Mosaic Patterned Surfaces toward Generating Hardly‐Volatile Capsular Droplet Arrays for High‐Precision Droplet‐Based Storage and Detection

Mosaic Patterned SurfacesIn article number 2206274, Yanjun Hu, Lin Li, and co‐workers report a mosaic patterned surface‐based chip that acquires mutually independent and hardly‐volatile capsular droplet arrays. The concept shows high compatibility and practicability, paving the way for the new microfluidic chips used in COVID‐19 diagnosis and other high‐precision detection.

Mosaic Patterned Surfaces toward Generating Hardly-Volatile Capsular Droplet Arrays for High-Precision Droplet-Based Storage and Detection.

Precise detection involving droplets based on functional surfaces is promising for the parallelization and miniaturization of platforms and is significant in epidemic investigation, analyte recognition, environmental simulation, combinatorial chemistry, etc. However, a challenging and considerable task is obtaining mutually independent droplet arrays without cross-contamination and simultaneously avoiding droplet evaporation-caused quick reagent loss, inaccuracy, and failure. Herein, a strategy to generate mutually independent and hardly-volatile capsular droplet arrays using innovative mosaic patterned surfaces is developed. The evaporation suppression of the capsular droplet arrays is 1712 times higher than the naked droplet. The high evaporation suppression of the capsular droplet arrays on the surfaces is attributed to synergistic blocking of the upper oil and bottom mosaic gasproof layer. The scale-up of the capsular droplet arrays, the flexibility in shape, size, component (including aqueous, colloidal, acid, and alkali solutions), liquid volume, and the high-precision hazardous substance testing proves the concept's high compatibility and practicability. The mutually independent capsular droplet arrays with amazingly high evaporation suppression are essential for the new generation of high-performance open-surface microfluidic chips used in COVID-19 diagnosis and investigation, primary screening, in vitro enzyme reactions, environmental monitoring, nanomaterial synthesis, etc.

Towards integrated production of an influenza A vaccine candidate with MDCK suspension cells.

Seasonal influenza epidemics occur both in northern and southern hemispheres every year. Despite the differences in influenza virus surface antigens and virulence of seasonal subtypes, manufacturers are well-adapted to respond to this periodical vaccine demand. Due to decades of influenza virus research, the development of new influenza vaccines is relatively straight forward. In similarity with the ongoing coronavirus disease 2019 pandemic, vaccine manufacturing is a major bottleneck for a rapid supply of the billions of doses required worldwide. In particular, egg-based vaccine production would be difficult to schedule and shortages of other egg-based vaccines with high demands also have to be anticipated. Cell culture-based production systems enable the manufacturing of large amounts of vaccines within a short time frame and expand significantly our options to respond to pandemics and emerging viral diseases. In this study, we present an integrated process for the production of inactivated influenza A virus vaccines based on a Madin-Darby Canine Kidney (MDCK) suspension cell line cultivated in a chemically defined medium. Very high titers of 3.6 log10 (HAU/100 µl) were achieved using fast-growing MDCK cells at concentrations up to 9.5 × 106 cells/ml infected with influenza A/PR/8/34 H1N1 virus in 1 L stirred tank bioreactors. A combination of membrane-based steric-exclusion chromatography followed by pseudo-affinity chromatography with a sulfated cellulose membrane adsorber enabled full recovery for the virus capture step and up to 80% recovery for the virus polishing step. Purified virus particles showed a homogenous size distribution with a mean diameter of 80 nm. Based on a monovalent dose of 15 µg hemagglutinin (single-radial immunodiffusion assay), the level of total protein and host cell DNA was 58 µg and 10 ng, respectively. Furthermore, all process steps can be fully scaled up to industrial quantities for commercial manufacturing of either seasonal or pandemic influenza virus vaccines. Fast production of up to 300 vaccine doses per liter within 4-5 days makes this process competitive not only to other cell-based processes but to egg-based processes as well.

High cell density perfusion process for high yield of influenza A virus production using MDCK suspension cells.

Similar to the recent COVID-19 pandemic, influenza A virus poses a constant threat to the global community. For the treatment of flu disease, both antivirals and vaccines are available with vaccines the most effective and safest approach. In order to overcome limitations in egg-based vaccine manufacturing, cell culture-based processes have been established. While this production method avoids egg-associated risks in face of pandemics, process intensification using animal suspension cells in high cell density perfusion cultures should allow to further increase manufacturing capacities worldwide. In this work, we demonstrate the development of a perfusion process using Madin-Darby canine kidney (MDCK) suspension cells for influenza A (H1N1) virus production from scale-down shake flask cultivations to laboratory scale stirred tank bioreactors. Shake flask cultivations using semi-perfusion mode enabled high-yield virus harvests (4.25 log10(HAU/100 µL)) from MDCK cells grown up to 41 × 106 cells/mL. Scale-up to bioreactors with an alternating tangential flow (ATF) perfusion system required optimization of pH control and implementation of a temperature shift during the infection phase. Use of a capacitance probe for on-line perfusion control allowed to minimize medium consumption. This contributed to a better process control and a more economical performance while maintaining a maximum virus titer of 4.37 log10(HAU/100 µL) and an infectious virus titer of 1.83 × 1010 virions/mL. Overall, this study clearly demonstrates recent advances in cell culture-based perfusion processes for next-generation high-yield influenza vaccine manufacturing for pandemic preparedness. KEY POINTS: • First MDCK suspension cell-based perfusion process for IAV produciton was established. • "Cell density effect" was overcome and process was intensified by reduction of medium use and automated process control. • The process achieved cell density over 40 × 106 cells/mL and virus yield over 4.37 log10(HAU/100 µL).