6.1 Cell Biology Methods

Question 1

What is tissue culture?

Answer 1

A terminology for procedures used for maintenance and growth of
cells or organs in an artificial and aseptic environment.

Question 2

What is an explants culture?

Answer 2

the culture of the whole or garments of an organ to study the development.

Question 3

What helped scientist improve tissue culture?

Answer 3

1. The use of antibiotics to control contamination;
2. Development of chemically defined, nutrient-rich media to grow cell
   lines;
3. The use of trypsin (protease) and collagenase to remove cells from the
   tissue and the plastic vessel

Question 4

What are trypsin and collagenase?

Answer 4

digestive enzymes used to break down small tissue pieces into even smaller size.

These enzymes degrade proteins that hold cells together but don’t’ degrade proteins that are inside the cell.

Question 5

Cells are incubated in what type of atmosphere?

Answer 5

one that contains about 5% carbon dioxide b/c it mimics the conditions found in the body more than ambient air.

Question 6

What are the advantages of cell cultures and what can be studied with cell cultures?

Answer 6

Advantage: cells are generally much more accessible for studies like microscopy than they would be in an animal and it’s possible to grow large quantities of an individual cell type for analysis.

-because cells properties including shape, genes, proteins, cell origin and function are maintained in a culture, scientist can study the biological activity of these cells and their proteins, gene expression patterns, as well as what these cells might have been doing in the animals own organs.

Question 7

What are the disadvantages of cell cultures?

Answer 7

-in many cases, the cells don’t behave in a culture exactly the way they do in the animal and therefore, scientist always have to consider whether their results reflect natural cell behaviors or, instead, artifacts caused by the cell culture environment.

Question 8

What are primary cells and why are they used by scientist?

Answer 8

• primary cells are cells obtained directly from tissues of an animal
• they are used by scientist who are concerned about the effects of long-term cell culture on the behavior of cells.

Question 9

What are the disadvantage of primary cells?

Answer 9

-The isolation of cells from tissues is time consuming and the
source of tissues may be rare.

-In addition, primary cells change dramatically over time as they
adapt to a cell culture condition.

-Primary cells also have a finite life span

Question 10

How often do cultured cells divide? what does this mean?

Answer 10

it divides about once every 24 hours. This means each division is a new generation of cells.

Question 11

What happens when cells in a dish covered the entire surface of the dish? what is the removal process called?

Answer 11

When cells grow over the entire surface of the dish they must be removed from the bottom of the dish and a portion of them added to a new dish in order to seed new growth. This is called passing cells.

Question 12

How many generations do primary cells usually survive?

Answer 12

about 25-50 cell generations and after that almost all of the cells die.

Question 13

How can cells be “immortalized”?

Answer 13

many cancer cells are immortal so introducing cancer-causing genes (oncogenes or telomerase genes) into them can immortalize non-cancerous cells

Question 14

What is the issue with non-cancerous cells being made immortal?

Answer 14

some of the cancer genes (oncogenes or telomerase) dramatically change the behavior of the cells they are expressed in.

Question 15

What are HeLa cells?

Answer 15

are human cervical carcinomas cells isolated from a woman
named Henrietta Lax who died of her cancer in 1951. It’s thought that more of Ms. Lax cells had
been produced after her death than were in her body while she was alive although this is possibly
an urban-legend. However, what’s interesting is that her cells were propagated without her
knowledge or consent and there was a subsequent lawsuit which was decided against her ruling
that her cells were no longer her property once they had been removed as a consequence of a
medical procedure for which she had given her consent. So there are some interesting ethical
issues that are around in cell biology

Question 16

What is contact inhibition of growth? &anchorage-dependence of growth?

Answer 16

this means cells don’t normally continue to divide when they are surrounded by others cells and they have to be attached to a surface in order to survive

Question 17

Which cells escaper contact inhibition of growth?

Answer 17

cancer cells and cells that have some active oncogenes expressed in them. Thy are anchorage dependent

Question 18

How could you increase the productivity of a culture chamber?

Answer 18

if you could get cells to lose their anchorage dependence but continue to produce useful proteins products

Question 19

What cells lack anchorage dependence ?

Answer 19

Blood cells (lymphoblast cells) lack anchorage dependence and can grow suspended in a flask that is shaking.

Question 20

Give a summary of primary cells

Answer 20

From living organism – normal cells

Have to isolate from living organism.

Have a finite lifespan.

Undergo replicative cell senescence.

Question 21

Give a summary of normal cells

Answer 21

Contact inhibited
o Grow as mono-layer on a plastic dish
o Reach confluence
o Anchorage-dependent

Question 22

Give a summary of cell lines

Answer 22

Easier to obtain (access)
o “Immortalized” cell line can grow continuously.
o Cell line can have abnormal chromosome number.

Question 23

Give a summary of transformed cells

Answer 23

Not contact inhibited
o Can grow as multi-layer on a plastic dish.
o Form foci
o Anchorage-dependent – can grow in suspension or in soft agar

Question 24

What are hybridomas?

Answer 24

Hybridomas are cells created by the fusion of an antibody-producing
cell and a tumor cell. It is then necessary to pick out the cells that produce useful quantities of antibodies from those that dont

Question 25

How are antibody-producing cells isolated to form hybridomas?

Answer 25

The antibody-producing cells are isolated from the spleen of an immunized
mouse or rat and, on their own, these cells would die shortly after they were isolated from the
animal. However, they are then chemically fused to tumor cells which are immortalized and the
resulting hybrid cells have capabilities of both precursor cells. In other words, they both produce
antibodies and grow indefinitely

Question 26

How are cells that produce useful quantities of antibodies separated from those that don’t?

Answer 26

cells are first separated into groups and then the groups are screened to see if any of them are producing a desired antibody. If one group is found that does this, then the group is then further diluted and divided and placed into a multi-well plates. Single cells in a well replicate until they fomr enough cells to allow more screening for antibodies.

Question 27

What are cloned cells?

Answer 27

a population of cells derived from a single pre-cursor cell and all of the cells are genetically identical

Question 28

What are monoclonal antibodies?

Answer 28

antibodies produced by cloned cells

Question 29

What are multipotent stem cells?

Answer 29

can differentiate into several other cell types. Typical of adult stem cells. used for wound healing or tissue regeneration.

Question 30

What are pluripotent stem cells?

Answer 30

can differentiate into all other cell types. Typical of embryonic stem cells

Question 31

Why is it likely that stem cells will soon be created from a patients adult stem cells in the near future?

Answer 31

More recently, it has been
discovered that stem cells can develop from other cell types by turning on a limited set of genes
which predispose cells to become stem cell types.

Question 32

What is one of the major challenges of working with stem cells?

Answer 32

the culture condition used to grow them must be carefully controlled b/c any changes in their environment may trigger developmental progression and reduce pluripotency in ways that are not productive

Question 33

What are embryonic stem cells?

Answer 33

they are harvested from the inner mass of the early embryo and can proliferate indefinitely.

They have the ability to give rise to different differentiated cells after treatment with retinoic acid, hormones, growth factors, etc

Question 34

What is the first step in isolating specific cell types from a mixture of cells?

Answer 34

the first step is to create a suspension of individual cells that are all separated from one another. This is achieved by digestion any cell-to-cell connections with digestive enzymes of some sort. (trypsin, collagenase)

Question 35

What are the different techniques used to separate cell types from a mixture of cells?

Answer 35

1) Cell suspension by enzymatic digestion
2) Cell Sorting
3) Laser Capture microdissection
4) Use an unusual characteristic of specific cells.

Question 36

What are the different techniques for cell sorting?

Answer 36

1) By cell size: Gravity sedimentation through a density gradient
2) By Fluorescence activated cell sorter
3) by magnetic beads

Question 37

How can cells be sorted by size? (aka what is gravity sedimentation)

Answer 37

by placing a mixture of cells at the top of a solution that has different densities and allowing the
cells to settle through it and this can work surprisingly well for separating simple cell mixtures.
However, it is too crude to separate complex mixtures where many cells are similar in size.

Question 38

How do magnetic beads help sort cells?

Answer 38

using an antibody or some reagent that sticks tightly to the cell you want.

You can then attach the antibodies or binding molecule to the surface of tiny magnetic beads and these are stirred w/a cell suspension and then an opposing magnetic field is applied to the cell suspension to pull out any cells that have been attached to a bead.

Question 39

What is a difficulty with using magnetic beads to sort cells?

Answer 39

you do need a reagent that

sticks to the desired cell type with high affinity and these are sometimes hard to come by.

Question 40

How can cells be sorted by fluorescence activated cell sorter?

Answer 40

this method requires that the cells you are
trying to isolate have some sort of unique fluorescent property, like a dye or molecule associated with them, which can also be something like a fluorescent antibody that detects a unique cell
surface protein.

The cell suspension is then placed in a chamber with an ultra-fine nozzle which drips cells, one cell at a time past a fluorescence detector. The detector is sensitive enough to detect the fluorescence of individual cells.

The droplets containing the cells pass an electrically charged surface and the magnitude and polarity of the charge can be modified by the machines
electrical system. The result and changes of this charge shift the path of the falling droplets in
one direction or another and you can place test tubes in the path of the falling droplets to catch
the cells that have the right fluorescence properties and exclude the other ones DNA can be marked by fluorescence. 2N, N 4N cells are sorted by the amount of fluorescence

Question 41

How can Laser Capture Microdissection be used to sort/isolate cells?

Answer 41

what you do is you create a thin layer of tissue, usually by sectioning it with a razor blade, and then you embed the tissue in a special plastic matrix which can be cut with a laser (this kills the cells.)

The specimen is viewed under a microscope and there is a software system which is used to trace the outline of a particular group of cells or an area of tissue on the computer screen and the computer then drives the laser which cuts the desired area free of the surrounding tissue and this area can be as small as just a few cells in a very complex tissue.

Question 42

What is the advantage of using laser capture microdissection?

Answer 42

the advantage of this method is that you can isolate very
small sections of cells from very complicated tissues. For example, you could remove and
analyze cells from very small tumors in a complex organ like the brain.

Question 43

What is the disadvantage of using laser capture microdissection?

Answer 43

this method has not been used to isolate live cells and so the method is really used to study proteins or gene expression patterns in cells that are fixed and dead.

Question 44

What is differential adhesion?

Answer 44

some cells are highly resistant to pH changes
while other are not and, therefore, raising or lowering the pH of culture media for some period of time might kill off all of the undesired cells and leave only the desired cell-type behind.

Cells that don’t have adherent properties are usually washed away from the dish in the isolation process.