/. Embryol exp. Morph. Vol. 59, pp. 145-155, 1980
Printed in Great Britain © Company of Biologists Limited 1980
145
Changes in the surface coat of mesenchymal cells
of mouse limb buds after enzymatic cell separation
EVAMARIA KOHNERT-STAVENHAGEN
AND BERND ZIMMERMANN1
From the Institutfur Toxikologie und EmbryonaJpharmakologie der
Freien Universitdt Berlin {Project: Prof. Dr H.-J. Merker)
SUMMARY
Isolation of cells is nowadays performed by enzymatic means. The influence of such
enzymes on the surface coat of mesenchymal and blastemal cells during the dissociation of
limb buds from 11 -day-old mouse embryos was studied electron microscopically after staining
with ruthenium red. EGTA or collagenase failed to bring about cell separation. The surface
coat seemed to be unchanged after collagenase treatment. After EGTA an increase in extracellular filaments was observed. The proteases a-chymotrypsin, dispase II, papain, pronase P
and trypsin (0-2%, 37 °C, 20 min) succeeded in completely dissociating limb buds. Apart
from single granules, there was a detachment of the surface coat from the cells in all cases
studied. Hyaluronidase led to only partial separation, but the detachment of the surface coat
was almost complete, indicating a GAG-rich surface layer on these cells.
INTRODUCTION
Enzymatic dissociation of an organ into single cells is a common means for
the preparation of primary cell cultures from solid organs (Moscona, 1952;
Lasfargues, 1956; Rinaldini, 1958). In most cases proteases such as trypsin or
pronases, or specific enzymes such as elastase (Rinaldini, 1959) or collagenase
and hyaluronidase (Seglen, 1972) are used. Ca2+ deficiency with EDTA or
EGTA may also lead to organ dissociation. In a number of cases a chelating
agent is combined with a protease, or a Ca2+- and Mg2+-free medium is used
(Moscona, 1952; Wiepjes & Prop, 1970).
During organ isolations not only disintegration of the intercellular matrix but
also detachment of the cellular contacts (Fukuyama, Black & Epstein, 1974)
and formation of microvilli on the cells (Follet & Goldman, 1970) are observed.
In addition, detachment of glycosaminoglycans (GAG) and glycoproteins from
the surface coat of the cells takes place (Kraemer & Tobey, 1972; Ceriani,
Petersen & Abraham 1978). Concomitantly the electrophoretic motility of
freshly isolated cells changes (Barnard, Weiss & Ratliffe, 1969; Maslow, 1970)
as well as cell adhesion, cellular growth (Poste, 1971) and the reaggregation
behaviour of the cells (Wiseman & Hammond, 1976).
1
Author's address: Institut fur Toxikologie und Embryonalpharmakologie, Garystrasse
1-9, D-l000 Berlin 33, Germany.
146
E. KOHNERT-STAVENHAGEN AND B. ZIMMERMANN
The surface coat, consisting of oligosaccharide chains of membrane-integrated
glycoproteins, of glycolipids, and of membrane-associated proteoglycans and
GAGs (Rambourg, 1971; Bretscher, 1973; Sturgess, Moscarello & Schachter,
1978), can be observed by different methods (e.g. ruthenium red) and has been
seen in all cells so far studied (Rambourg, Neutra & Leblond, 1966; Rambourg
& Leblond, 1967; Luft, 1966; Behnke, 1968; Friess & Liebich, 1972; Latta,
Johnston & Stanley, 1975).
Numerous functions are attributed to this cell surface coat during cell recognition, adhesion and migration as well as in the communication between
cells and extracellular spaces (cf. Marchesi, Ginsburg, Robbins & Fox, 1978;
McClain & Edelman, 1978).
The surface coat is of great significance in the behaviour of the cells during
morphogenesis and in vitro. Therefore we investigated the surface coat of the
cells after isolation by electron microscopy. We performed these studies on
embryonic mesenchymal and blastemal cells of mouse limb buds. In this tissue
the surface coat is expected to be of great morphogenetic significance. Furthermore, the morphology of this layer is not as yet exactly understood.
MATERIALS AND METHODS
NMRT mice were kept at a reversed day/night cycle. The day of conception
was considered as day 0*of development. The upper limb buds of 11-day-old
mouse embryos were cut off at the trunk, rinsed once in Hanks BSS and subsequently incubated for 20 min in 20 ml enzyme solution at 37 °C in a water
bath at a shake frequency of 90/min. For cell isolation the suspension was
pipetted four to five times, made up with Hanks BSS to double its original
volume and centrifuged at 50 x g for 10 min. The cell sediment was washed
once in Hanks BSS and then fixed in ruthenium red/glutaraldehyde. Ruthenium
red was used for demonstration of the surface coat, because it leads to intensification of contrast of glycosaminoglycans and proteoglycans in the electron
microscope (Luft, 1966; Rambourg, 1971; Kelley & Lauer, 1975).
Fixation was done according to Luft (1972): 1 h in 1 % glutaraldehyde with
1-5 g ruthenium red/1 in 0-1 M cacodylate buffer pH 7-4, rinsing in 0-1 M cacodylate buffer with 800 mg ruthenium red/1, postfixation for 1 h in 1 % OsO4
with 400 mg ruthenium red/1 in 0*1 M cacodylate buffer. This was followed by
thoroughly rinsing in 0-9% NaCl, dehydration in acetone and embedding in
Mikropal (Ferak, Berlin, Germany). Thin sections were prepared on an LKBmicrotome; post-staining was performed with lead citrate and uranyl acetate in
aqueous solution. Pictures were made using a Siemens 101 and a Zeiss EM9
electron microscope.
Effects of enzymes on surface coats of mouse mesenchymal cells
147
The following solutions were used
Ca- and Mg-free solution (CMF: 8 0 g/1 NaCl, 0-3 g/1 KC1, 0-05 g/1
NaHPO 4 .H 2 O, 0-025 g/1 KH 2 PO 4 1-0 g/1 NaHCO 3 , 2-0 g/1 glucose (Wiepjes
and Prop, 1970). 2mM EGTA (Serva, Heidelberg, Germany) in CMF; 0-2%
a-chymotrypsin, 45 U/mg (Serva, Heidelberg, Germany) in CMF; 0-2% collagenase, 0-15 U/mg (Boehringer, Mannheim, Germany) in Hanks BSS; 0-2%
dispase II, 0-5 U/mg (Boehringer, Mannheim, Germany); 0-2 % hyaluronidase,
250USP-U/mg (Merck, Darmstadt, Germany) in CMF; 0-2% papain, 3-5 m
Anson-U/mg (Merck, Darmstadt, Germany) in CMF; 0-2% pronase P, 45000
PUK-U/mg (Serva, Heidelberg, Germany) in CMF; 0-2% trypsin 1:250,
4 U/mg (Serva, Heidelberg, Germany) in CMF.
RESULTS
Treatment of 11-day-old mouse limb buds with Ca2+- and Mg2+-free solution
(CMF) failed to bring about cell separation. The electron microscopical picture
corresponded to that of untreated controls: in the peripheral and distal regions
of the limb buds loosely packed, undifferentiated mesenchymal cells with
irregular nuclei, organelle-poor, ribosome-rich cytoplasm and irregular cell
processes were observed (Fig. 1 a).
At higher magnification a surface coat could be recognized on the cell membrane of these cells. This surface coat either formed a regular layer or consisted
of isolated granules or plaques (Fig. \b). Indentations, augmentation and
intensification of staining was often observed on the membrane.
At high magnification a surface coat of about 25 nm thickness became
recognizable. It clearly shows a filamentous structure linked to the membranous bi layer. More concentrated areas of surface coat alternated with less
concentrated ones (Fig. 1 c).
In the central regions of the limb buds the formation of the blastema was
already completed at this stage. The cells were densely packed; many ribosomes
were still to be seen in the cytoplasm, and mitochondria and rough endoplasmic
reticulum occurred to an ever increasing extent (Fig. 2a).
At higher magnification single granules of ruthenium-red-stainable material
were visible at membranes in the intercellular space. Between membranes of
adjacent cells a thin layer of surface coat was recognizable (Fig. 2b). After
treatment with 2 ml EGTA in CMF cell separation was not successful. But in
contrast to controls, electron-dense, about 5 nm-thick filaments were seen,
which extended far into the extracellular space and inserted into adjacent cells
(Fig. 3).
Treatment with various proteases (a-chymotrypsin, dispase II, papain,
pronase P, trypsin) in all cases led to complete isolation. The electron
microscopical picture of the cell membrane was uniform. There was an almost
148
E. KOHNERT-STAYENHAGEN AND B. ZIMMERMANN
Effects of enzymes on surface coats of mouse mesenchymal cells
149
complete loss of the surface coat. But single 25 nm granules with fine radiating
filaments were still present (Fig. 4). Lesions of the membranous bilayer were
never detected.
After trypsin treatment one peculiarity was striking. While, after the other
proteases part of the surface coat material was distributed in small fragments
almost evenly in the extracellular space, after trypsin an almost complete
detachment of the surface coat over wide areas seemed to have taken place.
Under these conditions, a continuous surface coat layer, which was detached
from the cell membrane over long distances, was often seen (Fig. 5). After an
0-1 % trypsin concentration this effect was especially pronounced.
After hyaluronidase, dissociation of the blastema was incomplete. Even after
thorough pipetting tissue fragments remained. The single cells that were
obtained after sedimentation of these fragments from the supernatant showed
on the cell membrane clearly visible remainders of the ruthenium-red-positive
surface coat, which were thinner (15 nm) than those on untreated cells (Fig. 6).
After collagenase treatment dissociation of the blastema was not observed.
There were units of mesenchymal and blastemal cells as in controls. Their
surface coat was unchanged.
DISCUSSION
An electron-dense, about 25 nm-thick, surface coat is seen on the cell membrane of blastemal and mesenchymal cells of 11-day-old mouse embryos after
ruthenium-red staining. It seems certain, that this ruthenium-red stainable
coat is strongly correlated to the biochemical findings on the membrane glycoproteins and GAGs. This surface coat either consists of a continuous layer, or
granules and plaques. According to what is known of the structure of the
surface coat, sensitivity towards proteolytic treatment is to be expected
(Bretscher, 1973; Sturgess et al. 1978).
In our experiments, too, this layer disappears after cell isolation with the
different proteases. However, attribution of the isolated cells to the mesenchymal or blastemal part is no longer possible. In all cells investigated the same
FIGURE 1
(a) Mesenchymal cells of limb buds from 11-day-old mouse embryos. The irregularly shaped cells have an oval, sometimes lobular, nucleus, homogenous cytoplasm
and coarse cell processes. N = nucleus, C = cytoplasm, M = unstructured matrix,
x 3600.
(b) At higher magnification after ruthenium red staining, an irregular, partly continuous (arrow heads), partly granular (arrows) surface coat is observed, which is
concentrated in cell indentations (dark arrows). N = nucleus, C = cytoplasm,
x 9200.
(c) At high magnification the filamentous structure of the surface coat can be
demonstrated (arrows). Besides loosely packed areas, aggregations of the surface
coat (arrow heads) are seen. The average thickness is about 25 nm. x 150000.
150
E. KOHNERT-STAVENHAGEN AND B. ZIMMERMANN
Fig. 2(a). Blastema of the limb bud from an 11-day-old mouse embryo. Cells with
round-oval nuclei (N) in close contact, only little extracellular space (arrow heads):
x 3600. (b) At higher magnification surface coat granules are detected at the
membrane with empty extracellular space (arrows) and in a uniform layer between
the cells (arrowheads): N = nucleus, C = cytoplasm, M = mitochondria (x 19000).
Effects of enzymes on surface coats of mouse mzsenchymal cells
151
effect is seen: proteases cause complete dissociation of limb buds. Except for a
few granules, the ruthenium-red-positive surface coat disappears. We do not
assume that cell isolation is only possible when the whole surface coat is
destroyed by proteolytic treatment. Detachment of the surface coat should
rather be considered as an additional effect of the proteases, since trypsin
treatment at + 4 °C also causes dissociation of the different tissues without,
however, reducing the surface coat which can be demonstrated with ruthenium
red (Caravita & Zacchei, 1974). This is also supported by the observation that
with trypsin treatment at low temperatures ( + 4 °C, +15 °C) number and size
of colonies are increased after culture of isolated cells (McKeehan, 1977). It is
possible that under these conditions only substances which are responsible for
adhesion are disintegrated. Investigations on other cell types support this
assumption. Although extensive detachment of the surface coat can be achieved
with trypsin (Onodera & Sheinin, 1970; Anghileri & Dermietzel, 1976; Cox
Bour & Haenelt, 1977; Vogel, 1978) or papain (Forstner, 1971), in certain
systems complete detachment of the surface coat cannot be reached. Some
glycoproteins on the membrane of erythrocytes (Jackson, Seegrest & Marchesi,
1971) as well as on the membrane of fibroblasts in vitro (Huet & Herzberg, 1973;
Kelley & Lauer, 1975) are resistant towards trypsin treatment. These differences
may be attributed to a different concentration or activity of trypsin (Huggins,
Chestnut, Durham & Caraway, 1976; Friedlander & Fischman, 1977) and to
a different composition of glycoproteins in the surface coat of various cell types.
In the cells investigated in the present studies, surface structures which do not
detach after proteases and therefore possibly represent glycolipids could be
demonstrated in all cases.
The resistance of the surface coat towards chelating agents has been described
by many authors (Huet & Herzberg, 1973; Kelley & Lauer, 1975; Cox et al.
1977; Vogel, 1978). It can therefore be assumed that the components of the
surface coat are not linked via ionic linkages but covalently bound (Vogel &
Dolde, 1979). The formation of fine filaments described here after EGTA
indicates that Ca 2+ may have a function during inner organization and stabilization of the surface coat. This is also supported by the fact that about 90 % of
the total Ca2+ of HeLa cells are localized in the cell coat (Borle, 1968).
Hyaluronidase caused detachment of a large amount of the surface coat
without yielding satisfactory cell isolation. Cell adhesion in the limb bud
blastema therefore does not primarily depend upon the effect of GAGs, but a
large proportion of the surface coat seems to consist of GAGs. In other cells,
too, above all in monolayer cells in vitro, GAGs were observed in the surface
coat (Vogel & Kelley, 1977; Kraemer & Barnhart, 1978, Barnhart, Cox &
Kraemer, 1979). The high percentage of hyaluronidase-soluble material in the
surface coat of limb bud cells seems to be interesting in view of the findings of
Toole and co-workers (Toole, 1972; Toole, Jackson & Gross 1972) indicating
that hyaluronate stimulates cell migration in the mesenchyme and inhibits
152
E. KOHNERT-STAVENHAGEN AND B. ZIMMERMANN
Effects of enzymes on surface coats of mouse mesenchymal cells
Fig. 7. An untreated mesenchymal cell shows a continuous layer of ruthenium redstainable material of about 20 nm thickness, x 56000.
FIGURES
3-6
Fig. 3. Mesenchymal cells of limb buds treated with 2 mu EGTA for 20 min at 37 °C.
The surface coat seems to be thicker and more loosely packed. Fine filaments are
clearly seen. These protrude into the extracellular space and insert into adjacent
cells (arrows). C = cytoplasm, M = unstructured matrix, x 56000.
Fig. 4. Treatment with 0-2 % a-chymotrypsin led to complete dissociation into single
cells. Except for a few granules (arrows) complete detachment of the surface coat is
seen. Similar results are obtained with papain, pronase P, dispase II and trypsin.
C = cytoplasm with free ribosomes. x 56000.
Fig. 5. This picture shows partial detachment (arrow) of a continuous layer (arrow
heads) of surface coat material after treatment with 0-2 % trypsin. C = cytoplasm.
x 19000.
Fig. 6. Treatment with 0-2 % hyaluronidase does not cause complete detachment of
the surface coat. Small amounts of the surface coat material are still recognizable
on the cell membrane (arrow heads). C = cytoplasm, x 56000.
153
154
E. KOHNERT-STAVENHAGEN AND B. ZIMMERMANN
differentiation. Consequently, a high concentration of hyaluronic acid may be
expected in the surface coat of undifferentiated cells. Collagen, on the other
hand, does not play an essential role for these cells; collagenase therefore does
not have any effect on tissue dissociation or on the surface coat.
Our thanks are due to Mrs Heidi Kruger and Mrs Angelika Steuer for their excellent
assistance in doing the electron microscopic sections and to Prof. H.-J. Merker for his helpful
advice in preparation of this manuscript. This work was supported by grants from the
Deutsche Forschungsgemeinschaft awarded to the Sonderforschungsbereich 29.
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(Received 5 September 1979, revised 11 March 1980)