CO2-Free On-Stage Incubator for Live Cell Imaging of Cholangiocarcinoma Cell Migration on Microfluidic Device

Abstract

Long-term live cell imaging requires sophisticated and fully automated commercial-stage incubators equipped with specified inverted microscopes to regulate temperature, CO₂ content, and humidity. In this study, we present a CO₂-free on-stage incubator specifically designed for use across various cell culture platforms, enabling live cell imaging applications. A simple and transparent incubator was fabricated from acrylic sheets to be easily placed on the stages of most inverted microscopes. We successfully performed live-cell imaging of cholangiocarcinoma (CCA) cells and HeLa cell dynamics in both 2D and 3D microenvironments over three days. We also analyzed directed cell migration under high serum induction within a microfluidic device. Interesting phenomena such as "whole-colony migration", "novel type of collective cell migration" and "colony formation during cell and colony migration" are reported here for the first time, to the best of our knowledge. These phenomena may improve our understanding of the nature of cell migration and cancer metastasis.

Keywords: cell migration; cholangiocarcinoma (CCA); live-cell imaging; microfluidics; on-stage incubator.

Figure 1

(A) Inverted microscope mounted with stage incubator and camera; (B) stage incubator design; (C) side view; and (D) front view of the incubator.

Figure 2

(A) Design illustration of the microfluidic device. (B) Top view of round micropillars on a silicon wafer. Green arrows show the fluidic path. (C) Cross-sectional view of round PDMS micropillars with scanning electron microscope (SEM).

Figure 3

Blocking methods: (A) inlets of microfluidic device blocked with sterile stainless-steel pins. (B) Inlets of microfluidic device blocked with tips of the micropipette tips and molten wax on their top.

Figure 4

On-stage incubator temperature profile: (A) change in incubator temperature with time after turning on; (B) temperature fluctuations of the incubator over the time of 12 days.

Figure 5

Growth curve of CCA cells in the standard CO₂ incubator and CO₂-free on-stage incubator: (A) trypan blue dye-exclusion method; (B) MTT assay. Error bars show ±1 standard deviation (n = 3). The data sets were significantly not different from day 1 to day 5 but on days 6 and 7 the data sets were significantly different, in both the trypan blue dye-exclusion method and the MTT assay for both the CO₂ and CO₂-free on-stage incubators. The p-value is <0.05 for both growth assays.

Figure 6

CCA colony dynamics in 2D: (A) CCA colony migration and dissociation; red arrow indicating the colony of interest (B) CCA colony migration and merging into another colony forming a bigger colony, blue arrow indicating the colony of interest. Also, see Supplementary Videos S3 and S4, respectively.

Figure 7

Single cell and colony dynamics in 3D microenvironment of Hela Red arrows shows the colony of interest (A) and CCA, blue arrow shows the colony of interest (B). Also, see Supplementary Videos S5 and S6, respectively.

Figure 8

CCA cell migration in a microfluidic device: (A–E). Cells are numbered and marked with colored circles (each color represents a different image time). The circled part of the images was extracted by clipping off the rest and overlaying the extracted circles over the previous image time.

Figure 9

Time-lapse of medium evaporation using blocking method 1 (A) with sterile stainless-steel pins compared with blocking method 2 (B) with sterile cut tips of the 200 µL micropipette tips and filling with molten wax. The red arrows represent the meniscus of the medium evaporation with respect to time.