Effects of chemical and physical methods on decellularization of murine skeletal muscles

Abstract

Volumetric muscle loss causes functional weakness and is often treated with muscle grafts or implant of biomaterials. Extracellular matrices, obtained through tissue decellularization, have been widely used as biological biomaterials in tissue engineering.Optimal decellularization method varies among tissues and have significant impact on the quality of the matrix. This study aimed at comparing the efficacy of four protocols, that varied according to the temperature of tissue storage and the sequence of chemical reagents, to decellularize murine skeletal muscles. Tibialis anterior muscles were harvested from rats and were frozen at -20°C or stored at room temperature, followed by decellularization in solutions containing EDTA + Tris, SDS and Triton X-100, applied in different sequences. Samples were analyzed for macroscopic aspects, cell removal, decrease of DNA content, preservation of proteins and three-dimensional structure of the matrices. Processing protocols that started with incubation in SDS solution optimized removal of cells and DNA content and preserved the matrix ultrastructure and composition, compared to those that were initiated with EDTA + Tris. Freezing the samples before decellularization favored cell removal, regardless of the sequence of chemical reagents. Thus, to freeze skeletal muscles and to start decellularization with 1% SDS solution showed the best results.

Decellularization for the retention of tissue niches

“…Most protocols that use EDTA also include trypsin to help cleave the bonds between cells. 37 Though most protocols pair EDTA with trypsin, its versatility allows it to also be paired with ionic detergents and non-ionic detergents. 37 , 167 Miranda et al 37 coupled EDTA with Tris buffer and used it for murine skeletal muscle decellularization by induction of an osmotic shock.…”

“… 37 Though most protocols pair EDTA with trypsin, its versatility allows it to also be paired with ionic detergents and non-ionic detergents. 37 , 167 Miranda et al 37 coupled EDTA with Tris buffer and used it for murine skeletal muscle decellularization by induction of an osmotic shock. This approach, while successful, was improved with additional treatment by ionic and nonionic detergents but was still less effective than standard ionic or nonionic protocols.…”

“…This approach, while successful, was improved with additional treatment by ionic and nonionic detergents but was still less effective than standard ionic or nonionic protocols. 37 , 167 …”

Decellularization Strategies for Regenerating Cardiac and Skeletal Muscle Tissues

“…In the final stages of the protocol, once a tissue has been decellularized, washes with sterilizing and anti-mycotic agents such as penicillin and streptomycin, ( Merritt et al, 2010 ; Kasukonis B. et al, 2016 ; Kasukonis B. M. et al, 2016 ; Jin et al, 2021a ; Jin et al, 2021b ), peracetic acid, ( Wolf et al, 2012 ; Zhang et al, 2016 ; Choi et al, 2019 ), and ethanol ( Choi et al, 2019 ) are used. Lastly, many groups administer EDTA in series or in combination with the first few decellularizing agents to help prevent clot formation as the blood is removed from the tissue vasculature ( Gillies et al, 2011 ; Miranda et al, 2021 ). Although these supplementary chemicals contribute to a more complete decellularization, they may also impact the ECM retention, myofiber morphology, and overall structural integrity of decellularized tissues.…”

Characterization of Decellularized Extracellular Matrix from Milkfish (Chanos chanos) Skin

“…A previous study reported that among the decellularization agents used in their study, TX100 produced the best ECM in terms of structure, toxicity, cell adhesion, and mechanical and physical properties [ 7 ]. On the other hand, SDS is already known to be an essential reagent for the decellularization of numerous organs [ 12 ].…”