# Lab-on-Chip Culturing System for Fungi--Towards Nanosatellite Missions
## 2 Materials and Methods
### Loc--Construction and Technology
Two channels with dimensions of \(3.5\times 30\) mm\({}^{2}\) and a depth of \(550\) um were designed in the lower substrate. In the first channel, the fungi nourishment is supplied (medium channel), while the second channel provides an appropriate gas atmosphere (gas channel).The fungi culture is also established and developed in the gas channel. In the upper glass substrate, the inlet/outlet holes enabling the introduction of the culture medium (diameter 1 mm) and gas diffusion (diameter 2 mm) were designed. A matrix of microchannels connecting the medium and gas channels was also applied in the upper glass substrate (7 connecting channels), whose dimensions are \(8.8\times 1\) mm\({}^{2}\) and the depth is \(20\) um [61]. This solution is proposed to ensure the constant gas exchange within the area of the medium channel, simultaneously preventing excessive \"flooding\" of the gas chamber. Figure 1: Scheme of the lab-on-chip for fungi culture: (**a**) top view, (**b**) cross-section, (**c**) general view. As the translation of macroscale methodologies towards microscale lab-on-chip approach is always not trivial, the matter of dry, \"non-immersed\" fungi spores is a significant novelty, which may open the way towards other applications, suggesting indirect creation of chemical gradients within the culturing habitat. Moreover, use of all-glass technology is still unpopular with scientists dealing with microfluidics, thus we hope that this work is a good example of its application, which may shortly change the attitude to glass microengineering, by and large. Computer simulations were carried out to support and initially assess the correctness of the proposed solution. For this purpose, COMSOL Multiphysics 4.3 software was used. It is a software that enables simulation in a very wide range of changes in parameters and materials [62; 63; 64]. The Laminar Flow module was selected for the simulation. It enables conducting research in a microscale [65; 66]. The equations used in this module are the Navier-Stokes, dedicated to conservation of momentum and the continuity equation for conservation of mass. The definition of laminar flow based on the Reynolds number was also used. The following boundary conditions were adopted: at the inlet of the liquid channel (according to the Figure 1), the liquid flow velocity was 200 uL/min, which was set in accordance with the planned in the experiment, while an atmospheric pressure was established at the outlet of the liquid channel and at the inlet and outlet of the gas channel. Water was chosen as the liquid and air as the gas. Next, the project was meshed. A uniform mesh for the entire model was used herein (Figure 2). As a result, it could be observed that the spread of the liquid in the medium channel was as expected (Figure 3a). At the same time, the correct working of the channels connecting both chambers was confirmed, the task of which is to provide humidity in the gas chamber, but not to flood it with nutrient solution. The gas channel remained almost dry (Figure 3b). Figure 2: Liquid flow distribution in a lab-on-chip. We also carried out simulations with the use of three different number of mesh elements (coarse, normal, fine) at which was measured the point of flow in the liquid and gas channel (Table 1). It can be observed that the increasing number of mesh elements does not have a significant effect on the final result. On that basis, it can be concluded that the proposed design of the lab-on-chip provides the correct environment for fungi cultivation. Next, microengineering processes were used to fabricate the lab-on-chip (Figure 4). Two borosilicate glass substrates (BOROFLOAT(r) 33 Schott, Mainz, Germany) of standard dimensions 50 \(\times\) 25 \(\times\) 1.1 mm served as the basic spatial structures. Designs for the masks were prepared according to the geometry of the individual channels utilising the CAD technique.