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Abstract Brief pulses of electric field (electroporation) and/or tensile stress (mechanoporation) have been used to reversibly permeabilize the plasma membrane of mammalian cells and deliver materials to the cytosol. However, electroporation can be harmful to cells, while efficient mechanoporation strategies have not been scalable due to the use of narrow constrictions or needles which are susceptible to clogging. Here we report a high throughput approach to mechanoporation in which the plasma membrane is stretched and reversibly permeabilized by viscoelastic fluid forces within a microfluidic chip without surface contact. Biomolecules are delivered directly to the cytosol within seconds at a throughput exceeding 250 million cells per minute. Viscoelastic mechanoporation is compatible with a variety of biomolecules including proteins, RNA, and CRISPR-Cas9 ribonucleoprotein complexes, as well as a range of cell types including HEK293T cells and primary T cells. Altogether, viscoelastic mechanoporation appears feasible for contact-free permeabilization and delivery of biomolecules to mammalian cells ex vivo. 

Cryptosporidium hominisis a serious cause of childhood diarrhea in developing countries. The development of therapeutics is impeded by major technical roadblocks including lack of cryopreservation and simple culturing methods. This impacts the availability of optimized/standardized singular sources of infectious parasite oocysts for research and human challenge studies. The humanC.hominisTU502 isolate is currently propagated in gnotobiotic piglets in only one laboratory, which limits access to oocysts. Streamlined cryopreservation could enable creation of a biobank to serve as an oocyst source for research and distribution to other investigators requiringC.hominis. Here, we report cryopreservation ofC.hominisTU502 oocysts by vitrification using specially designed specimen containers scaled to 100 μL volume. Thawed oocysts exhibit ~70% viability with robust excystation and 100% infection rate in gnotobiotic piglets. The availability of optimized/standardized sources of oocysts may streamline drug and vaccine evaluation by enabling wider access to biological specimens.