Comparison of two methods for prolong storage of decellularized mouse whole testis for tissue engineering application: An experimental study

Abstract

Background: Biological scaffolds are derived by the decellularization of tissues or organs. Various biological scaffolds, such as scaffolds for the liver, lung, esophagus, dermis, and human testicles, have been produced. Their application in tissue engineering has created the need for cryopreservation processes to store these scaffolds.

Objective: The aim was to compare the two methods for prolong storage testicular scaffolds.

Materials and methods: In this experimental study, 20 male NMRI mice (8 wk) were sacrificed and their testes were removed and treated with 0.5% sodium dodecyl sulfate followed by Triton X-100 0.5%. The efficiency of decellularization was determined by histology and DNA quantification. Testicular scaffolds were stored in phosphate-buffered saline solution at 4°C or cryopreserved by programmed slow freezing followed by storage in liquid nitrogen. Masson's trichrome staining, Alcian blue staining and immunohistochemistry, collagen assay, and glycosaminoglycan assay were done prior to and after six months of storage under each condition.

Results: Hematoxylin-eosin staining showed no remnant cells after the completion of decellularization. DNA content analysis indicated that approximately 98% of the DNA was removed from the tissue (p = 0.02). Histological evaluation confirmed the preservation of extracellular matrix components in the fresh and frozen-thawed scaffolds. Extracellular matrix components were decreased by 4°C-stored scaffolds. Cytotoxicity tests with mouse embryonic fibroblast showed that the scaffolds were biocompatible and did not have a harmful effect on the proliferation of mouse embryonic fibroblast cells.

Conclusion: Our results demonstrated the superiority of the slow freezing method for prolong storage of testicular scaffolds.

Keywords: Mouse.; Scaffold; Testis; Cryopreservation.

Figure 1

Characterization of testicular scaffolds. Macroscopic images showed that scaffolds were completely translucent (A) while native testes were opaque (B). Histological comparison of scaffolds (C) and native testes (D) by H&E staining exhibited the elimination of the cells. Original magnification 100x. DNA quantification confirmed the removal of 98% of the DNA from the tissue (E). a indicated significant difference with native testis (p $<$ 0.05). Masson's trichrome staining showed collagen preservation in scaffolds (F) and native testes (G). Alcian blue staining confirmed GAGs retention in scaffolds (H) and native testes (I). Original magnification 100$\times$.

Figure 2

Quantification of collagen and GAGs content in native and scaffolds. Collagen in native and scaffolds was quantified using the Sircol assay (A). Quantification of GAGs in native and scaffolds using the Blyscan assay (B). Results are presented as Means $\pm$ SD (p $<$ 0.05).

Figure 3

Immunohistochemical and ultrastructural analyses of testicular scaffolds and native testes. Representative images of fibronectin expression in scaffolds (A) and native testis (B), collagen IV expression in scaffolds (C) and native testis (D), and laminin expression in scaffolds (E) and native testis (F). Original magnification 100$\times$.

Figure 4

Evaluation of scaffold cytocompatibility. The result of the MTT test did not show any significant difference in the optical density (OD) values, meaning that the cells proliferated at a rate similar to that of the controls.

Figure 5

Characterization of testicular scaffolds after storage. Representative image of macroscopic appearance (A&B), H&E staining (C&D), Masson's trichrome staining (E&F), Alcian blue staining (G&H) of scaffolds after storage at 4°C or cryopreservation by slow freezing method, respectively. The images confirmed the superiority of the slow freezing method in the preservation of the ECM component. Original magnification 100$\times$.

Figure 6

Quantification of collagen and GAGs content in scaffolds after storage. Collagen in 4°C-stored and frozen-thawed scaffolds were quantified using the Sircol assay (A). Quantification of GAGs in 4°C-stored and frozen-thawed scaffolds using the Blyscan assay (B). Results are presented as Mean $\pm$ SD (p $<$ 0.05).

Figure 7

Immunohistochemical analyses of frozen-thawed scaffolds. Representative images of fibronectin, collagen IV, and laminin expression in frozen-thawed (A-C) and 4°C-stored scaffolds (D-F). Original magnification 100$\times$.

Figure 8

Evaluation of cell attachment to the frozen-thawed scaffold. Spermatogonial cells were seeded directly on the frozen-thawed scaffolds to study cell attachment. Cell culture was carried out for up to seven days. Spermatogonial-cell attachment and infiltration as seen after seven days by DAPI staining (A) and H&E staining (B). Original magnification 200$\times$.