Formation of organoid-like structures in the decellularized rat testis

Abstract

Objectives: In testis, the extracellular matrix (ECM) in addition to the supportive role for cells in the seminiferous epithelium, is also essential for the accurate functioning of these cells. Thus, using a decellularized testicular ECM (DTECM), as a scaffold for three-dimensional (3D) culture of testicular cells can mimic native ECM for studying in vitro spermatogenesis.

Materials and methods: The rat testis was decellularized via perfusion of 0.5% sodium dodecyl sulfate (SDS) for 48 hr, followed by 1% Triton X-100 for 6 hr, and then 1% DNase I for 1 hr. The efficiency of decellularization was evaluated by histology, immunohistochemistry (IHC), scanning electron microscopy (SEM), and MTT test. The prepared scaffolds were recellularized with testicular cells and cultured and assessed with hematoxylin-eosin (H&E) staining after two weeks.

Results: Based on the H&E image, no trace of cell components could be observed in DTECM. IHC images demonstrated collagen types I and IV, laminin, and fibronectin were preserved. Masson's trichrome and alcian blue staining revealed that collagen and glycosaminoglycans (GAGs) were retained, and the SEM image indicated that 3D testicular architecture remained after the decellularization process. Based on the results of the MTT test, DTECM was cytocompatible, and H&E images represented that DTECM supports testicular cell arrangements in seminiferous tubule-like structures (STLSs) and organoid-like structures (OLSs).

Conclusion: The results showed that the applied protocol successfully decellularized the testis tissue of the rat. Therefore, these scaffolds may provide an appropriate vehicle for in vitro reconstruction of the seminiferous tubule.

Keywords: Decellularization; Extracellular matrix; Organoid; Seminiferous tubule; Testis.

Figure 1

Culture pieces of Cell-loaded scaffolds on agarose gel

Figure 2

Appearance changes of rat testis during decellularization. At the beginning of the experiment, the testis was opaque and pink (A), after 24 hr it was brighter (B), and post 48 hr was completely whitish translucent (C)

Figure 3

Histological evaluation of decellularized testicular ECM (DTECM). H&E staining comparison of decellularized (A) and native testes (B) exhibited the elimination of the cells and preserved extracellular matrix (ECM) architecture. Masson’s trichrome staining showed collagen fiber was preserved after the decellularization process (C) similar to the intact testis (D). Alcian blue staining demonstrated the preservation of GAGs following decellularization process (E) intact testis stained as control (F)

Figure 4

Immunohistochemistry (IHC) of extracellular matrix (ECM) proteins in decellularized testicular ECM (DTECM). Collagen type I (A and B), collagen type IV (C and D), fibronectin (E and F), and Laminin (G and H) were detected in decellularized testes (left series) with similar patterns as in native testes (right series)

Figure 5

SEM image of the 3D structure of decellularized testicular ECM (DTECM). The image indicates the preservation of the 3D testicular architecture in the decellularized scaffold, seminiferous tubules are shown without any cell (A). Image of intact testis used as control (B)

Figure 6

DNA content and cytocompatibility assessment of decellularized testicular ECM (DTECM). DNA quantification confirmed that the decellularization process almost removed the entire DNA content from the decellularized tissue (A). DAPI staining of the DTECM section also confirms this (B). The native tissue was stained for control (C). Optical density (OD) values, did not indicate the cytotoxic effect of decellularized scaffold on the proliferative activity of testicular cells (D). The cells kept a normal proliferation rate compared with the controls

Figure 7

Neonatal rat testicular cells after two weeks’ culture

Figure 8

H&E staining showed testicular cells were located on the basement membrane and the seminiferous tubule-like structures (STLSs) were formed in some sections (A (B: higher magnification) and C) and the organoid-like structures (OLSs) were observed in other sections (D)

Figure 9

A: Data showed that 20% and 45% of the segments contained seminiferous tubule-like structure (STLS) and organoid-like structures (OLS), respectively. B: diameters of STLSs and OLSs were significantly different from that of seminiferous tubules in the adult rat testis (P<0.05), while not significantly differing from the diameter of seminiferous tubules in neonate rats (P>0.05). Data were represented by mean ± SD after three repeats