Biology subjects

Koltay, P.

Publications and source records attributed to Koltay, P..

3 recordsLinked to original sources

bioRxiv2022

A drop-on-demand bioprinting approach to spatially arrange multiple cell types and monitor their cell-cell interactions towards vascularization based on endothelial cells and mesenchymal stem cells

Spheroids, organoids, or highly-dense cell-laden droplets are often used as building blocks for bioprinting, but so far little is known about the spatio-temporal cellular interactions post printing. We present a drop-on-demand approach to study the biological interactions of such building blocks in micrometer dimensions. Droplets (containing approximately 700 cells in 10 nl) of multiple cell types are patterned in a 3D hydrogel matrix with a precision of less than 70 m. It is applied to investigate interactions of cell types relevant for vascularization approaches. We show that a gap of 200 m between droplets containing endothelial cells (HUVECs) and adipose-derived mesenchymal stem cells (ASCs) leads to decreased sprouting of HUVECs towards ASCs and increased growth from ASCs towards HUVECs. For mixed aggregates containing both cell types, cellular interconnections of ASCs with up to approximately 0.8 millimeter length and inhibition of HUVEC sprouting are observed. When ASCs are differentiated into smooth muscle cells (SMCs), HUVECs display decreased sprouting towards SMCs in separate aggregates, whereas no cellular interconnections or inhibition of HUVEC sprouting are detected for mixed aggregates. These findings demonstrate that this approach acts as a new tool to investigate cell-cell interactions of different cell types in 3D bioprinted constructs.

bioengineering↗

bioRxiv2022

Integrated workflow of down-scaled Smart-seq2 and absolute quantitative, single-cell digital PCR validates lack of representability of low-abundant transcripts

Single-cell RNA sequencing (scRNA-seq) can unmask transcriptional heterogeneity facilitating the detection of rare subpopulations at unprecedented resolution. In response to challenges related to coverage and quantity of transcriptome analysis, the lack of unbiased and absolutely quantitative validation methods hampers further improvements. Digital PCR (dPCR) represents such a method as we could show that the inherent partitioning enhances molecular detections by increasing effective mRNA concentrations. We developed a scRT-ddPCR method and validated it using two breast cancer cell lines, MCF7 and BT-474, and bulk methods. ErbB2, a low-abundant transcript in MCF7 cells, suffers from dropouts in scRNA-seq and thus calculated fold changes are biased. Using our scRT-ddPCR, we could improve the detection of ErbB2 and based on the absolute counts obtained we could validate the scRNA-seq fold change. We think this workflow is a valuable addition to the single-cell transcriptomic research toolbox and could even become a new standard in fold change validation because of its reliability, ease of use and increased sensitivity.

molecular biology↗

bioRxiv2020

Characterization of CRISPR/Cas9 RANKL knockout mesenchymal stem cell clones based on single-cell printing technology and emulsion coupling assay as a low-cellularity workflow for single-cell cloning

The homogeneity of the genetically modified single-cells is a necessity for many applications such as cell line development, gene therapy, and tissue engineering and in particular for regenerative medical applications. The lack of tools to effectively isolate and characterize CRISPR/Cas9 engineered cells is considered as a significant bottleneck in these applications. Especially the incompatibility of protein detection technologies to confirm protein expression changes without a preconditional large-scale clonal expansion, creates a gridlock in many applications. To ameliorate the characterization of engineered cells, we propose an improved workflow, including single-cell printing/isolation technology based on fluorescent properties with high yield, a genomic edit screen (surveyor assay), mRNA rtPCR assessing altered gene expression and a versatile protein detection tool called emulsion-coupling to deliver a high-content, unified single-cell workflow. The workflow was exemplified by engineering and functionally validating RANKL knockout immortalized mesenchymal stem cells showing altered bone formation capacity of these cells. The resulting workflow is economical, without the requirement of large-scale clonal expansions of the cells with overall cloning efficiency above 30% of CRISPR/Cas9 edited cells. Nevertheless, as the single-cell clones are comprehensively characterized at an early, highly parallel phase of the development of cells including DNA, RNA, and protein levels, the workflow delivers a higher number of successfully edited cells for further characterization, lowering the chance of late failures in the development process. Author summaryI completed my undergraduate degree in biochemistry at the University of Ulm and finished my master's degree in pharmaceutical biotechnology at the University of Ulm and University of applied science of Biberach with a focus on biotechnology, toxicology and molecular biology. For my master thesis, I went to the University of Freiburg to the department of microsystems engineering, where I developed a novel workflow for cell line development. I stayed at the institute for my doctorate, but changed my scientific focus to the development of the emulsion coupling technology, which is a powerful tool for the quantitative and highly parallel measurement of protein and protein interactions. I am generally interested in being involved in the development of innovative molecular biological methods that can be used to gain new insights about biological issues. I am particularly curious to unravel the complex and often poorly understood protein interaction pathways that are the cornerstone of understanding cellular functionality and are a fundamental necessity to describe life mechanistically.

cell biology↗