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Application Notes

Observing a Vascularized Tumor Spheroid on a Chip with a Confocal Microscope

Studies show tumors induce blood vessel growth to support vigorous tumor activity. These blood vessels serve as tumor lifelines and play an important role in the tumor microenvironment (TME) to support this activity. Until now, tumor responses to biochemical and biomechanical stimuli were evaluated under static conditions and failed to incorporate the effects of blood flow to tumors. In this study, we present a tumor-on-a-chip platform that enables the evaluation of a tumor microenvironment with media flow through a perfusable vascular network and measure the effects of drug administration incorporated in the blood flow.

3D Observation of the Tumor Spheroid in the Microfluidic Device

Figure 1. Microfluidic device and tumor model.

Figure 1. Microfluidic device and tumor model.
(Left) Image of the microfluidic device, (right) tumor model with a perfusable vascular network.

In this study, we recapitulated the tumor microenvironment, including the vascular network, by coculturing human umbilical vein endothelial cells (HUVEC) with spheroids containing human breast adenocarcinoma cells (MCF-7) in microfluidic devices. To confirm the perfusability of a vascular network in the microfluidic device, we observed if fluorescent beads (green) continuously passed through the vascular network and spheroid using the FV3000RS confocal microscope. 3D cultured samples are difficult to image due to the thickness of the mass. Higher excitation light intensity is typically required to produce a fluorescent signal strong enough to be detected. However, higher excitation light also increases phototoxicity, resulting in damage to the cells. To overcome this, we used the FV3000 confocal microscope, which incorporates Olympus’ TruSpectral detection technology and high-sensitivity GaAsP detectors, to capture weak fluorescent signals and minimize the laser power.


(a) Projection image of the tumor spheroid. Scale bar: 200 μm, objective: UPLSAPO10X2.


(b) 3 plane image (x-y、x-z、y-z) of white frame in (a). Scale bar: 20 μm, objective: UPLSAPO40X2.

Figure 2. The tumor spheroid and its vascular network.
Nuclear: cyan (405 nm, Hoechst 33342), RFP-HUVEC: magenta (561 nm, RFP), E-cadherin: yellow(640 nm, Alexa Fluor 633).
(a) Projection image of the tumor spheroid. Scale bar: 200 μm, objective: UPLSAPO10X2.
(b) 3 plane image (x-y、x-z、y-z) of white frame in (a). Scale bar: 20 μm, objective: UPLSAPO40X2.

Fast Imaging of Blood Flow Using the Resonant Scanner

We also examined the flow in the vascular network of the tumor spheroid using fluorescent microbeads (green, diameter: 3.1 μm). When observing fast blood flow, the scanning speed of the normal galvanometer scanner may not be enough. In this experiment, images were captured using the Olympus FV3000RS confocal microscope equipped with a high-speed resonant scanner. We confirmed that when fluorescent microbeads were injected into channel 3 of the microfluidic device, the microbeads passed through the luminal structures of the spheroid and reached channel 1, indicating the perfusability of the engineered vascular network. Upon confirming the validity of this engineered tumor spheroid model for studying the effects of blood flow on the TME, it was clarified in subsequent experiments that the efficacy of drugs in the TME, including the blood vessel network, varies depending on the presence or absence of intravascular flow. We expect that our three-dimensional model can contribute to drug development as a transvascular drug administration model in the future.

Video: flow of fluorescent beads inside blood vessels and tumor spheroids constructed on a microfluidic device.
Imaging condition: 65 msec/frame. Scale bar: 100 μm.

Comment by Dr. Yokokawa

The novelty of this study was a perfusable vascular network constructed in a tumor spheroid. It was important to visualize how the vascular network connected the spheroid and microfluidic channels. Moreover, it was essential to simultaneously observe the vascular lumen and the flow through the vasculature to demonstrate the perfusability. The high-speed resonant scanner installed in the FV3000 confocal microscope enabled the imaging of the vascular network (RFP-labeled) and the dynamic flow of microbeads (green).

Dr. Ryuji Yokokawa

Dr. Ryuji Yokokawa1

Dr. Yuji Nashimoto

Dr. Yuji Nashimoto2

This application note was prepared with the help of the following researchers:
Department of Micro Engineering, Kyoto University1
Frontier Research Institute for Interdisciplinary Sciences (FRIS), Tohoku University2

Nashimoto Y, Okada R, Hanada S, Arima Y, Nishiyama K, Miura T, Yokokawa R. Biomaterials. 2020, Jan;229:119547. "Vascularized cancer on a chip: The effect of perfusion on growth and drug delivery of tumor spheroid.” DOI: 10.1016/j.biomaterials.2019.119547

How the FV3000 Confocal Microscope Facilitated Our Experiment

Fully Spectral System with High-Efficiency GaAsP Detectors Provides High Sensitivity for Live-Cell Imaging

TruSpectral detection technology

The FV3000 confocal microscope series features Olympus’ TruSpectral detection technology, which diffracts light via transmission through a volume phase hologram unit. This technology enables a much higher light throughput than conventional spectral detection units with reflection-type gratings. The FV3000 microscope’s two-channel high-sensitivity spectral detector (HSD) uses TruSpectral technology with Peltier-cooled GaAsP PMTs for a high-quantum efficiency of 45% with a high signal-to-noise ratio. This combination of detection technologies enables powerful high-sensitivity detection and minimizes the laser power needed for live tissue observation.

Two Scan Units Options

Video: Platelets bound to a thrombosis in the blood vessel of a mouse. Images taken at 30 fps in full frame using a resonant scanner with 2-channel GaAsP PMTs.

Image data courtesy of: Dr. Takuya Hiratsuka, Dr. Michiyuki Matsuda, Graduate School of Biostudies, Kyoto University.

Choose between two scan units: a traditional galvanometer scanner (FV3000) or a hybrid galvanometer/resonant scanner (FV3000RS). A resonant scanner enables you to capture 30 frames per second with a full field of view at 512 × 512 pixels, or up to 438 frames per second by clipping down in Y to capture critical live physiological events, such as calcium ion flux.

Products related to this application

Confocal Laser Scanning Microscope


  • Available with either galvanometer-only (FV3000) or hybrid galvanometer/resonant (FV3000RS) scanner configurations
  • Highly efficient and accurate TruSpectral detection on all channels
  • Optimized for live cell imaging with high sensitivity and low phototoxicity
  • Inverted and upright frame options to suit a variety of applications and sample types

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