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Florian, D.

Publications and source records attributed to Florian, D..

2 recordsLinked to original sources

Rapid Prototyping of Cell Culture Microdevices Using Parylene-Coated 3D Prints

Fabrication of microfluidic devices by photolithography generally requires specialized training and access to a cleanroom. As an alternative, 3D printing enables cost-effective fabrication of microdevices with complex features that would be suitable for many biomedical applications. However, commonly used resins are cytotoxic and unsuitable for devices involving cells. Furthermore, 3D prints are generally refractory to elastomer polymerization such that they cannot be used as master molds for fabricating devices from polymers (e.g. polydimethylsiloxane, or PDMS). Different post-print treatment strategies, such as heat curing, ultraviolet light exposure, and coating with silanes, have been explored to overcome these obstacles, but none have proven universally effective. Here, we show that deposition of a thin layer of parylene, a polymer commonly used for medical device applications, renders 3D prints biocompatible and allows them to be used as master molds for elastomeric device fabrication. When placed in culture dishes containing human neurons, regardless of resin type, uncoated 3D prints leached toxic material to yield complete cell death within 48 hours, whereas cells exhibited uniform viability and healthy morphology out to 21 days if the prints were coated with parylene. Diverse PDMS devices of different shapes and sizes were easily casted from parylene-coated 3D printed molds without any visible defects. As a proof-of-concept, we rapid prototyped and tested different types of PDMS devices, including triple chamber perfusion chips, droplet generators, and microwells. Overall, we suggest that the simplicity and reproducibility of this technique will make it attractive for fabricating traditional microdevices and rapid prototyping new designs. In particular, by minimizing user intervention on the fabrication and post-print treatment steps, our strategy could help make microfluidics more accessible to the biomedical research community.

bioengineering

Comparative analysis reveals the species-specific genetic determinants of ACE2 required for SARS-CoV-2 entry

Coronavirus interaction with its viral receptor is a primary genetic determinant of host range and tissue tropism. SARS-CoV-2 utilizes ACE2 as the receptor to enter host cell in a species-specific manner. We and others have previously shown that ACE2 orthologs from New World monkey, koala and mouse cannot interact with SARS-CoV-2 to mediate viral entry, and this defect can be restored by humanization of the restrictive residues in New World monkey ACE2. To better understand the genetic determinants behind the ability of ACE2 orthologs to support viral entry, we compared koala and mouse ACE2 sequences with that of human and identified the key residues in koala and mouse ACE2 that restrict viral receptor activity. Humanization of these critical residues rendered both koala and mouse ACE2 capable of binding the spike protein and facilitating viral entry. The single mutation that allowed for mouse ACE2 to serve as a viral receptor provides a potential avenue for the development of SARS-CoV-2 mouse model.

microbiology