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

ABSTRACT: Introduction: The application of single-cell RNA sequencing has greatly improved our under-standing of various cellular and molecular mechanisms involved in physiological and pathophysi-ological 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 effec-tively used for leukemia blast samples. Its ease of implementation holds the potential to simplify the integration of this technique into routine clinical practice.

ORGANISM(S): Homo sapiens

PROVIDER: GSE237239 | GEO | 2023/08/17

REPOSITORIES: GEO

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Project description:Background: The cryopreservation of semen is crucial for conserving genetic diversity and reproductive success in endangered species like the Siberian sturgeon. However, this process can cause cryodamage, affecting quality and protein profile of spermatozoa. While cryoprotectants like dimethyl sulfoxide (DMSO) and methanol (MeOH) facilitate post-thaw motility recovery, DMSO-preserved spermatozoa exhibit reduced fertilizing ability. This study investigates how DMSO and MeOH impact the proteome of Siberian sturgeon spermatozoa and examines semen quality parameters. Two complementary approaches of quantitative proteomics, liquid chromatography-mass spectrometry (LC-MS) and two-dimensional difference in gel electrophoresis (2D-DIGE), were used to analyze the proteomic profiles of fresh and cryopreserved spermatozoa, as well as the extracellular medium (EM; n=7 for each group). Results: Cryopreservation led to a decline in motility parameters (MOT, VCL, PROG) and viability, along with an increase in ROS levels, membrane fluidity, and acrosome damage. Despite similar quality parameters between DMSO and MeOH-preserved sperm, DMSO-preserved sperm showed dramatically lower fertilization success (6.2% vs 51.2%). A total of 224 and 118 differentially abundant proteins in spermatozoa cryopreserved with MeOH and DMSO, respectively, were identified compared to fresh samples, with 342 and 363 proteins released into the EM. The most affected proteins by cryopreservation included H2A, CABYR, PYGM, ENO3, DBI and LTA4H. Additionally, 36 and 39 uniquely altered sperm-leakage proteins were identified for MeOH and DMSO cryopreserved samples, respectively. Bioinformatic analysis showed that MeOH-specific proteins were related to chromosomal structure and mitochondrial functionality, while DMSO-specific proteins were mainly involved in acrosome reaction, zona pellucida binding, flagella structure, and nuclear pore organization. These proteins are potentially involved in sturgeon sperm fertilizing ability. The expression of six proteins was verified by western blot analysis. Conclusions: This study provides the first comprehensive proteomic characterization of Siberian sturgeon spermatozoa after cryopreservation with DMSO and MeOH, revealing insights into proteomic changes that affect fertilizing ability and aiding conservation efforts for this endangered species.

Project description:Cerebrospinal fluid (CSF) matrix biomarkers have become increasingly valuable surrogate markers of neuropsychiatric diseases in research and clinical practice. In contrast, CSF cells have been rarely investigated due to their relative scarcity and fragility, and lack of common collection and cryopreservation protocols, with limited exceptions for neurooncology and primary immune-based diseases like multiple sclerosis. The advent of a microfluidics-based multi-omics approaches to studying individual cells has allowed for the study of cellular phenotyping, intracellular dynamics, and intercellular relationships that provide multidimensionality unable to be obtained through acellular fluid-phase analyses. challenges to cell-based research include site to- site differences in handling, storage, and thawing methods, which can lead to inaccuracy and inter-assay variability. In the present study, we performed single-cell RNA sequencing (10x Genomics) on fresh (Samples labeled as FRE) or previously cryopreserved human CSF samples from three alternative cryopreservation methods: Fetal Bovine Serum with Dimethyl sulfoxide (FBS/DMSO, Samples labeled as FBS), FBS/DMSO after a DNase step (a step often included in epigenetic studies, samples labeled as DNA), and cryopreservation using commercially available Recovery©media (Samples labeled as REC). In comparing relative differences between fresh and cryopreserved samples, we found little effect of the cryopreservation method on being able to resolve donor-linked cell type proportions, markers of cellular stress, and overall gene expression at the single-cell level, whereas donor-specific differences were readily discernable. We further, demonstrate the compatibility of fresh and cryopreserved CSF immune cell sequencing using biologically relevant sexually dimorphic gene expression differences by donor. Our findings support the utility and interchangeability of FBS/DMSO and Recovery cryopreservation with fresh sample analysis, providing a methodological grounding that will enable researchers to further expand our understanding of the CSF immune cell contributions to neurological and psychiatric disease.

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

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