# Integrated cryopreservation-thawing-transplantation platform for neural stem cell-based spinal cord injury repair

## TL;DR

A new platform for spinal cord injury repair integrates cryopreservation, thawing, and transplantation of neural stem cells to improve cell survival and function.

## Contribution

Development of an integrated cryopreservation-thawing-transplantation platform that maintains cell viability and modulates the immune response for spinal cord injury repair.

## Key findings

- The CTT platform maintains high NSC viability and stemness post-cryopreservation and thawing.
- The platform modulates the inflammatory microenvironment by promoting macrophage polarization toward tissue repair.
- Minimally invasive injection of the platform restores motor function in SCI rats through neurogenesis and remyelination.

## Abstract

Spinal cord injury (SCI) repair lacks clinically validated restorative therapies. Transplantation of exogenous neural stem cells (NSCs) offers significant potential for therapeutic applications; however, challenges remain, including substantial cell loss, uncontrolled differentiation, and limited tissue integration within inflammatory microenvironments. Furthermore, the workflow associated with traditional NSC transplantation—including cryopreservation, thawing, transportation, and injection—remains fragmented, resulting in systemic limitations. These issues manifest as reduced cell viability and stemness, an elevated risk of contamination, and dosing inaccuracies. All these significantly impede clinical translation. An integrated system for NSC preservation, transport, and transplantation is required to meet the following criteria: (i) maintenance of high cell viability and stemness post-cryopreservation and thawing; (ii) modulation of the acute-phase immune microenvironment; (iii) regulation of the differentiation fate of transplanted NSCs; (iv) injectable, standardized, and closed-system operation. To meet these requirements, we established a comprehensive cryopreservation, thawing, and transplant (CTT) integrated platform. Utilizing the bioactive material PM-BMH@Exo, this platform enables seamless end-to-end workflow integration through a mechanism that preserves bioactivity. It not only ensures high viability retention and directed differentiation of NSCs but also effectively mitigates the rapid viability decline of cells observed after traditional cryopreservation. Furthermore, the system enables closed-loop operations spanning cryopreservation, thawing, and minimally invasive injection. It breaks through systemic bottlenecks from multi-step procedures, comprehensively enhancing the timeliness and standardization of therapeutic interventions. We systematically evaluated the system's feasibility and efficacy via in vitro and in vivo experiments. This study presents a technologically viable and clinically compatible pathway with potential applications for SCI repair.

Image 1

•BMH@Exo alters the inflammatory microenvironment associated with SCI by facilitating macrophage polarization towards a tissue-repair phenotype, thereby creating more favorable conditions for NSCs engraftment.•HUCMSC-Exo, an essential element of the CTT platform, promotes the differentiation of NSCs into neurons through activation of the PI3K-Akt and calcium signaling pathways.•The development of a three-dimensional culture system utilizing Poly (D, L-lactic acid) porous microspheres offers an optimal microenvironment to support the growth, proliferation, and differentiation of NSCs.•Minimally invasive injection of the CTT platform rebuilds neural circuits through neurogenesis and remyelination, thereby restoring motor function in SCI rats.•The end-to-end CTT workflow integrates cryostorage, thawing, transport, and injection, addressing the fragmentation of conventional NSC-based SCI therapies.

BMH@Exo alters the inflammatory microenvironment associated with SCI by facilitating macrophage polarization towards a tissue-repair phenotype, thereby creating more favorable conditions for NSCs engraftment.

HUCMSC-Exo, an essential element of the CTT platform, promotes the differentiation of NSCs into neurons through activation of the PI3K-Akt and calcium signaling pathways.

The development of a three-dimensional culture system utilizing Poly (D, L-lactic acid) porous microspheres offers an optimal microenvironment to support the growth, proliferation, and differentiation of NSCs.

Minimally invasive injection of the CTT platform rebuilds neural circuits through neurogenesis and remyelination, thereby restoring motor function in SCI rats.

The end-to-end CTT workflow integrates cryostorage, thawing, transport, and injection, addressing the fragmentation of conventional NSC-based SCI therapies.