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

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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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&

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

The Use of Co-Culturing in Solid Substrate Cultivation and Possible Solutions to Scientific Challenges

This perspective systematically summarizes the use of solid substrate co‐cultures in agriculture, food, plant, and industrial biotechnology applications. The summarization is organized by organism, i.e. fungus, bacteria, yeast and then co‐cultivation of either two or three organisms. Generally, in solid substrate co‐culture, the organisms synergistically penetrate and degrade the solid substrate, thereby increasing product yield and productivity over a monoculture. Efforts to increase co‐culture performance include optimizing process parameters (pH, temperature, moisture, and oxygen demand) and defining the acceptable types of substrate. Scientific challenges exist in understanding the interactions between microbial stains, such as viability, suite of products, and bio‐transformations. The perspective details possible solutions to these challenges and highlights future research directions for co‐cultures using either solid or liquid fermentation

Pancreatic stone protein/regenerating protein is a potential biomarker for endoplasmic reticulum stress in beta cells

Endoplasmic reticulum (ER) stress in beta cells is an important pathogenic component of both type 1 and type 2 diabetes mellitus, as well as genetic forms of diabetes, especially Wolfram syndrome. However, there are currently no convenient ways to assess ER stress in beta cells, raising the need for circulating ER stress markers indicative of beta cell health. Here we show that pancreatic stone protein/regenerating protein (PSP/reg) is a potential biomarker for ER stressed beta cells. PSP/reg levels are elevated in cell culture and mouse models of Wolfram syndrome, a prototype of ER stress-induced diabetes. Moreover, PSP/reg expression is induced by the canonical chemical inducers of ER stress, tunicamycin and thapsigargin. Circulating PSP/reg levels are also increased in some patients with Wolfram syndrome. Our results therefore reveal PSP/reg as a potential biomarker for beta cells under chronic ER stress, as is the case in Wolfram syndrome