Explant in natura and cryopreserved myometrial tissue for in vitro cell culture of Primary Smooth Muscle from Myometrium of Ovis Aries (PSMo24) v1

Since the first in vitro cell culture was conducted in 1907 by the American Ross Granville Harrison to the present day, cell culture has established itself as an essential tool for basic and applied research, covering areas such as cell biology, molecular biology, physiology, animal and human biotechnology, among others. Cell culture is widely recognized as the standard in cell biology studies due to its ability to mimic the cellular environment in a way similar to the in vivo state. In the context of studying primary smooth muscle from myometrium cell, beyond their reproductive importance, these cells share physiological mechanisms with other cell types, allowing for a more comprehensive understanding of cellular function and specific muscle cells. One of the main challenges in establishing primary culture is the immediate acquisition of uterine tissue. To overcome this limitation, this study describes the development of a methodology for primary smooth muscle from myometrium of Ovis Aries (PSMo24) and establish a protocol for cryopreservation of myometrial tissue followed by subsequent cell explantation. This approach aims to maximize the uterus’ biological potential and minimizing tissue waste, which is traditionally used only in fresh form. Moreover, given the restrictions on animal’s experimentation, tissue culture stands out as an alternative method, providing valuable knowledge to evaluate substances. This contributes to the reduction or replacement of animal experimentation, aligning with ethical and scientific principles in biological studies. For the establishment of PSMo24 cell culture, uterine tissue samples from Ovis Aries were collected and subjected to enzymatic digestion. Simultaneously, fragments of myometrial tissue were cryopreserved, thawed, and explanted for cell culture. Ten cell passages were performed, along with quantification, morphology analysis, cytotoxicity assessment, antioxidant capacity evaluation, and RNA extraction. Here, we validated the protocol for PSMo24 from fresh cells and cryopreserved tissue for explant by demonstrating the morphology, viability, and cell proliferation, indicating new possibilities for their aplication in studies using reproductive and muscle cells, as well as in pharmacological or bioproducts tests in animals and humans.