A Fast and Accessible Methodology for Micro-Patterning Cells on Standard Culture Substrates Using Parafilm™ Inserts

Micropatterning techniques provide direct control over the spatial organization of cells at the sub-mm scale. Regulation of these spatial parameters is important for controlling cell fate and cell function. While micropatterning has proved a powerful technique for understanding the impact of cell organization on cell behaviour, current methods for micropatterning cells require complex, specialized equipment that is not readily accessible in most biological and bioengineering laboratories. In addition, currently available methods require significant protocol optimization to ensure reliable and reproducible patterning. The inaccessibility of current methods has severely limited the widespread use of micropatterning as a tool in both biology and tissue engineering laboratories. Here we present a simple, cheap, and fast method to micropattern mammalian cells into stripes and circular patterns using Parafilm™, a common material found in most biology and bioengineering laboratories. Our method does not require any specialized equipment and does not require significant method optimization to ensure reproducible patterning. Although our method is limited to simple patterns, these geometries are sufficient for addressing a wide range of biological problems. Specifically, we demonstrate i) that using our Parafilm™ insert method we can pattern and co-pattern ARPE-19 and MDCK epithelial cells into circular and stripe micropatterns in tissue culture polystyrene (TCPS) wells and on glass slides, ii) that we can contain cells in the desired patterns for more than one month and iii) that upon removal of the Parafilm™ insert we can release the cells from the containment pattern and allow cell migration outward from the original pattern. We also demonstrate that we can exploit this confinement release feature to conduct an epithelial cell wound healing assay. This novel micropatterning method provides a reliable and accessible tool with the flexibility to address a wide range of biological and engineering problems that require control over the spatial and temporal organization of cells

Selection of aptamers for a protein target in cell lysate and their application to protein purification

Functional genomics requires structural and functional studies of a large number of proteins. While the production of proteins through over-expression in cultured cells is a relatively routine procedure, the subsequent protein purification from the cell lysate often represents a significant challenge. The most direct way of protein purification from a cell lysate is affinity purification using an affinity probe to the target protein. It is extremely difficult to develop antibodies, classical affinity probes, for a protein in the cell lysate; their development requires a pure protein. Thus, isolating the protein from the cell lysate requires antibodies, while developing antibodies requires a pure protein. Here we resolve this loop problem. We introduce AptaPIC, Aptamer-facilitated Protein Isolation from Cells, a technology that integrates (i) the development of aptamers for a protein in cell lysate and (ii) the utilization of the developed aptamers for protein isolation from the cell lysate. Using MutS protein as a target, we demonstrate that this technology is applicable to the target protein being at an expression level as low as 0.8% of the total protein in the lysate. AptaPIC has the potential to considerably speed up the purification of proteins and, thus, accelerate their structural and functional studies

Monkeypox: a systematic review of epidemiology, pathogenesis, manifestations, and outcomes

Introduction. Since May 2022, an unusually large number of new monkeypox infections-a previously rare viral zoonotic disease, mainly reported from central and western Africa has been reported globally, and the World Health Organization (WHO) declared a global health emergency in July 2022. We aimed to systematically review the monkeypox virus epidemiology, pathogenesis, transmission, presentations, and outcomes. Materials and methods. Our aim is to systematically review the epidemiology, pathogenesis, manifestations, and outcomes of Monkeypox disease. We searched the keywords in the online databases of PubMed, Embase, Scopus, and Web of Science and investigated all English articles until December 2022. In order to ascertain the findings, this study adheres to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) checklist. In order to optimize the quality, this review study benefits from the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) checklist. To minimize any probable bias risk, we utilized the Newcastle-Ottawa Scale (NOS) risk assessment tool. Results. The most prevalent symptoms were rash and fever. The infection was accompanied by different complications such as, but not limited to, encephalitis (mainly in children), septicemia, bacterial cellulitis, retropharyngeal and parapharyngeal abscesses, etc. A wide range of hospitalization from 3.7% to 100% has been reported. The mortality rate ranged from 0% to 23%, which mainly occurred in infants and children. High mortality of the monkeypox rate was reported among pregnant women. The mortality rate of monkeypox is lower among women and those who received the smallpox vaccine compared to men and those who did not receive the vaccine. A wide range of the overall second-rate attack was reported, which is more pronounced in unvaccinated patients. Conclusion. In our systematic review of 35 studies on monkeypox, we cast light on the existing evidence on its epidemiology, pathogenesis, manifestation, and outcomes. Further studies are needed to elucidate the natural history of the disease in various patients’ population, as well as detailing the monkeypox attack rate

Engineering Tissue Patterning: Rules Governing Gene Expression Patterning and Compartment Boundary Formation in vitro

Engineered bioartificial tissues represent one possible solution for overcoming the devastating shortage of organs and tissues, which causes hundreds of Canadian deaths every year. Despite considerable efforts however, the promise of bioengineering complex solid organs, such as liver and kidney, remains unfulfilled partly due to the challenges of organizing multiple cell components comprising such tissues ex vivo. One promising avenue of designing biomimetic tissues with improved organization is recapitulating certain aspects of developmental biology. In an embryo, cells within a developing tissue are commonly organized in a process of tissue patterning. During tissue patterning, expression of certain key genes is spatially patterned in initially naïve cells, leading to the formation of distinct phenotypic cell domains. Following this patterning process, separation of cells from distinct phenotypic domains is commonly ensured by implementation of compartment boundaries. To successfully recapitulate tissue patterning ex vivo therefore, it is important to study the rules governing gene expression patterning and boundary formation in vitro. In this work the question of how tissue pattering can be engineered in vitro is explored. This thesis describes methodology for patterning gene expression in vitro, outlines design principles governing generation of gene expression patterns with sharp interfaces, and explores the rules governing compartment boundary formation in vitro. The work presented here suggests that simply spatially controlling cell organization at the stage of developing an engineered tissue is not sufficient. Instead, to obtain multicomponent tissues with organizational stability it is necessary to incorporate long-term instructive cues into the design of bioartificial tissues.Ph.D

A simple and rapid method for generating patterned co-cultures with stable interfaces

In native tissues, different cell types are organized into defined structures and architectures that are critical for correct tissue function. In vitro cellular patterning methods enable control over the spatial organization of cells, permitting, to some extent, the reproduction of native tissue structures and the generation of a more "in vivo-like" culture platform. While this is advantageous for applications such as drug screening, existing patterning methods are time-consuming, labor-intensive, and low-throughput. Here, we describe a novel medium-throughput patterning strategy for generating spatially controlled co-cultures of two cell types based on differential deposition of BSA solution in a tilted plate. Our method allows generation of homotypic and heterotypic co-cultures that are stable for at least seven days in culture. The reproducibility and consistency of this patterning technique, together with its low cost and ease of use, make it a promising cell culture platform for medium- to high-throughput screening using high-content imaging.We acknowledge R. Sodi and SI Ontario for technical assistance. This work was funded by a Natural Science and Engineering Research Council of Canada (NSERC) Discovery grant and a Connaught Early Researcher Award to A.M. and an NSERC graduate scholarship and CIHR Training Program in Regenerative Medicine Fellowship to S.J. The authors have no conflict of interests to declare. S.J. and K.L. designed the project, conducted experiments, analyzed the data and wrote the manuscript, A.M. designed the project, analyzed the data, and wrote the manuscript

A simple and rapid method for generating patterned co-cultures with stable interfaces

In native tissues, different cell types are organized into defined structures and architectures that are critical for correct tissue function. In vitro cellular patterning methods enable control over the spatial organization of cells, permitting, to some extent, the reproduction of native tissue structures and the generation of a more “in vivo—like” culture platform. While this is advantageous for applications such as drug screening, existing patterning methods are time-consuming, labor-intensive, and low-throughput. Here, we describe a novel medium-throughput patterning strategy for generating spatially controlled co-cultures of two cell types based on differential deposition of BSA solution in a tilted plate. Our method allows generation of homotypic and heterotypic co-cultures that are stable for at least seven days in culture. The reproducibility and consistency of this patterning technique, together with its low cost and ease of use, make it a promising cell culture platform for medium- to high—throughput screening using high-content imaging