Enhancing cell and gene therapy manufacture through the application of advanced fluorescent optical sensors

Cell and gene therapies (CGTs) are examples of future therapeutics that can be used to cure or alleviate the symptoms of disease, by repairing damaged tissue or reprogramming defective genetic information. However, despite the recent advancements in clinical trial outcomes, the path to wide-scale adoption of CGTs remains challenging, such that the emergence of a “blockbuster” therapy has so far proved elusive. Manufacturing solutions for these therapies require the application of scalable and replicable cell manufacturing techniques, which differ markedly from the existing pharmaceutical incumbent. Attempts to adopt this pharmaceutical model for CGT manufacture have largely proved unsuccessful. The most significant challenges facing CGT manufacturing are process analytical testing and quality control. These procedures would greatly benefit from improved sensory technologies that allow direct measurement of critical quality attributes, such as pH, oxygen, lactate and glucose. In turn, this would make manufacturing more robust, replicable and standardized. In this review, the present-day state and prospects of CGT manufacturing are discussed. In particular, the authors highlight the role of fluorescent optical sensors, focusing on their strengths and weaknesses, for CGT manufacture. The review concludes by discussing how the integration of CGT manufacture and fluorescent optical sensors could augment future bioprocessing approaches

Improving effect of metal and oxide nanoparticles encapsulated in porous silica on fermentative biohydrogen production by Clostridium butyricum.

peer reviewedaudience: researcher, professional, student, popularizationThis paper investigated the enhancement effect of nanometre-sized metallic (Pd, Ag and Cu) or metallic oxide (Fe(x)O(y)) nanoparticles on fermentative hydrogen production from glucose by a Clostridium butyricum strain. These nanoparticles (NP) of about 2-3nm were encapsulated in porous silica (SiO(2)) and were added at very low concentration (10(-6)molL(-1)) in batch hydrogen production test. The cultures containing iron oxide NP produced 38% more hydrogen with a higher maximum H(2) production rate (HPR) of 58% than those without NP or with silica particles only. The iron oxide NP were used in a 2.5L sequencing-batch reactor and showed no significant effect on the yields (established at 2.2mol(hydrogen)mol(glucose)(-1)) but an improvement of the HPR (+113%, reaching a maximum HPR of 86mL(hydrogen)L(-1)h(-1)). These results suggest an improvement of the electron transfers trough some combinations between enzymatic activity and inorganic materials.Etude de la production d'hydrogène par les bactéries anaérobies chimiotrophes (dark-fermentation

Intracellular processing of silica-coated superparamagnetic iron nanoparticles in human mesenchymal stem cells

Silica-coated superparamagnetic iron nanoparticles (SiMAGs) are an exciting biomedical technology capable of targeted delivery of cell-based therapeutics and disease diagnosis. However, in order to realise their full clinical potential, their intracellular fate must be determined. The analytical techniques of super-resolution fluorescence microscopy, particle counting flow cytometry and pH-sensitive nanosensors were applied to elucidate mechanisms of intracellular SiMAG processing in human mesenchymal stem cell (hMSCs). Super-resolution microscopy showed SiMAG fluorescently-tagged nanoparticles are endocytosed and co-localised within lysosomes. When exposed to simulated lysosomal conditions SiMAGs were solubilised and exhibited diminishing fluorescence emission over 7 days. The in vitro intracellular metabolism of SiMAGs was monitored in hMSCs using flow cytometry and co-localised pH-sensitive nanosensors. A decrease in SiMAG fluorescence emission, which corresponded to a decrease in lysosomal pH was observed, mirroring ex vivo observations, suggesting SiMAG lysosomal exposure degrades fluorescent silica-coatings and iron cores. These findings indicate although there is a significant decrease in intracellular SiMAG loading, sufficient particles remain internalised (>50%) to render SiMAG treated cells amenable to long-term magnetic cell manipulation. Our analytical approach provides important insights into the understanding of the intracellular fate of SiMAG processing, which could be readily applied to other particle therapeutics, to advance their clinical translation

Mapping the Pharyngeal and Intestinal pH of <i>Caenorhabditis elegans</i> and Real-Time Luminal pH Oscillations Using Extended Dynamic Range pH-Sensitive Nanosensors

Extended dynamic range pH-sensitive ratiometric nanosensors, capable of accurately mapping the full physiological pH range, have been developed and used to characterize the pH of the pharyngeal and intestinal lumen of <i>Caenorhabditis elegans</i> in real-time. Nanosensors, 40 nm in diameter, were prepared by conjugating pH-sensitive fluorophores, carboxyfluorescein (FAM) and Oregon Green (OG) in a 1:1 ratio, and a reference fluorophore, 5-(and-6)-carboxytetramethylrhodamine (TAMRA) to an inert polyacrylamide matrix. Accurate ratiometric pH measurements were calculated through determination of the fluorescence ratio between the pH-sensitive and reference fluorophores. Nanosensors were calibrated with an automated image analysis system and validated to demonstrate a pH measurement resolution of ±0.17 pH units. The motility of <i>C. elegans</i> populations, as an indicator for viability, showed nematodes treated with nanosensors, for concentrations ranging from 50.00 to 3.13 mg/mL, were not statistically different to nematodes not challenged with nanosensors up to a period of 4 days (<i>p</i> < 0.05). The nanosensors were also found to remain in the <i>C. elegans</i> lumen >24 h after nanosensor challenge was removed. The pH of viable <i>C. elegans</i> lumen was found to range from 5.96 ± 0.31 in the anterior pharynx to 3.59 ± 0.09 in the posterior intestine. The pharyngeal pumping rate, which dictates the transfer of ingested material from the pharynx to the intestine, was found to be temperature dependent. Imaging <i>C. elegans</i> at 4 °C reduced the pharyngeal pumping rate to 7 contractions/min and enabled the reconstruction of rhythmic pH oscillations in the intestinal lumen in real-time with fluorescence microscopy

Three-dimensional principal component scores plot for PC 1, 2 and 3 for the ToF-SIMS spectra (no. m/z peaks = 822) of partially exsheathed L₃ <i>N</i>. <i>americanus</i> in both negative (no. m/z peaks = 429) and positive polarity (no. m/z peaks = 393).

<p>PCA analysis was conducted on 24 different regions of interest (cuticle = 12, sheath = 12).</p