High cell density optimization strategies for continuous bioprocesses using perfusion bioreactors

Intensified perfusion bioprocesses enable high cell density cultures with higher volumetric productivity and are a promising alternative to the fed-batch technology most commonly used in current biopharmaceutical production processes. Some of the key challenges when working with extremely high cell density cultures are high oxygen demand, consequent generation of shear stress, and foam by spargers, resulting in technical difficulties to maintain the bioprocesses. Our study demonstrates the optimization of bioreactor parameters and culture conditions enable very high cell densities using perfusion systems for intensified processes. One of the parameter focused in our study is to improve bioreactor performance measuring dissolved oxygen (DO) to determine volumetric mass transfer coefficient (kLa) using static gassing out method in EX-CELL® Advanced HD Perfusion Medium to understand the mass transfer as a function of agitation speed and aeration rate using spargers. We evaluated bioreactor process parameters such as agitator speed, gassing rate, properties of the medium, anti-foam agents, surface active solutes that affect kLa to enable higher cell density suspension cultures while maintaining high viability. Our study showed that aeration rate has larger effect on kLa than agitation rate and gives us tool to predict Kla requirements at specific cell densities in the perfusion bioreactor. With the above mentioned optimized kLa conditions, we made improvements on CHOZN®GS cell line for dynamic perfusion (no bleed) bioprocess and showed how changes in the process can enable the increase of cell density by 3-fold, reaching densities above 250x106vc/mL with 2vvd (CSPR\u3c10pL/cell/d) - while maintaining or increasing viability

Do more with less: Fit-for-purpose tools to speed up upstream process development for continuous biomanufacturing

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A scalable single-use perfusion system: Are single-use bioreactors suitable & scalable?

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Targeting cellular calcium homeostasis to prevent cytokine-mediated beta cell death

AbstractPro-inflammatory cytokines are important mediators of islet inflammation, leading to beta cell death in type 1 diabetes. Although alterations in both endoplasmic reticulum (ER) and cytosolic free calcium levels are known to play a role in cytokine-mediated beta cell death, there are currently no treatments targeting cellular calcium homeostasis to combat type 1 diabetes. Here we show that modulation of cellular calcium homeostasis can mitigate cytokine- and ER stress-mediated beta cell death. The calcium modulating compounds, dantrolene and sitagliptin, both prevent cytokine and ER stress-induced activation of the pro-apoptotic calcium-dependent enzyme, calpain, and partly suppress beta cell death in INS1E cells and human primary islets. These agents are also able to restore cytokine-mediated suppression of functional ER calcium release. In addition, sitagliptin preserves function of the ER calcium pump, sarco-endoplasmic reticulum Ca2+-ATPase (SERCA), and decreases levels of the pro-apoptotic protein thioredoxin-interacting protein (TXNIP). Supporting the role of TXNIP in cytokine-mediated cell death, knock down of TXNIP in INS1-E cells prevents cytokine-mediated beta cell death. Our findings demonstrate that modulation of dynamic cellular calcium homeostasis and TXNIP suppression present viable pharmacologic targets to prevent cytokine-mediated beta cell loss in diabetes.</jats:p

Insulin signalling regulates Pink1 mRNA localization via modulation of AMPK activity to support PINK1 function in neurons

Mitochondrial quality control failure is frequently observed in neurodegenerative diseases. The detection of damaged mitochondria by stabilization of PTEN-induced kinase 1 (PINK1) requires transport of Pink1 messenger RNA (mRNA) by tethering it to the mitochondrial surface. Here, we report that inhibition of AMP-activated protein kinase (AMPK) by activation of the insulin signalling cascade prevents Pink1 mRNA binding to mitochondria. Mechanistically, AMPK phosphorylates the RNA anchor complex subunit SYNJ2BP within its PDZ domain, a phosphorylation site that is necessary for its interaction with the RNA-binding protein SYNJ2. Notably, loss of mitochondrial Pink1 mRNA association upon insulin addition is required for PINK1 protein activation and its function as a ubiquitin kinase in the mitophagy pathway, thus placing PINK1 function under metabolic control. Induction of insulin resistance in vitro by the key genetic Alzheimer risk factor apolipoprotein E4 retains Pink1 mRNA at the mitochondria and prevents proper PINK1 activity, especially in neurites. Our results thus identify a metabolic switch controlling Pink1 mRNA localization and PINK1 activity via insulin and AMPK signalling in neurons and propose a mechanistic connection between insulin resistance and mitochondrial dysfunction

Development of a highly concentrated perfusion medium supplement to decrease media demand leveraging a newly designed 250 mL single use perfusion bioreactor

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A soluble endoplasmic reticulum factor as regenerative therapy for Wolfram syndrome

Endoplasmic reticulum (ER) stress-mediated cell death is an emerging target for human chronic disorders, including neurodegeneration and diabetes. However, there is currently no treatment for preventing ER stress-mediated cell death. Here, we show that mesencephalic astrocyte-derived neurotrophic factor (MANF), a neurotrophic factor secreted from ER stressed cells, prevents ER stress-mediated β cell death and enhances β cell proliferation in cell and mouse models of Wolfram syndrome, a prototype of ER disorders. Our results indicate that molecular pathways regulated by MANF are promising therapeutic targets for regenerative therapy of ER stress-related disorders, including diabetes, retinal degeneration, neurodegeneration, and Wolfram syndrome

Following the TraCS of exoplanets with Pan-Planets: Wendelstein-1b and Wendelstein-2b

Hot Jupiters seem to get rarer with decreasing stellar mass. The goal of the Pan-Planets transit survey was the detection of such planets and a statistical characterization of their frequency. Here, we announce the discovery and validation of two planets found in that survey, Wendelstein-1b and Wendelstein-2b, which are two short-period hot Jupiters that orbit late K host stars. We validated them both by the traditional method of radial velocity measurements with the HIgh Resolution Echelle Spectrometer (HIRES) and the Habitable-zone Planet Finder (HPF) instruments and then by their Transit Color Signature (TraCS). We observed the targets in the wavelength range of $4000 - 24000$ Angstr\"om and performed a simultaneous multiband transit fit and additionally determined their thermal emission via secondary eclipse observations. Wendelstein-1b is a hot Jupiter with a radius of $1.0314_{-0.0061}^{+0.0061}$ $R_J$ and mass of $0.592_{-0.129}^{+0.165}$ $M_J$, orbiting a K7V dwarf star at a period of $2.66$ d, and has an estimated surface temperature of about $1727_{-90}^{+78}$ K. Wendelstein-2b is a hot Jupiter with a radius of $1.1592_{-0.0210}^{+0.0204}$ $R_J$ and a mass of $0.731_{-0.311}^{+0.541}$ $M_J$, orbiting a K6V dwarf star at a period of $1.75$ d, and has an estimated surface temperature of about $1852_{-140}^{+120}$ K. With this, we demonstrate that multiband photometry is an effective way of validating transiting exoplanets, in particular for fainter targets since radial velocity (RV) follow-up becomes more and more costly for those targets.Comment: 14 pages, 12 figures. Accepted for publication in A&

Rpl13a small nucleolar RNAs regulate systemic glucose metabolism

Small nucleolar RNAs (snoRNAs) are non-coding RNAs that form ribonucleoproteins to guide covalent modifications of ribosomal and small nuclear RNAs in the nucleus. Recent studies have also uncovered additional non-canonical roles for snoRNAs. However, the physiological contributions of these small RNAs are largely unknown. Here, we selectively deleted four snoRNAs encoded within the introns of the ribosomal protein L13a (Rpl13a) locus in a mouse model. Loss of Rpl13a snoRNAs altered mitochondrial metabolism and lowered reactive oxygen species tone, leading to increased glucose-stimulated insulin secretion from pancreatic islets and enhanced systemic glucose tolerance. Islets from mice lacking Rpl13a snoRNAs demonstrated blunted oxidative stress responses. Furthermore, these mice were protected against diabetogenic stimuli that cause oxidative stress damage to islets. Our study illuminates a previously unrecognized role for snoRNAs in metabolic regulation

Insights into molecular mechanisms of disease in Neurodegeneration with Brain Iron Accumulation; unifying theories.

Neurodegeneration with brain iron accumulation (NBIA) is a group of disorders characterised by dystonia, parkinsonism and spasticity. Iron accumulates in the basal ganglia and may be accompanied by Lewy bodies, axonal swellings and hyperphosphorylated tau depending on NBIA subtype. Mutations in 10 genes have been associated with NBIA that include Ceruloplasmin (Cp) and Ferritin Light Chain (FTL), both directly involved in iron homeostasis, as well as Pantothenate Kinase 2 (PANK2), Phospholipase A2 group 6 (PLA2G6), Fatty acid hydroxylase 2 (FA2H), Coenzyme A synthase (COASY), C19orf12, WDR45 and DCAF17 (C2orf37). These genes are involved in seemingly unrelated cellular pathways, such as lipid metabolism, Coenzyme A synthesis and autophagy. A greater understanding of the cellular pathways that link these genes and the disease mechanisms leading to iron dyshomeostasis is needed. Additionally, the major overlap seen between NBIA and more common neurodegenerative diseases may highlight conserved disease processes. In this review, we will discuss clinical and pathological findings for each NBIA-related gene, discuss proposed disease mechanisms such as mitochondrial health, oxidative damage, autophagy/mitophagy and iron homeostasis and speculate potential overlap between NBIA subtypes