BPS2026 – Assembling large numbers of size-controlled giant unilamellar vesicles for biophysical applications

Giant unilamellar vesicles (GUVs) are micron size model cell systems. Typical applications are currently limited to studies requiring tens to hundreds of GUVs, such as membrane phase behavior, protein interactions, and synthetic cell reconstitution. Studying the biophysics of systems that involve interactions of collections of cells such as in tissues, blood flow, and clot formation require large numbers of GUVs. For example, a typical hemostatic microfluidic experiment requires 4 mL of solution at concentrations of 300,000 synthetic platelets per microliter. However, assembling such large numbers of GUVs, especially in solutions of physiological ionic strengths, is challenging with current methods. Here, we report a novel workflow using PAPYRUS (paper-abetted lipid hydration in aqueous solutions) that produces > 10 9 GUVs in 70 mL of buffer (10 4 GUVs per microliter) ranging from 1–85 μm within 2.5 h in a reclosable bag and commonly available laboratory tools. The method facilities assembly of membranes with saturated lipids and high cholesterol contents that required high assembly temperatures. Unlike, in 48-well plates, use of reclosable bags requires the use of a fraction of lipids with charged headgroups, demonstrating a previously unknown compositional restriction when scaling up assembly. Using synthetic platelets as our model, we further process the GUV suspension to obtain 99.8% of the GUVs < 10 μm and no GUVs were larger than 20 μm, which is the upper limit of circulating leukocytes (∼20 μm in diameter). Centrifugation and solvent exchange increase the concentration to 300,000 per microliter in physiological saline. We obtain sufficient quantities to potentially perform 11 in vitro microfluidic experiments or 70 in vivo mouse experiments in <3 h. The process can be further scaled by using multiple bags in parallel.