Keep your cells at 37°C ± 0.5°C outside the CO2 incubator
Test different conditions in the same system
Designed for plates, dishes and perfused chips
Beta Packs, such as the Live Cell Imaging pack, are an assembly of instruments that include Elveflow’s catalog products and, at least, one beta instrument, in this case, the stage top incubator. It has recently been developed by our Innovation team, and it is in testing phase. If you are interested in this beta pack, its applications, or possible variations to better fit your needs, contact our experts!
Our stage top incubator allows you to perform long-term live cell imaging experiments on top of the microscope stage at ease. Designed to fit k-frame microscope stages, the internal chamber fits up to three parallel chips. The ITO glass ensures homogeneous temperature across the microfluidic devices and the frame was made so you don’t need to worry about keeping your reservoirs heated. Your media will arrive at the right temperature on top of your cells regardless of whether you leave the reservoir at room temperature.
As a proof-of-concept of the suitability of the stage top incubator for live cell imaging, we cultured mouse microglial cells for 48 hours under continuous perfusion on top of the microscope stage. The media flow control was done with the OB1 pressure driven flow controller and MFS flow sensor, as shown below.
Microglial cells, as other cell types from the brain parenchyma, are very sensitive to shear stress as they are not usually exposed to flow in their physiological environment. Thus, to prevent the negative effects of shear stress, we employed the µ-Slide III 3D Perfusion and a microfluidic resistance to ensure a low and stable flow.
– Stage top incubator
– OB1 pressure driven flow controller
– MFS flow sensor
– u-Slide III 3D Perfusion
Details of the experimental design can be found on the Specifications tab.
Primary microglial cells were isolated from mice pups brains according to the protocol published in Sepulveda et al, Glia, 2016.
Primary mouse microglial cells were prepared and kindly provided by Patrick P. Michel and Rocio Gimenez, from the Brain and Spine Institute in the frame of the LOCAI project.
Cells were passaged after 1 week of isolation onto ibidi chips (µ-Slide III 3D Perfusion, cat. 80376) at a density of 150 000 cells/mL. Cell medium was supplemented with FBS and PS. HEPES in powder was added to a final concentration of 20 mM. Once the HEPES dissolved, the pH was corrected and brought to 7.2 using Na 1 N, then the media was filtered using a 0,22 filter. Cells were left to attach overnight in an incubator at 37°C and 5% CO2 prior to live cell imaging.
Live cell imaging allows for the study of the dynamic aspect of cellular processes. In vitro models, however, need to be under strict environmental control. Parameters such as pH, temperature and oxygen levels are critical for robust and reliable results. Whether the experimental paradigm requires shear stress, continuous perfusion or static conditions, the need for accurate temperature regulation is the same.
The videos below are 8h time-lapses of primary microglial cells taken immediately after stimulation with lipopolysaccharide (LPS). These videos were recorded using microglial cells seeded onto microfluidic chips and either left under static, non-perfused conditions (video 1) or connected to a pumping system for continuous perfusion along the experiment (1 µL/min, video 2).
Video 1.- Microglial cells seeded in a µ-Slide III 3D Perfusion chip well and maintained in the stage-top incubator on the microscope frame in static conditions for 8 hours after LPS stimulation. Cells were challenged with 10 ng/mL LPS at t = 0 h. Micrographs in phase contrast were acquired every 30 minutes for 8 hours. Image acquisition was made using an AxioVert inverted microscope using a 20x magnification objective (Zeiss).
Video 2.- Microglial cells seeded in a µ-Slide III 3D Perfusion chip well and maintained in the stage-top incubator with continuous perfusion at a flow rate of 1 µL/min. Cells were challenged with 10 ng/mL LPS at t = 0 h. Micrographs in phase contrast were acquired every 20 minutes for 8 hours. Image acquisition was made using an AxioVert inverted microscope using a 20x magnification objective (Zeiss).
In both cases, the incubator allowed for the recording of microglial cells. Micrographs taken every 20 or 30 minutes show the highly dynamic and motile nature of these cells. Importantly, microglial cells, as other cell types from the brain parenchyma, are not exposed to mechanical stresses under physiological conditions. They require, however, nutrient delivery and removal of waste metabolites for survival as in physiological conditions.
The stage top incubator presented here allows for flexibility in designing experiments, including when deciding to apply specific mechanical stress linked to flow. Several off-the-shelf chips with different geometries will allow to extend the design possibilities to address either physiological or pathophysiological conditions using the tissue or cell type of choice
Contact us for expert help in microfluidics!
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