/. Embryol. exp. Morph. Vol. 36, 1, pp. 175-181, 1976
Printed in Great Britain
175
A simplified procedure for preparing
myogenic cells for culture
By ARNOLD I. CAPLAN 1
From the Biology Department, Case Western Reserve University, Cleveland
SUMMARY
A procedure involving mechanical agitation referred to as vortexing is compared to a trypsin procedure for obtaining myogenic cells for culture. The vortex procedure appears to be
at least as useful as the trypsin procedure and has several advantages including speed, the
elimination of chemical disruptive agent, elimination of collagen coating of culture dishes and
earlier onset of fusion.
INTRODUCTION
In an attempt to obtain muscle cells from earlier than 11- to 12-day chick limbs
a variety of agents and techniques were used. The most simple and successful
approach employing mechanical agitation is reported here. This procedure
has several advantages over the widely used trypsin procedure (Konigsberg,
McElvain, Tootle & Herrmann, 1960; Coleman & Coleman, 1968): myoblasts
can be isolated from as early as 6- to 7-day chick limbs; the procedure uses no
chemical disruptive agents; the process of fusion and multinuclear cell maturation starts within a 12- to 24-h period after plating rather than experiencing a
50- to 70-h lag, which allows one to minimize fibroblast obscurement of these
events and eventual fibroblast overgrowth; and plating can be done on plastic
tissue culture dishes in the absence of added collagen.
METHOD
The embryo is isolated from the egg through the air space using sterile
techniques and placed into a Petri dish with Tyrode's balanced salt solution.
The legs are removed with watchmaker forceps by cutting at slightly above
the knee joint. The foot, and if possible (depending on the age of the embryo), the
skin is removed. No other cutting is required; we currently grossly remove the
cartilage or bone core and cut the soft tissue into smaller segments. From 10
to 15 legs are placed in 1 ml of plating medium in a 10 ml sterile plastic disposable
test tube (Falcon 2001) and agitated on a 'vortex Jr' mixer for 30 to 60 s. From
3 to 4 ml of medium is then added and mixed by the brief action of the vortex
mixer. The chunks are allowed to settle to the bottom of the tube; the super1
Author's address: Biology Department, Case Western Reserve University, Cleveland,
Ohio 44106.
176
ARNOLD I. CAPLAN
natant is pipetted into a glass cylinder, the bottom of which is fitted with 20 /mi
pore size Nitex filter cloth (Cahn, Coon & Cahn, 1967). This Nitex-fitted tube
is placed in a 30 ml conical centrifuge tube which functions to catch the medium
and single cells which pass through the filter. The cells are counted and diluted
to the desired levels and plated on 35 mm plastic Petri dishes (Falcon 3001).
These manipulations are referred to as procedure Bx in Table 1.
The experiments discussed in this communication were arranged as follows:
legs were obtained from chick embryos after 5, 6, 7, 8, 9 and 10 days of incubation and on each day split into two groups. The first group was vortexed as
described above and the single cells plated. The chunks were recovered and
incubated in Ca-Mg-free Tyrode's at 37 °C for 1 h; these chunks were then
placed in 1 ml of plating medium and vortexed. The cells were passed through a
Nitex filter, counted, diluted and plated. This is referred to as procedure B2 in
Table 1.
The second group of limbs was placed in 0-2% trypsin-0-1 % EDTA in
Ca-Mg-free Tyrode's at 37 °C for 30 min to 1 h. The chunks were then dropped
through a 10 ml column of plating medium which served to wash and inactivate
the trypsin on the limbs; the top 9 ml of the medium were drawn off and
discarded. After vortexing for 5-15 sec, the cells were filtered through Nitex,
counted and plated. This is referred to as procedure A in Table 1 and represents
the standard dissociation procedure as used to obtain stage 24 limb mesodermal
cells for culture (Caplan, Zwilling & Kaplan, 1968; Caplan, 1970, 1971a, b;
Schacter, 1970). Cells from each procedure were plated at densities of 0-1, 0-25,
0-5, 1-0 and 2-0 x 106 cells/35 mm plastic dish and only equal density plates were
compared. Data for Table 1 and most of the observations reported here are from
monitoring plates initially seeded with 0-5 x 106 cells.
Two culture medias seem to give similar results: Eagle's MEM with 7 %
horse serum, 3 % fetal calf serum and 5 % embryo extract and 40 % 199
in Hanks salts with 10 % horse serum and 5 % embryo extract. The Eagle's
medium was used in the experiments reported here. Cultures were fed every
other day by completely replacing the 1-5 ml of medium. Cultures were viewed
daily using phase optics and were fixed with Bouin's and stained with Harris
hematoxylin at the end of the culture period. In all cases reported in Table 1,
comparisons are made after 5 days in culture.
Terms in Table 1: ' M N C (multinuclear cell) is a single cell with two or more
nuclei; 'straps' refers to a cell with many nuclei whose striations are easily
observable in unfixed or fixed-stained material; 'contraction' refers to the observed contraction of a multinuclear cell. This is the least important criterion
because of the fact that contraction is spontaneous and relatively infrequent
and thus difficult to score. Many cells appear to have contractile machinery,
but no serious attempt has been made to activate this machinery and therefore
these observations should be treated lightly. The value of these observations
is to demonstrate that some cells can contract.
New procedure to obtain myoblasts
111
Table 1. Muscle development in vitro as a function of the age of the embryonic
chick leg and the method used to obtain myoblasts for culture. Details found in the
text
Age of embryo Procedure
MNC
Observed straps
Bt
+
+++
B2
A
B2
A
Bi
B2
A
Bi
B2
Day 6 A
Day 7
Day 8
Day 9
Contraction
Cartilage
+
++to+ + +
0
+to++
+++
+ *,0
++
+
+
+
0
0
++
+
++
++
++++
+++
+++
++++
+++
+
+
++++
++
++
++++
+++
+
0 to+
++++
+
+
++++
+++
0
0
0
0
0
0
0
+
Very few definitely present: few in number across the plate; represents about
1-5 % of cells.
++
Few in number: represents 10-15 % of the cells.
+++
Moderate number: about 50 % of cells.
+ + + + Very large number: greater majority of cells (70-90 %).
* Cells were observed to secrete cartilaginous matrix material, but in this one case 5 x 106
cells were plated on a 35 mm Petri dish.
RESULTS
The three basic procedures for preparing single cell suspensions are compared
in Table 1. Chick legs were used in these experiments and were obtained from
embryos after 6, 7, 8 and 9 days of incubation. A is a trypsin procedure and B
is a vortex procedure; Bx represents vortexing without prior incubations, while
B2 represents vortexing with prior incubation in Ca-Mg-free Tyrode's after the
limb material had been subjected to procedure Bx.
Procedure A
As can be seen in Table 1, multinuclear cells can be observed in material
prepared from any day by any procedure. As had been pointed out previously
(Schacter, 1970; Caplan, 1970), myogenic elements can be observed in material
from legs of embryos after 5 days of incubation (Stage 24, Hamburger & Hamilton, 1951) by a procedure similar to procedure A. These multinuclear cells remain
small (less than 8-10 nuclei) in cells obtained from day 6 or 7 material by the
procedure involving the use of trypsin. These multinuclear cells do form longitudinal striations, but in the material from these early stages large straps are
rare and have not been seen to contract with any regularity. Cells from 8-day
embryos fuse, forming multinuclear cells with cross-striations; these can be seen
to contract more frequently than material from earlier stages. By day 9, the
cells obtained maturate into typical contracting straps.
12
EMB 36
178
ARNOLD I. CAPLAN
Table 2. The number of cells liberated as a function of the age of the embryonic
chick limb and the methods used to free the cells. Details included in the text
Days
incubated
6
7
8
9
12
Stage
Number
of limbs
Procedure Bx
number of cells
recovered
Procedure A
number of cells
recovered
26-27
28
29
30-31
38
35
20
20
20
2
1-25 xlO 6
5 0 x 106
14-5 x 10G
7-5 x 106
21 x 106
30 x 106
lOxlO 6
5x10°
7xlO 6
30 x 10°
The trypsin procedure gives various results in regard to the appearance of
muscle phenotypes. This variation is related to the age and batch of trypsin
used to obtain cells. The more active the trypsin (in terms of converting the tissue
chunks from a firm, compact piece to a soft, easily dispersible piece), the smaller
the number of observed muscle straps. It is currently our view that treatment
with relatively inactive trypsin is overcome by the vortexing of this material in
that the myogenic cells are released by mechanical agitation. Active trypsin
treatment seems to destroy or abuse myogenic cells, as evidenced by the limited
size of multinuclear cells as well as the decrease in the total number of nuclei
present in muscle cells.
Procedure B
Procedure B1 or vortexing in complete plating medium releases cells from all
the variously staged limbs so far examined. The total number of cells released
by procedure B1 is approximately equal to that obtained by procedure A as seen
in Table 2. The quantity of myogenic cells obtained by procedure B r is greater
than that obtained using procedure A as indicated in Table 1 and as judged from
the percentage of nuclei which are observed in multinuclear cells after 3 days in
culture. This is especially apparent when 6- or 7-day-old leg material is prepared.
Unlike the trypsin treatment, cells prepared using the vortex method form
typical straps even from material from early stages. Fusion of myoblasts starts
after 12-24 h in culture and is usually completed about 24 h later. Because of
the early fusion phenomenon the fibroblast population is low during this
developmental phase. In our hands the vortex technique does not seem to
abuse myogenic cells and allows for a moderately high percentage of myogenic
cells; in some preparations from 8-day leg almost 9 5 % of the cells plated
appeared to be myogenic.
New procedure to obtain myoblasts
179
DISCUSSION
A new simplified method for obtaining myoblasts from chick limb material is
presented. This method is compared to a trypsin procedure used to obtain
mesoderm cells from limb-buds of 4- to 7-day-old embryos (Caplan et ah 1968;
Caplan, 1970, 1971a, b; Schacter, 1970). This trypsin procedure seems to abuse
or even selectively destroy myogenic cells and accounts for the variability in
observing myogenic cells from limb-bud mesoderm preparations (Schacter,
1970; Caplan, 1970). The advantages of the vortex procedure are (a) speed:
incubation and selection procedures (Richler & Yaffe, 1970) are not employed;
(b) large numbers of myogenic cells can be obtained from relatively young chick
embryos; (c) the procedure uses no chemical disruptive agent; (d) the process of
fusion and multinuclear cell maturations starts within 12-24 h after plating;
(e) fibroblast obscurement of fusion is minimized and (/) plating can be done on
plastic dishes in the absence of added collagen.
Trypsin procedures using 0-05 to 0-5 % trypsin have been reported elsewhere
(Konigsberg et al. 1960; Coleman & Coleman, 1968). The vortex procedure
seems to eliminate the need to use trypsin and, as has been recently shown by
Hosick & Strohman (1971), proteolytic enzymes have a dramatic effect on the
cell's protein synthesizing machinery. The effects of vortexing have not been
analyzed but, considering the increased proportions of myogenic cells liberated
and their earlier time of initiation of fusion, the vortex method does not appear
to be as abusive as the trypsin procedure.
The comparisons presented here utilized a relatively high level of trypsin
(0-2 %) compared to that normally used by, say, Coleman & Coleman (1968)
who employ 0-05 % for 10 min. In a few preliminary experiments using this
lower level of trypsin, vigorous vortexing or trituration was necessary to release
myogenic cells. The trypsin at this level seems to be of limited value in freeing
myogenic cells. At the very least, the vortex method is comparable to this low
trypsin procedure.
We have recently used collagen-coated Petri dishes and myogenic cells liberated by the vortex procedure, and note very limited difference in the developmental progression starting with myoblast and ending with cross-striated muscle
straps. As usual, the collagen coating prevents early lifting and peeling of the
strap-containing sheet of cells, and for this reason we now routinely coat culture
dishes with collagen. Of value is the fact that the collagen coating does not seem
to be necessary for the early events involving muscle development in culture.
Considering the evidence that collagen is necessary for myoblast attachment to
the substrate (Konigsberg, 1965; Hauschka & Konigsberg, 1966; Hauschka,
1968), I would predict that the vortex procedure provides such collagen as needed
for these early events.
A basic assumption in this report is that the absence of multinuclear cells
reflects the absence of myogenic cells. This may not be the case, as pointed out
180
ARNOLD I. CAPLAN
by Nameroff & Holtzer (1969), who demonstrated that non-proliferating
heterotypic and homotypic cells interfere with processes involved in muscle
formation. It is possible that the spectrum of cells released by the trypsin
procedure is different from those cells released by vortexing. This spectrum of
trypsin-released cells may inhibit events involved in muscle formation by
directly interacting with myogenic cells or even by selectively overgrowing myogenic cell population. Our present view is that trypsin, especially in the rather
high levels used, is selectively detrimental to myogenic cells; we cannot, however,
exclude the other possibility raised above.
I report the vortex procedure for releasing myogenic cells because of its simplicity and obvious advantages. Analysis using this technique has been conducted in my laboratory since 1970 and has been used by others since its
inception (Tepperman, Essien & Heywood, 1975; Thi Man & Cole, 1974;
Heywood, Havaranis & Herrmann, 1973).
Supported by grants from National Institutes of Health HD-35609 and HD-07209, The
National Foundation and the Muscular Dystrophy Association of America.
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NAMEROFF,
(Received 7 February 1976)