Biology subjects

Langer, K.

Publications and source records attributed to Langer, K..

5 recordsLinked to original sources

bioRxiv2022

High-throughput cell spheroid production and assembly analysis by microfluidics and deep learning

3D cell culture models are an important tool in translational research but have been out of reach for high-throughput screening due to complexity, requirement of large cell numbers and inadequate standardization. Here, we present a high-throughput workflow to produce and characterize the formation of miniaturized spheroids using deep learning. We train a convolutional neural network (CNN) for cell ensemble morphology classification, benchmark it against more conventional image analysis, and characterize spheroid assembly determining optimal surfactant concentrations and incubation times for spheroid production for three cell lines with different spheroid formation properties. Notably, this format is compatible with large-scale spheroid production and screening. The presented workflow and CNN offer a template for large scale minispheroid production and analysis and can be extended and re-trained to characterize morphological responses in spheroids to additives, culture conditions and large drug libraries.

bioengineering↗

bioRxiv2021

Analysis of M4 transmembrane domains in NMDA receptor function: a negative allosteric modulation site at the GluN1 M4 is determining the efficiency of neurosteroid modulation

Ionotropic glutamate receptors (iGluRs) are tetrameric ligand-gated ion channels that play a crucial role in excitatory synaptic transmission in the central nervous system. Each subunit contributes with three transmembrane domains (M1, M3, and M4) and a pore loop (M2) forming the channel pore. Recent studies suggest that the architecture of all eukaryotic iGluRs derives from a common prokaryotic ancestral receptor that lacks M4 and consists only of the transmembrane domain segments M1-M3. Although a crucial contribution of M4 to the assembly and trafficking of iGluRs is suspected, the role of this additionally evolved domain in receptor function remains controversial. Here, we investigated how deletions and mutations of M4 in members of the NMDA receptor subfamily, the conventional heteromeric GluN1/GluN2 and glycine-gated GluN1/GluN3 NMDA receptors, affect expression and function in Xenopus oocytes. We show that deletion of M4 in the GluN1, GluN2, or GluN3 subunit, despite retained receptor assembly and cell surface expression, results in nonfunctional membrane receptors. Coexpression of the corresponding M4 domains as an isolated peptide in M4-deleted receptors rescued receptor function of GluN1/GluN2A NMDARs without altering the affinity of glutamate or glycine. Substitution of non-conserved residues and insertion of interhelical disulfide bridges confirmed the proximity of positions M813 and F817 in M4 of GluN1 to residues of the TMs of neighboring subunits. Electrophysiological analyses of agonist-induced receptor function and its modulation by the neurosteroid pregnenolone sulfate (PS) at mutations of the GluN1-M4/GluN2/3-TM interface indicate a crucial role of interdomain interactions in the functional coupling of M4 to the nuclear receptor and the modulatory effect of PS. Our results show that although the M4 domains in NMDA receptors are not important for receptor assembly and surface expression, residues at the subunit interface are substantially involved in M4 recognition to the core receptor and regulation of PS efficacy. Because mutations in the M4 of GluN1 specifically resulted in loss of PS-induced inhibition of NMDA receptor currents, our results point to distinct roles of M4s in NMDA receptor modulation and highlight the importance of evolutionarily newly evolved M4s for selective in vivo modulation of glutamate- and glycine-activated NMDA receptors by steroids. Highlights- The role of the M4 transmembrane domain in the assembly and function of ionotropic glutamate receptors remains controversial - Here we show that deletion of M4 in glutamate-gated GluN1/GluN2A and glycine-gated GluN1/GluN3A receptors results in nonfunctional NMDA receptors with retained surface expression - The functional loss in M4-deleted GluN1/GluN2A receptors is rescued without affecting agonist affinity by a M4 transmembrane domain of the respective subunit expressed as an isolated peptide - Specific interactions in the M4 interfaces with the M1 and M3 domain of the adjacent subunit are required for the recognition of the isolated M4 and the functional rescue - Finally, the M4 domain-interfaces of GluN1 determine the negative modulatory effect of pregnenolone sulfate in glutamate-gated GluN1/GluN2A and glycine-gated GluN1/GluN3A NMDA receptors

neuroscience↗

bioRxiv2019

Doxorubicin-loaded human serum albumin nanoparticles overcome transporter-mediated drug resistance

Resistance to systemic drug therapies is a major reason for the failure of anti-cancer therapies. Here, we tested doxorubicin-loaded human serum albumin (HSA) nanoparticles in the neuroblastoma cell line UKF-NB-3 and its ABCB1-expressing sublines adapted to vincristine (UKF-NB-3rVCR1) and doxorubicin (UKF-NB-3rDOX20). Doxorubicin-loaded nanoparticles displayed increased anti-cancer activity in UKF-NB-3rVCR1 and UKF-NB-3rDOX20 cells relative to doxorubicin solution, but not in UKF-NB-3 cells. UKF-NB-3rVCR1 cells were resensitised by nanoparticle-encapsulated doxorubicin to the level of UKF-NB-3 cells. UKF-NB-3rDOX20 cells displayed a more pronounced resistance phenotype than UKF-NB-3rVCR1 cells and were not re-sensitised by doxorubicin-loaded nanoparticles to the level of parental cells. ABCB1 inhibition using zosuquidar resulted in similar effects like nanoparticle incorporation, indicating that doxorubicin-loaded nanoparticles circumvent ABCB1-mediated drug efflux. The limited re-sensitisation of UKF-NB-3rDOX20 cells to doxorubicin by circumvention of ABCB1-mediated efflux is probably due to the presence of multiple doxorubicin resistance mechanisms. So far, ABCB1 inhibitors have failed in clinical trials, probably because systemic ABCB1 inhibition results in a modified body distribution of its many substrates including drugs, xenobiotics, and other molecules. HSA nanoparticles may provide an alternative, more specific way to overcome transporter-mediated resistance.

pharmacology and toxicology↗

bioRxiv2019

DRAFT - Robotic automation of production and the recovery of cell spheroids

Droplet microfluidics enables high throughput cell processing, analysis and screening by miniaturizing the reaction vessels to nano-or pico-liter water-in oil droplets, but like many other microfluidic formats, droplet microfluidics have not been interfaced with or automated by laboratory robotics. Here we demonstrate automation of droplet microfluidics based on an inexpensive liquid handling robot for the automated production of human scaffold-free cell spheroids, using pipette actuation and interfacing the pipetting tip with a droplet generating microfluidic chip. In this chip we produce highly mono-disperse 290m droplets with diameter CV of 1.7%. By encapsulating cells in these droplets we produce cell spheroids in droplets and recover them to standard formats at a throughput of 85000 spheroids per microfluidic circuit per hour. The viability of the cells in spheroids remains high after recovery only decreased by 4% starting from 96% after 16 hours incubation in nanoliter droplets. Scaffold-free cell spheroids and 3D tissue constructs recapitulate many aspects of functional human tissue more accurately than 2D or single cell cultures, but assembly methods for spheroids, e.g. hanging drop micro-plates, has had limited throughput. The increased throughput and decreased cost of our method enables spheroid production at the scale needed for lead discovery drug screening and approaches the cost where these micro tissues could be used as building blocks for organ scale regenerative medicine.

bioengineering↗

bioRxiv2018

Incorporation of doxorubicin in different polymer nanoparticles and their anti-cancer activity

Nanoparticles are under investigation as carrier systems for anti-cancer drugs. They have been shown to accumulate in cancer tissues through the enhanced permeability and retention (EPR) effect, to reduce toxicity to non-target tissues, and to protect drugs from preliminary inactivation. However, nanoparticle preparations are not commonly compared for their anti-cancer effects at the cellular level. Here, we prepared doxorubicin-loaded nanoparticles based on poly(lactic-co-glycolic acid) (PLGA), polylactic acid (PLA), and PEGylated PLGA (PLGA-PEG) by solvent displacement and emulsion diffusion approaches. The resulting nanoparticles covered a size range between 73 and 246 nm. PLGA-PEG nanoparticle preparation by solvent displacement resulted in the smallest nanoparticles. In PLGA nanoparticles, the drug load could be optimised using solvent displacement at pH7 reaching 53 {micro}g doxorubicin/mg nanoparticle. In addition, these PLGA nanoparticles displayed sustained doxorubicin release kinetics compared to the more burst-like kinetics of the other preparations. In neuroblastoma cells, doxorubicin-loaded PLGA-PEG nanoparticles (presumably due to their small size) and PLGA nanoparticles prepared by solvent displacement at pH7 (presumably due to their high drug load and superior drug release kinetics) exerted the strongest anti-cancer effects. In conclusion, doxorubicin-loaded nanoparticles made by different methods from different materials displayed substantial discrepancies in their anti-cancer activity at the cellular level. Optimised preparation methods resulted in PLGA nanoparticles characterised by increased drug load, controlled drug release, and high anti-cancer efficacy. The design of drug-loaded nanoparticles with optimised anti-cancer activity at the cellular level is an important step in the development of improved nanoparticle preparations for anti-cancer therapy.

pharmacology and toxicology↗