Single-Cell Transcriptome Analysis of Acute Myeloid Leukemia Cells Using Methanol Fixation and Cryopreservation

Author:

Madaci Lamia1ORCID,Gard Charlyne1,Nin Sébastien1,Sarrabay Alexandre1,Baier Céline2,Venton Geoffroy13,Rihet Pascal1ORCID,Puthier Denis1ORCID,Loriod Béatrice1,Costello Régis13

Affiliation:

1. TAGC, TGML, INSERM, UMR1090, Aix-Marseille University, Parc Scientifique de Luminy, 13009 Marseille, France

2. Advanced BioDesign, Parc Technologique de Lyon, 655 Allée des Parcs, 69800 Saint Priest, France

3. Hematology and Cellular Therapy Department, Conception University Hospital, 13005 Marseille, France

Abstract

Introduction: The application of single-cell RNA sequencing has greatly improved our understanding of various cellular and molecular mechanisms involved in physiological and pathophysiological processes. However, obtaining living cells for this technique can be difficult under certain conditions. To solve this problem, the methanol fixation method appeared as a promising alternative for routine clinical use. Materials and Methods: In this study, we selected two AML samples that had been fixed in methanol for 12–18 months. Once the cells were rehydrated, these samples were subjected to single-cell RNA sequencing. We then compared the results obtained from these samples with those obtained from the same samples cryopreserved in DMSO. Results: We used a previously validated methanol fixation protocol to perform scRNA-seq on DMSO cryopreserved cells and cells fixed in methanol for more than one year. Preliminary results show that methanol fixation induces some genetic and transcriptional modification compared with DMSO cryopreservation but remains a valuable method for single-cell analysis of primary human leukemia cells. Conclusions: The initial findings from this study highlight certain resemblances in methanol fixation over a 12-month period and cryopreservation with DMSO, along with associated transcriptional level modifications. However, we observed genetic degradation in the fixation condition when extending beyond one year. Despite certain study limitations, it is evident that short-term methanol fixation can be effectively used for leukemia blast samples. Its ease of implementation holds the potential to simplify the integration of this technique into routine clinical practice.

Funder

Inserm, GIS IBiSA, Aix-Marseille Université

Publisher

MDPI AG

Subject

General Medicine

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