The isolation of living cartilage cells (chondrocytes)
Jürgen Rohwedel and Charly Kruse, Institute for Medicine, Molecular Biology
at the University of Lübeck, 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
Embryonic stem cells
from mouse were
cultivated as so called
„embryoid
bodies“ (EBs). Fig. 1
shows these EBs at
low magnification.
After 2 - 3 weeks of
cultivation, cartilage cells develop and form areas of high
cell density, so-called cartilage "nodules“ (N), which are
illustrated in figures
1, 2 and 3 in different
degrees of magnification.
Fig. 3 shows a nodule
in relation to the
Filtertip MDS (F) and
the MicroChisel (M).
Devices
1 Eppendorf Micro-Dissector
2 TransferMan NK 2
Consumables
Eppendorf MicroChisel
Eppendorf Filtertips
MDS, inner diameter
150 µm
Microdissection procedure
Two micromanipulators were used to move both the cutting
tool with MicroChisel and the micropipette with the
Filtertips (MDS).
The nodule (N) can be isolated from the surrounding cells
by using the MicroChisel (M), which vibrates with an
ultrasonic frequency (Fig. 4). After the separation is completed,
the nodule is pipetted with the Filtertip MDS (F)
and transferred to another petri dish for further cultivation.
Downstream applications
After 24 hours, the first cells begin to grow fom the
nodule (Fig. 5).
Fig. 6 shows an enlargement of the framed sector of Fig. 5.
Figures 7 and 8 show the very same nodule after a period
of 3 days, distinctly revealing successful separation of
the cells that are now multiplying.
References
[1] 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.
PPMD step by step
Mounting the tools
The cutting tool (plus MicroChisel) and the aspirating tool
(plus Filtertip MDS) can be mounted on the right or the
left-hand side of the microscope, depending upon the
preferences of the user.
Mount the cutting tool with the MicroChisel at a steep
angle of approx. 40 – 45°. According to experiences with
several types of tissues, we recommend to mount the
aspiration tool, the micropipette, at an angle of approx. 40°.
The angles can vary according to the 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
“nodule” (N) (Fig. 2), which shows a size of app. 150 µm.
(The size of a cell area can be easily judged by using the
functions of the TransferMan NK 2. Set the coordinates
to “0” - operating manual page 90. Measure the size
with the help of the coordinates shown on the manipulator
´s display 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 app. 50 %.
Pressing the appropriate, e.g. right foot control activates
the MicroChisel. 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.
As the cartilage is a three-dimensional, rather thick area,
the frequency will be optimized in the high range in order
to be able to penetrate the strong structure.
Aspiration side
Bring the Filtertip MDS into focus. Choose the magnification
according to the tissue you are working on.
Place the hand control in a reachable position in front of
you: The left-hand key of the three-key hand control is
used to perform a step-by-step aspiration. In this application
the step-by-step mode is preferable to the continuous
aspiration to prevent the uptake of too much media.
The green LED marked “Uptake/Step” and located between
the volume and speed regulators lights up until aspiration
is finished. The volume should be set to approx. 10
– the speed to 3 - 5.
If you want to release your collected sample, the righthand
key of the hand control has to be hold down.
The corresponding LED remains lit for as long as the key is
held down. You can observe, that the value in the display
(Dispensed Volume) increases in size. In this way sensitive
samples, e.g. isolated cells, can be dispensed gently.
● Preadjust everything with a control cell, if possible.
● Set the "Dispensed volume" of the pipette to approx.
80 % before you start by pressing the left key of the
hand control. Otherwise capillary forces might be
deceiving.
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 display “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
while the aspirating pipette is in HOME-position.
● In order to achieve a complete and atraumatic resolving
of the adherent growing cell you cut into the plastic of the
dish; define a Z-limit at that level. The oscillation resonance
enables the resolving of the cell area from the dish.
The ideal way to do this is to move the MicroChisel above
the cells - first from left to right.
● Stop the cutting and start again from left to right below
the cells.
● Resolve the nodule carefully from the dish by scraping
without the oscillating function.
● Put the cutting tool a little bit backwards.
● Lower the aspirating tool immediately after cutting.
● Move the Filtertip MDS to the nodule.
● Aspirate the nodule with the micropipette in a stepwise
mode (Uptake/ Step) by using the left key of the
hand control.
● Option: Alternatively the CellTram vario can be used.
● Move up the aspirating tool and the cutting tool manually.
● Exchange the petri dish.
● Transfer the nodule to another dish by first bringing the
aspirating tool manually down and then releasing the cell
carefully by pressing the right key of the hand control
using a low speed between 3 and 5.
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 aspirating tool is not working adequate.
● Please check the condition of the black O-ring in the
nose cone of the adapter (operating manual page 15).
(You can do this with the help of a thin metal tool, which
is available e.g. in the O-ring set, ord. number 5176
196.000.)
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.
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