The obtainment of animals more resilient to climate changes, and with a lower environmental impact, would be greatly desirable. Based on this, during the last years, particular attention has been dedicated to the development and further improvement of a sustainable animal agriculture with positive socioeconomic and environmental impacts. In this perspective, new biotechnological approaches, such as assisted reproductive technologies (ART) and genetic selection (GS), were introduced and used simultaneously in livestock breeding programs, including in the bovine species (Mueller and Van Eenennaam, 2022). Nevertheless, the possibility of these techniques to improve genetic gain is limited by the average age of an animal when replacement offspring are born (Kasinathan et al., 2015). It is therefore clear that the greatest improvement in accelerating the genetic selection can be achieved by shortening the generation interval (Cenariu et al., 2012; Pasquariello et al., 2024). To this aim, different strategies have been proposed in both male and female domestic ruminants, however, a substantial shortening of the interval is still far away.

In the male, one promising approach is represented by the in vitro propagation and differentiation of spermatogonia into mature sperm or even in vitro recreation of whole spermatogenesis from embryonic stem cells. To date, a variety of culture systems as well as different medium compositions to enhance spermatogonial stem cell (SSC) viability, proliferation (Fath-Bayati et al., 2023; van Maaren et al., 2023) and differentiation in vitro (Cho and Easley, 2023; Kulibin and Malolina, 2023; Salem et al., 2023; Damyanova et al., 2024) have been developed. However, the complete spermatogenesis ex vivo has been obtained only in the murine species (Perrard et al., 2016), while, in cattle and in other domestic species, it is possible only to propagate spermatogonia without inducing an effective meiotic division. This is mainly due to the complexity of the process, during which male germ cells differentiate into mature spermatozoa, thank to well-orchestrated interactions among hormones, growth factors, cytokines, and extracellular matrix (ECM)-derived bio-mechanical and bio-chemical cues. In addition, the lack of knowledge on niche microenvironment, nutritional requirements, as well as on the multiple regulatory machinery driving self-renewal, proliferation, and differentiation, in ruminates, has significantly hindered progresses in this field. It is therefore desirable to developed reliable 3D in vitro models able to faithfully mimic the architecture and the physiological microenvironment of the native testicular tissue, bridging the gap between the in vivo complexity and the over-simplified conventional two-dimension (2D) in vitro cultures.

To date, several 3D platforms for testicular bioengineering, including testicular organoids (Richer et al., 2020), hydrogel bioreactors (Perrard et al., 2016), and synthetic, natural or decellularized scaffolds (Horvath-Pereira et al., 2023) have been developed in human, mouse and rat. In contrast, to our knowledge, no cell engrafted 3D scaffolds have been developed in the bovine species and only one study reported the differentiation of gonocytes into presumptive spermatids through the use the alginate encapsulation technique (Lee et al., 2001). In this scenario, the generation of a bovine decellularized testicular 3D scaffold represents a promising option, since it accurately replicates the in vivo topography and the complex milieu of the native tissue, thus promoting the necessary interactions between cells and their surrounding microenvironment. In addition, the preserved extracellular matrix (ECM), obtained through the decellularization process, provides essential biomechanical and biochemical cues that encourage the correct cell growth, differentiation, and function.

In the present study, we generate bovine testicular bio-scaffolds for the creation of reliable 3D artificial models. In particular, we test three different decellularization protocols and identify the protocol that better preserves intact ECM composition and microstructure, while efficiently eliminating cells. We then repopulate the generated testicular bio-scaffolds with fibroblasts isolated from bovine testes and monitor the scaffold ability to sustain cell adherence and proliferation.

All reagents were purchased from Thermo Fisher Scientific unless otherwise indicated.

In the present manuscript, we generate a testicular bio-scaffold, able replicate the in vivo topography and the bio-mechanical and bio-chemical stimuli derived from the native ECM. The decellularized bio-scaffold successfully encourages cell homing, colonization and growth, demonstrating its ability to sustain testicular cell adherence and proliferation.

At the end of the three-step decellularization process, macroscopic evaluations revealed a color change from red to white in all the obtained bio-scaffolds, regardless of the protocol used. This suggests the occurrence of a significant reduction in the cellular components. Indeed, similar color variations were previously reported by other Authors, which applied decellularization protocols to different tissue, including heart (Rajabi-Zeleti et al., 2014), lung (Lecht et al., 2014), liver (Ghiringhelli et al., 2021; Lee et al., 2017), kidney (Yu et al., 2014), muscle (Aulino et al., 2015), trachea (Baiguera et al., 2014; Pennarossa et al., 2021b), esophagus (Sjöqvist et al., 2014), urinary tissue (Singh et al., 2018), arteries (Kajbafzadeh et al., 2017), derma (Gilpin and Yang, 2017), intestine (Arcuri et al., 2024), ovary (Laronda et al., 2015; Laronda, 2020; Pennarossa et al., 2020; Pennarossa et al., 2022) and vagina (Zhang et al., 2017), and that resulted in a significant decrease in cell content. This was confirmed by our H&E staining that demonstrated a decrease of basophilic color in all the three experimental groups and by DAPI staining, showing a significant decrement in cell nuclei in Decell-A, Decell-B, and Decell-C samples. All these morphological observations were further corroborated by cell density analysis experiments that indicated a statistically significant cell number reduction in all experimental groups, when compared with the untreated tissues (CTR). However, while the cellular compartment was reduced in all three groups, it is interesting to note that Decell-B and Decell-C protocols allowed a more efficient and significant reduction in cell number, compared to Decell-A. Altogether, these results demonstrate that the correct combination of a freeze-thaw cycle, with sequential incubations with SDS and Triton X-100, allows for an efficient cell removal only when the native tissue is exposed to SDS for at least 12 h, as in the protocols Decell-B and Decell-C. This is in agreement with previous studies which demonstrated SDS ability to successfully eliminate the cellular compartment from the native tissues (Scarrit et al., 2015; Singh et al., 2023), selecting in a tissue-specific manner the appropriate concentration and time of exposure (Gilpin and Yang, 2017).

It is however important to note that a fundamental aspect in the decellularization process is also the maintenance of the original ECM microstructures, including fibers and macromolecules. The histochemical analysis carried out in our study, demonstrated the preservation of intact elastic fibers, GAGs and collagen in Decell-A and Decell-B. This is in agreement with Kiani et al. (2021) and Khazaei et al. (2023) that described the generation of rat and calf testis scaffolds, respectively, and demonstrated the persistence of the major ECM proteins at the end of the decellularization process. In contrast, elastic and collagen fibers, as well as GAGs appeared to be significantly reduced when SDS exposure is prolonged (Decell-C). All these morphological observations were also confirmed by stereological analysis, which revealed no significant changes for collagen, elastin, and GAG content among the untreated tissue, Decell-A and Decell-B groups. In contrast, Decell-C showed a statistically significant decrement in the ECM components when compared to the CTR. Although several different explanations can be hypothesized, we suggest that the prolonged exposure to SDS used in the Decell-C protocol, while ensuring an efficient removal of the cellular compartment, may exert a detrimental effect and cause damages to structural proteins, such as collagen fibers and GAGs. This is in line with previous observations that described SDS disruptive side effects on collagen fibers in porcine urinary bladder (Faulk et al., 2014), caprine pancreas (Singh et al., 2023) and many other tissues (Crapo et al., 2011; Keane et al., 2015). It is also in agreement with Kasturi and Vasanthan and Moffat et al. that reported SDS ability to damage GAGs during the decellularization processes of different organs, including liver, pericardium, articular cartilage, heart, and kidney (Moffat et al., 2022; Kasturi and Vasanthan, 2023). All these observations clearly point to the need of a strategy that sets a fine tuning of the SDS conditions to ensure, in a species-specific and organ-specific way, the efficient removal of cells, while preserving an intact ECM structure.

A crucial point for the use of a bio-scaffold in tissue engineering is its ability to encourage cell adhesion, homing, and growth (Chan and Leong, 2008; Lynch et al., 2021; Pennarossa et al., 2021a). To address this point, we isolated fibroblasts from bovine testis and used them to repopulate the generated decellularized bio-scaffolds. The results obtained demonstrated a rapid engrafting process with cells that adhered and colonized the matrix within 24 h from seeding. In addition, H&E, DAPI staining and cell density analysis showed a linear increment in cell number and a homogenous and steady distribution of the cell population onto Decell-A and Decell-B scaffolds for as long as 7 days, when culture was arrested. It is also interesting to note that, although the number of cells identified in Decell-A should be affected by cellular residues present at the end of the decellularization process, the increasing number observed during the culture period demonstrates the presence of proliferating cells which derive from reseeding process. Altogether, these results indicate the bio-scaffold ability to host cells and to encourage their proliferation possibly via bio-mechanical and bio-chemical stimuli. They also exclude the persistence of toxic carry-overs from the decellularization processes, may impair the subsequent recellularization and biocompatibility, both in vitro and in vivo (Morris et al., 2017).

Overall, the results obtained suggest the potential of the bio-scaffold here described to sustain cell adherence and proliferation. In particular, the cell type selected for repopulation experiments well fits with the possible use of iPS-derived or chemically reprogrammed cells, for regenerative experiments of the testicular tissues. Although, these results are still preliminary, they pave the way toward the use of decellularized testicular bio-scaffolds in the field of reproductive biology and biotechnology as suitable artificial niches for ex vivo culture of testicular cells and to reproduce spermatogenesis in vitro.