No abstract

Maximizing the speed and efficiency at which single cells can be liberated from tissues would dramatically advance cell-based diagnostics and therapies. Conventional methods involve numerous manual processing steps and long enzymatic digestion times, yet are still inefficient. In previous work, we developed a microfluidic device with a network of branching channels to improve the dissociation of cell aggregates into single cells. However, this device was not tested on tissue specimens, and further development was limited by high cost and low feature resolution. In this work, we utilized a single layer, laser micro-machined polyimide film as a rapid prototyping tool to optimize the design of our microfluidic channels to maximize dissociation efficiency. This resulted in a new design with smaller dimensions and a shark fin geometry, which increased recovery of single cells from cancer cell aggregates. We then tested device performance on mouse kidney tissue, and found that optimal results were obtained using two microfluidic devices in series, the larger original design followed by the new shark fin design as a final polishing step. We envision our microfluidic dissociation devices being used in research and clinical settings to generate single cells from various tissue specimens for diagnostic and therapeutic applications.

Conventional biomedical research is mostly performed by utilizing a two-dimensional monolayer culture, which fails to recapitulate the three-dimensional (3D) organization and microenvironment of native tissues. To overcome this limitation, several methods are developed to fabricate microtissues with the desired 3D microenvironment. However, they tend to be time-consuming, labor-intensive, or costly, thus hindering the application of 3D microtissues as models in a wide variety of research fields. In the present study, we have developed a pressure-assisted network for droplet accumulation (PANDA) system, an easy-to-use chip that comprises a multichannel fluidic system and a hanging drop cell culture module for uniform 3D microtissue formation. This system can control the desired artificial niches for modulating the fate of the stem cells to form the different sizes of microtissue by adjusting the seeding density. Furthermore, a large number of highly consistent 3D glomerulus-like heterogeneous microtissues that are composed of kidney glomerular podocytes and mesenchymal stem cells have been formed successfully. These data suggest that the developed PANDA system can be employed as a rapid and economical platform to fabricate microtissues with tunable 3D microenvironment and cellular heterogeneity, thus can be employed as tissue-mimicking models in various biomedical research.

The assembly of enterovirus requires the cleavage of P1 polyprotein by protease 3CD into individual structural proteins. Two recombinant baculoviruses were constructed to encode P1 and 3CD of enterovirus 71 (EV71), respectively. The expressed 3CD successfully cleaved P1 in vitro and in vivo. Also, the co-infection in insect cells resulted in crystalline virus-like particle structures morphologically resembling the authentic EV71 aggregates, which are reported for the first time.