Culture surfaces induce hypoxia-regulated genes in human mesenchymal stromal cells.

Culturing human Mesenchymal stromal cells (hMSCs) in vitro in hypoxic conditions resulted in reduced senescence, enhanced pluripotency and altered proliferation rate. It has been known that in vitro hypoxia affects expression of cell surface proteins. However, the impact of culture surfaces on the hypoxia-regulated genes (HRG) have not yet been reported. This study utilized Next-Generation sequencing to analyse the changes in the gene expression levels of HRG for hMSCs cultured on different culture surfaces. The samples, which were cultured on four different synthesized surfaces (treatments) and tissue culture plate (control), resulted in a difference in growth rate. The sequencing results revealed that the transcription of a number of key genes involved in regulating hypoxic functions were significantly altered, including HIF2A, a marker for potency, differentiation, and various cellular functions. Significant alternations in the expression levels of previously reported oxygen-sensitive surface proteins were detected in this study, some of which closely correlate with the expression levels of HIF2A. Our analysis of the hMSCs transcriptome and HRG mapped out a list of genes encoding surface proteins which may directly regulate or be regulated by HIF2A. The findings from this study showed that culture surfaces have an impact on regulating the expression profile of HRG. Therefore, novel culture surfaces may be designed to selectively activate HIF2A and other HRG and pathways under in vitro normoxia. The understanding of the crosstalk between the regulating genes of hypoxia and culture surfaces may be utilized to strengthen desired hypoxic functions.

Electrosprayed genipin cross-linked alginate-chitosan microcarriers for ex vivo expansion of mesenchymal stem cells.

Mesenchymal stem cells (MSCs) are potential therapeutic candidates, owing to their ability to differentiate into several cell types. However, the gap between availability and demand of MSCs requires alternative expansion methods from 2D culture flasks. Microcarriers are a promising approach for MSC expansion due to their high surface area-to-volume ratio. However, current commercial microcarriers do not provide the highest cell yield due to low cell attachment efficiencies and difficulty in cell detachment. This study developed a hydrogel-based microcarrier from genipin cross-linked alginate-chitosan beads. Alginate beads were produced by electrospraying before being coated with chitosan and cross-linked in genipin. The degree of cross-linking was determined through fluorescence reading of the genipin-chitosan conjugates. MSCs cultured on these microcarriers had a 26% higher cell attachment and twice the proliferation rate compared to the commercial microcarrier Cytodex 1. Cells easily detached from the hydrogel beads and did not require extended incubation periods or intense agitation during cell harvest. There was no significant difference in gene expression between the two microcarriers for the positive MSC surface markers CD-90, CD-105, and CD-73 as well as showing either low or no signal for negative MSC surface markers CD-45 and CD-34. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 122-133, 2019.

A narrative review of the success of intramuscular gluteal injections and its impact in psychiatry.

There are 12 billion injections given worldwide every year. For many injections, the intramuscular route is favoured over the subcutaneous route due to the increased vascularity of muscle tissue and the corresponding increase in the bioavailability of drugs when administered intramuscularly. This paper is a review of the variables that affect the success of intramuscular injections and the implications that these success rates have in psychiatry and general medicine. Studies have shown that the success rates of intended intramuscular injections vary between 32 and 52%, with the rest potentially resulting in inadvertent subcutaneous drug deposition. These rates are found to be even lower for certain at-risk populations, such as obese patients and those on antipsychotic medications. The variables associated with an increased risk of injection failure include female sex, obesity, site of injection, and subcutaneous fat depth. New guidelines and methods are needed in order to address this challenge and ensure that patients receive optimum care. Looking forward, the best way to improve the delivery of intramuscular injections worldwide is to develop uniform algorithms or innovative medical devices to confirm or guarantee successful delivery at the bedside.

Aligned electrospun fibers for neural patterning.

OBJECTIVES: To test a 3D approach for neural network formation, alignment, and patterning that is reproducible and sufficiently stable to allow for easy manipulation. RESULTS: A novel cell culture system was designed by engineering a method for the directional growth of neurons. This uses NG108-15 neuroblastoma x glioma hybrid cells cultured on suspended and aligned electrospun fibers. These fiber networks improved cellular directionality, with alignment angle standard deviations significantly lower on fibers than on regular culture surfaces. Morphological studies found nuclear aspect ratios and cell projection lengths to be unchanged, indicating that cells maintained neural morphology while growing on fibers and forming a 3D network. Furthermore, fibronectin-coated fibers enhanced neurite extensions for all investigated time points. Differentiated neurons exhibited significant increases in average neurite lengths 96 h post plating, and formed neurite extensions parallel to suspended fibers, as visualized through scanning electron microscopy. CONCLUSIONS: The developed model has the potential to serve as the basis for advanced 3D studies, providing an original approach to neural network patterning and setting the groundwork for further investigations into functionality.

Engineered method for directional growth of muscle sheets on electrospun fibers.

Research on the neuromuscular junction (NMJ) and its function and development spans over a century. However, researchers are limited in their ability to conduct experimentation on this highly specialized synapse between motor neurons and muscle fibers, as NMJs are not easily accessible outside the body. The aim of this work is to provide a reliable and reproducible muscle sheet model for in vitro NMJ study. A novel culture system was designed by engineering a method for the directional growth of myofiber sheets, using muscle progenitor cells cultured on electrospun fiber networks. Myoblastic C2C12 cells cultured on suspended aligned fibers were found to maintain directionality, with alignment angle standard deviations approximately two-thirds lower on fibers than on regular culture surfaces. Morphological studies found nuclei and cytoskeleton aspect ratios to be elongated by 20 and 150%, respectively. Furthermore, neurons were shown to form innervation patterns parallel to suspended fibers when co-cultured on developed muscle sheets, with alignment angle standard deviations three times lower compared with those on typical surfaces. The effect of agrin on samples was quantified through the slow release of agrin medium, encapsulated in alginate pellets and imbedded within culture chambers. Samples exposed to agrin showed significantly increased percentage of AChR-covered area. The developed model has potential to serve as the basis for synaptogenesis and NMJ studies, providing a novel approach to bio-artificial muscle alignment and setting the groundwork for further investigations in innervation. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 1165-1176, 2018.