Permeabilization of Adhered Cells Using an Inert Gas Jet

Various cell transfection techniques exist and these can be broken down to three broad categories: viral, chemical and mechanical. This protocol describes a mechanical method to temporally permeabilize adherent cells using an inert gas jet that can facilitate the transfer of normally non-permeable macromolecules into cells. We believe this technique works by imparting shear forces on the plasma membrane of adherent cells, resulting in the temporary formation of micropores. Once these pores are created, the cells are then permeable to genetic material and other biomolecules. The mechanical forces involved do run the risk of permanently damaging or detaching cells from their substrate. There is, therefore, a narrow range of inert gas dynamics where the technique is effective. An inert gas jet has proven efficient at permeabilizing various adherent cell lines including HeLa, HEK293 and human abdominal aortic endothelial cells. This protocol is appropriate for the permeabilization of adherent cells both in vitro and, as we have demonstrated, in vivo, showing it may be used for research and potentially in future clinical applications. It also has the advantage of permeabilizing cells in a spatially restrictive manner, which could prove to be a valuable research tool.     

Cover Image,Volume 116, Number 5, May 2019

Human pluripotent stem cell-derived endothelial cells (hPSC-ECs) present an attractive alternative to primary EC sources for vascular grafting. However, there is a need to mature them towards either an arterial or venous subtype. A vital environmental factor involved in the arteriovenous specification of ECs during early embryonic development is fluid shear stress; therefore, there have been attempts to employ adult arterial shear stress conditions to mature hPSC-ECs. However, hPSC-ECs are naïve to fluid shear stress, and their shear responses are still not well understood. Here, we used a multiplex microfluidic platform to systematically investigate the dose-time shear responses on hPSC-EC morphology and arterial-venous phenotypes over a range of magnitudes coincidental with physiological levels of embryonic and adult vasculatures. The device comprised of six parallel cell culture chambers that were individually linked to flow-setting resistance channels, allowing us to simultaneously apply shear stress ranging from 0.4 to 15 dyne/cm ². We found that hPSC-ECs required up to 40 hr of shear exposure to elicit a stable phenotypic change. Cell alignment was visible at shear stress <1 dyne/cm ², which was independent of shear stress magnitude and duration of exposure. We discovered that the arterial markers NOTCH1 and EphrinB2 exhibited a dose-dependent increase in a similar manner beyond a threshold level of 3.8 dyne/cm ², whereas the venous markers COUP-TFII and EphB4 expression remained relatively constant across different magnitudes. These findings indicated that hPSC-ECs were sensitive to relatively low magnitudes of shear stress, and a critical level of ~4 dyne/cm ² was sufficient to preferentially enhance their maturation into an arterial phenotype for future vascular tissue engineering applications.     

Sugarcane Molasses: A Cheap Carbon Source for Calcite Production in Different Class of Soils using Stimulation of Indigenous Urease-producing Bacteria

Abstract

In this research, we investigated the abilities of three different concentration of sugarcane molasses as a carbon source to stimulate indigenous bacterial growth in different classes of soil, namely poorly graded sand (SP), silty sand (SM), and clayey sand (SC) (according to the Unified classification system). A total of 7, 10, and 15 days after the treatment, direct shear tests were performed on the untreated and treated samples. The calcite content on all direct shear samples was determined to further correlate it with the strength gains in the treated samples. The scanning electron microscopy (SEM) images, EDX analysis, and X-ray diffraction (XRD) patterns were taken before and after treatment for all samples to analyze the microbial-induced calcite precipitation (MICP) process. The SP soil samples showed the highest strength gains and also highest calcite content as compared with other two soil type. The peak cohesion intercept for SP-treated samples increased by 2.7–5.5 times as compared to the untreated samples for molasses concentration of 1–3?g/L, respectively. The treated samples became more dilative with the increase in molasses concentration. The sample with highest molasses concentration showed stiffer behavior in shear than the samples with lower concentration.     

Rac1 mediates laminar shear stress-induced vascular endothelial cell migration

The migration of endothelial cells (ECs) plays an important role in vascular remodeling and regeneration. ECs are constantly subjected to shear stress resulting from blood flow and are able to convert mechanical stimuli into intracellular signals that affect cellular behaviors and functions. The aim of this study is to elucidate the effects of Rac1, which is the member of small G protein family, on EC migration under different laminar shear stress (5.56, 10.02, and 15.27 dyn/cm²). The cell migration distance under laminar shear stress increased significantly than that under the static culture condition. Especially, under relative high shear stress (15.27 dyn/cm²) there was a higher difference at 8 h (P < 0.01) and 2 h (P < 0.05) compared with static controls. RT-PCR results further showed increasing mRNA expression of Rac1 in ECs exposed to laminar shear stress than that exposed to static culture. Using plasmids encoding the wild-type (WT), an activated mutant (Q61L), and a dominant-negative mutant (T17N), plasmids encoding Rac1 were transfected into EA.hy 926 cells. The average net migration distance of Rac1Q61L group increased significantly, while Rac1T17N group decreased significantly in comparison with the static controls. These results indicated that Rac1 mediated shear stress-induced EC migration. Our findings conduce to elucidate the molecular mechanisms of EC migration induced by shear stress, which is expected to understand the pathophysiological basis of wound healing in health and diseases.     

Computational fluid modeling and performance analysis of a bidirectional rotating perfusion culture system

A myriad of bioreactor configurations have been investigated as extracorporeal medical support systems for temporary replacement of vital organ functions. In recent years, studies have demonstrated that the rotating bioreactors have the potential to be utilized as bioartificial liver assist devices (BLADs) owing to their advantage of ease of scalability of cell‐culture volume. However, the fluid movement in the rotating chamber will expose the suspended cells to unwanted flow structures with abnormally high shear conditions that may result in poor cell stability and in turn lower the efficacy of the bioreactor system. In this study, we compared the hydrodynamic performance of our modified rotating bioreactor design with that of an existing rotating bioreactor design. Computational fluid dynamic analysis coupled with experimental results were employed in the optimization process for the development of the modified bioreactor design. Our simulation results showed that the modified bioreactor had lower fluid induced shear stresses and more uniform flow conditions within its rotating chamber than the conventional design. Experimental results revealed that the cells within the modified bioreactor also exhibited better cell‐carrier attachment, higher metabolic activity, and cell viability compared to those in the conventional design. In conclusion, this study was able to provide important insights into the flow physics within the rotating bioreactors, and help enhanced the hydrodynamic performance of an existing rotating bioreactor for BLAD applications. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1002–1012, 2013     

Effects of boron on growing pullets

The effects of dietary boron on bone ash content and on the ultimate shear force, stress, and fracture energy of the tibia, femur, humerus, and radius from white Leghorn pullets were investigated. There was a significant increase in the shear force of the tibia and femur for pullets supplemented with 50 and 100 mg/kg of dietary boron. There was a significant increase in the shear stress of the tibia at 50 and 100 mg/kg of boron, and also an increase in shear fracture energy at 50 and 100 mg/kg boron for the femur. Tibia bone ash content increased significantly at 50, 100, and 200 mg/kg boron with the highest value at 50 mg/kg. Even though there was not a significant increase in body wt at 50 and 100 mg/kg boron, the pullets fed these supplements were consistently heavier than the control group.