Chapter 4- culturing and visualizing cells Flashcards
Robert Hooke
Used a microscope to look at a cork, coined the term cell since the rectangles he saw reminded him of monk cells.
Antoni van Leeuwenhoek
Observed microorganisms under a microscope- these became the first descriptions of live cells.
Schleiden and Schwann
Proposed that cells constitute the fundamental units of life in plants, animals, and single celled organisms.
Culturing definition
Isolated cells can be maintained in the laboratory under conditions that permit their survival and growth.
Advantages to culturing (3)
- Specific types of cells can be grown in culture, without other types of cells growing with them.
- Experimental conditions can be better controlled
- A single cell can be grown into a colony of many identical cells (clones). The clones are genetically identical cells.
Disadvantages to culturing
Cultured cells are not in their native setting- it’s not the same thing as the environment cells would have inside an animal.
Culture of animal cells requires (3)
Nutrient rich media, special solid surfaces, and for conditions to match the cell’s natural environment as closely as possible. This includes temperature, pH, and access to nutrients.
Culture medium
A nutrient rich liquid added to the dish/flask with the isolated cells.
How is temperature and humidity of the culture controlled?
The cultures are kept in incubators where temperature and humidity can be controlled.
How do researchers guard against the contamination of cell culture?
This includes adding antibiotics to the culture to reduce bacterial contamination and adding reagents to the culture medium.
Which nutrients does a culture medium need to supply? (4)
- 9 amino acids must be included in the media, as these amino acids can’t be synthesized by adult vertebrae cells.
- 3 other amino acids that are only made in specialized animal cells, so they are typically included in the media as well.
- Glutamine- serves as a nitrogen source
- Vitamins, salts, fatty acids, glucose, and serum (this is the fluid remaining after blood plasma clots).
Why is serum important for cultured cells?
Serum contains protein factors like insulin, transferrin (supplies iron), and growth factors. Certain cell types will require specialized growth factors that aren’t included in serum.
Why are cellular adhesion molecules important?
Most animal cells will only grow attached to a solid surface. CAMS bind to other cells or components of the extracellular matrix like collagen. The solid growth surface will usually be coated with extracellular matrix proteins so the cells will attach to them. These proteins can also come from the serum.
How long does it take for a cell colony to grow?
A single cell cultured on a glass or plastic dish will proliferate to form a mass (colony) in 4-14 days.
Primary cells
Cells isolated directly from tissues. Normal animal tissues or whole embryos are used to establish primary cell cultures.
How are cells prepared for a primary culture?
The interactions between cells and between the cell and the extracellular matrix must be broken.
To do this, tissue fragments are treated with a protease (like a collagen hydrolyzing enzyme) and a divalent cation chelator (which gets rid of free calcium in the medium). Once the cells are released, they can be put in dishes with the medium.
What is the importance of fibroblasts?
Fibroblasts are found in connective tissue. They produce extracellular matrix components, like collagen, that will bind to cells. Fibroblasts divide more rapidly than other cells and must be removed when other types of cells are being cultured.
Cell senescence
When cells are removed from an embryo or adult animal and cultured, the adherent cells will divide a finite number of times and then stop growing. Fetal fibroblasts will divide 50 times before stopping.
Cell strain
A lineage of cells originating from one initial primary culture. Cell strains can usually be frozen and resume growth after they thaw.
Under appropriate conditions, which cells are able to grow indefinitely?
Embryonic stem cells
How can cells be transformed so that they will grow indefinitely?
Cells that undergo an oncogenic transformation are able to grow indefinitely. This is observed in tumor cells, or in normal cells that mutate spontaneously. This is important because biologists want to be able to maintain cell cultures for more than 50 doublings.
Cell line
A culture of cells with an indefinite life span and is considered immortal. HeLa cells were the first cell line, and came from cervical cancer.
Aneuploid
Cells in immortal lines often have chromosomes with abnormal DNA sequences. These cells are said to be aneuploid.
A line with a single copy of most genes is useful for genetic analysis
Flow cytometer
Flows cells past a laser beam that measures the light they scatter and the fluorescence they emit. Therefore, it can quantify the cells in the mixture that are expressing the fluorescence protein.
What is the difference between a flow cytometer and FACS?
FACS can analyze the cells and separate them into another culture dish, a flow cytometer can only analyze the cells.
FACS
Fluorescence activated cell sorter- capable of both analyzing the cells and separating the fluorescent cells into another culture dish.
This procedure is frequently used to purify different types of white blood cells
Why is FACS used to purify WBCs?
Each white blood cell has distinctive proteins on its surface. The proteins will bind to their specific monoclonal antibodies. Biologists can add fluorescent dye linked to the antibody that goes with the specific cell surface protein. For example, only T cells have CD3 and Thy1 proteins, so they can be separated from a mixture based on these proteins.
Uses of flow cytometry (4)
- Measures a cell’s DNA content
- Determines the size and shape of a cell from the amount of scattered light
- Identifies specific cell types in a population
- Used to study signaling pathways in single cells.
How does flow cytometry measure a cell’s DNA content?
Measures a cell’s DNA content from the amount of fluorescence emitted from the DNA binding dye- these measurements are used to follow replication of DNA as the cell progresses through the cell cycle.
How does flow cytometry distinguish cell types in a population?
Identifies specific cell types in a population by using antibodies that are specific to a cell type. Fluorescent molecules are attached to the antibodies. Distinguishes cell types in blood samples
How is flow cytometry used to study signaling pathways in cells?
Stimulation of some signaling pathways causes the phosphorylation of specific proteins. Flow cytometry quantifies the phosphorylation levels of specific proteins in individual cells. Fluorescent reporter molecules are linked to phospho-specific antibodies. Flow cytometry will measure the phosphorylation of the proteins to assess the activation of signaling pathways.
How are epithelial cells grown in culture?
Many cell types function only when they are linked to other cells. Epithelial cells cover the internal and external surfaces of organs. They function by transporting molecules across the epithelial sheet. They are unable to do this when stored on plastic or glass. Therefore, there are containers with porous surfaces that act as a basal lamina. Epithelial cells can attach to it and form a two dimensional sheet.
Which cell line is used to study epithelial cells?
The Madin-Darby canine kidney (MDCK) cell line is used to study the formation/function of epithelial sheets.
How are cells able to grow three dimensionally?
Under appropriate conditions, MDCK cells can form a tubular structure that looks like the duct of a secretory gland or a tubular organ. The apical side lines the lumen, the basal side is in contact with the extracellular matrix.
How are adult stem cells different from embryonic stem cells?
Adult stem cells are necessary for tissue repair but are not pluripotent, like embryonic stem cells. Embryonic stem cells are able to give rise to any tissue. The division/differentiation of adult cells is controlled by many factors, like interactions with the cell matrix and other cells, response to soluble factors, and changes in gene expression.
How are adult stem cells used to make organoids?
Adult stem cells have been used to make intestinal organoids that had the crypt and villus structures of a normal small intestine. Brain organoids have also been developed, and have a similar structure to the fetal brain. Currently, organoids advance to the stage of development seen in a fetus but don’t continue to the organization seen in adult organs. These results show that cells have an intrinsic program to divide, differentiate, and organize themselves into complex structures.
Uses for organoids (4)
- Cells can be followed more easily in an organoid than in a living animal- we can examine early human brain development in more detail.
- The stem cells that are used to make organoids can be manipulated by introducing mutations and then seeing how they affect the development of an organoid.
- Might be able to test the effects of drugs on organoids, which is easier than it is for animals.
- Tumor cells from a patient can be used to make a tumor organoid in culture and use it to test the efficacy of drugs before they’re given to the patient.
Which problem of organ transplant can be resolved by organoids?
This solves the problem of immune rejection and can be used in the future to make synthetic organs.
Antibodies
Proteins secreted by white blood cells, and they have a high affinity for their antigen that they are secreted in response to.
Epitope
An epitope is a small region that contains a small amino acid sequence. Any antibody producing B lymphocyte is capable of making a single type of antibody that binds to a specific epitope on an antigen.
Polyclonal antibodies
Antigen exposure usually causes multiple different B lymphocyte clones that produce different antibodies. Most antigens contain multiple epitopes, and polyclonal antibodies are able to bind to different epitopes.
Monoclonal antibodies
Activated B lymphocytes form a clone of cells in the spleen or lymph nodes, and each cell produces identical antibodies. These antibodies will only bind to one epitope.
How are we able to produce monoclonal antibodies?
We need a line of immortal antibody producing cells. We can do this injecting an animal with an antigen and waiting for it to produce the B lymphocytes that will bind to that antigen. We extract these cells and mix them with multiple myeloma cells to form a hybridoma.
Hybridoma cells
These cells are screened with assays to determine which ones produce the monoclonal antibody of interest. Hybridoma cells are immortal, and each one produces the monoclonal antibody encoded by its B lymphocyte parent. We can grow the hybridoma cells indefinitely to mass produce the monoclonal antibody.
3 ways that hybridoma cells can be used
- They can be grown to mass produce the antibody of interest
- They can be injected into organisms to study cancer
- They can be frozen for later use
Which experiments can monoclonal antibodies be used for? (4)
- Affinity chromatography- to isolate and purify proteins.
- Immunofluorescence microscopy- to locate proteins in cells
- Immunoblotting to identify proteins in cell fractions
- They can bind to and inactive toxins in bacterial diseases
How are cell biological processes studied with cultured cells?
One way to understand a biological process is to interfere with part of the cell and assess the outcome- what will go wrong when a part of the cell is “broken”? Researchers can rely on naturally occurring genetic lesions like with genetic diseases, or they can manipulate cultured cells to interfere with the expression of specific components
How are drugs used in cell biological research?
We can analyze biological processes by treating cells with drugs that will bind to certain cell components and activate/inactivate them.
What is the resolution of a conventional light microscope?
2 micrometers
What are two major decisions to make when using a microscope?
- What microscope do I use?- all microscopes are designed for a different purpose
- How do I prepare the tissue/cells?
Resolution
The ability to distinguish between 2 closely positioned objects
How do we generate contrast in a microscope? (3)
- Dyes
- Manipulating light
- Computers
Categories of dyes (2)
- Calorimetric- absorb light and have color as a consequence
- Fluorescent dyes- absorb light- emit light at a different wavelength
Bright field microscopy/compound light microscopy
The oldest microscope technique, but the most popular. Uses calorimetric dyes. It has an objective lens and projection lens for viewing specimens, and the objective lens is used to collect light.
What type of experiment could bright field microscopy be used for?
Viewing live cells. However, these microscopes can’t see the fine details of cells, and the cell must be prepared so they can be visible. This is because live cells don’t absorb light.
Light microscopy protocol (5 steps)
The cells are fixed, dehydrated, embedded in paraffin, and sectioned using a microtome. They are then stained with a dye that allows the main structures to be visible.
What are fixatives used for?
Fixes the tissue as if it was in the lifelike state. Formaldehyde is a common fixative.
Formaldehyde
A fixative that cross links amino acids on adjacent molecules. It stabilizes protein-protein and protein-nucleic acid interactions and makes the molecules more stable for other procedures
How are cells/tissues dehydrated?
They are soaked in alcohol-water solutions. Ethanol is used to replace the water, and xylene is used to replace the ethanol- it is an organic compound that is compatible with the embedding medium.
Cryosectioning
This is an alternative to paraffin embedding. Samples can be quickly frozen in liquid nitrogen without dehydrating, and are then cut using a cryostat (similar to a microtome). It can be used to view the fine details of a cell under a microscope.
Cryosection applications
Allows for quick tissue biopsy in the operating room. Mohs surgery is used to treat skin cancer and takes a circular biopsy. Takes small amounts of tissue several times and views them using cryosections to determine if there are clear margins
Cryostat
Used for cryosectioning, basically a microtome but in a freezer
Phase contrast microscopy
An optical-microscopy technique that converts phase shifts in light passing through a transparent specimen to brightness changes in the image. Phase shifts themselves are invisible, but become visible when shown as brightness variations. The darkness or brightness of a region of a specimen depends on the refractive index of that region, or how much the light bends when it passes through the specimen.
What is phase contrast microscopy used for?
It’s used for observing single cells or thin cell layers, but not thick tissues. It’s most useful for examining the location and movement of larger organelles in live cells
What causes fibrosis in the lungs of covid patients?
Due to the storm of chemicals from the virus- lymphocytes load the lungs. Fibrosis means that there is lots of collagen due to inflammation and the lungs aren’t able to exchange oxygen correctly.
DIC microscopy
The light is split into two components and recombined after it passes through the specimen to observe the interference pattern. It generates contrast based on the refractive index of the specimen and its medium. DIC images represent thin optical sections of a specimen, and a 3D image can be created when the sections are reconstructed.
What do phase microscopy and DIC microscopy have in common?
They are both used in live cell microscopy. This is where the same cell is viewed at regular intervals over time to generate a movie of cell movement.
How do the images from bright field microscopy, phase microscopy, and DIC microscopy compare?
In bright field microscopy, the cells are barely visible as no special methods have been used to generate contrast. In phase microscopy, there is a bright halo surrounding the image. In DIC microscopy, there appears to be a shadow to one side due to differences the refraction index.
What can DIC microscopy be used for?
It is most often used to view extremely small details and thick objects. It can define the outlines of large organelles as well. Also important for single cell electrophysiology, like with patch clamping
Hematoxylin and eosin stain
Hematoxylin binds to basic amino acids, and eosin binds to acidic molecules, like DNA and the side chains of aspartate and glutamate. Because they have different binding processes, these stains allow different types of cells to be distinguished visually.
Patch clamping
Patch clamp records the ion flux to a single ion channel in the cell membrane. Used for action potential readings in single cell electrophysiology. DIC microscopy is important for this.
Dark field microscopy
Describes an illumination technique used to
enhance the contrast in unstained samples. It works by illuminating the sample with
light that will not be collected by the objective lense and thus will not form part of the image. This produces the classic appearance of a dark, almost black background
with bright objects on it
Why use dark field microscopy instead of bright field microscopy?
Good candidates for darkfield observation often have refractive
indices very close in value to that of their surroundings and are difficult to image
with conventional bright field techniques
Applications of dark field microscopy
Used for live, unstained specimens. Used in transmission electron microscopes, imaging of atoms and other small particles, as in microbiology.
Polarizing light microscopy
Polarizing light to analyze “highly ordered parallel structures”. Ex- collagen, microtubules, and microfilaments. A light source going through a polarizer creates polarized light (sort of like sunglasses work). Light goes through an analyzer and we can see the highly ordered structures in cells. This method is qualitative.
What is polarizing light microscopy used for?
Can be diagnostic for looking at fibrosis- the collection of collagen fibers present in diseases. These fibers are highly ordered.
Qualitative
We get an image
Two major limitations of conventional fluorescence microscopy
- The observer sees a blurred image due to the superposition of fluorescent images from molecules at different depths of the cell
- For thick specimens, images must be taken at different levels of the specimen and be reconstructed.
Deconvolution microscopy
Uses a computer to remove fluorescence from the out of focus parts of the sample and increase practical resolution.
Confocal microscopy
A microscope that has a microlens array disc incorporated into it and is designed to
visualize dynamic changes in cells revealed by fluorescent dyes with a minimal level of
photobleaching. They collect a series of vertical images and use them to create a 3D representation of the sample. Includes spinning disk and laser scanning confocal microscopy.
Point scanning confocal microscopy
Uses a point laser light source at the excitation wavelength to scan the focal plane, collect the fluorescence in a photomultiplier tube, and create a high resolution 2D or 3D image.
