Preparation of 3D Decellularized Matrices from Fetal Mouse Skeletal Muscle for Cell Culture

To our knowledge, this protocol is the first to produce decellularized matrices from fetal skeletal muscle, providing a novel approach to study myopathies that start developing in utero. This protocol generates a developmental and tissue-specific metrics that can be used to study muscle cell behavior and interactions with extracellular matrix in a controlled environment. LAMA2-CMD is a congenital muscle dystrophy that starts manifesting at birth.

This model can help unravel the pattern mechanisms involved in its onset and lead to new target therapies. This protocol can be adapted to different tissues and disease models. Different complexity levels can be achieved depending on matrix and cell types, demonstrating the versatility of the system.

This is a very straightforward protocol. The most challenging part is the collection and the handling of the samples. But, with practice, technical skills can easily be improved.

Begin by transferring one euthanized fetus at a time to a Petri dish containing ice-cold PBS. After excising the skin and limbs, cut through the ventral side of the ribcage. Remove the sternum and underlying organs.

Position the fetuses dorsal side up and remove the cervical portion of the vertebral column. Next, excise the dorsal fat deposits and the deep back muscle connective tissue. Now, anchor the ribcage using surgical forceps.

With a microscalpel, carefully scrape to detach the deep back muscles from the surrounding tissue. Store the tissues in PBS in a 12-well cell culture plate at four degrees Celsius for future use. For a longer period, store the tissue in an empty microcentrifuge tube at minus 80 degrees Celsius.

On the first day, use whole epaxial muscle masses. Add three milliliters of hypotonic buffer containing 1%penicillin and streptomycin to each well of a 12-well plate. Add a muscle tissue fragment to each well and incubate overnight for 18 hours with agitation.

On the second day, remove the buffer with a fine-tip pipette. Now wash the samples three times with three milliliters of PBS with a one-hour agitation each time. After discarding the PBS, incubate the samples in three milliliters of 0.05%SDS detergent solution for 24 hours with agitation.

On the third day, use a fine tip-pipette to remove the SDS detergent and wash the fragments thrice with three milliliters of hypotonic wash buffer with a 20-minute agitation each time. Replace the solution of the wells with two milliliters of DNase solution and incubate the tissue fragments at 37 degrees Celsius for three hours with agitation. After removing the DNase solution, wash the fragments thrice with three milliliters of PBS with a 20 minute agitation each time, leaving the last wash overnight.

To a T-25 flask seeded with subconfluent C2C12 cells, add 500 microliters of trypsin and resuspend in one milliliter of complete medium. Mix 10 microliters of the suspension with 10 microliters of trypan blue dye and load into a hemocytometer to count cell number and estimate viability. In a laminar flow hood, place the decellularized matrices, or dECMs, in a Petri dish containing PBS with 1%penicillin and streptomycin.

Using a microscalpel and tweezers, separate the matrices into small pieces. Transfer the fragments into the wells of a 96-well plate, placing three to four pieces per well. Add 200 microliters of prewarmed complete culture medium into each well and incubate the plate for two hours at 37 degrees Celsius with 5%carbon dioxide.

Discard the medium in the well plate and add 200 microliters of complete culture medium containing 50, 000 viable C2C12 cells to each well. Incubate the well plate for two days. Transfer the dECMs with the cells to a 48 well-plate containing 400 microliters of complete culture medium.

Aspirate the medium carefully every two days to prevent matrix detachment and replenish with the fresh medium until the eighth day. For differentiation, replace the complete culture medium with a differentiation medium and incubate for four days until the 12th day. The muscle tissue was reddish immediately post-isolation and turned white due to cell lysis after incubation in hypotonic buffer.

SDS causes the muscles to turn transparent. After DNase treatment, a smaller, transparent dECM was obtained. DNA quantification reveals a nearly 100%decrease in the dECMs compared to the native tissue.

The dECMs and native tissue displayed similar tubular staining for laminin alpha two and laminin protein. Western blot analysis of the native tissue displayed two bands for the laminin alpha two subunit, while three smaller bands were detected in the dECM. For total laminins, dECM samples showed protein fragmentation when compared to the native tissue.

Fibronectin and collagen I were present in the interstitial space between cells of the native tissue and the dECMs. Similar fibronectin bands were present in both samples, indicating that it is not affected by decellularization. Fewer collagen I bands were observed in the dECMs.

For collagen IV, both samples showed similar tubular staining. However, the molecular weight of the dECM bands was lower. C2C12 cells colonized and proliferated in the dECMs and fused into multi-nucleated myotubes.

These expressed myosin-heavy chain protein after four days of incubation in differentiation medium. Intracellular and pericellular staining was observed for laminin alpha two chain, total laminins, and fibronectin. Decellularized fetal mouse skeletal scaffolds can be used to grow cells in co-culture systems, bringing it closer to the in vivo situation, and therefore contributes to understanding muscle disease.