Download Automated dissociation of skeletal muscle tissue Isolation of satellite

Isolation of functional satellite cells using
automated tissue dissociation and magnetic cell separation
Janina Kuhl, Christoph Hintzen, Andreas Bosio, and Olaf Hardt
Miltenyi Biotec GmbH, Bergisch Gladbach, Germany
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Introduction
One of the most commonly used experimental models in tissue
regeneration are satellite cells. However, the analysis of their
molecular and functional characteristics is frequently hampered by
the use of heterogeneous cell populations, as oftentimes the
observed effects are caused by contaminating cell populations
rather than the target fraction. To circumvent this issue, it is necessary
to purify the satellite cells prior to downstream analysis.
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Isolated satellite cells can be expanded
and differentiated into mature myotubes
Satellite cells isolated by this novel method have major advantages
over unseparated cells: they can be plated and directly give rise to
homogenous myoblast cultures without the need for pre-plating
steps or selection media to deplete fibroblasts. The isolated cells can
be efficiently expanded in vitro and reliably differentiate into
multinuclear, contracting myotubes after three days of induction
with horse serum.
A
10³
10³
10²
10²
nGFP
Phase contrast
Transmitted light
nGFP/Phase contrast
10¹
90.55 %
1
0
-1
-1 0 1
10¹
10²
FSC
FSC
10³
C
Phase contrast
Conclusion and outlook
After 6 days in culture
MyoD
DAPI
DAPI/MyoD
10¹
1
0
92.13 %
-1
-1 0 1
10¹
10²
Phase contrast
We developed an easy and fast method for the dissociation of skeletal
muscle tissue as well as a cell isolation method that allows for an
accurate downstream analysis of satellite cells. Isolation of the cells
avoids analytical bias caused by contamination with non-target cells.
Reliable methods for the dissociation, analysis, and isolation of
satellite cells from wild type and transgenic mouse models, in
combination with molecular and functional downstream examination,
will provide new insight into the biology of this highly interesting
subpopulation.
References
1. Sacco, A. et al. (2008) Nature 456: 502–506.
10³
PE
PE
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The purification of target cell populations is crucial for reliable
downstream analysis, e.g., by transcriptome or proteome profiling,
as otherwise it is hard to tell whether observed effects can be
attributed to the cells of interest or are caused by contaminating cell
populations. Based on published expression profiles¹ we determined
markers to set up an optimal panel for the identification of satellite
Lin+
Isolation of satellite cells
cells. Moreover, we established a protocol for the isolation of satellite
cells by magnetic cell separation (MACS® Technology). This protocol
is based on the depletion of cells expressing lineage markers (CD31,
CD45, CD11b, Sca-1), with an optional additional positive selection
step based on integrin-alpha-7 expression to further increase the
purity of satellite cells.
10³
C
After 7 days in culture and 3 days of differentiation
MyoD/Actinin
10³
10¹
1
0
-1
-1 0 1
10³
ITGA7-PE
10³
10²
10¹
1
0
-1
-1 0 1
12.78 %
10¹
10²
ITGA7-PE
ITGA7-PE
10³
90.70 %
10¹
10²
10³
ITGA7-PE
10³
CD34-APC
CD34-APC
CD34-APC
CD34-APC
10²
10¹
1
0
-1
-1 0 1
9.85 %
10¹
10²
Lin–
76.85 %
10²
Lin+
10²
Lin*-FITC
Lin*-FITC
Magnetic Isolation of negative
fraction, i.e., satellite cells
Lin*-FITC
Lin*-FITC
0.20 %
Magnetic labeling of
lineage cells
Figure 4
Lin–
PIPI
SSC
SSC
DAPI/MyoD
Lin+
based on the gentleMACS™ Dissociator. The process allowed for the
complete dissociation (B) of skeletal muscle tissue, with low amounts
of debris (A, left) and cell viabilities (A, right) above 90%. The
improved marker preservation enabled the downstream identification
of lymphocyte- as well as muscle- and endothelial-derived cell
lineages. A completely automated process was implemented
subsequently, based on the gentleMACS Octo Dissociator (C), which
enables enzymatic dissociation directly on the instrument through
integrated heater modules.
B
Figure 2
DAPI
Automated dissociation of skeletal muscle tissue
A
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using StemMACS™ Transfection Reagent. With this method,
transfection efficiencies higher than 75% could be achieved per
treatment. In addition, we tested up to four consecutive transfections
and did not observe cell death or other negative effects.
Lin–
MyoD
A prerequisite for the efficient isolation of cell populations from
solid tissue is a reliable method for the dissociation of the respective
tissue. We have screened multiple types of enzymes and enzyme
combinations in order to optimize cell yield and viability after
dissociation. By using high-purity enzymes, high epitope stability
was maintained, which is essential for the reliable identification and
isolation of target cell populations from dissociated tissue. After
determining the optimal enzyme components and concentrations,
we developed an automated procedure for all mechanical steps,
Figure 1
We developed a method to transfect cultured myoblasts with
artificial mRNAs, allowing for the effective modulation of this stem
cell population without the risk of permanent genetic modification.
As a proof of concept, 100 ng of nuclear GFP mRNA per well was
transfected into myoblasts (cultured in in 96-well plates for five days)
After 3 days in culture
Results
1
Modulation of satellite cells by mRNA transfection
10¹
1
0
-1
-1 0 1
83.92 %
10¹
10²
10³
ITGA7-PE
ITGA7-PE
*CD31/CD45/CD11b/Sca-1
Figure 3
MyoD/Actinin/DAPI
Phase contrast