Conditioning period impacts the morphology and proliferative effect of extracellular vesicles derived from rat adipose tissue derived stromal cell

Abstract

A serum-free conditioning period is a crucial step during small extracellular vesicle (sEV) preparation ranging from 12 to 72h. There is a paucity of knowledge about downstream effects of serum-free conditioning on sEVs and the optimal duration of the conditioning period. The aim of this study was to investigate the influence of the serum-free conditioning period on the sEVs derived from primary adipose stromal cells (AdSCs) and their regenerative potential. Primary AdSCs were conditioned in serum-free medium for 72h. Conditioned medium was collected and refreshed every 24h obtaining three fractions, namely sEVs released after 24h (early), 24h to 48h (intermediate) and 48h to 72h (late). After sEV enrichment with ultracentrifugation, the sEV fractions were analyzed by their size, phenotypic expression, and morphology. Proliferation assays of primary Schwann cells after treatment with sEVs were performed. Particles meeting criteria to be classified as sEVs were detected in all fractions. However, sEVs differed by their size and phenotypic expression. A long conditioning period led to a heterogenous population of larger sEVs and increased protein per particle ratio. Moreover, the expression of tetraspanines was affected. Lastly, the proliferative effect of sEVs on Schwann cells decreased with increasing conditioning period. In conclusion, particles meeting the criteria of EVs are released by primary AdSCs over 72h under serum free conditioning. Nonetheless, they significantly differ in their proliferative effect on Schwann cells cultures.

Keywords: Adipose derived stromal cells; Exosomes; Extracellular vesicles isolation; Nerve regeneration; Proliferation; Schwann cells.

Fig. 1

Conditioning and Isolation of sEVs released from primary rat AdSCs in three time-windows. After reaching a confluency of 60–70%, AdSCs in p2 and p3 were conditioned with serum-free growth medium for 24h. Each 24h of conditioning period the medium was collected, and medium was replaced. This step was repeated twice thus obtaining three fractions– 0h to 24h (early); 24h-48h (intermediate); 48h-72h (late). Isolation was performed with differential centrifugation including one filtration step through a 200nm PVDF filter. Pellets containing the three sEV fractions were suspended in the respective media depending on their use and stored at − 80 °C

Fig. 2

Viability and metabolic activity of AdSC cultured under serum-free conditions. Primary AdSCs (n = 3) were cultured without serum for 24h, 48h, and 72h. a Shows phase contrast micrographs of AdSCs of CTRL and after 24h, 48 h, and 72 h of serum-free culturing. b Displays the normalized viability of AdSC with (yellow line) and without serum (green line) (w/o FCS).The bar chart in c displays the comparison of cells cultured under serum-free conditions for 24h, 48h and 72h (n = 3, mean + SD) * p-value < 0.05; ** p-value < 0.01; ns: p-value > 0.05

Fig. 3

Quantification of sEVs. sEVs released in three-time windows were quantified by NTA in fluorescence mode, and micro-BCA protein assay for protein quantification. Histograms depict the mean ± SD with dots representing each single donor. a Particle quantification by NTA in fluorescence mode (n = 5). b Particles/cell ratio was calculated from particles measure in NTA fluorescence mode and respective viable cells at the end of conditioning period (n = 3). c Total yield amount of protein of sEV preparation per T175 flask (n = 5). d Particles per µg of protein were calculated from particles detected in NTA fluorescence mode and the respective amount of protein (n = 5). Whiskers displays the minimum and maximum range. The corpus gives the interquartile range with median inside the corpus. * p-value < 0.05; ** p-value < 0.01; ns: p-value > 0.05

Fig. 4

Size distribution and immunophenotype of sEVs. sEVs size distribution obtained by NTA in fluorescence mode and scatter mode. Histograms depicts the mean ± SD. Whiskers of boxplots display the minimum and maximum range. The corpus gives the interquartile range with a median inside the corpus. a, b Boxplots show the median size of particles in scatter mode and fluorescence mode as displayed, respectively. c Bar chart shows percentage inside each population of following size classes; exomere 0-45nm; small sEVs 45-105nm; large sEVs 105-165nm; > 165nm for particles larger. d–i Violin bars show ratio of CMG, CD9, CD63, CD81, Calnexin (CNX), CD90 positive events normalized on all detected particles determined by Imagestream IsX. j, k display the ratio of colocolized double or triple expressed tetraspanines (CD9, CD63 and CD81). l shows the mean standard deviation between single donors inside one EV preparation of all expression markers investigated before. n = 5; * p-value < 0.05; ** p-value < 0.01; *** p-value < 0.001; **** p-value < 0.0001; ns: p-value > 0.05

Fig. 5

Exemplary micrographs of sEV obtained with cryo-electron microscopy. In the right corner the source of exemplary pictures is displayed—early, intermediate (IM), late released sEVs. To avoid wrong conclusions, we only display the exemplary character without any interpretation about the whole population found in one group. a,d A bilayer membrane vesicles around 120nm found in early, and late sEV preparation. b Shows size heterogeneity found in intermediately released sEVs with a 21nm sized vesicle, complying with a ribosome (white arrowhead). c,f Show a multilayer vesicle or one vesicle entrapped in a larger one. e,f Shows partially aggregated, multiple vesicles. n = 1

Fig. 6

Atomic Force Microscopy of sEVs. Atomic Force Microscopy (AFM) pictures were taken of sEVs from early (a), intermediate (b) and late released (c) sEVs. All values are given in nm ± SD. n = 1

Fig. 7

Principal Component Analysis (PCA) of sEVs preparations and cell lysates. a Shows the loadings of variables used for PCA. b Distribution of donors and time releasing periods of sEVs preparation. n = 5

Fig. 8

Proliferation experiment. a Timeline of proliferation assay. Created with BioRender.com b Bar charts display the mean, whiskers display SD. All rates were normalized on control group (PBS only). Confluency (overgrown area/OA) shows the increase of overgrown area with primary Schwann cells (n = 5) measured after 24h and 48h of coculture. The proliferation rate (PF) of SCs was calculated by EdU positive/ (Sox-10 & DAPI) positive cells after 48h of coculture. For better clarity,” non-significant” labels were edited out. * p-value < 0.05; ** p-value < 0.01