In vitro reaggregation of dissociated mouse cerebellar cells

Copyright 0 1982 by Academic Press. Inc. All rights of reproduction in any form reserved 0014-4827/82/060285-12102.00/0

Experimental Cell Research 139 (1982) 285-296

IN VITRO

REAGGREGATION MOUSE

I. Demonstration

CEREBELLAR

OF DISSOCIATED CELLS

of Different Aggregation

Mechanisms

G. FISCHER and M. SCHACHNER Department of Neurobiology, University of Heidelberg, 0-4!200Heidelberg, Germany

SUMMARY Reaggregation of mechanically dissociated mouse cerebellar cells (M cells) was compared with cells that received an additional trypsinization either before (T cells) or after (MT cells) the dissociation step. Reaggregation behaviour was followed by measuring the number and size distribution of particles with a Coulter counter. Aggregation rates which were calculated as percentage of decrease of particles could be measured reproducibly. Since the percentage of very large particles (>lOO cells) formed during aggregation varied considerably from one experiment to the next, size distribution curves of particles were used more to distinguish qualitative differences in a less quantitative way. Whereas aggregation rates and size distribution of particles with M cells were almost identical when aggregation occurred in medium of high (1.1 mM) or low (0.1 mM) Ca9+ concentrations, T and MT cells aggregated better at high Ca*+ concentration. Their aggregation rates were reduced by approx. 50% at low Ca*+ concentrations and larger aggregates were hardly formed under these conditions. The aggregation rates of T and MT cells showed a clear dependence on CaP+concentration, being half maximal at approx. 0.1 mM CaZ+. The ability of M cells to aggregate at low or high Cal+ concentrations was influenced by subsequent trypsinization to produce MT cells. When the trypsin concentration was changed from 0.001 to 0.1% during this procedure the aggregation rates at high Ca*+ concentration were reduced to approx. 80% of the maximal value, whereas those at low Ca2+ concentrations were reduced to 35%. Variation of the Ca*+ concentration between 1.1 and 0.1 mM during the trypsinization step (0.015% trypsin) revealed no difference on the aggregation rates. We propose that M cells aggregate mainly or exclusively by a Ca2+-independent binding mechanism, whereas T or MT cells aggregate using a Cal+-dependent one which may be functionally silent in M cells.

Cell interactions mediated by cell surface molecules presumably underlie many developmental events in the formation of the nervous system. To study the molecular mechanisms involved in the recognition of different cell types at different developmental stages, several laboratories have resorted to simplified in vitro assay systemsr based mainly on the reaggregative or adhesive behaviour of dissociated cells [l-l 11. With the now almost classical paradigm of

neural retina cells from chicken, several adhesive mechanisms and molecular activities have been implicated. These include calcium-dependent and -independent mechanisms [ 12-181 and several distinct molecular species of adhesive or so-called recognition molecules [D-23] which were also found in other cell types [24-261. We have chosen to study cell interactions in the developing mouse cerebellum for several reasons. It contains only five neuExp Cell Res 139 (1982)

286

Fischer and Schachner

ronal cell types which are organized in relatively simple geometric arrays repeated throughout the cerebellar cortex [27-291. The connectivity of these cell types is well known and most transmitters have been identified. Some major events in neuron formation, migration and differentiation take place postnatally so they become more easily accessible to the investigator. ‘In particular, the migration of post-mitotic granule cells generated in the germinal external granular layer has been postulated to proceed along the surfaces of Bergmann glial processes in a form of contact guidance [30]. The availability of batch separation and immunoselection methods to isolate subsets of cerebellar cell types is within reach of possibilities [31-361 and will open a new era in the study of cell interactions in the cerebellar cortex. Also, a number of neurological mutants show selective neuronal death, perhaps based on developmental abnormalities in cell interaction [37, 381. The present study was undertaken to examine the mechanisms involved in the reaggregation of dissociated cerebellar cells from early postnatal mice using a quantitative assay for aggregate formation in a kinetic analysis. MATERIALS

with salt composition of Earle (BME-Earle’s) was supplemented with 10% horse serum (Seromed, Munchen).

Preparation of single cell suspensions Trypsinized cells (T cells) from &day-old mouse cerebellum were prepared as described previously [34). In short, the cerebellum was transferred into ice-cold M-HBSS where it was freed from the meninges. It was cut sagittally into three pieces and trypsinized for 13 min at room temuerature with 1% try&in (218 U/mg, Worthington, via Seromed! Mtinthen) and 0.1% DNase (1588 U/mg, Worthmgton, via Seromed), in M-HBSS. After washing three times with CMF-HBSS, cells were mechanically dissociated with tire-uolished Pasteur oinettes in BME-Earle’s containing’ 0.05% DNase and 0.25% glucose. After standing for 5 min at room temperature the single cell suspension was removed from large settled particles and centrifuged in the cold for 5 min at 109 g. The cells were resuspended in a mixture of 1 vol HBSS and 10 vol of CMF-HBSS containing 0.002% DNase and washed twice in the same medium. Finally they were resuspended in the aggregation medium. Mechanically dissociated cells (M cells) were prepared without the preceding trypsinization step. Cerebellum freed from the meninges was cut sagittally into five pieces and dissociated with fire-polished Pasteur pipettes in HBSS containing 0.002% DNase. After standing for 5 min at room temperature the cell suspension was removed from larger particles and washed twice as described for T cells. Mechanically dissociated cells with subsequent tryp sinization (MT cells) were obtained by mechanical dissociation as described for M cells, followed by two washes in HBSS and CMF-HBSS containing DNase as described for T cells for the removal of larger particles and cell debris. Trypsinization was cased out at a concentration of 0.015% for 15 min at room temperature. Afterwards cells were washed as described for T cells. Cell viability was measured by exclusion of trypan blue using a freshly prepared solution of 0.16% dye in physiological saline.

AND METHODS

Animals

Precoating

Throughout these experiments the mouse strain C57B1/6J was used to preparecerebellar cells. Animals were maintained in the breeding facilities of this department.

Costar plates (type 3524, Costar, Cambridge, Mass.) were precoated with BME-Earle’s supplemented with 10% horse serum for 1 h at room temperature or alternatively with BME-Earle’s containing 1 mg/ml bovine serum albumin (BSA, Serva, Heidelberg). The plates were then washed three times with distilled water. These procedures abolished the attachment of cells to the plastic surface in the subsequent aggregation period.

Media used fo# cell preparation and aggregation Hanks balanced ‘&t solution (HBSS) was prepared as usual and without calcium and magnesium (CMFHBSS) or with magnesium alone (M-HBSS). The concentration of CaZ+ in HBSS is 1.1 mM. Earle’s balanced salt solution (EBSS) was alternatively repared without calcium and magnesium (CMF-EB BS). Reduced calcium concentrations were compensated in -osmolarity by addition ofNa$30,. Basal medium Eagle Exp Cell Res 139 (1982)

Aggregation

of aggregation

vessels

of cells

If not otherwise stated, T, M, or MT cells were incubated at a concentration of 3x l(P particles/ml (counted in Coulter counter channels >16, see below), in 500 I.LI of HBSS containing 0.002% DNase in a

Reaggregation rotary water bath (Infers HT, Basle) at 85 rpm and 37°C. After incubation periods of 20-90 min aliquots of 50 ~1 were taken for Coulter counter measurements.

Coulter counter measurements Particle concentration and size distribution of particles in cell preparations and aggregation experiments were determined with a Coulter counter TA II eauiowd with, a’200 Mm capillary (Coulter Electronics G&H, Krefeld, FRG) using 10 d of the cell susoensions directly after prepar&on.or S@pI aliquots of the aggregation mixtures. These samples were diluted in 20 ml of a particle free 0.9% sodium chloride solution containing 5 ml/l of 37% formaldehyde. Particles can be counted according to size in 16 channels such that from one to the next channel the volume of counted particles increases by a factor of two. For calibration of measurements, latex beads with 13.7pm in diameter (Coulter Electronics) were used. Coulter counter measurements were adjusted in a way that these particles were counted equally well in channels 8 and 9. The lower border of channel 9 is therefore set to measure spherical cell body diameters of 13.7 pm. The lower borders of channels 3-16 are equivalent to 3.4- 4.3- 5.4-6.88.6- 10.8- 13.7- 17.3-21.727A - 34.5 - 43.5 - 54.8 - 69 pm in order of increasing channel number. For each measurement, 5OOQOparticles were counted in channels 3-16. Particles in channels 1 and 2 were not counted to reduce the large contribution of very small debris particles and electronic background noise to the. measurements. Cell debris was counted in channels 3 and 4. Low numbers in volume of particles in these channels throughout the entire aggregation time were taken as indication of the intactness of cells. From channel 5 onward small single cells, then small aggregates, larger single cells and larger aggregates were counted. In addition to the measurement of particle number in each channel the values of differential volumes (V,,,) and counting time was plotted. Vdifi is defined as the volume of particles in the different channels related to the total volume of particles as 100%. The aggregation rate R, designates the decrease in particles in channels 5-8 for T and MT cells and in channels 5-9 for M cells expressed in % as described below in Results.

RESULTS Comparison preparatioris

of T, M, and MT cell

Different cell dissociation procedures lead to preparations with a slightly different size distribution. With trypsinization procedures and subsequent mechanical dissociation (T cells) mainly single cells are obtained, whereas mechanical dissociation alone (M cells) leads to a mixture of single cells and

of dissociated .mouse cerebellar cells

287

smaller aggregates. The latter are dissociated into single cells by a subsequent trypsinization step (MT cells). Coulter counter analysis of cell preparations shows that significantly more larger particles are counted in M cell preparations compared to T or MT cells as shown in fig. 1a, where particle concentration is plotted semilogarithmically over channel number. The number of particles counted per second (ZV) corresponds to the concentration of particles in the aggregation mixture. w shows higher values for M cells in channels 7-12, whereas T or MT cells display a more pronounced peak in channel 6. This difference in size distribution of particles becomes even more evident when values for differential volumes (Vd& of particles are compared (fig. lb). Both T and MT cells show a high and narrow peak in channels 5-7. The peak of M cells is broader and shifted to higher channel numbers indicating that M cell preparations contain relatively more small aggregates. It seems pertinent to state that the size distribution of cerebellar cells varies from small neurons (granule, stellate, and basket cells with a cell body diameter of about 6-g pm) to large neurons (Purkinje and Golgi type II cells with a cell body diameter of about 20 pm). Preparations of single cells from early postnatal mouse cerebellum consist of about 90% of small neurons, mainly granule cells [35]. Single cells are expected to be measured in channel numbers 5-7 which corresponds to the dominating particles in T and MT cell preparations. Aggregates of small cells cannot be discriminated from larger cells by the Coulter counter which measures solely particle volumes. One can therefore assume that starting with channel 7 small and progressively larger aggregates are counted. Cell viability as measured by exclusion of Exp Cell Res 13Y flY82J

288 N’ tow

Fischer and Schachner

t a

Vdiff (

channel

Fig. 1. Distribution of particles in preparations of M, MT, and T cells. Values are expressed as (a) number of particles counted per second W; or (b) differential volume of particles Vdiff as a function of channel

b

channel

number. A-A, M cells; O-O, MT cells; and X-X, T cells. Each value is the mean of eight independent experiments.

trypan blue was slightly higher in T-cell gregated single cells. Although a quantitapreparations than in M and MT cells (table tive determination is diffkult, proportions 1). Yield of single cells was highest in T-cell of dead vs live cells in aggregated and nonpreparations (table 1) when measured on aggregated forms were comparable. the basis of particle number in channels 5-16, since M-cell preparations contain Aggregation of M, MT, and T cells at more small aggregates. Compared with a T- high Ca2+ concentration (1 .l mM) cell preparation the number of cells may be As shown in fig. 2a, 6, for M cells, the equal. After 1 h of aggregation time (see aggregation of cells was followed by countbelow) trypan blue positive, dead cells were ing the particle distribution and their diffound among aggregated cells and non-agferential volumes (V&. The cells were incubated at 37°C and 85 r-pm in HBSS containing 1.1 mM Ca*+ at a concentration of Table 1. Viability and yield of M, T, and MT 3x lo6 particles per ml. The particles cells prepared from postnatal-day 6 mouse counted in the different channels per seccerebellum ond (fig. 2a) as well as their Vdiff values Particles/ (fig. 2b) were plotted as a function of chanViability Cell cerebellum preparation nel number. To count 50 000 particles, 2& cw (XlW 60 set were normally needed, depending on 8555 aggregation conditions and aggregation ET 9of5 z4.s T 90+5 %9 times. It is evident from fig. 2a, b that in channels 13 and 14 (fig. 2b), where after an Cells were prepared as described in Material and Methods. To determine cell yields particles were aggregation time of 60 min 14 and 6 %, counted in channels 5-8 for T and MT cells and in respectively, of all cells are counted, based channels 5-9 for M cells. Cell viability was determined by exclusion of trypan blue. on Vdiff values only about 4 or 1 particles Exp Cell Res 139 11982)

Reaggregation

of dissociated

mouse cerebellar cells

289

loo-

10 -

channel

channel

Fig. 2. Size and particle distribution of M cells in HBSS after various aggregation periods at high Cal+ concentrations (1.1 mM). (a) The number of particles counted per second W and (b) the differential volume of particles V dilf are plotted as a function of

channel number after different incubation oeriods: X-X, 0; O-O, 20; and A-A, 60 min when 1.5~ 106 particles per 500 ~1 of HBSS containing 0.002% DNase were incubated at 85 rpm and 37°C. Each value is the mean of eight independent experiments.

(fig. 2a) are counted per second. Even fewer particles are counted after 60 min of aggregation time for T cells under the same experimental conditions as for M cells. Based on the Vdirf value, 7 or 4.5% of all cells are counted in channels 15 and 16 with a counting rate of less than 0.5 particles per second. Although it would be desirable to calculate the aggregation of cells directly from Vdiff values (for they represent the disstribution of cells among particles of differing size) by quantifying the shift in Vdiff distribution curves to higher channel numbers, statistical problems would be encountered, for the variations in Nf and the Vdiff values in channels with high numbers is large, as indicated by standard deviations (fig. 3a, b). For quantitation of aggregation values we therefore calculated the decrease in particles counted per second in channels 5-8 (for T and MT cells) and in channels 5-9 (for M cells) taking into account that the size distribution of particles in M-cell preparations is more heterogeneous (fig. la, 6).

The percentage of decrease in particles is defined as the aggregation rate R,. R, is normally plotted as a function of aggregation time, as shown in fig. 4 for M, MT, and T cells incubated under the same conditions as for figs 2 and 3. Vdin distribution plots as shown in figs 2b and 3 b will be taken to represent tendencies in size distribution of particles formed after different aggregation periods. The aggregation of MT cells in HBSS containing high Ca2+ concentrations is shown in fig. 5a, b. MT cells were incubated under conditions comparable to M a& T cells before. By comparing the aggregation behaviour of the different cell preparations in high Ca*+ concentrations it is evident that M cells aggregate better than T or MT cells in several respects. Their aggregation rate is higher than that of the other cell preparations (see fig. 4) and no discrete peak of single cells remains in channels 5-9 (see fig. 2a, b). T cells seem to aggregate more Exp Cell Res 139 (19821

290

Fischer and Schachner

2

6

4

8

10

12

channel

Fig. 3. Size and particle distribution of T cells in HBSS after various aggregation periods at high Ca2+ concentrations (1.1 mM). (a) Number of particles counted per second N* and (b) their differential volumes are plotted as a function of channel numbers.

heterogeneously with respect to the size distribution of aggregates (fig. 3a) than the other cell preparations forming possibly more larger aggregates within 60 min than M or MT cells. However, all three cell preparations show an aggregation rate R, of more than 65 after 1 h of aggregation in HBSS-containing high Ca2+ concentrations .

11

16

channel

Incubation conditions were as described in caption to fig. 2. Incubation periods are: x-x, 0; O-0,-20; A-A, 60 min. Each value represents the mean of eight independent experiments. Bars indicate SD.

RP!

ao-

60-

Aggregation of M, MT, and T cells at low Ca2+ concentrations (0.1 mA4)

Similar aggregation experiments as those shown in figs 2-5 were carried out with all three different cell preparations at low concentrations of Ca2+ (0.1 mM). Whereas the aggregation behaviour of M cells remained unchanged under these conditions the aggregation of T and MT cells was remarkably reduced, as shown for Vdln values in fig. 6 for T cells and in fig. 7 for MT cells. Both formation of larger aggregates and aggregation rate R, (fig. 8) are reduced. Cell debris remained as low as during aggregation in HBSS with high Ca2+concentrations Exp Cell RPS 139 (1982)

c

20

40

60

+

+---c

[mini Fig. 4. Aggregation rates R, of M, T, and MT cells

in HBSS at high Cay+ concentrations (1.1 mM). Aggregation rates Rp are expressed as decrease in particle numbers @ in channels 5-g for O-O, T cells; A-A, MT cells and in channels 5-9 for x-x, M cells taking into account the different distribution of particles in the cell preparations (see fig. 1 and Methods). The Rp values are plotted as a function of incubation time. Incubation conditions for all three cell preparations were the same as described in caption to fig. 2. Each value represents mean of six independent experiments. Bars indicate SD.

Reaggregation

of dissociated mouse cerebellar cells VdiffA 30-

b

20-

1'

2

6

10

12

1L

16

29 1

TT

I7

2

4

6

8

10

12

lb

16

channel

Fig. 5. Size and particle distribution of MT cells in HBSS after various aggregation periods at high Cal+ concentrations (1.1 mM). (a) Number of particles counted per second NL and (b) their differential volumes are plotted as a function of channel number.

Incubation periods are: X-X, 0; O-O, 20; A-A, 60 min. Incubation conditions were the same as described in caption to fig. 2. Each value represents mean of six independent experiments. Bars indicate /> SD.

and is shown as Vdirr values in channels 3 and 4 in figs 6 and 7. When M, MT, and T cells are kept in HBSS at low Ca2+ concentrations for 1 h on ice they all aggregate normally after adjustment to high

Caz+ concentrations, as shown before it! figs 2-5. This indicates that the cells are not damaged in medium with low Caz+ con: centrations which might otherwise lead to inhibition of T- and MT-cell aggregation. It

Vdiff 30 t

vd,ff 30t

channel

of T cells in IIBSS after various periods at low Ca*+ concentrations (0.1 mM). Vain values are plotted as a function of channel number after X-X, 0; O-O, 20; and A-A, 60 min of incubation. Particles at a concentration of 1.5x106/ 500 ~1 medium contahring 0.002% DNase were incubated at 85 rpm and 3PC. Each value represents mean of four independent experiments. Bars indicate SD.

Fig.

6. Size distribution

Fig. 7. Size distribution of MT cells in HBSS after

various aggregation periods at low Caa+concentrations (0.1 mM). Vdln values are plotted as a function of channel number after X-X, 0; O-O, 30; A-A, 60 min of incubation and each represents mean value of four independent experiments. Bars indicate SD. Incubation conditions were the same as described in the caption to fig. 6. Exp Cell Res 139 (1982)

292

Fischer and Schachner

RP 80 t

0.2

0.6

0.6

1.0

1.2

Fig. 9. Influence of Ca*+ concentration on aggregation rates of M, T, and MT cells. Cells were incubated with a particle concentration of 1.5~ 106particles per 500 ~1 of HBSS at different Ca*+ concentrations (abscissa) for 30 min at 85 tpm and 37°C. Aggregation rates R, were related to maximal aggregation rates for each cell type as 100% (ordinate) for x--X, M cells; O-O, T cells; and A-A, MT cells. Fig. 8. Aggregation rates R, of M, T, and MT cells in HBSS at low Ca*+ concentrations (0.1 mM). Aggregation rates R, (see fig. 4) were calculated and plotted as a function of incubation time for X-X, M cells; O-O, T cells; and A-A, MT cells. Incubation conditions were the same as described in the caption to fig. 6. Each value represents the mean of four independent experiments. Bars indicate SD.

further indicates that small changes in pH, which might occur in normal atmosphere, do not influence the aggregation of cells in the assay. In addition, in experiments, where aggregation was carried out in a 5% CO, atmosphere the same aggregation rates were observed as in normal atmosphere. Dependence of the aggregation rate of T and MT cells on Ca2+ concentrations

To evaluate the aggregation rate of the different cell preparations as a function of Ca2+ concentrations, cells were aggregated in CMF-HBSS containing 0.05-2 mM in Ca2+. As shown in fig. 9 the aggregation rate of T and MT cells after 30 min of incubation varied with the Ca2+ concentration, whereas that of M cells remained nearly constant. The half-maximal aggregation rate is obtained at about 0.1 mM Ca2+ as Erp Cell Res 139 (1982)

indicated by an arrow. The aggregation rates at different Ca2+ concentrations are given in fig. 9 as percentage values of the maximal aggregation rate, being 68 for M cells, 59 for T cells, and 57 for MT cells after 30 min of aggregation time (see fig. 4). When cells were aggregated in Ca2+-free medium (CMF-HBSS) somewhat lower R, values were obtained than with 0.1 mM Ca2+. The formation of cell debris increased, however, after incubation periods of more than 30 min, indicating that cerebellar cells from early postnatal mice are not stable under these conditions. The results in fig. 9 indicate that T and MT cells aggregate predominantly with a Ca2+-dependent aggregation mechanism and are unable to form larger aggregates at low Ca2+ concentrations (compare figs 6 and 7), whereas M cells aggregate equally well at low and high Ca2+concentrations. Influence of trypsinization aggregation rate

on the

When the aggregation of T and MT cells is compared at high and low Ca2+ concen-

Reaggregation

of dissociated mouse cerebellar cells

293

Table 2. Influence

of Ca2+ concentration during trypsinization for preparation of MT cells on aggregation rates mM Ca2+ in aggregation medium

I 0

0,001

0.01

% Trypsik Fig. 10. Influence of trypsin concentration during cell

preparation on aggregation rates. M cells were trypsinized in HBSS containing 0.002% DNase and different concentrations of trvusin (ah&ml for 15 min at room temperature. After-washing twice they were incubated in HBSS with high (1.1 mM, x-x) or low (0.1 mM, O-O) Car+ concentrations at a particle concentration of 1.5~106/500 ul and 85 mm at 37°C. Aggregation rates of cells after 30 min -were related to those of M cells as 100% (ordinate). Values represent means of three independent experiments. The arrow indicates the trypsin concentration normally used to prepare MT cells.

Cell preparation

(% R, max)

M MT (1.1 mM Ca2+) MT (0.1 mM Ca2+)

100+ 4 87klO 90f13

1.1

0.1 (% R, max) 97f 5 54+ 7

51+10

M cells were trypsinized in either HBSS with high (1.1 mM) or low (0.1 mM) Ca*+ concentration for 15 min at room temnerature using 0.015% trvusin. APgregation rates were determinedafter 30 minof incubition time at 85 rpm, 37°C and a particle concentration of 1.5~ 106particles in 500 ~1 of HBSS with high (1.l mM) or low (0.1 mM) Ca*+ concentrations. Each aggregation rate was related to the maximal aggregation rate as 100% and represents mean value of three independent experiments.

maximal aggregation, being 68 for M cells after 30 min of aggregation in either low or high Ca2+concentrations. Formation of cell trations (see figs 4 and 8) it can be seen debris was low during the aggregation that the differences in aggregation are much period at both Ca2+ concentrations, even more pronounced at low Caz+ concentra- when cells were trypsinized with high contions than at high Ca2+ concentrations. To centrations of 0.1%. Trypsinization at conevaluate the influence of trypsinization con- centrations above 0.1% were not practicaditions on the aggregation behaviour these ble due to the increasing instability of cells were altered in two different ways. First, during incubations. When M cells are trypsinized with we varied the trypsin concentration between 0.001 and 0.1% at 1.1 mM Ca2+ 0.015% trypsin concentrations in HBSS and second, we lowered the Ca2+ concen- with either high (1.1 mM) or low (0.1 mM) tration from 1.1 mM to 0.1 mM at a trypCa2+concentrations to give MT cells, comsin concentration of 0.015% to see if there parable aggregation rates are measured for is a protective effect of Ca2+ during tryp- the different cell preparations (table 2). sinization procedures for the Ca2+-depend- These rates are approx. 90% of those of M cells when aggregation takes place in 1.1 ent aggregation mechanism. Fig. 10 illustrates how aggregation rates mM Ca2+ and about 50% of those of M at low Ca2+concentrations are decreased in cells when aggregation takes place in 0.1 cell preparations obtained in HBSS at mM Ca2+. Before trypsinization, M cells higher trypsin concentrations relative to ag- were equilibrated to high or low Ca2+ congregation rates observed at high Ca2+ con- centrations by washing twice in the correcentration. As in fig. 9, the aggregation sponding incubation medium. Trypsinizarates are given as percentage values of the tion in the absence of Ca’+ and presence of Exp Cell Res 13Y(IY82)

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Fischer and Schachner

EDTA (ranging from 0.1-l mM) was not possible due to the instability of cells. DISCUSSION Crucial to the investigation of the parameters which underlie the reaggregation of dissociated cells is the availability of a suitable assay system to quantitatively measure the number of aggregates and their size. It is possible to measure the degree of aggregation as a function of the decrease of single cells. This type of measurement can readily be performed by Coulter counter analysis [39, 401. An important facet of aggregation parameter is lost, however, when the size of aggregates cannot be taken into consideration [ 171. Measurement of light scattering was found to be a convenient way to follow aggregation of cells. This parameter is influenced more by large than by small particles [41, 421. In the present study the reaggregation behaviour of dissociated mouse cerebellar cells was measured with regard to number and size distributions of particles by Coulter counter analysis as described for the aggregation of tibroblasts [43] and Chinese hamster cells [44] where aggregates consisting of up to 20 cells were measured. In another case, clusters of 30 and 125 cells were measured selectively to characterize the aggregation of chick neural retina cells [17]. The Coulter counter TA II enabled us, however, to follow the aggregate formation of even very large aggregates, consisting of up to several hundred cells. Particle numbers of the whole spectrum of single cells, smaller, larger, and very large aggregates were measured as a function of aggregation time. However, the counting of large particles consisting of up to several hundred cells is not sufficiently reproducible, due to the

relatively small numbers of large aggregates, which are close to background levels. Nevertheless, their contribution to size distribution curves expressed as differential volume (Vdiff) is considerable (see fig. 2a, b and fig. 3a, b). The size distribution curves were therefore used in a more qualitative way to describe the aggregation behaviour of cells. For quantification, the decrease in small particles comprising single cells in very small aggregates (of up to 16 cells) was calculated in terms of aggregation rates (R,), as defined by the relative decrease in number of particles of small size during aggregation. The measurement of cell debris (very small particles in channels 3 and 4) was used to check the stability of cells under various aggregation conditions. The aggregation behaviour of dissociated cells from early postnatal mouse cerebellum was assayed with three different cell preparations: mechanically dissociated cells (M cells), or cells obtained by trypsinization of cerebellar tissue pieces before mechanical dissociation (T cells) or by trypsinization of mechanically dissociated cells (MT cells). Two distinct aggregation systems could be detected, depending on the calcium concentration used under the aggregating conditions. At high calcium concentrations (1.1 mM) only small differences between the cell preparations could be measured with respect to aggregation rates (fig. 4). M cells tended to aggregate in a narrower range of size distributions as compared with T or MT cells. With T- and MT-cell preparations, more cells tended to remain as single cells or to form smaller aggregates in a given time, which may be due to the alteration of surface properties of trypsinized cells (figs 2, 3, 5). In all three cell preparations, however, small aggregates are

Reaggregation

formed, before the larger ones become apparent, as reported for fibroblasts [43] and Chinese hamster cells [44]. The formation of larger aggregates was taken as a sign of favorable aggregation condition for all three cell preparations. At a low calcium concentration (0.1 mM), M cells aggregated almost as well in terms of aggregation rates and in size distribution of particles as described for high calcium concentrations. The aggregation rates of T cells and MT cells, however, decreased to about 50% of the values found at high calcium concentrations (fig. 8). These differences in aggregation behaviour under different calcium concentrations were seen most prominently after short aggregation periods. Analysis of the size distribution curves for aggregates revealed that formation of very large aggregates was almost completely inhibited and that the numbers of larger, medium-sized particles were greatly reduced (figs 6, 7). When T or MT cells are used at low Ca*+ concentrations, aggregation does not seem to be stable enough to yield larger aggregates. Therefore a low calcium concentration is favorable only for the aggregation of M cells but not for either T or MT cells. This became even more evident when the aggregation rates were measured in dependence of the Caz+ concentration (0.05 mM to 2 mM). Whereas the aggregation rates of M cells remained almost constant, those of T and MT cells increased from about 30% at 0.05 mM to about 85 % at 0.4 mM of the maximal values at around 0.6 mM and higher (fig. 9). It therefore seems that M cells can aggregate with a Ca2+-independent mechanism, whereas MT and T cells use a Ca*+dependent one. The Ca*+-dependent mechanism was found to be more stable towards trypsinization (0.001-O. 1% trypsin) in the presence

of dissociated mouse cerebellar cells

295

of Ca*+ (1.1 mM) than was the Ca*+-independent one (fig. 10). Ca*+ may have a protective effect on the appropriate binding molecules even at low (0.1 mM) Ca2+ concentrations (table 2) which are not sufficient, however, to stabilize the formation of large aggregates via the Ca*+-dependent binding mechanism (figs 6, 7). A similar protective effect of Ca*+ on a Ca*+-dependent binding mechanism was described for a Chinese hamster cell line [13, 141and chick neural retina cells [14-161. Observations similar to ours were reported for the aggregation behaviour of a Chinese hamster fibroblast line [13, 141and embryonic chick neural retina cells [12, 1418]. These cells, when dissociated mechanically in the presence of 1 mM EDTA, aggregated by a calcium-independent binding mechanism. Trypsinization in the presence of calcium destroyed the calcium-independent binding mechanism, leaving intact the calcium-dependent one. This latter calcium-dependent mechanism was proposed to be functionally inactive prior to trypsinization [ 151. Trypsinization conditions using 0.001% trypsin in the presence of 1 mM EDTA selectively destroyed the calciumdependent binding mechanism, while trypsinization at a concentration of 0.01% in the presence of EDTA destroyed both binding mechanisms [15]. As mentioned under Results, a comparable trypsinization of mouse brain cells in the presence of EDTA was not possible due to the instability of the cells under these conditions. Experiments are underway to characterize these different binding mechanisms with immunological and biochemical methods. It would be of interest to see if binding molecules of mouse cerebellar cells will resemble those for other cell types [19231.Whatever their function may be in reguEm Cd Res 139 i/9(12)

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Fischer and Schachner

lating cell-cell contacts in developing tissue in situ, it seems possible that binding between cells may be regulated either by variation in the local Ca2+ concentration and/or intermediate modulation by proteolytic cleavage, as has been hypothesized previously [ 151. The authors are indebted to Roswitha Hehl and HansJoachim Sack for assistance during the initial stages of this work. The excellent technical assistance of Christa Raab is gratefully acknowledged. This work was supported by Fonds der Chemischen Industrie and Deutsche Forschungsgemeinschaft (Scha 185/S).

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E.rp Cd Res 139 (IY82J

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