Extracellular vesicles from human multipotent stromal cells protect against hearing loss after noise trauma in vivo

Abstract

The Effect of extracellular vesicles derived from human stromal cells on the inner ear has been investigated. The vesicles were characterized and tested in vitro in spiral ganglion neurons and in vivo in a mouse noise trauma model.

First‐in‐human intracochlear application of human stromal cell‐derived extracellular vesicles

“…The preparation of the batch that was used for injection contained 1.03 × 10 11 particles/ml with a diameter range of 110-130 nm as measured by nanoparticle tracking analysis (NTA, Model PMX110 from ParticleMetrix, Germany, Figure S Nanoparticle Tracking Analysis of UC-MSC-EVs). Surface profiling by multiplex flow cytometry demonstrated the presence of the tetraspanins CD9, CD63, CD81, as previously described (Warnecke et al, 2020) as well as MSC-EV markers CD29, CD44, CD49e and CD73 and revealed the absence of CD1/2/3/8/11c/14/19/20/24/25/31/40/45/56/69/86/133/142/209/326 and absence of the marker molecules HLA-ABC, HLA-DR, ROR1, SSEA-4 (Figure S Multiplex Marker Profiling of UC-MSC-EVs). Cryo-transmission electron microscopic imaging confirmed the presence of UC-MSC-EVs by visualization of double-layer lipid membranes around spherical objects (Figure 1b).…”

“…Total protein amount of the used UC-MSC-EVs was 3.7 mg/ml. The biological activity of this particular clinicalgrade EV preparation was confirmed through in vitro and in vivo testing using inner ear cell culture and a noise trauma mouse model (Warnecke et al, 2020).…”

“…After primary isolation of umbilical-cord (UC) derived MSCs from a single donor, cells were cultured in fibrinogen-depleted culture medium at 5% CO 2 and 37 • C as previously described (Warnecke et al, 2020). For the EV manufacturing process we have established a Master and a working cell bank from primary UC-MSCs.…”

“…In brief, no xenogenic substances have been employed throughout the entire manufacturing process and MSCs were expanded in the presence of human platelet lysate (Gimona et al, 2017;Laner-Plamberger et al, 2015;Rohde et al, 2019). The EV-batch used for the preclinical experiments was extensively characterized in Warnecke et al, 2020(Warnecke et al, 2020 and is identical to the one used for the patient application in this report. UC-MSC-EVs were prepared from conditioned medium by tangential flow filtration (TFF) and diafiltration, respectively, using a 100 kDa hollow fibre filter (Spectrum Labs).…”

“…This GMP unit has obtained a European manufacturing and distribution licence for the human investigational medicinal product 'MSC-EV' granted by the Austrian regulatory authorities. Rigorous and standardized characterization of UC-MSC-EVs was performed as published previously (Warnecke et al, 2020) and complied with the criteria of the MISEV2018 guidelines (Théry et al, 2018) where applicable for therapeutic preparations. The ready to use suspension of UC-MSC-EVs was stored at -80 • C and delivered to the operating room as a sterile solution in 500 µl vials (Figure 1a.…”