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A novel multicellular model of the adult mouse sinoatrial node retains spontaneous electrical activity and enables live investigation of the S100B-associated cell population

Georgiana Luisa BacaiD, Robert E. MonticoneiD, Bruce D. ZimaniD, Shah Md Toufiqur Rahman, Pathik ParekhiD, Sadia AfriniD, Christopher DunniD, Richard S. TelljohanniD, Dongmei YangiD, Kwan-Wood Gabriel LamiD, Peter Killeen, Dimitrios TsitsipatisiD, Ana‐Maria ZăgreaniD, Myong‐Hee SungiD, Nigel H. GreigiD, Supriyo DeiD, Allison B. HermaniD, Payel SeniD, Rafael de CaboiD, Edward G. LakattaiD

Abstract

Approximately half of the adult sinoatrial node (SAN) consists of non-myocyte populations, indicating that cardiac pacemaking depends on interactions within a multicellular tissue rather than on pacemaker cardiomyocytes alone. Among these, an S100B-associated cell population has been implicated in pacemaker function, yet its identity and physiological roles remain poorly understood. These cells are rare and dispersed throughout the small, structurally complex SAN, making them difficult to observe repeatedly while preserving the native multicellular environment. Here, we established a dissociated multicellular culture of adult mouse SAN tissue on soft collagen-gelatin hydrogels that retains spontaneous electrical activity and permits longitudinal live imaging of S100B-associated cells. Using an S100B-EGFP+ reporter, we identified at least six reproducible morphological and behavioral phenotypes, including migration, proliferation, phagocytic behavior, and spontaneous self-organization into three-dimensional clusters. Cultures remained spontaneously electrically active for more than 10 days in vitro, with peak activity around day 10. This multicellular culture model bridges the gap between intact SAN preparations and isolated-cell cultures, allowing repeated observation of rare S100B-associated cells within a spontaneously active multicellular environment. HighlightsO_LIThe platform enables longitudinal live imaging of rare S100B-associated cells within an diverse multicellular SAN culture. C_LIO_LILive imaging reveals at least six reproducible morphological and behavioral phenotypes of S100B-associated cells. C_LIO_LIDissociated multicellular SAN cultures remain spontaneously electrically active for more than 10 days in vitro. C_LI

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Claude Opus 5.5 · xhigh effort · 8 Nov 2026 · from the full text of version 1 (20 Jul 2026) · CC0 1.0

Dissociated adult mouse sinoatrial node cultures stay spontaneously electrically active beyond ten days on MEA arrays and, on compliant hydrogels, permit live tracking of heterogeneous S100B-EGFP-labeled cells.

Non-myocyte cells occupy nearly half of human sinoatrial node (SAN) tissue volume and shape pacemaking, yet endogenous S100B-expressing cells are poorly defined, and current in vitro models either isolate single populations or use stem-cell-derived pacemaker-like cells, losing native context. This work offers an adult SAN culture that keeps diverse cell types together for longitudinal imaging and network electrophysiology.

Key results

  1. ShownMulticellular SAN cultures show spontaneous Ca2+ transients by day 5 and unstimulated multi-unit MEA spiking through day 14; firing rate and network coherence peak at day 10, then amplitude and synchrony partly decline.

    3Brain CorePlate CMOS MEA (4,096 electrodes), recordings on days 7, 10, 12, 14: mean firing rate maximal at day 10 vs day 7 (p < 0.001), lower at days 12 and 14 (p < 0.01 to p < 0.001 vs day 10); inter-spike interval shorter at day 10 vs day 7 (p < 0.001); Calbryte 520 Ca2+ transients by day 5; one-way ANOVA with Bonferroni. · Microelectrode array recordings; Multicellular SAN cultures remain spontaneously…; Figure legends

  2. SuggestedLive time-lapse imaging separates S100B-EGFP+ cells into at least six recurring morphological and behavioral classes (elongated, spindle, amoeboid, long-extension networking, stationary perinuclear-bright, small granular), with mitosis, migration, process remodeling and debris engulfment observed.

    Qualitative classification from live confocal imaging of EGFP plus SPY650-DNA nuclei, with long-term runs of 24–72 h at 5 to 15 minute intervals; no counts or proportions per class are reported, and identities rest on morphology, behavior and reporter intensity (Table 1, Figs. 2, 3). · Time-lapse and live nuclear imaging; Dissociated SAN cultures contain a…; Figure legends; Tables

  3. ShownS100B-EGFP+ cells form a minor but distinct population, roughly 9% of single cells in dissociated SAN, at a fraction similar to that in right atrium.

    Flow cytometry with a threshold at the 99.9th percentile of paired non-transgenic controls: SAN mean 9.1% (range 6.4–10.6%, n = 6); right atrium mean 8.8% (range 7.4–10.1%, n = 6); controls ≤0.1% of single cells (0.11% in each tissue); per-sample gated events 18,044–40,187. · Flow cytometry; S100B-EGFP+ cells comprise a defined fraction of…; Figure legends

  4. SuggestedWithout scaffolds or patterning cues, a subset of cells self-organizes, typically within three to five days, into roughly spherical three-dimensional clusters containing HCN4+ cells plus S100B-EGFP+ cells at core and periphery; some clusters contract spontaneously.

    Clusters were absent right after seeding and appeared progressively; confocal thickness approximately 17 to 32 µm; contraction was not causally tied to the S100B-EGFP+ population; no cluster counts or frequencies are reported. · Spontaneous formation of S100B+-enriched…; Discussion

Methods, data and reproducibility

For each preparation, SAN tissue pooled from two S100B-EGFP reporter mice was collagenase-dissociated and cultured on compliant collagen/gelatin hydrogels. Cultures were then characterized by live time-lapse and Ca2+ imaging, immunofluorescence and flow cytometry, plus high-density CMOS MEA recordings on separate cultures.

  • SAN tissue from Kosmos S100B-EGFP reporter mice (B6;D2-Tg(S100B-EGFP)1Wjt/J), male and female, aged 12–24 weeks; two mice pooled per dissociation · mouse · n = n = 6 SAN samples (flow cytometry); other assays not stated · JAX #005621 (mouse strain)
  • Right atrial tissue from reporter mice, dissociated for flow cytometry comparison · mouse · n = n = 6
  • Wild-type C57BL/6J mice used for platform optimization and as non-reporter flow cytometry gating controls · mouse · n = one control baseline per tissue · JAX #000664 (mouse strain)
  • Separate dissociated SAN cultures grown directly on CMOS MEA arrays for extracellular recordings · mouse
  • Intact micro-dissected SAN explants cultured on ~1 kPa hydrogels for up to seven days · mouse
Methods & tools
Softview collagen/gelatin hydrogel plates (Matrigen SF-35-20, ~6–14 kPa), Calbryte 520 AM calcium indicator, SPY650-DNA live nuclear probe (Spirochrome), NucBlue nuclear counterstain, Immunofluorescence for βIII-tubulin, GFAP, Iba1, c-Kit and HCN4, 3Brain CorePlate high-density CMOS MEA (4,096 electrodes), BD flow cytometer with FACSDiva 9.0.1, FlowKit (Python) for gating and quantification, Propidium iodide viability staining, Zeiss AxioExaminer D1 with sCMOS camera, Keyence BZ-X810 microscope, Zeiss LSM 880 confocal microscope
Reagents & environment
Flow acquisition in BD FACSDiva 9.0.1; gating and quantification in Python with FlowKit (FlowKit and Python versions not stated).
Reproducibility gaps
Data only on reasonable request to the corresponding author; detailed protocols (digestion, substrate preparation, imaging settings) deferred to peer-reviewed publication; no code repository given.
Limitations (6)
  • Dissociation and culture alter cell phenotypes; the model is characterized functionally and morphologically, and molecular equivalence to native SAN tissue is not asserted. · stated by the authors
  • Cultures are mixed populations without lineage tracing, so the S100B-associated subtypes in Table 1 are provisional and inferred from morphology, behavior and reporter brightness; formal identity would need single-cell RNA sequencing. · stated by the authors
  • Extracellular MEA signals are network-level and cannot be assigned to a cell type, so any link between S100B+ cells and recorded activity is correlative; because the arrays are opaque, MEA cultures were separate from the imaged dishes. · stated by the authors
  • All data come from adult mouse SAN, so relevance to human tissue is unestablished, and the explant outgrowth findings are descriptive only. · stated by the authors
  • Replicate numbers for MEA cultures, imaging fields and cluster measurements are not reported, phenotype classes are not quantified, and flow gating relied on a single non-transgenic control per tissue, which makes reproducibility hard to judge. · noted in this summary
  • The reporter is driven by the human S100B promoter, and no antibody co-stain confirming endogenous S100B protein in EGFP+ cells is described; nuclear EGFP may reflect free EGFP diffusion rather than S100B localization. · noted in this summary

Who should read:Read this if you study sinoatrial node biology, cardiac glia or non-myocyte niches and need an imageable adult multicellular culture system whose MEA-grown cultures stay spontaneously active beyond ten days.

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