Fluctuations of Dry and Total Mass of Cells Exposed to Different Molecular Weights of Polyethylene Glycol

Abstract

Almost all aspects of cellular and multicellular activities depend on macromolecular interactions. [1] In this context, macromolecular displacement refers to the effect exerted by high in vivo concentrations of molecules in the cytoplasm. [2] Such crowding in living systems has major implications for all cellular activities and biochemical processes such as enzyme activity and protein stability. [1] Macromolecular solutions such as biopolymers, like polyethylene glycol (PEG) or proteins, have been used to replicate the crowded environment inside living cells. The advantage of well-defined molecules like PEG is that, unlike in vivo, they are easy to handle and chemically and physically well-defined to study protein interactions, enzymatic processes, [1] and cell membrane fusion. [3] PEG, for example, acts simply by volume exclusion, resulting in an osmotic force that fuses membranes in a dehydrated region; thus, the role of various lipids and fusion proteins has also been studied previously. [3] Recently, the mechanism of cellular uptake of PEG has been well described by Wang and his team. [3] The uptake of PEGylated nanoparticles occurs rapidly by endocytosis, while pure PEG does not form nanoparticles. [4] Also different mechanisms for cell entry have been described: Low molecular weight (MW) PEG up to 2000 Da (at various concentrations from 50 to 1000 μM) can enter the cell by passive diffusion; [4] however, with slower kinetics for higher MW PEG (2000 Da) than for lower MW PEG (750 Da). [4] On the contrary, the contribution of passive diffusion to the uptake of higher MW PEG is low and the uptake occurs mainly by endocytosis, suggesting aggregation of higher MW PEG. [4] Another study by Yang et al. [4] investigated the distribution, trafficking and exocytosis of PEG in vitro. After incubation of 3 Â 10 7 cells with 5 mM PEG of 5000 Da, a total amount of 62 μg PEG accumulated inside the cells. Specifically, internalized PEG is first transported to endosomes and from there to lysosomes. [5] After, it can either be transferred into the cytosol, endoplasmic reticulum, and mitochondria or leave the cells by exocytosis, [5] or is degraded by hydrolytic and proteolytic activities. [6] During this intracellular redistribution process, PEG has a very high residence time in cells, which can even exceed 96 h. [5] When the concentration of extracellular solutes increases by PEG addition, water is forced out of the cell by osmotic pressure which leads to a decreased cell volume with strong effects on the cells' physiological processes, cytotoxicity, and mechanics. [7,8] PEG is considered to be almost nontoxic in medical applications; [9] however, cytotoxicity of different MWs and concentrations of PEG has been reported in various crowding studies, [9][10][11][12] mainly due to hyperosmotic stress. [9] Metabolically activated low MW PEG (200-400 Da) has also been reported to have genotoxic effects on numerous cell lines. [13] While the mechanism and cytotoxicity of PEG internalization are well studied, and first studies hav...

Two‐Photon Laser Printing to Mechanically Stimulate Multicellular Systems in 3D

“…To characterize the mechanical properties of the hydrogels where the cells grow on and in, respectively, micro‐indentation measurements were performed using a Pavone nanoindentation platform (Optics11 Life) according to a previously published method. [ 54 ] The measurements of the HYDROBIO INX N100 (BIO INX) structures were carried out using a cantilever with 0.250 N m −1 stiffness and a 9 µm tip radius, while for the HYDROBIO INX N400 (BIO INX) a cantilever with 0.032 N m −1 stiffness and a 24 µm tip radius was used. During the probe calibration and measurements, the structures were kept in cell culture media.…”

2-photon laser printing to mechanically stimulate multicellular systems in 3D

“…To characterize the mechanical properties of the hydrogels where the cells grow on and in, respectively, micro-indentation measurements were performed using a Pavone nanoindentation platform (Optics11 Life) accordingly to a previous published method[54]. The measurements of the HYDROBIO INX N100 (BIO INX) structures were carried out using a cantilever with 0.250 N/m stiffness and a 9 μm tip radius, while for the HYDROBIO INX N400 (BIO INX) we used a cantilever with 0.032 N/m stiffness and a 24 μm tip radius.…”

Multimaterial 3D Laser Printing of Cell-Adhesive and Cell-Repellent Hydrogels

“…Mechanical characterization: Indentation measurements were carried out using a Pavone Optics11 Life device according to a previously published method. [23] A one-piece optical probe consisting of an optical fiber, a cantilever, and a spherical tip was approached onto printed hydrogel blocks in DMEM buffer. Cantilever deflection upon surface contact was detected with an interferometer, allowing for precise displacement measurements.…”