The isolation of cellular aggregates
Charli Kruse, Institute for Medicine, Molecular Biology at the University of Lübeck, Germany
Nada Pavlovic, Eppendorf AG, Hamburg, Germany
Introduction
With the help of the Eppendorf MicroDissector, which
uses the so called Piezo-Power Microdissection (PPMD),
the isolation of dense cell areas from living tissue for further
individual cultivation has been successfully shown by
Rohwedel and Kruse.
Materials and methods
Cell cultivation
Primary cells from rat were cultivated. After approx. 3
weeks three-dimensional cellular aggregates, growing
adherent, are developing.
Aggregates with a size of about 150 µm were chosen and
isolated for further cultivation.
Devices
Eppendorf MicroDissector
2 TransferMan® NK 2
1 CellTram vario
Consumables
Eppendorf MicroChisel
Eppendorf CustomTip number 02/223 (type I, diameter 150 μm ID, shaft angle 35°)
Microdissection procedure
The cellular aggregate can be isolated from the surrounding
cells by using the MicroChisel, which is vibrating with
an ultrasonic frequency.
After the separation is completed, the aggregate is aspirated
into a glass capillary by applying a vacuum with the
manual piston pump CellTram vario and transferred to
another petri dish.
Downstream applications
After 48 hours the first cells begin to grow from the transferred
aggregate revealing successful separation of the
cells that are now multiplying.
PPMD step by step
Mounting the tools
The cutting tool (plus MicroChisel) and the capillary holder
(CellTram vario) can be mounted on the right or the
left-hand side of the microscope, depending on the
users preferences.
Mount the cutting tool with the MicroChisel at a steep
angle of approx. 40 – 45°.
According to experiences with several tissues, we
recommend to mount the capillary at an angle of 35°.
The angles can vary according to capillary angle, type of
petri dish and amount of medium, which is used. In this
case standard dishes were used with a diameter of 5 cm
and 4 ml standard culture medium.
Cutting side
Focus on the MicroChisel. Choose the magnification
according to the tissue you are working on. Target on an
area of high cell density and choose a cell aggregate,
which shows a size of approx. 150 µm (Figure 1).
(Sizes of a cell can be easily judged by using the functions
of the TransferMan NK 2. Set the coordinates to “0”
- operating manual page 90. Measure the cell size with
the help of the coordinates by moving the mounted tool
e.g. MicroChisel along the area of interest.)
Setting the parameters
The frequency is set to a high level of approx. 40 - 55 kHz,
in correspondence with the interaction between the Micro-
Chisel and the tissue. Set the amplitude to approx. 50 %.
The MicroChisel is activated by pressing the appropriate e.g. right foot control.
What should be seen:
● The orange LED marked as “External control” lights up.
● Under the microscope you will see the MicroChisel
oscillating and can now fine-tune the frequency and
amplitude by observing the interaction of the MicroChisel
with the tissue.
● Irregular or very strong swinging of the MicroChisel
should be avoided.
● Setting a Z-limit by pressing the “Limit” button
(TransferMan NK 2) is very practical to control the
height, where the cutting is taking place.
Aspiration side
Bring the glass capillary into focus.
Choose the magnification according to the tissue you are
working on. Before starting with the microdissection procedure
the micropipette can be transferred to a “parking”
position a little bit above the cells of interest.
Hint: Positions can be stored using the buttons on the
manipulator´s control board “Pos 1 – 3”.
Microdissection and collection of the nodule
● Lower the MicroChisel to the petri dish bottom.
● Trigger the oscillation by pressing the appropriate foot
control.
● Start to microdissect the cell area at a high frequency.
● In order to achieve a complete and atraumatical resolving
of the adherent growing cell aggregate you cut into
the plastic of the dish; define a Z-limit (Eppendorf
TransferMan NK2) at that level. The ideal way to do that is
to move the MicroChisel above the cell - first from left to
right (Figure 2).
The oscillation resonance enables the resolving of the cell
from the dish.
● Now stop the cutting and start again from left to right
below the cell (Figure 3).
● Now resolve the cell aggregate carefully from the dish
without the oscillating function by scraping.
● Put the cutting tool a little bit backwards.
● Lower the glass capillary (CellTram vario) immediately
after cutting (Figure 4).
● Move the capillary to the cut cell aggregate.
● Aspirate this area with the CellTram vario by moving
the piston counterclockwise (Figure 5).
● Move up the capillary and the cutting tool manually.
● Exchange the petri dish.
● Transfer the aggregate to another dish by first bringing
the capillary manually down and release the cells carefully
by moving the piston of the CellTram vario clockwise
(Fig. 6). Observe the cells after 24 (Figure 7) and 48 hours
(Figure 8).
Remarks and troubleshooting
Although the MicroChisel oscillates well, the cutting outline is not correct.
● The angle of mounting of the cutting tool may not be steep enough (app. 40 - 45°).
● The MicroChisel has to be replaced; it might be blunt.
The cell doesn't resolve from the bottom.
● Cut into the plastic of the dish. By that technique the
oscillation of the MicroChisel helps to resolve the cells
from the bottom.
The cell can´t be loosened from the bottom.
● Cut into the plastic of the dish. By that technique the
oscillation of the MicroChisel helps to loosen the cells
from the bottom.
References
Kramer et al. (2000): Embryonic stem cell-derived chondrogenic
differentiation in vitro: activation by BMP-2 and
BMP-4. Mechanisms of Development 92, 193 – 205.
back to top