The elastomer gasket G is mounted between the lid F and the circular glass cover E. Cells are cultured onto a circular substrate, 19 mm in diameter (Figure 1B, part H), composed of any material thinner than 500 µm. Unlike culture chips, there are no constraints on the choice of the mechanical, physical, and chemical properties of the substrate. To study 3-D tissue in reflection mode, the substrate is simply replaced by the sample of interest with an additional elastomer spacer (Figure 1B, part S) and a coverslip. The optional elastomer spacer S creates an additional cavity useful for the study of 3-D tissue. One side of the tissue sample is in contact with this additional cavity while the other side is in contact with the cavity formed by the silicone ring B. In this configuration, an additional stainless steel ring the same height as the elastomer spacer is mounted around it. Medium, dye, or gas can be injected or circulated as desired within the additional cavity through the two Teflon capillaries connected to the protective thick silicone tubes. The incubator base (Figure 1B, part I), machined in stainless steel, closes one side of the system. The substrate or the tissue sample is clamped between the base I and the silicone ring B. The base is fixed against the container A with three countersunk M2 screws. A platinum resistance temperature sensor (Figure 1B, part J) (Cat. no. 1266941; Farnell, Leeds, UK) is fixed on the edge of the silicone ring within a small drop of elastomer. The sensor, protected by a thin layer of elastomer, comes directly in contact with the substrate or the tissue sample. The thermal regulator (Figure 1B, part K) is a central-hole Peltier module (Cat. no. RH1.4–14–06L; Melcor, Draycott, UK), the upper side of which is connected to an aluminum heat sink. The thermal regulator K is mounted on top of the lid F (with a thin layer of thermal grease in between) and powered through a proportional integral derivative (PID) controller (Cat. no. MTTC-1410; Melcor) with a feedback loop based on the signal given by the temperature sensor J.Preparation of the incubator
Prior to first use, the upper elements of the mµi (A–G and J) are put together in an easy-to-handle assembly. Afterward this assembly is unmounted every ten experiments to change the membrane C. On this occasion, all the above mentioned parts are thoroughly cleaned, first with pure water, then with ethanol, and finally brushed with soap. Parts are again cleaned with water, ethanol, and finally rinsed with water. Parts are then reassembled and autoclaved.
If required, the mµi can also be operated in an “open-dish” version on an inverted microscope (Figure 2A). For this purpose, one removes the membrane C, the rings D, the elastomer gasket G, and the glass E from the upper assembly. One can then access the sample through the aperture of the lid F.Results and discussion The mµi for observation of cell monolayers
Cells can be seeded and grown within the incubator or transferred for microscopy. Cells cultured de novo in our micro-incubator are maintained in the same culture conditions from start to finish, even during microscopic observations; this allows the user to avoid alterations in cell behavior caused by any sharp changes of environment during transfer from the laboratory incubator into the microscope incubator. To culture cells de novo in the mµi, the user places the substrate for cell growth at the center of the base I and secures it to the upper assembly with screws. If the cell-growth substrate can bear high temperatures, the entire assembly can be sterilized by autoclaving. If the substrate is sensitive to temperature, the incubator base I and the upper assembly should be pre-sterilized first and assembled with the substrate in a sterile environment after autoclaving. Cells suspended within culture medium are injected into the silicone ring B cavity through the capillaries and allowed to adhere to the substrate at 37°C. Air contained in the cavity needs to be removed if the mµi is to be used on an upright microscope. Cells can be cultured in the mµi for several days until they reach confluency (Supplementary Materials, section 1). Although culture conditions are better controlled with a culture de novo in the mµi, it may be useful to prepare several cell cultures in a standard incubator and then place the best sample into the mµi on its initial substrate without any passage. Substrates needs only to be thinner than 500 µm and circular (19 mm in diameter); complex substrates can be used. Patterned substrates (7) or composite substrates (Supplementary Materials, section 1) can easily be implemented into the mµi. Deformable or stretchable substrates are also possible for the mµi. Cells attached onto a stretchable substrate in the mµi can be stressed in a well-controlled manner within a defined environment (Supplementary Materials, section 2).