Human Skeletal Myocytes
|FIGURE 1 Myocytes and myotubules in various
stages of differentiation.
(A) Individual myocytes
prior to differentiation.
(B) Myotubules showing
the classic "peas in a pod" appearance of
multiple nuclei within single cells. Note that
mononucleated as well as
multinucleated cells in the culture.
(C) Striations in differentiated myotubules.
Human muscle tissue consists of many cell types,
including adipocytes, fibroblasts, nerve cells, and
stromal-vascular components. In the past, biochemical
studies of human muscle used organ culture or dissociated
monolayers of primary cells. By the nature of
theses techniques, both methods would result in contamination
of the muscle cells with other cell types. In
1981, Blau and Webster developed a method to maximize
proliferation and differentiation of muscle satellite
cells to produce cultures of pure human muscle cells.
Subsequently, serum-free media were developed to
optimize the growth of human myocytes without differentiation
(Ham et al.
, 1988). It then became feasible
to use a sequential two-media approach to grow and
differentiate human myocytes. With these techniques,
and more recent modifications, it is now possible to
study human muscle myocytes without the confounding
complication of contamination by other cellular
components of muscle tissue.
There are many reasons why it is valuable to be able
to study myocytes in isolation. Some investigators
have considered the potential of human myoblasts in
gene therapy. This exploits the important characteristic
of muscle cells; the progeny of a single cell can be
taken full circle from the animal to the culture dish and
then back to the animal where they fuse into mature
myofibers of the host (Blau et al.
, 1993). In fact, this
quality of myoblasts affords itself as a way to develop
gene- and cell-based therapies for many genetic
disorders, including Duchenne muscular dystrophy
(Gussoni et al.
, 1997). A number of investigators have
been studying the effects of insulin on multiple aspects
of metabolism in human muscle cells as it relates to
type 2 diabetes mellitus and other insulin-resistant states (Borthwick et al.
, 1995; Park et al.
, 1998; Gaster et al.
, 2002). Maintenance of muscle cells under controlled
conditions permits evaluation of the contribution
of the components of the type 2 diabetic
environment, such as hyperglycemia, hyperinsulinemia,
and hyperlipidemia, to the metabolic behaviors of
muscle as compared to the intrinsic or genetic properties
of muscle. The culture conditions can also be
manipulated to reproduce acquired behaviors. As a
final example of how human myoblast cultures can be
employed, investigators have also looked at the development
of factors during the maturation of human
myocytes to myotubes (Gunning et al.
, 1987). Therefore,
the ability to investigate human muscle cells in
vitro has a great importance in discovering clinically
significant in vivo
Investigators have discovered that the differentiation
of myoblasts to myotubes while in culture involves
changes in gene expression (Gunning et al.
, 1987). Using
the technique illustrated in this article, characterization of
the muscle cells in terms of the biochemical markers they
express during the fusion process has been described
previously. Some of the changes in gene expression in
major markers that are expressed include increases in
sarcomeric specific c~-actin protein (3.5-fold), the
muscle-specific isoform of creatinine phosphokinase
(CPK-M) mRNA (2-fold), and CPK-M enzyme activity
(6-fold). The process of myoblast fusion into myotubes
can also be observed structurally through fluorescent
micrographs of cells stained for nuclei, as seen in Fig. 1
(Henry et al.
, 1995). A more complete list of changes in
biochemical and histologic markers following differentiation
of myocytes into myotubules is presented in
Table I. It is important to monitor a number of these
markers when manipulating conditions or comparing
cells from different individuals.
II. MATERIALS AND
Hams F10 media (Cat. No. 9056), custom ATV (Cat.
No. 9920), α-MEM (Cat. No. 9142), Fungibact (Cat. No.
9350), penicillin/streptomycin (pen/strep) (Cat. No.
9366), and glutamine (Cat. No. 9317) are from Irvine
Scientific (Santa Ana, CA). The SKBM and bullet kit (Cat. No. 3160) are from BioWhittaker (Walkersville,
MD). To make SKGM, the components of the bullet kit
are added to one bottle of SKBM, along with 10ml Fungibact
and 5 ml glutamine. The insulin bullet can be
added or omitted depending on the final purpose for
which the cells will be utilized (see later).
Fetal bovine serum (FBS) is from Gemini (Calabasas,
CA) (Cat. No. 100-106). The Hams F10 media,
SKGM, and α-MEM should all be stored at 4°C. Stocks
of custom ATV, fetal bovine serum, glutamine, Fungibact,
and pen/strep should be stored at-20°C. To
make fusion media, add fetal bovine serum to a final
concentration of 2%, 10ml of pen/strep, and 5ml of
glutamine to 500ml of α-MEM.
The following were all obtained from Fisher Scientific:
6-well tissue culture plates (Cat. No. 08772-1G),
12-well tissue culture plates (Cat. No. 08772-3A), 24-
well tissue culture plates (Cat. No. 07200-84), 100-mm
culture dishes (Cat. No. 08772-E), sterile scalpel (Cat.
No. 08927-5A), sterile pasteur pipettes (Cat. No. 13678-
20B), sterile flasks (Cat. No. 100429-A), 15-ml conical
polypropylene tubes (Cat. No. 145970C), 50-ml conical
tubes (Cat. No. 1495949-A), 5-cc pipettes (Cat. No.
13675-22), 10-cc pipettes (Cat. No. 13675-20), 25-cc
pipettes (Cat. No. 13655-30), p200 pipette tips (Cat. No.
02681422), and pl00 pipette tips (Cat. No. 02681422).
No special treatment or substrate is needed for the cells
to attach to the plastic. Cells are grown and maintained
in a humidified, 37°C incubator under 5% CO2
The following techniques for the growth of human
skeletal muscle cultures were established through
modifications of previously described methods (Blau
and Webster, 1981; Sarabia et al.
A. Cell Isolation
All steps are to be performed under sterile
B. Cell Culture
- Collect muscle tissue in a 50-ml conical tube in
20ml cold (4°C) Hams F10 media: approximately
100-200mg of tissue is needed.
- Aspirate media. Wash tissue three times with
chilled (on ice) Hams F10 to remove blood.
- Transfer tissue in a small volume (~5ml) of
Hams F10 to a 100-mm culture dish.
- Mince tissue with sterile scalpels. Make pieces
as small as possible.
- Transfer tissue and media back to centrifuge
tube and aspirate media with a sterile Pasteur pipette. Because the tissue will settle out on its own, the sample
does not need to be centrifuged.
- Add 20ml of trypsin/EDTA (custom ATV) and
transfer to a small flask (e.g., 50-ml Erlenmeyer flask)
containing a stir bar.
- Stir 20-30min at room temperature in a sterile
hood. Collect supernatant and place on ice.
- Add 20ml ATV to flask and repeat step 7 two
more times. Pool the supernatants together on ice.
- To the pooled supernatants add FBS to a final
concentration of 10%.
- Centrifuge cells for 5 min at 1600 rpm (550g) at
- Aspirate supernatant and add 20ml of SKGM
[SKBM + bullet kit (with or without insulin) + 10ml
Fungibact + 5ml glutamine] to cell pellet. Pipette up
and down gently to resuspend cells.
- Transfer media with cells into two 100-mm
dishes. Mark plates with subject identifier and place in
incubator at 37°C 5% CO2.
- Change SKGM media approximately every 3
days. Continue for 2-3 weeks.
- During the next 2-3 weeks, the muscle cells will
grow attached to the surface of the culture dish. Eventuallythe cells will form a confluent layer on the
bottom of the dish.
C. Cell Fusion/Differentiation
- When the cells are 60-70% confluent, aspirate
media and rinse the cells once with 5 ml of custom
ATV and aspirate.
- Place another 5 ml custom ATV on plates, aspirate
after 30s, and then incubate for 5 min at 37°C.
- Check cells under a microscope to see if they are
detached (cells will look rounded).
- If cells are detached, add 5 ml SKGM to rinse plate,
collect cells, and transfer to a 15-ml centrifuge
tube. If they are only partially detached, remove
the attached cells by pipetting up and down gently.
- Add 5ml SKGM media to trypsinized cells and
centrifuge at room temperature for 5min at
1600 rpm (550 g).
- Aspirate supernatant and resuspend cells in up to
5 ml SKGM media (not more than 6 ml, depending
on volume needed) by pipetting up and down
- Count cells in a hemocytometer chamber.
- Plate cells:
- 100-mm dishes: 60,000 cells per plate (approximately
60 µl cell suspension)
- 6-well dishes: 20,000 cells per well (approximately
- 12-well dishes: 6000 cells/well (approximately
- 24-well dishes: 3000 cells/well (approximately
10 µl per well)
- Cover wells with SKGM: 10 ml per 100-mm dish,
2ml for each well per 6-well plate, and 1 ml for
each well per 12-well and 24-well plates.
- Change the SKGM every 2-3 days.
- When cell cultures are 80-90% confluent, aspirate
media and rinse two times with α-MEM/pen-strep/
5mM glutamine/2% FCS (fusion media).
- Add fusion media to wells: 10ml per 100-mm
dish, 2ml/well per 6-well plate, and 1 ml/well per 12-
well and 24-well plates.
- Culture at 37°C, 5% CO2.
- Change media every 48 h.
- Fusion/differentiation is complete by 96 h.
The technique just described has been modified primarily
to investigate the metabolic characteristics of
human muscle cells. The system has been employed to
study insulin action on glucose uptake and glycogen
synthesis, fatty acid uptake and oxidation, insulin signaling,
and regulation of gene expression. Other investigators
have employed different techniques for the
culture of human muscle cells; however, the focuses of
those investigations were not on hormone action or
metabolic activities (Rando and Blau, 1994; Webster
and Blau, 1981), which is why the types of media
employed differ from those described in this article.
The major differences from the procedure described
here are that growth media include Hams F-10 with
0.5% chick embryo extract and 20% fetal calf or horse
serum. Furthermore, fusion media contain Dulbecco's
modified Eagle's media with 2% horse serum. It is
uncertain how the constituents of these other media
would differ in their impact on human skeletal muscle
metabolism when compared to the defined SKGM and
fusion media described in this article.
In the system described here, cells are passed only
a single time before terminal differentiation and are
not maintained over multiple passages. Our experience
is that both the extent of differentiation (percentage multinucleated cells) and the insulin responsiveness
for metabolic events are diminished in a passagedependent
manner. Other investigators have
maintained cultures from a single subject for a greater
number of passages (as high as 15) (Halse et al.
- It is critical to use sterile technique to avoid cell
contamination. Bacteria or fungal contamination can
lead to altered metabolic activity of the muscle cells.
- Omit insulin from the SKBM if planning to investigate
the effect of insulin on muscle cultures. If
the insulin bullet is added, the final concentration
(-30µM) is high enough to produce a state of insulin
- To avoid excessive cell damage from the trypsin,
do not overincubate with ATV.
- Change media every 48 h to avoid exhaustion of
growth factors and glucose. This can alter the metabolic
activity of the muscle culture.
- Treatment of cells can be performed either during
the fusion/differentiation period or at completion. If
treatment is done during differentiation, then the
extent of differentiation must be monitored for each
new manipulation. This can be done with careful
checking of differentiation by following muscle
markers mentioned in Table I.
- It is important to pass the cells at 60-70% confluency.
Beyond that point spontaneous fusion may
begin in adjacent cells, reducing subsequent
- If the fusion media and SKBM are used more
than 10 days after assembly, it will be necessary to supplement
media with additional glutamine.
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