Isolation and Characterization of Primary DMD Pig Muscle Cells as an In Vitro Model for Preclinical Research on Duchenne Muscular Dystrophy
Abstract
:1. Introduction
2. Materials and Methods
2.1. Animals
2.2. Isolation of Different Muscle Groups
2.3. Isolation of Satellite Cells
- Using a laminar flow cabinet, solution A was removed from the sample.
- Muscle tissue was separated from connective and fat tissue using a sterile scalpel.
- Then, remaining muscle was weighed, a minimum of 3 g, but if possible, 5–20 g of muscle were used for each primary culture.
- The piece of muscle was cut into very small pieces.
- The cut muscle pieces were magnetically stirred for 20 min at 37 °C in a 500 mL Schott® bottle in 25 mL of Hank’s balanced salt solution (HBSS) containing 0.2% collagenase I, 0.01% DNase I, and 0.025% trypsin.
- Following, the bottle was chilled in an ice bath for 2 min.
- The supernatant was transferred into fresh tubes and mixed with an equal amount (1:1) of growth medium (α-MEM, 10% FCS, 1% Glutamax, 0.2% amphotericin B, 0.2% gentamicin). For the remaining muscle piece, the steps from the magnetic stirring to the transfer of the supernatant were repeated two times.
- This mix was filtered through a 100 µm micro strainer and then centrifuged (800× g, 10 min, 4 °C).
- The supernatant was discarded and the pellet was resuspended in 15 mL of growth medium (4 °C).
- The suspension was then filtered through a 70 µm micro strainer and centrifuged (800× g, 10 min, 4 °C).
- The supernatant was discarded and the pellet resuspended in 12 mL of PBS.
- The suspension was slowly and carefully given on top of a step gradient [(50-mL tube containing 3 mL of a 60% Percoll-solution (Easycoll and PBS, lower layer) and 35 mL of a 20% Percoll-solution (upper layer)].
- This 50 mL tube was centrifuged (4800× g, 45 min, 4 °C, no brake)
- The boundary layer between the 20% and the 60% Percoll-solution was taken out by a sterile Pasteur pipette (approx. 8–12 mL), transferred into a new sterile tube, and mixed with 15 mL of growth medium. This suspension was centrifuged (500× g, 10 min, 4 °C), the supernatant was discarded, and the pellet resuspended in 10 mL growth medium.
- Depending on the size of the pellet, approx. 1 mL cell-pellet was transferred with 18 mL of additional growth medium into one 75 cm2 cell culture bottle.
- The newly harvested satellite cells were grown in growth medium in a 37 °C incubator with 5% CO2 for 24 h. The growth medium was changed after one day.
- A mycoplasma infection test was made regularly with the cells, using the MycoAlert™ Mycoplasma Detection Kit (LT07-118, Lonza, Basel, Switzerland).
2.4. Cell Growth, Storage, and Differentiation
2.5. Western Blot
2.6. Immunofluorescence Staining
3. Results
3.1. Characterization of Cell Growth and Myogenic Content
3.2. Differentiation into Myotubes
3.3. Coating of Dishes
3.4. Utrophin Expression
4. Discussion
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Conflicts of Interest
References
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Donandt, T.; Hintze, S.; Krause, S.; Wolf, E.; Schoser, B.; Walter, M.C.; Meinke, P. Isolation and Characterization of Primary DMD Pig Muscle Cells as an In Vitro Model for Preclinical Research on Duchenne Muscular Dystrophy. Life 2022, 12, 1668. https://doi.org/10.3390/life12101668
Donandt T, Hintze S, Krause S, Wolf E, Schoser B, Walter MC, Meinke P. Isolation and Characterization of Primary DMD Pig Muscle Cells as an In Vitro Model for Preclinical Research on Duchenne Muscular Dystrophy. Life. 2022; 12(10):1668. https://doi.org/10.3390/life12101668
Chicago/Turabian StyleDonandt, Tina, Stefan Hintze, Sabine Krause, Eckhard Wolf, Benedikt Schoser, Maggie C. Walter, and Peter Meinke. 2022. "Isolation and Characterization of Primary DMD Pig Muscle Cells as an In Vitro Model for Preclinical Research on Duchenne Muscular Dystrophy" Life 12, no. 10: 1668. https://doi.org/10.3390/life12101668