Analysis of the Growth of Muscle
Myoblasts in the Rotary Cell
Culture System
Abstract
Our previous experiments found that large multinucleated cells appeared when muscle
myoblast cells were grown in Synthecon’s Rotary Cell Culture System (RCCS). The
purpose of this study was to determine if these multinucleated cells exhibited
characteristics of differentiating muscle cells. A mouse muscle myoblast cell line, C2C12,
was cultured as three-dimensional aggregates (without support structures) in both the
RCCS and Petri dishes coated to prevent cell adhesion to the culture vessel. Cells were
cultivated in growth medium with no replacement of serum type or serum deprivation. For
this study, differentiation was defined as: 1) fusion of single myoblasts to form
multinucleated cells (syncitia); and 2) expression of myosin, a muscle specific protein.
Cell aggregates were fixed at day 3 and 7, embedded, and sectioned. Evaluation of H & E
stained sections revealed higher numbers of multinucleated cells (syncitia) in the RCCS
cultured cells than Petri dish suspension controls at both time points assessed.
Quantification of muscle specific myosin using specific staining (immunofluorescence)
revealed that RCCS cultured aggregates exhibited: 1) myosin distribution over greater
areas; 2) more intense staining in myosin expressing areas compared to the suspension
control; 3) the majority of syncitia and muscle specific myosin positive areas were on or
near the aggregate periphery. These studies show: 1) Multinucleated muscle myoblasts
exhibit characteristics of differentiation in the BR; 2) differentiation does not require
conditions such as serum deprivation, changes in serum types, or attachment to a
surface; and 3) this is a good model test system for studying muscle differentiation.
Background/Introduction
In vivo differentiation of muscle myoblast cells is thought
to require 1) adhesion (sticking) to a surface or substrate
and 2) removal of growth factors from the culture
medium.
Extra large cells have been observed on the periphery of
aggregates cultured in Synthecon’s Rotary Cell Culture
System (BR) without artificial substrate or the
removal/depletion of growth factors.
The purpose of this study was to assess if these muscle
myoblasts present in aggregates exhibited
characteristics of differentiating skeletal muscle cells.
Methods-Cell Culture
A)
B)
C)
Mouse myoblasts C2C12 (ATCC) were cultivated in growth medium (GM) consisting of Minimal
Essential Medium plus 10% Fetal Bovine Serum and antibiotics (1% Penicillin/Streptomycin).
A)
Mouse myoblasts growing in monolayer tissue culture. Note that each cell contains a single
nucleus (white arrows). Round cells are in the process of division (red arrows). Monolayer
cells were removed from the substrate by trypsin/EDTA, counted and seeded into 10 ml
disposable vessels.
B)
The Rotary Cell Culture System (Synthecon, Inc-Houston, TX) was used to culture cells in a
constant state of free fall to create 3D aggregates.
C)
Cells grown without support structures in a 10 ml vessel (BR). Controls consisted of cells
seeded into 60 mm Petri dishes coated to prevent cells from adhering to the culture surface
and remain in suspension like those in the BR (not shown).
Cell Localization With Culture Method
monolayer
suspension
Petri dish
Petri dish
Coated to prevent adhesion
Trypsinized cells
BR
3D
Cells are dispersed in GM following trypsin removal. After seeding, cells in monolayer culture
settle, attach to the vessel bottom, and flatten. If attachment is prevented by coating the bottom
surface of the vessel, cells remain suspended, spherical in shape, and attach to each other.
During BR culture, cell interaction and colocalization is facilitated and 3D aggregates form.
Methodology
Bioreactor cultureBR
BR
Fix 3 day aggregates
Culture Period:
3 Days in GM
SC
Suspension cultureSC
Stain: Myosin
OR
Fresh GM, continue
culture, and fix 6/7 day
aggregates
Paraffin embed and
section
Stain: H&E
Results: Cell Aggregation with Culture Time
A)
2D Graph 3
Single Cells Remaining in Culture
100
B)
% Single Cells
80
Graph of aggregate *
studies
60
40
*
20
C)
0
l
o
ntr
Co
0.5
ur
ho
Control
Bioreactor
Suspension Control
r
ou
1h
r
ou
2h
Treatment
r
ou
3h
r
ou
6h
D)
r
ou
9h
* only 2 replicates
* only 1 replicate
Cell aggregates formed in BR and SC culture environments with time. Graph represents mean percent single cells of 3 replicate
experiments with a minimum of 500 events counted per treatment per replicate. The percent of single cells remaining in culture was
used as a measure of cell aggregation. The SC’s at the 6 and 9 hour time points consisted of very large aggregates and few single cells.
Thus 500 events could not be counted in all replicates and were excluded from statistical evaluation. The control (black) consisted of an
aliquot of cells used to seed the culture vessels (0 treatment, 0 hour).
Two way ANOVA statistical analysis of the data (BR vs SC, Time) revealed a statistically significant interaction between Treatment and
Time (p = <0.001). Multiple comparisons vs control (Holm-Sidak method) revealed significant differences: 1) between BR and SC at all
time points ( p<0.001), 2) within the SC at all time points (p<0.001) and 3) within the BR at 2 and 3 hours (p=0.006, p<0.001, respectively).
Photos-(A) suspension culture 6 hr; (B) greater than 6hr; (C) BR aggregates and (D) close-up view of BR aggregates.
Results: Aggregate Viability
A)
B)
D)
C)
A)
B)
C)
D)
Thin sections of aggregates with H &E staining revealed absence of dead cells (necrosis).
Confocal microscopy image of a smaller aggregate stained with the Live/Dead assay (Molecular Probes).
Green fluorescent staining indicates that cells are alive while red cells are dead.
Confocal microscopy cross section of the image shown above at a depth of 25 m. No dead cells were
observed.
Confocal cross-section view of another aggregate. Three dead cells (stained red) are evident around the
periphery of aggregate.
Results: C2C12 Fusion/Differentiation
A)
B)
A) Monolayer culture: Arrows highlight some of
the maturing muscle cells characterized by
an elongated shape and many nuclei
(original mag 200X).
B) BR culture: Aggregated myoblasts fused to
form multinucleated cells known as syncitia
in BR without stimulation (400X). Few
syncitia were seen in SC aggregates (not
shown).
C)
C) H&E staining of 7 day aggregate (400X). Red
arrows indicate multinucleated syncitia.
Results: BR Culture Increases Syncitia Formation
Effects of Bioreactor Culture on Differentiation:
Syncitia Formation
Number of Syncitia per Field (Corrected)
10
8
Eight day BR aggregate. Note large cells
at periphery.
6
4
2
0
s
ay
3D
Bioreactor, 3 days
Suspension Control, 3 days
Bioreactor, 6+ days
Suspension Control, 6+ days
s
ay
3D
s
D
6+
ay
D
6+
s
ay
Treatment
Eight day BR aggregate sectioned
and stained with H & E.
The number of syncitia are increased in BR and with time in culture. H&E stained sections were used to quantify the number of syncitia per
field of view. Two way ANOVA (BR vs SC, Time) revealed a statistically significant interaction between Treatment and Time (p = <0.001).
Multiple Pairwise comparisons (Holm-Sidak method) revealed significant differences: 1) between BR and SC at 6+ days ( p<0.001); 2) within
the SC (p=0.001); and 3) within the BR (p<0.001). Graph represents the mean number of syncitia per field of view; bars represent standard
error. A minimum of 59 fields were counted per treatment (3 replicate experiments).
Results: Skeletal muscle specific myosin
expression increased in BR culture and with time
Effects of Bioreactor Culture on Differentiation:
Myosin Expression
200x103
Thresholded Area in Pixels
150x103
7 day SC aggregate sectioned and stained with
MHC antibody and secondary (red).
100x103
50x103
0
ay
BR
3D
BR
6/7
y
Da
SC
3D
Treatment
Bioreactor, 3 Days
Bioreactor, 6/7 Days
Suspension Control, 3 Days
Suspension Control, 6/7 Days
ay
y
SC
6/7
Da
7 day BR aggregate treated as above. Note
the increased expression of MHC.
Differentiation as determined by myosin heavy chain (MHC) expression is increased in BR and with time in culture. Thin sections were
deparaffinized, rehydrated, and stained using an antibody to MHC (fast and neonatal) (MY-32, Sigma) and appropriate secondary. Images
were collected using a Nikon Epi 800 with a CoolPix and analyzed with Metamorph software.
Two way ANOVA (BR vs SC, Time) revealed a statistically significant interaction between Treatment and Time (p = 0.009). Multiple Pairwise
comparisons (Holm-Sidak method) revealed significant differences: 1) between BR and SC at 6+ days (p<0.001) and 2) within the BR
(p<0.001). Graph represents the mean number of pixels per field of view; bars represent standard error. A minimum of 31 fields were
counted per treatment (2 independent experiments).
Conclusions
 Multinucleated myoblast cells grown in the Rotary Cell Culture System (BR)
were found to exhibit characteristics of differentiating muscle cells:
 Increased muscle myoblast cell fusion was observed as evidenced by a
significantly increased number of multinucleated synctia in the BR compared to
the control SC.
 Enhanced muscle myoblast differentiation occurred as indicated by increased
myosin heavy chain expression in the BR compared to the control SC.
 No solid supports (scaffolding or beads) or special differentiation medium is
needed for this fusion and differentiation to occur.
 BR culture conditions reduce cell death (necrosis) in the interior of
multicellular tissue constructs when compared to tissue constructs produced
in suspension in coated Petri dishes.
 This model test system is suitable for assessing cellular and molecular
alterations which occur during muscle myoblast differentiation and fusion.