Characterization of polymer‐polymer aqueous two‐phase system droplets for 3D culture future applications

Abstract

BACKGROUND: Cell-based high-throughput platforms using aqueous two-phase systems (ATPS) have led to the construction of 3D cultures using ATPS in which stem cells are confined into polymer-polymer droplets, giving rise to viable spheroids for their further proliferation and differentiation. This work aims to characterize polymer-polymer ATPS droplet generation based on diameter size, morphology and uniformity, using basic laboratory equipment.RESULTS: Dextran (DEX), including DEX 500 000 and 70 000 g mol −1 , as well as UCON were used as the droplet phase (0.5, 1, 2 ∼L). Polyethylene glycol (PEG), including PEG 8 000, 10 000 and 35 000 g mol −1 , as well as DEX 75 000 g mol −1 and Ficoll were used as the bulk phase (50, 100 ∼L). Different ATPS compositions (molecular weights and concentrations) with phosphate-buffered saline as ATPS solvent and construction strategies (droplet added, immersed or covered) were evaluated in 96-well plates. ATPS droplets were generated in PEG-DEX ATPS. Diameter size and morphology were susceptible to ATPS compositions, construction strategies and DEX droplet phase volumes with no significant difference in PEG bulk phase volumes, whereas higher uniformity of ATPS droplets was correlated to lower PEG bulk volumes. Therefore, 1 ∼L DEX droplets immersed at the bottom of a well filled with 50 ∼L PEG resulted in uniform ATPS droplets with a mid-range diameter of 1.35 ± 0.23 mm. CONCLUSIONS: According to the results, this work demonstrates simplified and practical alternatives for the generation of reproducible PEG-DEX ATPS droplets that could be adapted for the encapsulation of different cell types for further 3D culture applications.