Manufacturing, quality control, and GLP-grade preclinical study of nebulized allogenic adipose mesenchymal stromal cells-derived extracellular vesicles

Abstract

Background Human adipose stromal cells-derived extracellular vesicles (haMSC-EVs) have been shown to alleviate inflammation in acute lung injury (ALI) animal models. However, there are few systemic studies on clinical-grade haMSC-EVs. Our study aimed to investigate the manufacturing, quality control (QC) and preclinical safety of clinical-grade haMSC-EVs. Methods haMSC-EVs were isolated from the conditioned medium of human adipose MSCs incubated in 2D containers. Purification was performed by PEG precipitation and differential centrifugation. Characterizations were conducted by nanoparticle tracking analysis, transmission electron microscopy (TEM), Western blotting, nanoflow cytometry analysis, and the TNF-α inhibition ratio of macrophage [after stimulated by lipopolysaccharide (LPS)]. RNA-seq and proteomic analysis with liquid chromatography tandem mass spectrometry (LC–MS/MS) were used to inspect the lot-to-lot consistency of the EV products. Repeated toxicity was evaluated in rats after administration using trace liquid endotracheal nebulizers for 28 days, and respiratory toxicity was evaluated 24 h after the first administration. In vivo therapeutic effects were assessed in an LPS-induced ALI/ acute respiratory distress syndrome (ARDS) rat model. Results The quality criteria have been standardized. In a stability study, haMSC-EVs were found to remain stable after 6 months of storage at − 80°C, 3 months at − 20 °C, and 6 h at room temperature. The microRNA profile and proteome of haMSC-EVs demonstrated suitable lot-to-lot consistency, further suggesting the stability of the production processes. Intratracheally administered 1.5 × 10^8 particles/rat/day for four weeks elicited no significant toxicity in rats. In LPS-induced ALI/ARDS model rats, intratracheally administered haMSC-EVs alleviated lung injury, possibly by reducing the serum level of inflammatory factors. Conclusion haMSC-EVs, as an off-shelf drug, have suitable stability and lot-to-lot consistency. Intratracheally administered haMSC-EVs demonstrated excellent safety at the tested dosages in systematic preclinical toxicity studies. Intratracheally administered haMSC-EVs improved the lung function and exerted anti-inflammatory effects on LPS-induced ALI/ARDS model rats.

1. Introduction

Mesenchymal stromal cells (MSCs) exist in various adult mesenchymal tissues. They are multipotent and have immunomodulatory abilities [1]. Acute respiratory distress syndrome (ARDS) and acute lung injury (ALI) are life threatening clinical syndromes with high morbidity and mortality; however, there are limited effective clinical interventions for the treatment of ALI/ARDS [2]. Preclinical studies in mice, rats, and sheep have shown that MSC treatments inhibit lung damage, reduce inflammation, suppress immune responses, and promote alveolar fluid clearance, suggesting that MSCs can alleviate ARDS/ALI [3–11]. To date, more than 50 trials have been conducted using MSCs as therapeutic agents in ARDS/ALI (ClinicalTrials.gov). Multiple studies have revealed the safety of MSCs in ARDS treatment, and MSCs may reduce the mortality rate of patients with ARDS [12–15].

However, due to their cell size, MSCs are typically intravenously administered, and the effective dose of MSCs is relatively high (5–1.25 × 10^7/treatment) [16]. It was reported that some of the biological functions of MSCs are mediated by secreted extracellular vesicles (EVs) and mesenchymal stromal cells-derived extracellular vesicles (MSC-EVs) have shown beneficial effects in ARDS treatment [17–19]. EVs are bilayer membrane vesicles secreted by almost all cell types and play important roles in cell–cell communication. EVs are classified by physical characteristics (size or density, such as small EVs, and medium/large EVs); biochemical composition (CD63+/CD81+-EVs; etc.); or by their origin.