INTERNALIZATION OF F-ACTIN MONOMERS INTO 3T3 FIBROBLASTS VIA THERMAL INKJET PRINTING FOR INVESTIGATION OF CYTOSKELETON INCORPORATION AND MECHANICS

ABSTRACT We will review the conversion of a standard thermal inkjet printer into a bioprinting system and the effects of printing F-actin monomers with cells. The use of any printing system along with biological material or for biological or medical use has been termed bioprinting. Bioprinting has been used in vascular grafts, scaffold design, gene transfection, micro patterning and many other applications and is very diverse. Specifically we will look at the internalization of F-actin monomers into 3T3 fibroblasts as a result of cell membrane disruption from thermal inkjet printing. If the actin monomers were internalized and then incorporated into the cytoskeleton, further investigation of cytoskeleton organization, construction and response to mechanical loading from atomic for microscopy could be conducted. First, a bioprinter had to be modified from a standard printer. An HP Deskjet 500C and an HP Deskjet 500 were used. The only difference is that the HP Deskjet 500C is a color printer and has a different type of cartridge. Both the printers themselves and the ink cartridges that accompanied them had to be modified to accommodate cells and F-actin monomer solution. The printer and cartridges were customized for the application of printing cells. A proof of concept was performed first to see if the converted HP Deskjet 500 could indeed print viable cells without any marked decrease in viability and function. After finding that the cells that were being printed were not only viable, but also continued to grow until confluence it was decided to print the cells along with the fluorescently tagged F-actin monomers to see if monomers could be internalized by the printed cells. Fluorescence microscopy of the confirmed that the monomers could be internalized by the cell before the damage to the cell membrane could be repaired

A Long-term Co-perfused Disseminated Tuberculosis-3D Liver Hollow Fiber Model for Both Drug Efficacy and Hepatotoxicity in Babies

AbstractTreatment of disseminated tuberculosis in children≤6years has not been optimized. The pyrazinamide-containing combination regimen used to treat disseminated tuberculosis in babies and toddlers was extrapolated from adult pulmonary tuberculosis. Due to hepatotoxicity worries, there are no dose–response studies in children. We designed a hollow fiber system model of disseminated intracellular tuberculosis with co-perfused three-dimensional organotypic liver modules to simultaneously test for efficacy and toxicity. We utilized pediatric pharmacokinetics of pyrazinamide and acetaminophen to determine dose-dependent pyrazinamide efficacy and hepatotoxicity. Acetaminophen concentrations that cause hepatotoxicity in children led to elevated liver function tests, while 100mg/kg pyrazinamide did not. Surprisingly, pyrazinamide did not kill intracellular Mycobacterium tuberculosis up to fourfold the standard dose as monotherapy or as combination therapy, despite achieving high intracellular concentrations. Host-pathogen RNA-sequencing revealed lack of a pyrazinamide exposure transcript signature in intracellular bacteria or of phagolysosome acidification on pH imaging. Artificial intelligence algorithms confirmed that pyrazinamide was not predictive of good clinical outcomes in children≤6years who had extrapulmonary tuberculosis. Thus, adding a drug that works inside macrophages could benefit children with disseminated tuberculosis. Our in vitro model can be used to identify such new regimens that could accelerate cure while minimizing toxicity