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Efficient and gentle delivery of molecules into cells with different elasticity via Progressive Mechanoporation.

ABSTRACT: Intracellular delivery of cargo molecules such as membrane-impermeable proteins or drugs is crucial for cell treatment in biological and medical applications. Recently, microfluidic mechanoporation techniques have enabled transfection of previously inaccessible cells. These techniques create transient pores in the cell membrane by shear-induced or constriction contact-based rapid cell deformation. However, cells deform and recover differently from a given extent of shear stress or compression and it is unclear how the underlying mechanical properties affect the delivery efficiency of molecules into cells. In this study, we identify cell elasticity as a key mechanical determinant of delivery efficiency leading to the development of "progressive mechanoporation" (PM), a novel mechanoporation method that improves delivery efficiency into cells of different elasticity. PM is based on a multistage cell deformation, through a combination of hydrodynamic forces that pre-deform cells followed by their contact-based compression inside a PDMS-based device controlled by a pressure-based microfluidic controller. PM allows processing of small sample volumes (about 20 μL) with high-throughput (>10 000 cells per s), while controlling both operating pressure and flow rate for a reliable and reproducible cell treatment. We find that uptake of molecules of different sizes is correlated with cell elasticity whereby delivery efficiency of small and big molecules is favoured in more compliant and stiffer cells, respectively. A possible explanation for this opposite trend is a different size, number and lifetime of opened pores. Our data demonstrates that PM reliably and reproducibly delivers impermeable cargo of the size of small molecule inhibitors such as 4 kDa FITC-dextran with >90% efficiency into cells of different mechanical properties without affecting their viability and proliferation rates. Importantly, also much larger cargos such as a >190 kDa Cas9 protein-sgRNA complex are efficiently delivered high-lighting the biological, biomedical and clinical applicability of our findings.

SUBMITTER: Uvizl A

PROVIDER: S-EPMC8204113 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:The quality of the animal-human relationship and, consequently, the welfare of animals can be improved by gentle interactions such as stroking and talking. The perception of different stimuli during these interactions likely plays a key role in their emotional experience, but studies are scarce. During experiments, the standardization of verbal stimuli could be increased by using a recording. However, the use of a playback might influence the perception differently than "live" talking, which is closer to on-farm practice. Thus, we compared heifers' (n = 28) reactions to stroking while an experimenter was talking soothingly ("live") or while a recording of the experimenter talking soothingly was played ("playback"). Each animal was tested three times per condition and each trial comprised three phases: pre-stimulus, stimulus (stroking and talking) and post-stimulus. In both conditions, similar phrases with positive content were spoken calmly, using long low-pitched vowels. All tests were video recorded and analyzed for behaviors associated with different affective states. Effects on the heifers' cardiac parameters were assessed using analysis of heart rate variability. Independently of the auditory stimuli, longer durations of neck stretching occurred during stroking, supporting our hypothesis of a positive perception of stroking. Observation of ear positions revealed longer durations of the "back up" position and less ear flicking and changes of ear positions during stroking. The predicted decrease in HR during stroking was not confirmed; instead we found a slightly increased mean HR during stroking with a subsequent decrease in HR, which was stronger after stroking with live talking. In combination with differences in HRV parameters, our findings suggest that live talking might have been more pleasurable to the animals and had a stronger relaxing effect than "playback." The results regarding the effects of the degree of standardization of the stimulus on the variability of the data were inconclusive. We thus conclude that the use of recorded auditory stimuli to promote positive affective states during human-animal interactions in experimental settings is possible, but not necessarily preferable.

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Efficient and gentle delivery of molecules into cells with different elasticity via Progressive Mechanoporation.

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