Abstract Human tissues and organs are inherently heterogeneous, and their functionality is determined by the interplay between different cell types, their secondary architecture, and gradients of signalling molecules and metabolites. To mimic the dynamics of native tissues, perfusion bioreactors and microfluidic devices are widely used in tissue engineering (TE) applications for enhancing cell culture viability in the core of 3D constructs. Still, most in vitro screening methods for compound efficacy and toxicity assessment include cell or tissue exposure to constant and homogeneous compound concentrations over a defined testing period. Moreover, a prevalent issue inhibiting the large-scale adoption of microfluidics and bioreactor is the tubing dependence to induce a perfusion regime. Here, we propose a compartmentalized rotational (CR) 3D cell culture platform for a stable control over gradient tissue culture conditions. Using the CR bioreactor, adjacent lanes of constructs are patterned by controlled flow dynamics to enable tissue stratification. Numerical and experimental simulations demonstrate cell seeding dynamics, as well as culture media rotational perfusion and gradient formations. Additionally, the developed system induces vertical and horizontal rotations, which increase medium exchange and homogeneous construct maturation, allowing both perfused tubing-based and tubing-free approaches. As a proof-of-concept, experiments and accompanying simulation of cellular inoculation and growth in 3D scaffold and hydrogel were performed, before the examination of a blood-brain-barrier model, demonstrating the impact of a heterotypic culture on molecular permeability under mimetic dynamic conditions. Briefly, the present work discloses the simulation of 3D dynamic cultures, and a semi-automated platform for heterotypic tissues in vitro modelling, for broad TE and drug discovery/screening applications.