4.8

pointdepressedMécanique

22 févr. 2014 (il y a 3 années et 7 mois)

96 vue(s)

Rozaleth Role


Tissue Culture


Assignment 18


Apr. 8, 2010


F15


Cell Separation


After cloning the cells, selecting and separating the cells that are healthy and viable is the next step.
There are 4 well known separation techniques:


-
Cell Density or
specific gravity


-
Affinity of antibodies to cell surface epitopes


-
cell size


-
light scatter or fluorescent emission as sorted by flow cytometry

The first 2 techniques are inexpensive and requires a not much high level of technology. The second
2, howeve
r, requires a lot of money and high technology is needed.


Separation using cell density depends on the gravity of the cells. So, this type of separation is done
by the centrifugation of the cells at low gravity with conventional equipment. Using a density

medium can also help with separation through cell density. This medium should be nontoxic and is
not viscous at high density. The media also should have a little more osmotic pressure compared to
other types of media. Density medium may consist of serum a
lbumin, dextran, Ficoll or Percoll.


Variations can be done to acquire the best results. The position of the cells is important because its
position may affect the gradient during the formation of the cells by centrifugation. Other media
such as Ficoll ca
n be used. Ficoll is the preferred media because it can be autoclaved, is a little more
viscous than Percoll and may cause the cells to agglutinate which is helpful during cell separation.
Marker beads are used during Isopyknic sedimentation. With the mark
er beads present,
sedimentation may occur quicker and this may lead to higher yields of cells for a given volume of
gradient.


The following things may affect cell density:


-
the uptake of the medium used to form the gradient


-
position of the cells in th
e growth cycle


-
serum


Sedimentation is affected by cells size.
The cell size can verify the sedimentation velocity. Using unit
gravity sedimentation is not really the preferred technique because this type of sedimentation
cannot handle a large amount of
cells, cell separation does not work that much when cell size are
quire uniform and when the cell population is homogenous in size. Centrifugal elutriation requires a
specialized centrifuge and rotor. With a specialized centrifuge, the sedimentation rate i
ncreases and
the yield and resolution of cell separation is way better.


Another technique used to separate cells is the antibody
-
based technique. This is when antibodies
are presented in the solution with cells and whichever cell attaches to the binding
site, this lead to
separation. One way to do this is by immune panning. This is when the cells are placed in a dish
coated with antibodies. This may either be used to select a specific subset of cells or to remove
unwanted cells. Magnetic sorting uses anti
bodies that are joined with a ferritin bead or microbeads.
When the cells are placed in the solution with the beads, those with the antibody binding site will
attach to it. And with a magnet, those that are selected through the ferritin beads are separated
.
This allows the cells to be cultured or be processed without the need to remove the beads.


With fluorescence
-
activated cell sorting,
the cells are projected through a laser beam. By doing this,
the cells are then exposed to light and the light that ref
lects from it scatters. Using a photomultiplier,
the light scattered is detected and is then recorded.
With the help of a flow cytometer, the data
from the photomultiplier is processed and the flow cytometer analyzes the cell population. With a
fluorescenc
e
-
activated cell sorter, the signals emitted from the cells are sorted by placing them into
a collecting tube for the selected cells and those that are not selected are placed into waste.


Other techniques used are:


-
electrophoresis


done on either the F
icoll gradient or a curtain electrophoseis


-
affinity chromatography


use antibodies or plant lectins


-
concurrent distribution


used to purify murine ascite tumor cells


For a beginner that is practicing cell separation, the best technique to start with

is the density
gradient centrifugation. If the aim is to select cells that ha
ve

specific cell surface phenotype, immune
panning or MACS may be done. To acquire the purest cell population, FACS can be used and to have
a fast sorting of large amounts of cel
ls, use centrifugal elutriation.



F16


Characterization