Purpose In vitro millifluidic cultures with perfusion are essential tools to analyse and understand the interactions between cells, their matrix and multi-cell populations. The purpose of this paper is to focus on the design and development of a 3D-printed template that can be used for fabrication of a clear view poly (dimethyl siloxane) (PDMS) device. The major objective is to obtain a transparent device prototype that allows perfusion culture of two cell types for multiple days that can be imaged using laser scanning confocal microscopy. Design/methodology/approach The authors used a two-step approach for achieving the final geometric structure at a faster timeline and lower cost. The first part focuses on comparing the fidelity of the printing templates using fused deposition modelling (FDM) and stereolithography (SLA) printers for a range of dimensions. They then show that the complex geometry chip with connection chambers can be printed using low resolution low cost FormLab SLA printer. The final optimized design was then printed using high-resolution Projet 6000 SLA printer to obtain smoother structures. Findings In this work, the authors have shown that the FormLab SLA printer yields significantly lower error for printing complex design geometries as compared to FDM printer. Result shows that FormLab printer can be used to achieve a minimum dimension of 0.5 mm. They then use the printer to optimize the device dimension for the culture chip which requires several iterations of printing and experimenting. They showed the two-step protocol of printing the optimized template in a high-resolution SLA printer and further fabricating a clear view millifluidic PDMS device that is compatible confocal microscopy imaging. They used this culture chip for perfusion culture of two cell type, and the controlled fluidic exchange between the two chambers led to the formation of neuroglia junction. Originality/value One of the major bottlenecks for obtaining complex geometry in mili/microfluidic device by 3D printing is the need of multiple iterations on printing. This makes the tuning of dimension significantly expensive. Another challenge is to obtain a smooth surface of PDMS that leads to a leak proof clear view device compatible for laser based confocal imaging. The combination of two printers plays a crucial role for the rapid prototyping of the imaging device with flow control. The proposed approach lowers the cost for prototyping of in vitro culture chip with complex geometries to improve on biological research demanding multi-chamber fluidic device.