and pressure to sterilize items by
killing harmful microorganisms like bacteria, viruses, fungi, and spores. Autoclaves are often
used in healthcare, laboratory, food, and pharmaceutical industries. They can also be used for
industrial applications like curing composite resins and material testing in the aeronautical
industry.
There are different types of autoclaves, including horizontal, gravity displacement, and
vertical:
1. Horizontal autoclaves
These are floor-mounted units that are often used for medical, industrial,
research, and education purposes. They have rectangular pressure vessels that
are good for sterilizing flat trays of surgical instruments.
2. Gravity displacement autoclaves
Also known as gravity autoclaves, these are the most basic type of autoclave
and are commonly used in research laboratories. They use steam to displace
air in the chamber by gravity through a drain port.
3. Vertical autoclaves
Also known as top loading autoclaves, these generate steam to create pressure
and heat for sterilizing medical tools like flasks and beakers.

Bioreactor
A bioreactor is a type of fermentation vessel used to generate a variety of chemicals and biological
reactions. This is a closed container with aeration, agitation, temperature and pH control, and well-
placed drainage or overflow openings to remove waste biomass with its products from cultured
microorganisms’. enzymes, antibodies, vaccines, etc.
Different Types of Bioreactors for animal cell culture:
1. Stirred Tank Bioreactors:
A stirred tank bioreactor is a cylindrical vessel equipped with a motor-driven central shaft
that supports agitators (impellers) to mix the contents and enhance gas distribution. It is
widely used in industry due to its ease of operation, cost-effectiveness, simple cleaning
process, and efficient temperature control, making it ideal for fermentation and cell culture
processes.

2. Airlift Bioreactors:
An airlift bioreactor is a special tank that uses bubbles to move liquids around. It's split into
two parts: one where the liquid goes up with the bubbles, and one where it comes back down.
This helps mix everything evenly and is good for growing cells and making stuff like
medicine.
 3. Packed bed Bioreactors:
Packed bed bioreactors are like tall columns filled with small particles where microorganisms
or cells grow. Liquid passes through the packed particles, providing nutrients and removing
waste. They're used for various biological processes like fermentation or water treatment,
offering high surface area for efficient reactions and easy control over conditions.

4. Photobioreactors:
Photobioreactors are specialized tanks or containers where microorganisms like algae or
cyanobacteria grow using sunlight as their energy source. These reactors are designed to
optimize light exposure, temperature, and nutrient supply for efficient growth. They're used in
industries like biofuel production, wastewater treatment, and food supplements.

5. Fluidize bed bioreactors:

A fluidized-bed bioreactor is a type of vessel where solid particles are suspended and behave
like a fluid when liquid passes through them. Microorganisms attached to these particles
carry out biological processes such as fermentation or waste degradation. These bioreactors
offer efficient mixing and increased surface area for microbial activity, making them useful in
various industries for tasks like wastewater treatment and biofuel production.
Microfluidics
Microfluidics in tissue engineering involves the use of tiny fluid channels to precisely control
the cellular microenvironment, enhancing tissue growth and organization. This technology
enables the creation of complex tissue structures by mimicking physiological conditions. It
allows for high-throughput screening and real-time monitoring of cellular responses.
Microfluidics thus plays a crucial role in advancing regenerative medicine and developing
personalized medical treatments.

Applications of microfluidics
1. Point-of-Care Testing (POCT):
Provides rapid, on-site diagnostic results without the need for centralized laboratories,
enhancing immediate patient care. These devices are compact, user-friendly, and designed for
quick analysis of biological samples.
2. Lab-on-a-Chip (LOC):
Miniaturizes and integrates multiple laboratory processes onto a single chip for efficient and
comprehensive analysis. This technology allows complex biochemical reactions and analyses
to be conducted on a tiny, portable platform.
3. **High-Throughput Screening (HTS):
Facilitates the rapid screening of large libraries of drug compounds against target molecules
for therapeutic efficacy. Microfluidic HTS systems are efficient and cost-effective,
significantly accelerating the drug discovery process.
4. Single-Cell Analysis:
Allows for the detailed examination of individual cells, aiding in understanding cellular
behavior, genetics, and disease mechanisms. This application is crucial for research in cancer,
immunology, and personalized medicine.
5. Microfluidic Organ-on-a-Chip:
Mimics the physiological responses of human organs on a chip to study disease mechanisms
and drug responses. These models provide a more accurate representation of human biology
than traditional cell cultures or animal models.
6. PCR and DNA Analysis:
Conducts rapid and precise genetic analysis and amplification in a compact format,
enhancing molecular diagnostics. Microfluidic PCR devices offer high sensitivity and
specificity for detecting genetic material.
7. Cell Sorting:
Isolates and sorts cells using precise fluid control mechanisms for research and therapeutic
applications. This technique is essential for stem cell research, cancer treatment, and the
study of rare cell populations.
8. Drug Delivery Systems:
Enables controlled and targeted delivery of drugs to specific sites in the body, improving
therapeutic outcomes. Microfluidic devices can release drugs at a controlled rate and target
specific tissues, minimizing side effects.