A Rapid and Scalable Subcutaneously Administered Murine Thymus Micro-organoid for Generating Functional T cells

Gebremeskel, S.; Embile, I. B.; Bazhanov, N.; Fang, C.; Heard, A.; Chang, J.; Boyer, C.; Taruishi, M.; Baker, P.; Taylor, K. H.; Oheeron, P.; Khoja, H.

doi:10.1101/2025.09.05.671099

bioRxiv · 10.1101/2025.09.05.671099

A Rapid and Scalable Subcutaneously Administered Murine Thymus Micro-organoid for Generating Functional T cells

Gebremeskel, S.·Embile, I. B.·Bazhanov, N.·Fang, C.·Heard, A.·Chang, J.·Boyer, C.·Taruishi, M.·Baker, P.·Taylor, K. H.·Oheeron, P.·Khoja, H.

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Abstract

Thymic function can decline due to age-related involution, congenital disorders, acute infections, or chemo/radiation therapy. Decline in thymic function leads to decreased T cell production and weakened immunity. To address these thymic insufficiencies, we aimed to develop a transplantable and scalable micro-organoid system utilizing fibroblasts and thymic cells. We have developed a reliable and rapid method to generate thymic micro-organoids using selectively screened fibroblasts and murine thymic cells. The thymic micro-organoids are cryo-preservable, injectable, and give rise to T cells both in vitro and in vivo. Thymic organoids expressed key genes required to sustain T cell development and maturation: ccl25, dll-1, dll-4, foxn-1, il-7, scf. When injected into T cell-deficient Prkdcscid mice, the organoids gave rise to functional {beta}, {psi}8, natural killer T (NKT) cells, and FoxP3+ regulatory T cells. Organoid-derived T cells expressed a diverse T cell receptor (TCR) repertoire in vivo and responded to stimulation with anti-CD3/28, Concanavalin-A, or Phytohemagglutinin. Thymic organoids derived from pmel-1 thymocytes gave rise to V{beta}13+ T cells that delayed the growth of B16 melanoma and enhanced activation of T and NK cells. This approach presents a valuable tool for mechanistic studies and addressing current therapeutic gaps in diseases associated with thymic decline and insufficiencies.

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Gebremeskel, S., Embile, I. B., Bazhanov, N., Fang, C., Heard, A., Chang, J., Boyer, C., Taruishi, M., Baker, P., Taylor, K. H., Oheeron, P., Khoja, H.. 2025-09-08. A Rapid and Scalable Subcutaneously Administered Murine Thymus Micro-organoid for Generating Functional T cells. https://doi.org/10.1101/2025.09.05.671099

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