Embedded Files
Introduction
"Visible liver" - is a microfluidic device for long-term 3D liver tissue culture. Liver cells seeded in the channels of a scaffold self-assemble and form an array of 3-dimensional liver tissue units, mimicking in several ways the 3D microenvironment of liver. The tissue is imaged by high resolution confocal or two-photon microscopy while being perfused by cell culture medium. Because the tissue is primarily interrogated by optical means, the device maintains a relatively low number of cells (20 000 - 50 000). "Visible liver" microreactor can be used as a disease model in applications such as in vitro studies of early stages of tumor development in liver tissue which are challenging to perform in vivo. Another application is analyzing barriers to gene delivery in 3D tissues.
More information about the liver microbioreactor can be found in the following book chapter. [pdf]
Microreactor design
One of the key functional design criteria of the microreactor is accessibility of the 3D tissue structures to in situ optical microscopy. To provide this capability and allow using high numerical aperture objectives, a very thin (125 µm) polycarbonate optical window was used. In addition, the microreactor was designed in such a way that the distance from the top of the optical window to the tissue structures was only 750 µm (including the thickness of column of cell culture medium above the tissue). Finally, the top surface of the microreactor face was kept flat to facilitate optical scanning with very short working distance objectives. For this reason, fluidic inlets and outlets were brought to the side of the microreactor and custom-made low-profile fluidic connectors were used. The approach dictated employing a bonding step in the microreactor fabrication.
Design, 3D Model & Photo: Karel Domansky
Microreactor fabrication
The main body of the "Visible liver" microreactor was fabricated from polycarbonate by micromachining and thermal diffusion bonding. The technology was developed by the author at MIT and transfered to Eastern Plastics, Inc. (acquired by IDEX Corp.), where the polycarbonate body was fabricated. [more information]
The oval PVDF filter and silicone gaskets were punched. The insert for the O-ring was fabricated from stainless steel by CNC machining. The connectors were designed by the author and fabricated by CNC machining.
Tissue perfusion system
The microreactor is connected by gas-permeable tubing to two external peristaltic pumps that circulate cell culture medium between a reservoir and the microreactor.
Photo: Karel Domansky
Scaffolds for cell attachment and tissue formation
A variety of materials and microfabrication techniques can be used for manufacturing the scaffolds. In the photograph above, the scaffolds were made from silicon by deep reactive ion etching technique. The photograph below depicts a polyethylene terephthalate scaffold manufactured by laser micromachining. [more information]
SEM images courtesy of JP Sercel Associates.
Microreactor as a model for studying gene delivery in 3D tissues
After the liver tissue is transfected with replication deficient adenovirus carrying the gene for EGFP driven by the CMV promoter, 2-photon microscopy provides in situ 3D spacial information about the gene expression levels within the tissue. The 3D imaging involves the acquisition of a stack of xy planes, each plane corresponding to a different z-depth inside the channel. The number of cells stained with Ethidium Homodimer (red) provides a count for dead cells; cells stained with Hoechst (blue) give total cell count (within a reactor microchannel); cells expressing EGFP are green. Based on imaging of seven microchannels in two microbioreactors 60 h after infection initiation, the gene delivery efficiency was 45% and cell viability 84%. More information about studying gene delivery in 3D tissue can be found in journal article. [pdf]
Note: Images of tissue in a single 300 x 300 x 230-µm channel (silicon scaffold).
Photo and experimental data: Artemis Kalezi
Setup for 2-photon microscopy
Many biological studies require microscopical investigations lasting from several minutes up to several hours. The long imaging times can compromise integrity of the biological samples unless temperature and pH of the cell culture medium are adequately controlled. To maintain the physiological conditions, a large acrylic box ("incubator") around the microscope (left image) provides temperature control of the sample during 2-photon imaging. A secondary enclosure inside the acrylic box (right image) contains the microreactor (image insert with bottom view) and provides pH control. In addition, because the microreactor cultures are highly metabolically active, they must be perfused with cell culture medium during the imaging. To provide perfusion, the secondary enclosure also contains a reservoir with cell culture medium and pumps. In this way, tissue inside the microreactor is perfused with cell culture medium having physiological temperature and pH.
Photo: Karel Domansky
Fluorescent spectrometry of the liver tissue cultured in microreactor
Tissue in the "Visible liver" microreactor can be also optically interrogated with fluorescent spectrometry. As opposed to confocal or 2-photon microscopy mentioned above that aims at gathering local information from a small piece of tissue in a single channel, fluorescent spectrometry system depicted below integrates photons emitted from all channels of the scaffold. The technique can quickly provide information about the stage of the transfection and provide a reference time point for carrying out 2-photon imaging. More information can be found in the following book chapter. [pdf]
Design & Photo: Karel Domansky
Fluorescent emission spectrum of EGFP after AdEGFP addition (upper plot)
Fluorescent intensity versus time after AdEGFP addition (lower plot)
In collaboration with Artemis Kalezi.
Temperature, pH, and tissue perfusion rate during the spectrometry can be controlled with the environmental cartridge shown in the photograph below.
Design & Photo: Karel Domansky
Page updated
Report abuse