Mammalian cell culture is foundational to biomedical research, and the reproducibility of research findings across the sciences is drawing increasing attention. While many components contribute to reproducibility, the reporting of factors that impact oxygen delivery in the general biomedical literature has the potential for both significant impact, and immediate improvement. The relationship between the oxygen consumption rate of cells and the diffusive delivery of oxygen through the overlying medium layer means parameters such as medium depth and cell type can cause significant differences in oxygenation for cultures nominally maintained under the same conditions. While oxygenation levels are widely understood to significantly impact the phenotype of cultured cells in the abstract, in practise the importance of the above parameters does not appear to be well recognized in the non-specialist research community. On analyzing two hundred articles from high-impact journals we find a large majority missing at least one key piece of information necessary to ensure consistency in replication. We propose that explicitly reporting these values should be a requirement for publication.

Human male reproductive development has a prolonged prepubertal period characterized by juvenile quiescence of germ cells with immature spermatogonial stem cell (SSC) precursors (gonocytes) present in the testis for an extended period of time. The metabolism of gonocytes is not defined. We demonstrate with mitochondrial ultrastructure studies via TEM and IHC and metabolic flux studies with UHPLC-MS that a distinct metabolic transition occurs during the maturation to SSCs. The mitochondrial ultrastructure of prepubertal human spermatogonia is shared with prepubertal pig spermatogonia. The metabolism of early prepubertal porcine spermatogonia (gonocytes) is characterized by the reliance on OXPHOS fuelled by oxidative decarboxylation of pyruvate. Interestingly, at the same time, a high amount of the consumed pyruvate is also reduced and excreted as lactate. With maturation, prepubertal spermatogonia show a metabolic shift with decreased OXHPOS and upregulation of the anaerobic metabolism-associated uncoupling protein 2 (UCP2). This shift is accompanied with stem cell specific promyelocytic leukemia zinc finger protein (PLZF) protein expression and glial cell-derived neurotropic factor (GDNF) pathway activation. Our results demonstrate that gonocytes differently from mature spermatogonia exhibit unique metabolic demands that must be attained to enable their maintenance and growth in vitro. K E Y W O R D Smaturation, metabolism, prepubertal spermatogonia, reactive oxygen species 2 of 22 | VOIGT eT al. | MATERIALS AND METHODS | Animals and human subjects (samples)Testes were obtained by castration of 1-and 8-week-old pigs (Sunterra Farms Ltd; Acme, AB, Canada, and the University of Alberta, Edmonton, AB). All procedures were performed with approval and under the oversight of the Animal Care Committee and the Institutional Review Board of the University of Calgary. Human samples were obtained from a previous study 49 (approved by the Committee for Ethics in Medical Research at the Karolinska Institutet, 2009/716-31/2) and from the Fertility Preservation Program in Pittsburgh with approval from the University of Pittsburgh Institutional Review Board (Protocol #STUDY09020220.

Pectus carinatum (PC) presents itself as a protrusion on the chest wall of adolescent individuals. Current treatment for PC is performed with a Pectus carinatum orthosis (PCO) that applies a compressive force to the protrusion. While this treatment is accepted, the magnitude of compressive forces applied remains unknown leading to excessive or deficient compression. Although the need for this quantitative data is recognized, no studies reporting the data or methods are available. The purpose of this study was to design an accurate force measurement system (FMS) that could be incorporated into a PCO with minimal bulk. Components of the FMS were three-dimensional (3D)-printed and incorporated into an existing PCO design. The FMS was calibrated using a custom indenter that applied forces to the FMS in a controlled manner. Evaluation of the FMS on five human participants was also performed. A reliability measure of the FMS was calculated for analysis. The FMS was implemented into the PCO and able to withstand the applied forces. The calibration revealed an increase in load cell error with increased magnitude of applied force (mean error [SD] = 5.59 N [6.48 N]). Participants recruited to evaluate the FMS demonstrated reliable forces (R = 96%) with smaller standard deviations than those during the calibration. The FMS was shown capable of measuring PCO forces but requires further testing and improvement. This system is the foundational component in a wireless, minimalistic sensor system to provide real time force feedback to both the clinician and patient.