In this chapter we describe the basic methodologies regularly used in our laboratory for the isolation, culture and characterization of myogenic progenitors from adult mouse skeletal muscle. As detailed in the previous section, we also use contemporary approaches for satellite cell isolation that are based on fluorescence reporter expression and/or based on expression of cell surface antigens. However, such approaches require the availability of special resources and reagents. Here, we describe a basic and straightforward method that we frequently use to isolate and characterize satellite cell performance in culture. This procedure can be performed in any tissue culture facility, using wildtype and mutant mouse muscles of various ages (12
), and is suitable for satellite cell isolation from rat (62
) and chicken (46
) muscles. shows representative micrographs of myogenic cultures emanating from satellite cells isolated using our basic procedure from adult mouse hindlimb muscles.
Figure 3 Phase micrographs depicting the morphology of mouse myogenic cultures seeded on gelatin-coated (A-C) and Matrigel-coated (D) dishes. Cells were isolated by Pronase digestion and cultures were maintained in rich growth medium according to protocols detailed (more ...)
Our standard protocol for immuncytochemical analysis of satellite cell cultures provides quantitative insight into the “myogenicity” of the cell preparation (i.e., the presence and frequency of myogenic cells) and progression of satellite cell progeny from proliferation to differentiation and production of reserve cells. summarizes the source and characteristics of a set of monoclonal antibodies used in our laboratory for the analysis of myogenesis in primary cultures of mouse satellite cells, which are also applicable to rat satellite cells (8
). For analysis of chicken satellite cells, we rely on the same Pax7 and MF20 antibodies as in , but for the detection of myogenic regulatory factors we use rabbit polyclonal antibodies developed against the chicken proteins (21
Mouse monoclonal antibodies frequently used in our studies for analyzing progeny of mouse satellite cells as they transit through proliferation, differentiation and renewal.
In the following subheadings, we discuss some important considerations that should be taken in mind when establishing satellite cell primary cultures.
2.1. Strain and Age of Animals
The protocols in this chapter focus on the isolation and culture of myogenic progenitors from adult (3–6 month-old) C57BL/6 mice. Aged mice and other mouse strains have also been used in our studies following the same procedures (12
). However, muscles from younger mice may contribute more cells due to age-associated decline in satellite cells in some muscles (12
). Also, the contribution of nonmyogenic cells in the preparation may increase with age or in different mouse strains, and consequently, some conditions may need modification (i.e. duration of enzymatic digestion, extent of tissue trituration, cell straining conditions to remove debris, centrifugation speed of harvested suspension, etc.) to minimize the proportion of undesired cell types.
Herein we detail our standard procedure for the isolation of satellite cells from hindlimb muscles of adult mice. For this preparation, we typically pool the fast twitch muscles tibialis anterior (TA) and gastrocnemius from both hindlimbs, using one mouse per preparation. For additional details about TA and gastrocnemius anatomy and isolation procedures see Notes 1 and 2. This approach can also be used for isolating myogenic progenitors from limb, body and head muscles. However, the contribution of connective tissue and vasculature may vary between muscles, and the tissue isolation procedure should be modified accordingly to minimize cells derived from such structures. The purity of the resultant preparation of isolated satellite cells (and cultures emanating from this preparation) is directly dependent on the amount of effort spent meticulously cleaning the muscle of these additional structures.
2.3. Digestive Enzyme for Muscle Dissociation
Our procedure is based on cell dissociation from whole muscle using Pronase digestion (see item 7
in Subheading 3.4.
and items 4
in Subheading 4.1.
). Pronase (available from Calbiochem) consists of a mixture of proteases isolated from the extracellular fluid of Streptomyces griseus
. Due to its particular protease content, which includes several types of endo- and exopeptidases, Pronase has a broad activity (103
Pronase digestion may not be optimal for prospective satellite cell enrichment by antigen-based cell sorting because of extended digestion of surface antigens. However, myogenic cell preparations isolated by Pronase digestions show a lower level of nonmyogenic cells compared to that observed when collagenase or collagenase/dispase enzyme solutions are used. It is possible that certain nonmyogenic populations do not survive well after Pronase digestion and this may lead to the increased purity of these cultures.
2.4. Cell Yield, Choice of Culture Dish and Cell Seeding Density
Cell yields can vary depending on the age of the animal. Muscles from neonatal and young mice (1-month old or less) yield considerably more myogenic progenitors than muscles from adult mice. As mentioned before, variations are also observed when working with different muscles. For the mouse strain (C57BL/6) and hindlimb muscles (TA and gastrocnemius) used for the protocol described herein, each preparation typically yields 2–5 × 105 cells.
We commonly use 24-well or 35-mm culture dishes. We generally use 35-mm dishes for training or when performing single comparisons. In such cases, we initiate the cultures at 5–10 × 104 cells per plate. For multiple replicates across multiple time points, we use 24-well plates where starting cell density can be proportionally matched with that of the 35-mm plates based on surface area. Alternatively, seeding densities can be further reduced and depending on experimental goals, may range from 5 × 104 to 1 × 103 for primary cultures. While not further detailed below in the protocol section, it is noteworthy that we also use in some of our studies 48-well trays where we seed 2–10 cells per well; in such studies we aim to achieve clonal growth for monitoring progeny of individual satellite cells.
2.5. Culture Medium
The standard growth medium used for our mouse satellite cell cultures consists of high glucose Dulbecco's Modified Eagle Medium (DMEM) supplemented with 20% fetal bovine serum, 10% horse serum and 1% chicken embryo extract (CEE). This serum-rich growth medium supports both proliferation and differentiation of myogenic cells (12
). See Subheading 3.4.
and Notes 3–6
for details about recommended cell culture reagents, our protocol for preselection of optimal sera lots and preparation of CEE, and final medium preparation.
Some variations can be found from laboratory to laboratory with regard to the basic culture media (e.g., Ham's F10 instead of DMEM, or a mixture of the two), serum type and concentration, and source of growth factors (e.g., purified growth factors, especially fibroblast growth factor, instead of CEE). Differences in culture conditions may explain some divergences in satellite cell behavior among different laboratories. For example, some published protocols rely on first using serum-rich growth medium that supports proliferation followed by a switch to serum-poor medium to support differentiation. There are also reported variations in medium composition when preparing cultures from other species. For example, for primary cultures of chicken satellite cells we typically use medium containing 10% horse serum and 5% chicken embryo extract (21
To study the effects of specific growth factors on myogenic cell performance, we typically maintain the cells for 3 days in our standard rich growth medium to allow for optimal cell adherence, then switch the cells into serum-low (e.g., DMEM containing 2% horse serum) or serum-deprived media. Prior to switching to serum-low medium, the cultures are rinsed extensively with DMEM to remove traces of the rich medium that otherwise adhere to the cell layer and reduce the observed effect of the additives being examined.
2.6. Plate Coating Matrices
Adhesion of myogenic progenitors to cell culture dishes can be significantly improved by coating the plastic substrate with a variety of extracellular matrix constituents or derivatives. In addition to cell adhesion, matrix components can influence the extent of myogenic cell proliferation, differentiation and renewal (12
). In our laboratory, the main matrices used for coating tissue culture plates for satellite cell cultures are Matrigel and gelatin.
Matrigel is a solubilized basement membrane preparation extracted from the Engelbreth-Holm-Swarm mouse sarcoma, a tumor rich in extracellular matrix proteins. Its major component is laminin, followed by collagen IV, entactin, and heparan sulfate proteoglycan (107
). Matrigel is available from BD Biosciences and can be obtained in its standard format or in its growth factor reduced format. In our studies we have typically used the growth factor reduced format. Matrigel must be carefully handled on ice when aliquoting and coating tissue culture dishes with dilutions. For additional details on Matrigel source and handling, see item 8
in Subheading 3.4.
and Notes 7
for additional details.
Gelatin is produced by partial hydrolysis of type I collagen extracted from connective tissues. It can be purchased in a tissue culture grade powder form and easily reconstituted in water to the desired concentration. For specific details about gelatin source and our preparation of gelatin solution, see item 8 in Subheading 3.4. and Notes 9 and 10.
Gelatin is readily available, inexpensive and easy to use, which makes it an ideal product for training new team members and for use in standard cultures. However, long-term high-density myogenic cultures may spontaneously detach from plates coated with gelatin. In addition, satellite cell progeny typically demonstrate a more limited proliferative period, earlier differentiation, smaller myotubes and more meager development of reserve cells when grown on this substrate compared to Matrigel-coated dishes. Matrigel also allows a more even cell distribution upon initial cell plating compared to that observed when cells are seeded on gelatin-coated dishes. Additionally, when plated on Matrigel-coated dishes, myogenic progenitors can reach high cell densities and form complex myotube networks, typically without detaching from the substrate. The latter features have prompted us to use Matrigel especially when seeding cells at low density or when aiming to obtain single cell clones. Disadvantages of Matrigel include higher cost and the requirement for more careful handling. Other commercially available matrices that we have tested in pilot experiments that may provide reasonable alternatives include: (a) GelTrex (a Matrigel-like product from Invitrogen) and (b) Attachment Factor (Invitrogen), a ready made gelatin-based product.
2.7. Fixation and Immunostaining
For immunostaining analyses using the antibodies listed in , we typically fix the cultures with a paraformaldehyde-sucrose solution that is prepared in our laboratory. For further details about fixation approach and fixative composition, see Subheading 4.2. and Notes 11 and 12. It should be noted that fixatives should be optimized for preservation of both the cells and the antigens being analyzed. We perform all immunostaining steps in a manner that maintains sterility; handling antibodies strictly in the tissue culture hood minimizes possible bacterial contamination and helps maintain antibody stocks for years.