Case uitwerking
ALL cases from MBS1002 Biomedical Approaches
ALL cases from MBS1002 Biomedical Approaches in detail. Important aspects from lectures incorporated as well as feedback from the tutorial. Grade EXAM: 7,7
[Meer zien]Voorbeeld 10 van de 90 pagina's
Voorbeeld 10 van de 90 pagina's
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Voorbeeld van de inhoud
MBS 1002: Biomedical Approaches⇒ Case 1: How do cell cultures work
What are cell cultures?
Cell culture are laboratory methods that enable the growth of eukaryotic or prokaryotic cells in
physiological conditions. Involves creating a controlled environment that mimics the natural
conditions necessary for cell growth and propagation.
It allows for the manipulation of genes and molecular pathways, and provides a homogeneous and
well-defined system for studying cellular processes.
There are 2 methods of obtaining cells: cell bank or cell isolation;
- Cell bank → cell banking involves the preservation of cells for long-term storage to ensure
their availability for future use. Cells in a cell bank are typically stored at very low
temperatures, such as in liquid nitrogen, to maintain their viability over an extended period.
- Cell Isolation → obtaining specific cell types from tissues or complex cell mixtures. This can
involve enzymatic digestion, mechanical dissociation, or other methods to separate and
isolate cells of interest. After isolation, these cells can be cultured or used for various
experimental purposes.
There are 3 types of growth format; adhered, suspension, semi-adherent.
1. Adherent Culture: Involves growing cells as a monolayer attached to a solid surface (e.g.
tissue culture dish or flask). Most cells in the human body are adherent and require a surface
for attachment and growth. E.g. endothelial cells, spiked morphology.
Characteristics:
- Cells adhere to the culture substrate; flattened and spread-out morphology
- Adhesion is often mediated by interactions w/ extracellular matrix proteins or artificial
coatings on the culture vessel
- Typically for cell lines derived from tissues like fibroblasts, epithelial cells, and endothelial
cells
Advantages:
- Easy to observe and manipulate under a microscope
- Mimics natural environment of many cells in body
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2. Suspension Culture: Involves growing cells in a liquid medium without attaching to a solid
surface. Cells are free-floating, and this method is commonly used for cells that naturally
exist in a fluid environment, such as blood cells or certain types of immune cells. Cervical
shape.
Characteristics:
- Cells maintain a more spherical or irregular shape
- No substrate attachment, allowing cells to grow in suspension
- Cells can be kept in constant motion w/ the medium
Advantages:
- Well-suited for cell types that do not naturally adhere
- Allows for the production of cells in large quantities
- Easier harvesting of cells w/o the need for mechanical or enzymatic detachment → do not
need to stress (mechan/ enzym) them and therefore do not damage them
3. Semi-adherent culture: Involves cells that exhibit characteristics of both adherent and
suspension cultures. Some cells in the culture may adhere to the surface, while others
remain in suspension.
Characteristics:
- Cells may switch between adherent and suspension states based on condition or cell types
- Provides flexibility to study different phases of cell behavior
- E.g.s include certain cancer cells that can transition between adherent and non-adherent
states
Advantages:
- Allows for the study of dynamic changes in cell behavior
- Suitable for cell types that exhibit a spectrum of adhesion characteristics
General steps of cell culture
1. Set up a sterile environment: This includes working in a laminar flow hood, sterilizing all
equipment and surfaces, and wearing appropriate personal protective equipment; use of
antibiotics can help, but it is not recommended to use long-term
2. Prepare the culture medium: The culture medium is the nutrient-rich solution that provides
the necessary nutrients for cell growth. It is prepared by combining basal medium with
supplements such as fetal bovine serum (FBS), growth factors (GFs), and antibiotics.
3. Seed the cells (= placing cells onto cultured vessel): Cells are seeded onto a culture vessel,
such as a petri dish or a flask, containing the culture medium. The seeding density depends
on the specific cell type and experimental requirements.
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4. Incubate the cells: The culture vessel with the seeded cells is placed in an incubator set at
optimal conditions for cell growth, including temperature, humidity, and gas composition
(usually 5% CO2).
5. Monitor cell growth: The cells are regularly monitored for growth and confluency (= refers to
the extent to which the surface of a culture vessel, such as a tissue culture dish or flask, is
covered by adherent) cells using a microscope. The culture medium may need to be
replenished periodically to provide fresh nutrients.
6. Passage the cells: When the cells reach a certain confluency, they need to be passaged or
subcultured to a new culture vessel to prevent overcrowding. This involves detaching the
cells from the vessel surface using enzymatic or mechanical methods and transferring them
to a new vessel with fresh culture medium. → At 70-80% → change cells from dish.
Otherwise cells could degenerate or change into other cells. Main problem is when the
monolayer starts detaching.
7. Perform experiments or assays: Once the cells have reached the desired confluency or have
been passaged, they can be used for various experiments or assays to study specific research
questions or perform drug testing.
8. Maintain the cells: Continuous monitoring and maintenance of the cell culture is required to
ensure their health and viability. This includes regular feeding with fresh culture medium,
checking for contamination, and maintaining optimal culture conditions.
The freezing of the cells is done by using nitric nitrogen.
Cells are preserved in a medium called DMSO.
Thawing Cells: In cell culture, cells are often cryopreserved at low temperatures (usually in liquid
nitrogen) for long-term storage. When researchers need to use these cells, they initiate the thawing
process to bring the cells back to a viable, liquid state. This typically involves transferring the frozen
vial containing the cells from the freezer to a warm environment, such as a water bath or incubator,
to allow the cells to thaw gradually.
Thawing: DMSO is toxic to the cells so countering this is important with a new fresh medium during
thawing. The DMSO liquid needs to be immediately replaced with a new medium by centrifuging
after thawing, pipetting away and then adding new medium.
The use of antibiotics in cell culture is a common practice, particularly during the isolation and initial
stages of establishing a cell culture. However, as you've mentioned, the prolonged and frequent use
of antibiotics should be approached with caution due to the potential development of antibiotic
resistance and other considerations.
Isolation and Initial Culture:
● Antibiotics are often used in cell culture during the isolation process to prevent
contamination from bacteria or fungi that may be present in the initial tissue or
sample.
● This use of antibiotics helps establish a clean and uncontaminated primary cell
culture.
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Growth curve can be made when working with cells.; 70-80% cells do not have the
confluence anymore/ no more space → should subculture cells onto separate plate.
- plateau phase
- 70-80% confluence
- exponential growth
A laminar flow hood, also known as a laminar flow cabinet or clean bench, is a piece of laboratory
equipment that provides a controlled and sterile work environment, primarily used to handle
materials that are sensitive to contamination. The laminar flow hood works by directing a continuous,
smooth airflow in a horizontal or vertical direction over the work area. This airflow passes through a
HEPA (high-efficiency particulate air) or ULPA (ultra-low penetration air) filter, which removes
particles and microorganisms from the air. As a result, the work surface and any materials placed
within the hood remain clean and free from contaminants.
2 main types of laminar flow hoods:
- Horizontal Laminar Flow Hood: In this type, the filtered air flows horizontally over the work
surface, providing a barrier between the work area and the external environment. Personnel
typically work with their arms inside the hood to maintain the sterile environment.
- Vertical Laminar Flow Hood: In this type, the filtered air flows vertically from the top of the
hood to the bottom, creating a downward laminar airflow. This design is often used when the
work involves hazardous or volatile materials because it prevents the release of contaminants
into the laboratory environment.
Laminar flow hoods are essential for various applications, such as cell culture, microbiological
research, sample preparation, and any work that requires a sterile environment. They help prevent
contamination of samples and experiments, ensuring reliable and accurate results in scientific
research and industrial processes.
Basal medium provides (energy) essential nutrients, such as carbon, nitrogen, salts, vitamins, and
minerals, necessary for the organisms' survival and multiplication. Basal media are designed to be
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