Shear stress increases cytotoxicity and reduces transfection efficiency of liposomal gene delivery to CHO-S cells

Abstract

Animal cells in suspension experience shear stress in different situations such as in vivo due to hemodynamics, or in vitro due to agitation in large-scale bioreactors. Shear stress is known to affect cell physiology, including binding and uptake of extracellular cargo. In adherent cells the effects of exposure to shear stress on particle binding kinetics and uptake have been studied. There are however no reports on the effect of shear stress on extracellular cargo delivery to suspension cells. In this study, we have evaluated the effect of shear stress on transfection of CHO-S cells using Lipofectamine 2000 in a simple flow apparatus. Our results show decreased cell growth and transfection efficiency upon lipoplex assisted transfection of CHO-S while being subjected to shear stress. This effect is not seen to the same extent when cells are exposed to shear stress in absence of the lipoplex complex and subsequently transfected, or if the lipoplex is subjected to shear stress and subsequently used to transfect the cells. It is also not seen to the same extent when cells are exposed to shear stress in presence of liposome alone, suggesting that the observed effect is dependent on interaction of the lipoplex with cells in the presence of shear stress. These results suggest that studies involving liposomal DNA delivery in presence of shear stress such as large scale transient protein expression should account for the effect of shear during lipoplex assisted DNA delivery.

3D Printed Microfluidic Mixers—A Comparative Study on Mixing Unit Performances

“…According to simulations, the peak wall shear stress was 20.2 dynes cm −2 (Tesla‐like mixer) and 19.7 dynes cm −2 (HC mixer), respectively. Rawat and Gadgil reported that shear stresses of up to 220 dynes cm −2 lead to slightly lower cell growth, while 2000 dynes cm −2 lead to severe cell damage . Additionally, even shear stress of 2 dynes cm −2 lowered the transfection efficiency, thus shear stress should be kept at an absolute minimum to ensure reliable and efficient transfection in the target application.…”

“…Rawat and Gadgil reported that shear stresses of up to 220 dynes cm −2 lead to slightly lower cell growth, while 2000 dynes cm −2 lead to severe cell damage . Additionally, even shear stress of 2 dynes cm −2 lowered the transfection efficiency, thus shear stress should be kept at an absolute minimum to ensure reliable and efficient transfection in the target application. Therefore, the HC mixer design is most suitable for efficient mixing of CHO‐K1 cells and other biological applications, which require rapid sample mixing at low shear stress.…”

“…The motivation of this work and future application of the 3D printed mixers is the efficient and gentle mixing of CHO‐K1 (Chinese hamster ovary) suspension cells with DNA and transfection reagents for highly efficient and reproducible transient transfection. Passive micromixer designs were chosen as transfection efficiency and viable cell density were reported to decrease with higher shear stress in CHO‐S suspension cells and due to the generally low stress tolerance of CHO cells. To ensure that the channels cannot get blocked by the cells, with an average diameter of 16–24 µm, the mixer designs were scaled accordingly.…”

Micro Versus Macro – The Effect of Environmental Confinement on Cellular Nanoparticle Uptake

“…Shear stress was calculated using the equations from the manufacturer’s technical specifications τ=η×104.7×Φ or τ=η×906.0×Φ for 400 and 100 μm tall channels respectively where τ,η,Φ are shear stress (dyne/cm 2 ), dynamic viscosity (dyn.s/cm 2 ) and flowrate (mL/min) respectively. In the literature, the effect of shear stress on nanoparticle uptake ( Jurney et al, 2017 ), cytotoxicity ( Rawat and Gadgil, 2016 ) and molecular delivery ( Meacham et al, 2018 ) is very well established. However, the amount of shear stress in such investigations is typically on the order of 10–100 dyne/cm 2 , which is many orders of magnitude higher than the shear stress in this work.…”

Improved cultivation of CHO cells in bioreactor with reciprocal mixing

“…Turning or rotary paddles, which enable quick exchange of dissolved air, is also a common suspension culture system. Faster stirring improves the efficiency of mixing, although the shear stress increases, resulting in cell damage (Rawat, 2016). However, if stirring is slowed down, the efficiency of mixing decreases, and uniformity of the culture medium may be lost.…”