Facilitating long-term cell examinations and time-lapse recordings in cell biology research with CO2 mini-incubators

Abstract

In recent years, microscopy has revolutionized the study of dynamic living cells. However, performing long-term live cell imaging requires stable environmental conditions such as temperature, pH, and humidity. While standard incubators have traditionally provided these conditions, other solutions, like stagetop incubators are available. To further enhance the accessibility of stable cell culture environments for live cell imaging, we developed a portable CO₂ cell culture mini-incubator that can be easily adapted to any x-y inverted microscope stage, enabling long-term live cell imaging. This mini-incubator provides and maintains stable environmental conditions and supports cell viability comparable to standard incubators. Moreover, it allows for parallel experiments in the same environment, saving both time and resources. To demonstrate its functionality, different cell lines (VERO and MDA-MB-231) were cultured and evaluated using various assays, including crystal violet staining, MTT, and flow cytometry tests to assess cell adhesion, viability, and apoptosis, respectively. Time-lapse imaging was performed over an 85-h period with MDA-MB-231 cells cultured in the mini-incubator. The results indicate that this device is a viable solution for long-term imaging and can be applied in developmental biology, cell biology, and cancer biology research where long-term time-lapse recording is required.

Figure 1

(A) Exploded view of the mini-incubator, showcasing the individual components that can be assembled using screws. (B) Assembled view of the two main parts of the mini-incubator. (C) Mini-incubator placed on an inverted microscope stage.

Figure 2

A simplified control diagram showcasing the utilization of a microcontroller for temperature, CO₂ level, and humidity sensing and control.

Figure 3

Overview of the simulation conducted for the mini-incubator device: (A) analyzing the fluid velocity in the water jacket, (B) examining the temperature distribution in the internal slices, and (C) evaluating the temperature distribution on the top surface of the mini-incubator.

Figure 4

MTT assay results: (A) Optical density at 570 and 630 nm in incubator and mini-incubator. (B) Percentage of VERO cell survival in the mini-incubator compared to the conventional incubator. (p-value > 0.05, non-significant difference).

Figure 5

Crystal violet-stained MDA-MB-231 cells in two separate wells of the mini-incubator after 24 h of culture. The right images are shown at 4X magnification, with a scale bar of 500 µm, while the left images are at 10X magnification, with a scale bar of 200 µm.

Figure 6

Results of flow cytometry analysis comparing apoptosis levels in (A) the mini-incubator and incubator and (B) their percentage. (p-value > 0.05, non-significant difference).

Figure 7

Time-lapse imaging of MDA-MB-231 cells at (A) 30 min, (B) 24 h, (C) 48 h, and (D) 85 h, demonstrating the progression of cell growth and division over time.

Figure 8

Qualitative comparison of MDA-MB-231 cells cultured in (A) incubator and (B) mini-incubator after 4 days, displayed at different magnification levels.

Figure 9

Applications of mini-incubators.