This problem might be circumvented by using

This problem might be circumvented by using a scaffold that mimics the testicular architecture. Indeed, it is already common in tissue engineering to use scaffolds composed of biological extracellular matrix (ECM) (Brown and Badylak, 2014). In this context, we previously described the preparation of human decellularized testicular matrix (DTM) and its potential use as a scaffold (Baert et al., 2015).
Results
Discussion In the light of their nature, PTMCs and Sertoli transferase may have contributed directly to the reshaping of the scaffold. We show here that a number of in vivo testicular processes related to ECM (including the blood-testis barrier) remodeling were recapitulated in TOs derived from both young and adult cells (Díez-Torre et al., 2011; Mruk and Cheng, 2004; Siu and Cheng, 2004). First, tumor necrosis factor alpha, tissue inhibitor of metalloproteinase 1, IL-6, and monocyte chemotactic protein 1 were produced, as detected by way of membrane antibody arrays. Second, rearrangement of COL1 fibers from a testis-specific pattern to a widespread interconnecting network occurred with time. Third, functionally differentiated PTMCs producing ECM were detected, first as round and later as elongated ACTA2+ cells containing COL1 in their cytoplasm and interspersed between the COL1 fibers. Fourth, co-localization of SOX9, a marker of Sertoli cells, and the tight-junction protein ZO1 in certain cells in the TOs indicated production of protein components of the blood-testis barrier by Sertoli cells. Finally, cells other than PTMCs, possibly Sertoli cells (the other main source of testicular ECM), were found to express COL1. In addition, macrophages may have played a central role as well, especially since these cells participate in the degradation of ECM scaffolds (Valentin et al., 2009). Macrophages would be expected to be present in TOs formed from suspensions containing testicular cells, given that immune cells reside naturally in the testicular interstitium (DeFalco et al., 2015). Thus, remodeling of the scaffold might have involved interactions between PTMCs, Sertoli cells, and macrophages. This phenomenon may be relevant to the observation that degradation products of ECM components, so-called cryptic peptides, regulate tissue functions (Brown and Badylak, 2014). Nevertheless, the necessity of the natural testicular scaffold in TO formation is debatable, given that the spatial-temporal behavior and hormone and cytokine secretion profiles of testicular cells in scaffold-free TOs were comparable. The spheroid shape of both scaffold-based and scaffold-free TOs might be explained by contraction of ACTA2 proteins in PTMCs in response to androgens, as this occurs in vivo (Schlatt et al., 1993). Importantly, we found indications of Leydig cells effecting steroidogenesis in the TOs. The presence of cells expressing STAR and 3βHSD, which are critically involved in T biosynthesis, and, in particular, the detection of T in the medium suggest Leydig cell functionality throughout the entire culture period. Interestingly, physiological concentrations of gonadotrophins did not stimulate T production, nor did they influence the production of InhB, an indicator of Sertoli cell functionality. A possible explanation for this lack of effect could be that these cells became unresponsive to stimulation due to the age of the adult testicular cells used in this study (Haider et al., 2007). Alternatively, maximal stimulation was induced by gonadotrophin-like factors in the serum supplement used in the culture medium. Indeed, unresponsiveness of testicular cells to gonadotrophins has been reported before when serum was added to the culture medium (Roulet et al., 2006), while a gonadotrophin-induced increase in hormone production was detected in serum-free systems (Berensztein et al., 2000). Moreover, supplementation of the medium for mouse testicular organ culture with serum alone is sufficient to generate sperm, and, in this setup, lipid-rich albumin plays a critical role (Sato et al., 2011). Unfortunately, many other components of this serum replacement remain unknown. The maintenance of Leydig cells with testis-specific activity was further demonstrated by the production of MCSF, a cytokine important for germ-cell renewal (Martin and Seandel, 2013).