Microfluidic PDMS (Polydimethylsiloxane) Bioreactor for Large-Scale Culture of Hepatocytes

Abstract

Microfluidics could provide suitable environments for cell culture because of the larger surface-to-volume ratio and fluidic behavior similar to the environments in vivo. Such microfluidic environments are now used to investigate cell-to-cell interactions and behaviors in vitro, emulating situations observed in vivo, for example, microscale blood vessels modeled by microfluidic channels. These emulated situations cannot be realized by conventional technologies. In our previous works, microfluidic channels composed of two PDMS (poly(dimethylsiloxane)) layers were successfully used for Hep G2 cell culture. To achieve physiologically meaningful functions in vitro, a culture with a larger number of cells and higher density must be performed. This will require bioreactors with larger surface areas for cell attachment and sufficient amounts of oxygen and nutrition supply. For those purposes, we fabricated a bioreactor by stacking 10 PDMS layers together, i.e., four cell culture chambers, and a chamber dedicated to the oxygen supply inserted in the middle of the 10-stacked layers. The oxygen supply chamber is separated from the microfluidic channels for the culture medium perfusion by thin 300-microm PDMS walls. The high gas permeability of PDMS allows oxygen supply to the microfluidic channels through the thin walls. On the basis of the measurement of glucose consumption and albumin production, it is shown that cellular activity exhibits a gradual increase and saturation throughout the culture. We clearly observed that in the case of the microfluidic bioreactor for large-scale cultures, the oxygen chamber is indispensable to achieve longer and healthy cultures. In the present bioreactor, the cell density was found to be about 3-4 x 10(7) cells/cm(3), which is in the same order of magnitude as the conventional macroscale bioreactors. Consequently, by stacking single culture chambers and oxygen chambers in between, we could have a scalable method to realize the microfluidic bioreactor for large-scale cultures.

Cell handling using microstructured membranes

“…12,16,17 In contrast, most of the other elastomeric mechanical valves would seal upon the application of pressure through a thin membrane, and may introduce the challenge of maintaining the homeostasis of a cell suspension in the controlled channel. Gasses can diffuse easily through the membrane under the actuation pressure 18 and either diffuse in the cell suspension or, worse, form gas bubbles that can damage the cells. While a common solution for this problem is the filling of the control channels with liquid, recent results reported that the PDMS is permeable even to some commonly used liquids, 19 and raise the concern of maintaining the homeostasis of the medium around cells during pressure actuation.…”

Elastomer Application in Microsystem and Microfluidics

“…Permeability has been employed to fabricate non-contact pump for the manipulation of aqueous solutions within PDMS microfluidic devices 20 or supply oxygen for large-scale culture of hepatocytes 31 . On the other hand, PDMS permeability limits its application to microfluidic technology, in which coating such as glass-like layer using sol-gel chemistry is developed on PDMS to minimize permeability 32 .…”

Exploitation of physical and chemical constraints for three-dimensional microtissue construction in microfluidics

“…In another PDMS microbioreactor, rat hepatocytes attached to a porous PDMS membrane ͑5 ϫ 5 m pores͒ sandwiched between two perfusion chambers showed improved cell attachment, cell reorganization, albumin secretion, and ammonium removal. 175 In a series of studies, Leclerc et al [176][177][178] fabricated chamber based microbioreactors with 3D cellular aggregations, enhancement in glucose consumption, and albumin secretion for fetal human hepatocytes and HepG2 cells. Prokop et al 179 presented a NanoLiterBioReactor in which hepatocytes were trapped by 3 m sievelike features.…”