Automated Multichamber Time-lapse Videography for Long-term In Vivo Observation of Migrating Cells

Abstract

Aim: To observe and document the migration of living cells by time-lapse videography, we constructed a low-budget system based on a common inverted microscope.

Materials and methods: Long-term observation of six-well plates is enabled through maintenance of cell culture conditions (5% CO₂ in air at 37°C). Points of interest can be revisited in definable intervals with <1 μm repositioning error. Digital photographs from each programmed time point are paired with environmental data and combined into a record.

Results: We used this new chamber to observe the migration of various cell lines. The design represents a good compromise between low cost and good precision. Detailed analyses verified that the environmental conditions were appropriately maintained, enabling long-term observation of viable cells. The stimulating influence of irradiation with photons (radiotherapy) on cellular motility of glioblastoma cells is presented.

Conclusion: This study demonstrates that useful videographic systems can be constructed at low cost.

Keywords: Time-lapse videography; cellular motility; environmental chamber; live cell imaging; migrating cells.

Figure 1. Assembly of the videographic system. A: The system is based on a Zeiss Axiovert 25 microscope. A plate holder for standard multiwell plates is positioned precisely by two linear motors; reproducible focusing of points of interest is performed by a servo motor mounted to the manual drive of the microscope. B: The receptacle is designed to hold standard multiwell plates. The housing is sealed by transparent plates, and a constant atmosphere is secured by flooding the housing with 5% CO2 in air. Temperature is maintained by a water circuit in the aluminum frame. C: The system layout. The flowchart shows how the plate holder is controlled for data collection and how the environmental conditions are maintained.

Figure 2. The course of temperature during a single run. Temperature maintenance is achieved through the connected thermostat set to 37.0˚C. The temperature of the plate holder was continuously monitored by a PT-sensor located in the aluminum frame; values were paired and saved with every image over a period of 24 h

Figure 3. Monitoring pH levels via absorption maxima of phenol red in the cell culture medium. Acidic and basic peaks of the absorption spectrum were determined at 450 and 570 nm with an ELISA reader and their difference used as a measure for the pH value of the medium. A: Time course of the pH increase as a function of CO2 loss after plate transfer from 5% CO2 in the incubator to room conditions. B: Mean absorption value of the plates immediately after the run compared to reference values at defined pH. Data depicted are mean±standard error of the mean from six independent experiments.

Figure 4. U87 glioblastoma cells after 62 h in the environmental chamber. One image out of a series of 72 consecutive photos taken by the videographic system is shown

Figure 5. U373 glioblastoma cells were irradiated with 0.5 Gy photons. These cells and non-irradiated controls were transferred to the videographic system and observed over a period of 24 h. Three areas of interest were selected for each sample and photographed every 10 min. Randomly selected cells were tracked with ImageJ and analyzed with the ibidi migration tool. Typical migration patterns for both samples are shown. The columns represent means±standard error of the mean of 90-100 travelling cells. The level of significance was analyzed with Student’s t-test. ***p<0.001; ****p<0.0001