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Single-cell Microfluidic Analysis of Bacillus subtilis

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Systematic insights into cell density-dependent transcriptional responses upon medium replacements.

Pol Pérez-Rubio¹, Elianet Lorenzo Romero¹, Laura Cervera¹

¹Grup d'Enginyeria de Bioprocessos i Biocatàlisi Aplicada, Escola d'Enginyeria, Universitat Autònoma de Barcelona, Campus de Bellaterra, Cerdanyola del Vallès, Barcelona 08193, Spain.

Biomedicine & Pharmacotherapy = Biomedecine & Pharmacotherapie

|November 1, 2024

Summary

This summary is machine-generated.

Investigating media conditions in HEK293 cells reveals that removing extracellular vesicles (EVs) improves transfection efficiency, likely due to media changes, not cell responses. Histone gene upregulation suggests a new strategy for enhancing transient gene expression.

Keywords:

Cell density effect EV-depleted HEK293 Media replacement Transcriptomics

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Area of Science:

Cell Biology
Molecular Biology
Biotechnology

Background:

High cell density cultures face challenges with transfection efficiency and particle secretion.
The cell density effect impacts transient gene expression, necessitating mechanistic understanding.

Purpose of the Study:

To explore the impact of conditioned media on HEK293 cell transcriptome at varying densities.
To elucidate the role of extracellular vesicles (EVs), proteins, and RNAs in transfection efficiency.
To identify strategies for overcoming the cell density effect in gene expression.

Main Methods:

Comparative analysis of HEK293 cells cultured in fresh, exhausted, and EV-depleted media.
Transcriptome analysis to assess gene expression changes under different media conditions.
Proteomic characterization of EV-depleted media.

Main Results:

EV-depleted media improved transfection efficiency, primarily due to physicochemical alterations.
Downregulation of nucleocytoplasmic transport genes and upregulation of histone genes were observed in EV-depleted media.
EV-depleted media revealed pathways related to infection response and DNA breaks, and stimulated virion release.

Conclusions:

Manipulating histone levels is a potential strategy to enhance transient transfection.
Extracellular matrix proteins can inhibit transfection via proteoglycan competition.
HEK293 cells in depleted media exhibit an infection-like state, impacting cellular processes and virion release.