Fig. 1: Development of human testis-on-a-chip that demonstrates reciprocal endocrine crosstalk between Sertoli and Leydig cells in the testicular tissue and analysis of its physical properties.

The male seminiferous tubule is composed of two major endocrine cells, Sertoli and Leydig cells, which are reciprocally controlled by reproductive steroids and various endocrine factors. The Sertoli and Leydig cell chambers within the testis-on-a-chip were interconnected by vessel cell-coated media channels to allow reciprocal endocrine crosstalk. In addition, an immune cell (macrophage) chamber was incorporated into the chip platform to mimic the immune cell-interacting microenvironment of the seminiferous tubules (a). The casting mold for the human testis-on-a-chip platform that could properly mimic the structural features of the seminiferous tubules was fabricated using PLA-based 3D printing. Polydimethylsiloxane (PDMS) was then injected into the fabricated casting mold, and the synthesized chip platform was released from the mold after polymerization (b). The testis-on-a-chip platform was oval-shaped with a major axis length of 45 mm, center diameter of 30 mm, and height of 5.5 mm and was fabricated to mimic the microenvironment of the seminiferous tubules and endocrine crosstalk between the Sertoli and Leydig cell chambers (c). To reflect the multicellular complexity and physiological features of testicular tissue, various human testicular cellular components (Sertoli cells, Leydig cells, macrophages, and vascular endothelial cells) with a natural polymer mixture (collagen and hyaluronic acid) and blood coagulation factors (fibrinogen and thrombin) were loaded into the fabricated testis-on-a-chip platform. Two major human testicular cell types, Sertoli and Leydig cells, were isolated from a patient who underwent bilateral orchiectomy with complete spermatogenesis (d). The horizontal and vertical scanning electron microscopy (SEM) images of the fabricated natural polymer-based tissue architecture revealed a uniformly interconnecting microporous structure with different pore diameters ranging from 50 to 150 μm (e). The swelling behavior of the fabricated tissue architecture was evaluated by imaging all the samples in DW and PBS (pH 7.4) in each well at 37 °C. Nonabsorbed water was removed from the samples, and the hydrated samples were weighed to determine their water absorption potential (f). The mechanical strength of the fabricated natural polymer-based tissue architecture was measured by applying uniaxial compressive stress with a tensile strength testing machine (QM100S, QMESYS, Gunpo, Korea). The fabricated tissue architectures (10 mm in diameter and 10 mm in height) were analyzed to determine their mechanical strength. To determine the compressive stress at which the fracture occurred, compressive stress was applied at a loading rate of 5 mm/min until the samples were fractured (g). The rheological properties (viscosity) of the tissue architectures were analyzed by varying the shear rate from 1 s⁻¹ to 10 s⁻¹, which allows assessment of the shear thinning or thickening behavior (h). Significant differences are indicated as follows: *p < 0.05, **p < 0.005, and ***p < 0.001 (two-sample t test).