CELL SORTING
The multiparameter analysis of a heterogeneous cell suspension
allows to define subpopulations that can be physically separated
from the global population. For this purpose, separation criteria
are determined by the user (definition of the sorting areas of
interest) and any cell whose characteristics are between the
chosen values will be isolated.
Figure 1 : Principle of cell
sorting.
For this purpose, the liquid stream will be electrically charged,
then fractionated into a succession of droplets by vibrating the
nozzle with a piezoelectric system that will determine the frequency
of droplet formation (Figure 1). The droplet containing the desired
cell is deflected by passing through an electrostatic field and
collected in a collection vessel. If the cell belongs to an
unselected subpopulation or if the formed droplet does not contain a
cell, the liquid stream will not be charged and the droplet will be
discarded. Cell sorting requires a very high stability of the formed
jet to guarantee the purity of the harvested populations.
It is possible to sort, depending on the apparatus, from 1 to 6
different populations simultaneously in tubes or to recover the
cells directly in culture plates (6, 12, 24, 96 wells...) (Figure
2). The example of cells before and after sorting is shown in figure
3.
Figure 2 : The different ways of
sorting (on the left 4-way sorting + garbage can, on the right
sorting in 96-well plate)
Figure 3: Sorting of a
CD19+CD27+ population: Before sorting (top), After sorting
(bottom)
Limits of cell sorting :
Cells can be isolated with
purity levels above 99%. These cells can be re-cultured.
However, one should not forget the relative slowness of the
sorting: to obtain 106 cells from a population
initially representing 1% of the starting population, it would
take a relatively long time (9 hours and 15 minutes) if the
cells are very fragile, for example, whereas it takes only one
hour and 10 minutes if they are small and resistant (Table 3).
The high purity of populations sorted by FCM can therefore only
be achieved at the cost of a serious limitation of the number of
cells collected and constant critical monitoring during
separation.
Nozzle diameter
|
130
|
100
|
85
|
70
|
Pressure Psi (1atm=14,7 Psi)
|
10
|
25
|
45
|
70
|
Frequency (KHz)
|
11
|
34
|
49
|
90
|
Maximum speed (cells per second)
|
3000
|
7000
|
11000
|
24000
|
Cellules passées par heure en Millions
|
10,8
|
25,2
|
39,6
|
86,4
|
Time to sort 1 million cells representing
1% of the total
(if 100% sorting efficiency)
|
9h15
|
4h
|
2h30
|
1h10
|
Table 3 : Sorting constraints:
The diameter of the nozzle must be 3 to 5 times the size of
the cells. Fragile cells must be sorted at low pressure.
