Introduction

Live sample imaging has progressively become part of various cell morphology and structure characterization methods. From microrheology measurements over tens of minutes (1) to drug- or gene-delivery studies over tens of hours (2), long-term observations are required to characterize dynamic biological phenomena on a broad range of time scales.

To keep cells alive either ex vivo, (if part of a natural tissue) or in vitro, (if cultured and grown in two dimensions or three dimensions within a synthetic extracellular matrix), physiological conditions need to be maintained with respect to temperature, oxygen availability, and metabolic nutrients. Since the early 1950s, a variety of culture systems have been designed in parallel with improvements in microscopy and cell culture techniques (3). However, most of the available designs have been developed to meet a limited number of criteria (4-6). Here we present a modular microincubator (mµi) with extended versatility owing to its combination of a large set of features available only separately until now. The reusable mµi is optimized for mass and heat transfer, ease of assembly and cleaning, and modularity and robustness. In addition, the system offers the ability to (i) culture cells in monolayers or on synthetic or natural tissue matrices over extended time periods; (ii) image cells on any microscope, upright or inverted, owing to the use of a gas-permeable membrane to separate the culture medium from the ambient environment; (iii) control temperature at the sample level to prevent any light heating or any cooling if using oil immersion objectives; (iv) perfuse different media onto each side of thick tissue samples; and (v) perform micromanipulation in an “open-dish” format.

These features enable a broad spectrum of applications in experimental biophysics, as well as in the medical sciences, including cell stretching, cell motility along fibers, intracellular microrheology in variable environments and incorporation of fluorescently labeled nanocarriers in three-dimensional tissue cultures. A selection of different experiments chosen to demonstrate the capabilities of the mµi are described in detail in the Supplementary Materials that accompany this report.

Materials and methods

Incubator elements

The mµi is built from a series of different elements (Figure 1A), some which are optional and can be easily implemented depending on the specific needs of the user. The full height of the incubator is only 20 mm taking into account the thermal regulator. A file including the detailed drawings of the incubator is available online in the Supplementary Materials and an Autocad file is available upon request. The price for this mµi, comprising the temperature control system and the cost for the milling, is approximately €500, making the mµi much cheaper than many available commercial systems.

Figure 1. Schematics of the micro-incubator. (Click to enlarge)

Figure 1B illustrates the specific elements and their configurations in the mµi. An open stainless-steel container (height 7 mm, outer diameter 36 mm) (Figure 1B, part A), is machined to accommodate the silicone conical ring (flexiPERM ConA; Greiner Bio One, Stonehouse, UK) (Figure 1B, part B). The container A confers rigidity and conducts heat. The silicone ring B creates the cell growth and aeration cavities and acts as a gasket between the majority of the elements (Figure 2A). Two Teflon capillaries, 0.8 mm in diameter, are inserted in the silicone ring to allow injection or circulation of the culture medium. A syringe needle is used to pierce two small holes in the silicone ring through which the capillaries are inserted. The elasticity of the silicone ring ensures sealing around the capillaries. The portion of each capillary outside the ring is protected by thick mechanically resistant silicone tubes mounted around the capillary and clamped between the stainless steel ring A and the incubator lid (Figure 1B, part F). A permeable membrane (biofolie 25; Greiner Bio One) (Figure 1B, part C) separates the 2 mm–thick liquid cavity from the gaseous cavity. (The height of the liquid cavity is similar to the height of culture media normally above cells in routine non-perfused cell culture.) On the lower side of the membrane, the pH of the sterile medium is maintained at a physiological level due to the circulation on the upper side of the membrane of a 5% CO₂ atmosphere. The incubator can also be placed upside down and mounted on an upright microscope with the liquid cavity above the gaseous cavity (Figure 2). The membrane is clamped between two small stainless steel rings—a lower ring (Figure 1B, part D₁) and an upper ring (Figure 1B, part D₂)—pressed against the internal wall of the silicon ring B. The external conical shape of the rings defines the vertical position of the membrane/rings sandwich. A 1 mm–thick circular glass cover (Figure 1B, part E), cut from a standard microscope slide, is centered on top of the upper ring. The gaseous cavity is thus contained between the membrane and the glass. Gas circulation is carried out through two holes drilled in the lid F which sits on top of two apertures machined in the upper ring D₂. The gaseous cavity is partly sealed at the interface between the lid F and the silicon ring B and partly sealed at the interface between the lid F and an elastomer gasket (made using the Sylgard 184 polydimethylsiloxane kit; Dow Corning, Coventry, UK) (Figure 1B, part G). Lid F is fixed against the container A with three countersunk M2 screws.