In order to deepen the knowledge about the pathophysiological mechanisms of this myopathy and how it influences the PFM dysfunctions observed in pregnant women with GDM, the present investigation evaluated the feasibility of developing a three-dimensional (3D) cell culture model starting with progenitor muscle cells (PMC) isolated from the RAM of pregnant rats with hyperglycemia similar to GDM, as a new in vitro model for further investigations.
The Institutional Animal Care and Use Committee of our institution approved all animal experiments (protocol 1355/2020), in accordance with the Brazilian Council for Control of Animal Experimentation (CONCEA). PMC isolation protocol was based on previous studies. Both sides of the RAM from 10 pregnant rats were collected, totaling 1,814 grams of tissue per animal. The material was mechanically and enzymatically dissociated to release the different cell types that structure the muscle. A PMC pool was obtained from all animals, and 1x105 cells/flask were seeded and further cultivated under ideal conditions. Phenotypic characterization of the cells was performed by flow cytometry and images were obtained by inverted light microscopy.
Cells that adhered to the surface of the flasks, presented mesenchymal-like morphology, and formed typical colony units from the third day of culture were isolated and cultivated. Cells proliferated in vitro and were subcultured until the third passage. Cultures were phenotypically heterogeneous, composed mainly of PMC (Pax-7+ and MyoD+) and vascular cells (CD31+), which were maintained throughout the passages. Under differentiation media (maturation inducer), PMC became elongated, tubular and multinucleated, characteristics of muscle cells. After a long period in culture with differentiation medium, it was possible to observe contractions of these cells after stimulation (link or QR code of the video will be available).
An adapted protocol for isolating and cultivating PMC from RAM of hyperglycemic pregnant rats was stablished. In addition, the obtained model showed contraction after cultivation with differentiation medium. Further investigations with these cell cultures will be conducted to evaluate whether the changes caused by diabetic myopathy in vivo continue to affect the PMC in vitro. This model can significantly enrich the available methods to investigate and propose new treatments for UI.
Traditional (2D) cell cultures represent a crucial step towards the generation of 3D models, such as muscle organoids, which have greater complexity, structure, and the ability to mimic a certain degree of functionality of the organ under study. Muscle organoids represent an important tool for understanding the dysfunctions of RAM caused by diabetic myopathy, as well as a new and sophisticated model for studying muscle diseases.
Urinary incontinence
In vitro models
