Biology subjects

Chaleckis, R.

Publications and source records attributed to Chaleckis, R..

4 recordsLinked to original sources

bioRxiv2023

Modeling dry eye with an air-liquid interface in corneal epithelium-on-a-chip

Dry eye syndrome (DES) is a complex ocular condition characterized by an unstable tear film and inadequate tear production, leading to tissue damage. Despite its common occurrence, there is currently no comprehensive in vitro model that accurately reproduce the cellular characteristics of DES. Here we modified a corneal epithelium-on-a-chip (CEpOC) model to recapitulate DES by subjecting HCE-T human corneal epithelial cells to an air-liquid (AL) interface stimulus. We then assessed the effects of AL stimulation both in the presence and absence of diclofenac (DCF). Transcriptomic analysis revealed distinct gene expression changes in response to AL and AL_DCF, affecting pathways related to development, epithelial structure, inflammation, and extracellular matrix remodeling. Both treatments upregulated PIEZO2, linked to corneal damage signaling, while downregulating OCLN, involved in cell-cell junctions. They increased the expression of inflammatory genes (e.g., IL6) and reduced mucin production genes (e.g., MUC16), reflecting dry eye characteristics. TGFB1, crucial for corneal wound healing, was slightly downregulated in AL_DCF, potentially affecting wound healing processes rather than reducing inflammation by DCF. Metabolomic analysis showed increased secretion of metabolites associated with cell damage and inflammation (e.g., methyl-2-oxovaleric acid, 3-methyl-2-oxobutanoic acid, lauroyl-carnitine) in response to AL and even more with AL_DCF, indicating a shift in cellular metabolism. This study showcases the utilization of AL stimulus within the CEpOC as a comprehensive approach to faithfully reproduce the cellular characteristics of DES.

bioengineering↗

bioRxiv2023

Early Differentiation Signatures in Human Induced Pluripotent Stem Cells Determined by Non-Targeted Metabolomics Analysis

Human induced pluripotent stem cells (hiPSCs) possess immense potential as a valuable source for the generation of a wide variety of human cells yet monitoring the early cell differentiation towards a specific lineage remains challenging. In this study, we employed a non-targeted metabolomic analysis technique to analyze the extracellular metabolites present in samples as small as one microliter. The hiPSCs were subjected to differentiation by initiating culture under the basal medium E6 in combination with chemical inhibitors that have been previously reported to direct differentiation towards the ectodermal lineage such as Wnt/{beta}- catenin and TGF-{beta} kinase/activin receptor alone or in combination with bFGF, and the inhibition of glycogen kinase 3 (GSK-3), which is commonly used for the diversion of hiPSCs towards mesodermal lineage. At 0 hr and 48 hrs 107 metabolites were identified, including biologically relevant metabolites such as lactic acid, pyruvic acid, and amino acids. By determining the expression of the pluripotency marker OCT3/4, we were able to correlate the differentiation status of cells with the shifted metabolites. The group of cells undergoing ectodermal differentiation showed a greater reduction in OCT3/4 expression. Moreover, metabolites such as pyruvic acid and kynurenine showed dramatic change under ectodermal differentiation conditions where pyruvic acid consumption increased 1-2-folds, while kynurenine secretion decreased 2-folds. Further metabolite analysis uncovered a group of metabolites specifically associated with ectodermal lineage, highlighting the potential of our findings to determine the characteristics of hiPSCs during cell differentiation, particularly under ectodermal lineage conditions. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=84 SRC="FIGDIR/small/530741v1_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@3d680dorg.highwire.dtl.DTLVardef@17a5495org.highwire.dtl.DTLVardef@472d04org.highwire.dtl.DTLVardef@dc63cd_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2021

Spatiotemporal Determination of Metabolite Activities in Multiple Corneal Epithelium Barriers on a Chip

The corneal epithelial barrier maintains the metabolic activities of the ocular surface by regulating membrane transporters and metabolic enzymes responsible for the homeostasis of the eye as well as the pharmacokinetic behavior of drugs. Despite its importance, no established biomimetic in vitro methods are available to perform the spatiotemporal investigation of corneal metabolism and determine the transportation of endogenous and exogenous molecules. This study introduces multiple corneal epithelium barriers on a chip, namely, Cornea-Chip, which enables the spatiotemporal collection as well as analysis of micro-scaled extracellular metabolites from both the apical and basolateral sides of the barriers. Longitudinal samples collected during 48 h period were analyzed using untargeted liquid chromatography-mass spectrometry metabolomics method, and 104 metabolites were annotated. The shifts in extracellular metabolites and quantitative analysis of the mRNA associated with membrane transporters could allow the investigation of the correlation between the expression of and the secretion and transportation of metabolites across the polarized corneal epithelial barrier. The Cornea-Chip might provide a non-invasive, simple, and effectively informative method to determine pharmacokinetics and pharmacodynamics as well as to discover novel biomarkers for drug toxicological and safety tests as an alternative to animal experiments.

bioengineering↗

bioRxiv2020

Untargeted LC-MS metabolomics for the analysis of micro-scaled extracellular metabolites from hepatocytes

Metabolome analysis in micro physiological models is a challenge due to the low volume of cell culture medium (CCM). Here, we report a LC-MS-based untargeted metabolomics protocol for the detection of hepatocyte extracellular metabolites from micro-scale samples of CCM. Using a single LC-MS method we have detected 57 metabolites of which 27 showed >2-fold shifts after 72-hours incubation. We demonstrate that micro-scale CCM samples can be used for modelling micro-physiological temporal dynamics in metabolite intensities. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=62 SRC="FIGDIR/small/312520v3_ufig1.gif" ALT="Figure 1"> View larger version (11K): org.highwire.dtl.DTLVardef@1b307e3org.highwire.dtl.DTLVardef@cada64org.highwire.dtl.DTLVardef@1c7fb70org.highwire.dtl.DTLVardef@4cb37f_HPS_FORMAT_FIGEXP M_FIG Graphical Abstract C_FIG

bioengineering↗