Three‐dimensional electrospun gelatin scaffold coseeded with embryonic stem cells and sertoli cells: A promising substrate for in vitro coculture system

Abstract

In this study, we present an electrospun gelatin (EG) scaffold to mimic the extracellular matrix of the testis. The EG scaffold was synthesized by electrospinning and crosslinked with glutaraldehyde vapor to decrease its water solubility and degradation rate. The scanning electron microscope micrographs showed the homogenous morphology of randomly aligned gelatin fibers. The average diameter of gelatin fibers before and after crosslinking was approximately 180 and 220 nm, respectively. Modulus, tensile strength, and elongation at break values were as 161.8 ± 24.4 MPa, 4.21 ± 0.54 MPa, and 7.06 ± 2.12 MPa, respectively. The crosslinked EG showed 75.2% ± 4.5% weight loss after 14 days with no changes in the pH value of degradation solution. Cytobiocompatibility of the EG for sertoli cells and embryonic stem cells (ESCs)was determined in vitro. Sertoli cells were isolated from mouse testis and characterized by immunostaining and flow cytometry. The effects of EG on proliferation and attachment of both sertoli cells and ESCs were examined. The EG scaffolds exhibited no cytotoxicity for sertoli and ESCs. Both sertoli and ESCs were well attached and grown on EG. Coculture of sertoli and ESCs on EG showed better ESCs adhesion compared with ESCs alone. Our findings indicate the potential of EG as a substrate for proliferation, adhesion, and coculture of sertoli and ESCs and may be considered as a promising engineered microenvironment for in vitro coculture system with the aim of guiding stem cells differentiation toward sperm-producing cells.

Cross-Linking Strategies for Electrospun Gelatin Scaffolds

“…Among the chemical methods described in the previous sections, glutaraldehyde (GTA) vapor is the most widely described for the stabilization of gelatin nanofibrous matrices [54,70,79,80,81,82,83,84,85,86,87,88,89]. The nanofibrous gelatin samples are placed into an air-tight container filled with saturated GTA vapor, where GTA cross-linker molecules lead to the formation of covalent bonds among gelatin polymer chains.…”

Gelatin Electrospun Mat as a Potential Co-culture System for In Vitro Production of Sperm Cells from Embryonic Stem Cells

“…In our previously published article 33 , we fabricated an electrospun gelatin (EG) mat as a substrate for co-culture of Sertoli cells and ESCs. In this study, we evaluated the potential of this scaffold for guided differentiation of ESCs toward germ-like cells.…”

“…32 Recently, we fabricated a 3D electrospun gelatin (EG) mat and examined its mechanical and biological properties for Sertoli cells and ESCs. 33 In the current work, we hypothesized that the fabricated EG mat may be an excellent 3D system for coculture of Sertoli cells and ESCs, stimulating guided differentiation toward germline cells. Here, we expected that our electrospun scaffold cultured with Sertoli cells provide paracrine, cell−cell, and cell-3D matrix signaling cues for ESCs.…”

“…It may be a long way to develop a 3D scaffold precisely mimicking normal testis containing all the important signals required for differentiation and maturation of sperm cells, because the complete orchestrated signaling network involved in normal spermatogenesis is still not well-known 32 . Recently, we fabricated a 3D electrospun gelatin (EG) mat and examined its mechanical and biological properties for Sertoli cells and ESCs 33 . In the current work, we hypothesized that the fabricated EG mat may be an excellent 3D system for coculture of Sertoli cells and ESCs stimulating guided differentiation toward germline cells.…”

Advanced bioengineering of male germ stem cells to preserve fertility

“…Electrospun 3D scaffolds are known to be highly favorable for cell seeding, because they are thought to be more morphologically and structurally similar to ECM, compared to those synthesized by other approaches. 123 , 127 , 128 , 144 The effect of surface topography of these scaffolds on expansion of testicular cells has thus far not been reported. Fibrillar electrospun scaffolds have been reported to provide appropriate interactions mimicking the ECM and promoting appropriate migration and morphological alterations.…”