Formaldehyde-free fixation for safe, scalable and flexible single-cell multiomics studies

The development and commercialization of flexible single-cell multiomics methods has allowed for investigator selection and implementation of assays which provide the desired biological depth and context for their disease model of interest. Multiomic methods generally achieve best results with fresh, high-quality samples for efficient recovery of the native biological state. These dynamic samples, however, pose logistical challenges for larger clinical studies, limiting scope and reproducibility. Formaldehyde (PFA) fixation has been the standard for tissue preservation, due to its accessibility and stability enabling simplified clinical sample preparation and biobanking from large patient cohorts. The implementation of PFA, however, predates common knowledge of its carcinogenicity and nucleic acid crosslinking, limiting future transcriptomic and genomic study. Unlike tissue, fixation of single cell suspensions has not yet been standardized to this extent, offering a valuable opportunity to survey more safe and flexible solutions for sample preservation. To address this, we evaluated fixatives that have increasingly been investigated for these advantages over PFA with the goal of suggesting an alternative single-cell fixative solution for further investigation. We optimized each alternative fixation solution using various cellular and molecular analysis tools suggesting broad multiomic and clinical compatibility. Optimal formulations were used to treat peripheral blood monocytes (PBMCs) and their nuclei and analyzed by genetic (ATAC-seq), transcriptomic (WTA) and proteomic (CITE-seq) assays for multiomic evaluation against live and PFA controls. Our data identified a safe and stable novel fixative formulation for preserving PBMC samples throughout a mock biobanking process and gave broad multiomic compatibility comparable or exceeding that of PFA.This alternative and its novel preparation are underrepresented in academic and industry studies on single-cell fixation solutions at a critical time for large cohort studies. Further development, optimization and demonstration of this safer alternative is needed as these large single cell atlas and clinical studies become increasingly common. Such studies' data are shaping new virtual cell models and clinical standard operating procedures foundational to this field and necessitate further review of safer alternatives with more flexible assay compatibility. For Research Use Only. Not for use in diagnostic or therapeutic procedures.©2025 BD. All rights reserved. NPM-7460 (v1.0) 1125