Journal Article

O-052 Differential flow environments alter the cellular composition of clots by single cell-RNA seq and multiplex flow cytometry, and lead to differential clot contraction, kinetics, and dynamics in an in vitro cerebrovascular migration assay

Ghovvati, M, Tsukagoshi, E, Kaneko, N, Kawaguchi, R, Hinman, J, Imahori, T, Ghochani, Y

2025

Overview

IntroductionCerebral clots arise in various environments such as rapid flow in atherosclerosis, venous stasis, and turbulent flow of atrial fibrillation. Clot pathology evaluation by Martius, Scarlet, and Blue (MSB) staining doesn’t correlate with the etiology, and doesn't allow for a detailed analysis of white blood cells present. In this study, we investigated how clots formed in various flow environments (FE) affect the immune cells within them, using single-cell RNA-seq and flow cytometry. We then evaluated how various FEs lead to differential clot contraction, kinetics, and dynamics in 3D-printed in vitro cerebrovascular clot migration assays.MethodsClots were generated in vitro via a modified Chandler-Loop design (1) at 37°C at static condition, 10 rotations per minute (RPM, low FE) or 40 RPM (high FE) for 1 hour. Single-cell RNA-seq using the 10X Genomics platform was conducted and top 3000 highly variable features were used for principal component analysis (PCA). Multiplex flow cytometry was utilized to validate RNAseq cellular composition analyses. Cerebrovascular 3D-printed in vitro models were utilized to evaluate clot contraction, migration/location and flow obstruction qualities.ResultsTo characterize the WBC clot content, we performed scRNA-seq on clots generated at variable flow. UMAPs were applied to visualize the 20 clusters, and expression patterns were annotated for cellular identities (figure 1A). Enrichment of myeloid cells in the high flow clots vs. enrichment of lymphoid cells in the static clots along with cell identities was validated via multiplex flow cytometry (figure 1C). We next employed 3D-printed cerebral vasculature models to assess clot contraction, kinetics, and dynamics (figure 2), demonstrating significantly increased clot shrinkage (% original by weight) in static clots and significantly more flow obstruction in HF clots. Furthermore, the HF clots consistently migrated less and occluded more proximally than the static clots did (figure 2 B-C).Abstract O-052 Figure 1Abstract O-052 Figure 2ConclusionsOur studies demonstrate that FEs drive variable cellular composition of clots and lead to differential clot qualities that dictate clot migration/location, and extent of vascular occlusion. Understanding these differences can identify novel therapeutic strategies for variable stroke risk factors. Our future directions include functional evaluation of clot burden/qualities by targeting cell types and/or differentially expressed signaling pathways most upregulated at various FEs.DisclosuresY. Ghochani: None. M. Ghovvati: None. T. Imahori: None. E. Tsukagoshi: None. R. Kawaguchi: None. J. Hinman: None. N. Kaneko: None.

• Publisher: BMJ Publishing Group LTD
• Subject: Flow cytometry