Intracellular delivery of functional macromolecules, such as DNA and RNA, across the cell membrane and into the cytosol, is a critical process in both biology and medicine. Herein, we develop and use microfluidic chips containing post arrays to induce microfluidic vortex shedding , or μVS , for cell membrane poration that permits delivery of mRNA into primary human T lymphocytes. We demonstrate transfection with μVS by delivery of a 996-nucleotide mRNA construct encoding enhanced green fluorescent protein (EGFP) and assessed transfection efficiencies by quantifying levels of EGFP protein expression. We achieved high transfection efficiency (63.6 ± 3.44% EGFP + viable cells) with high cell viability (77.3 ± 0.58%) and recovery (88.7 ± 3.21%) in CD3 + T cells 19 hrs after μVS processing. Importantly, we show that processing cells via μVS does not negatively affect cell growth rates or alter cell states. We also demonstrate processing speeds of greater than 2.0 × 10 6 cells s −1 at volumes ranging from 0.1 to 1.5 milliliters. Altogether, these results highlight the use of μVS as a rapid and gentle delivery method with promising potential to engineer primary human cells for research and clinical applications.
Heart disorders are a major health concern worldwide responsible for millions of deaths every year. Among the many disorders of the heart, myocardial infarction, which can lead to the development of congestive heart failure, arrhythmias, or even death, has the most severe social and economic ramifications. Lack of sufficient available donor hearts for heart transplantation, the only currently viable treatment for heart failure other than medical management options (ACE inhibition, beta blockade, use of AICDs, etc.) that improve the survival of patients with heart failure emphasises the need for alternative therapies. One promising alternative replaces cardiac muscle damaged by myocardial infarction with new contractile cardiomyocytes and vessels obtained through stem cell-based regeneration. We report on the state of the art of recovery of cardiac functions by using stem cell engineering. Current research focuses on (a) inducing stem cells into becoming cardiac cells before or after injection into a host, (b) growing replacement heart tissue in vitro, and (c) stimulating the proliferation of the post-mitotic cardiomyocytes in situ. The most promising treatment option for patients is the engineering of new heart tissue that can be implanted into damaged areas. Engineering of cardiac tissue currently employs the use of co-culture of stem cells with scaffold microenvironments engineered to improve tissue survival and enhance differentiation. Growth of heart tissue in vitro using scaffolds, soluble collagen, and cell sheets has unique advantages. To compensate for the loss of ventricular mass and contractility of the injured cardiomyocytes, different stem cell populations have been extensively studied as potential sources of new cells to ameliorate the injured myocardium and eventually restore cardiac function. Unresolved issues including insufficient cell generation survival, growth, and differentiation have led to mixed results in preclinical and clinical studies. Addressing these limitations should ensure the successful production of replacement heart tissue to benefit cardiac patients.
A critical event for the ability of cells to tolerate DNA damage and replication stress is activation of the ATR kinase. ATR activation is dependent on the BRCT (BRCA1 C terminus) repeatcontaining protein TopBP1. Previous work has shown that recruitment of TopBP1 to sites of DNA damage and stalled replication forks is necessary for downstream events in ATR activation; however, the mechanism for this recruitment was not known. Here, we use protein binding assays and functional studies in Xenopus egg extracts to show that TopBP1 makes a direct interaction, via its BRCT2 domain, with RPA-coated singlestranded DNA. We identify a point mutant that abrogates this interaction and show that this mutant fails to accumulate at sites of DNA damage and that the mutant cannot activate ATR. These data thus supply a mechanism for how the critical ATR activator, TopBP1, senses DNA damage and stalled replication forks to initiate assembly of checkpoint signaling complexes.
Shewanella oneidensis MR-1 employs various methods of Extracellular Electron Transport (EET) to insoluble terminal electron acceptors, including graphite felt anodes of BioElectrochemical Systems (BESs). Fibers in the felt form a three dimensional meshwork within which microbes can form biofilms, as well as occupy the interstitial spaces as planktonic cells. Our results indicate that these interstices generated by the meshwork create a novel microenvironment where planktonic cells grow to higher density under certain conditions. When incubated anaerobically with 18 mM lactate and 30 mM fumarate, planktonic cell counts within the electrodes are ∼10-fold higher than bulk planktonic cell counts; a phenomenon we termed the "interstitial felt effect". Upon lowering both lactate and fumarate concentrations 10-fold, while bulk planktonic cell counts are stable, the interstitial felt effect disappears. This effect reappears when lactate and fumarate concentrations are lowered another 10-fold. Cyclic voltammetry experiments did not reveal any modification of the graphite fibers within the electrodes. A mutant strain lacking the primary flavin transporter gene, bfe, also expresses the interstitial felt effect. The interstitial planktonic microenvironment of electrically inert polyurethane sponge did not demonstrate this phenomenon. This study provides new insights into interactions of microbes with electrode materials that may help improve overall BES performance. Shewanella oneidensis MR-1 is a Gram-negative facultative gamma-proteobacterium with remarkable respiratory versatility. It can respire several soluble terminal electron acceptors including oxygen, fumarate, dimethylsulfoxide (DMSO), and, insoluble terminal electron acceptors such as iron and manganese oxides, which can be attributed to some of the 41 c-cytochromes encoded by the genome. 1,2The ability of bacteria to transfer electrons to insoluble substrates is accomplished through a process called Extracellular Electron Transport (EET).3,4 S. oneidensis MR-1 has been proposed to use direct and indirect mechanisms for EET: direct contact via outer membrane decaheme c cytochromes, 5-7 and via nanowires -periplasmic and outer membrane extensions to which decaheme c cytochrome complexes are localized; 8-10 and indirectly via secreted flavin molecules. 11,12 This EET capability of S. oneidensis MR-1 places it in the group of electrochemically active bacteria (EAB). Other examples from this group include delta-proteobacteria such as Geobacter metallireducens, phototrophic bacteria like Rhodopseudomonas palustris, and even a yeast, Pichia anomala.13 EAB can serve as redox catalysts in bioelectrochemical systems (BESs), which typically consist of an anodic and a cathodic chamber separated by a proton permeable membrane. Various types of BESs can be constructed, depending on the process and desired outcome.14 In microbial fuel cell (MFC) type BESs, EAB in the anodic chamber catalyze an oxidation reaction, for example oxidation of a specific carbohydrate, generating ele...
Recent studies have shown that treatments involving injection of stem cells into animals with damaged cardiac tissue result in improved cardiac functionality. Clinical trials have reported conflicting results concerning the recellularization of post-infarct collagen scars. No clear mechanism has so far emerged to fully explain how injected stem cells, specifically the commonly used mesenchymal stem cells (MSC) and endothelial precursor cells (EPC), help heal a damaged heart. Clearly, these injected stem cells must survive and thrive in the hypoxic environment that results after injury for any significant repair to occur. Here we discuss how ischemic preconditioning may lead to increased tolerance of stem cells to these harsh conditions and increase their survival and clinical potential after injection. As injected cells must reach the site in numbers large enough for repair to be functionally significant, homing mechanisms involved in stem cell migration are also discussed. We review the mechanisms of action stem cells may employ once they arrive at their target destination. These possible mechanisms include that the injected stem cells (1) secrete growth factors, (2) differentiate into cardiomyocytes to recellularize damaged tissue and strengthen the post-infarct scar, (3) transdifferentiate the host cells into cardiomyocytes, and (4) induce neovascularization. Finally, we discuss that tissue engineering may provide a standardized platform technology to produce clinically applicable stem cell products with these desired mechanistic capacities.
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