Introduction

Bilateral Vanishing Testis Syndrome is one of the most challenging problems of pediatric and adolescent urology. There are at least two major problems for patients with congenital or acquired anorchia: Long-life androgen replacement therapy and possible hepatic cancer occurrence as well as fertility issue. The purpose of present study was to describe a method to produce human sized decellularized testis scaffold as a primitive step for testis tissue engineering in such complex situation._x000D_

Methods

A total of 32 sheep testes were classified in 8 groups of 4. Seven decellularization protocols were proposed which were tested on groups 1-7, respectively and group 8 was left intact as the control group. The protocols consisted of 0.5%, 1%, 2% solution of sodium dodecyl sulfate (SDS), Trypsin-EDTA 0.5%, 1%, Triton X-100 1% and 2%, respectively. Then, using the best protocol obtained in phase I, the optimal timing was evaluated; ovine testes were decellularized by arterial perfusion and specimen were collected for evaluation at 4-6-8-10-12 hours after initiation of the procedure, respectively. _x000D_ To evaluate the efficacy of the protocol, histological examinations, scanning electron microscopy (SEM), magnetic resonance imaging (MRI) biochemical assays and evaluation of mechanical properties were performed. The patency of vascular network of the decellularized scaffold was examined by angiography. The cytotoxicity analyses of samples was performed by tetrazolium salt MTT (3-[4,5- dimethylthiazol- 2-yl]-2,5-diphenyltetrazolium bromide) assay. _x000D_

Results

Histological exams depict that treating the tissues with SDS 1% for 6-8 hours was found to be the best protocol for cell removal and preserving the extracellular matrix (ECM) components. The microstructure, ultrastructure and vascular integrity of the decellularized testis scaffold were well-preserved and confirmed by scaffold angiography, SEM and MRI. Moreover, MTT assay showed that the decellularized testis was not cytotoxic to cells and it is ready for recellularization

Conclusions

The SDS 1% with 6-8 hours perfusion based protocol is the best, effective, minimally invasive technique for creation of whole testis bioscaffold with well-preserved extra-cellular matrix structure and vascular network integrity. To the best of our knowledge the whole large size testis decellularization by this novel protocol has not been reported previously. This preliminary step may pave the road for slowing these problems in future. _x000D_

Funding

none