Most of the cells comprising tissues possess sites for interacting with the extracellular matrix (ECM), which are important for cell adhesion, migration, tissue formation, and intercellular communications [1], which in turn regulate cell proliferation, differentiation, tissue regeneration, and organ development. In stem cell culture systems, specific microenvironments, including the ECM, are necessary [2,3] for supporting the growth and differentiation of cells; indeed, the pluripotent characteristics of these cells may be altered in suitable microenvironments in stem cell culture [4].

Among the ECM soluble factors, gelatin has been used as the primary coating agent for the maintenance of mesenchymal stem cells and hematopoietic stem cells [5,6], as it is hydrophilic and has a low melting temperature [7]. In addition, gelatin is rich in the arginine-glycine-aspartic acid (RGD) sequence, which constitutes the interaction sites with integrins and promotes cell adhesion and migration [8]. Apart from its functions in cell culture systems, gelatin has been used in tissue engineering [9,10], regeneration therapy [11], and 3D printing as scaffolds [12].

Spermatogonial stem cells (SSCs) have the potential to proliferate and differentiate, resulting in the production of spermatozoa [13]. The process of spermatogenesis is regulated by various factors [14] that distinguish the microenvironment ‘niche’ [15]. The microenvironment of SSCs in the basal compartment of the seminiferous tubule provides cell signals via stimuli from the vascular network and the interstitial myoid and Sertoli cells [16]. In vitro culture systems of SSCs from mice [17], pigs [18], cows [19], sheep [20], and humans [21] have been developed on ECM-coated dishes in the presence of feeder cells.

Gelatin has been used as a primary ECM molecule for the derivation and culture of spermatogonial stem cells in canine [22], monkey [23], buffalo [24], mouse [25], and ovine [26]. Interaction between feeder cells and gelatin is critical for the migration of feeder cells on plates during pSSC colony formation [23]. However, details regarding the structure and characteristics of gelatin on the culture plate, such as pore size and the ability to form porous scaffolds, have not yet been defined for culturing pSGCs. Gelatin has changed its physical properties including swelling properties, water absorption, elastic modulus, and molecular transport, and these physical properties can be controlled by temperature. The gelling temperature of mammalian gelatin is ≤ 20 °C, while its melting temperature is ≥ 34 °C [27]. It has been reported that the gelatin solution has a semi-solid form below 30 °C and a cross-linked structure at 40–50 °C [28]. Interestingly, among the gelatins extracted from fish skins at 60, 80, and 95 °C, the strength of gelatin extracted at 80 °C was the highest [27]. Therefore, we aimed to identify the optimal topography of gelatin on culture dishes conducive to pSGC culture.