An improvised one-step sucrose cushion ultracentrifugation method for exosome isolation from culture supernatants of mesenchymal stem cells

Abstract

Background: Exosomes are nanovesicles (30-120 nm) of endosomal origin. These exosomes contain various functional proteins and RNAs that could be used for therapeutic purposes. Currently, having a standard method for exosome isolation retaining its biological properties with increased yield and purity is a major challenge. The most commonly used method is differential ultracentrifugation but it has its own disadvantages, which include high time consumption, low yield due to disruption of exosome integrity, and high protein contaminants. In this study, we have identified an improved method addressing these problems for exosome isolation using ultracentrifugation since it is cost-effective and used worldwide.

Method: We have compared differential ultracentrifugation with the modified method called one-step sucrose cushion ultracentrifugation for exosome isolation. The conditioned serum-free media from human mesenchymal stem cells cultured for 48 h was collected for exosome isolation. The cellular debris was removed by centrifugation at 300g for 10 min, followed by centrifugation at 10,000g for 30 min to remove microvesicles. Equal volumes of pre-processed conditioned media were used for exosome isolation by direct ultracentrifugation and one-step sucrose cushion ultracentrifugation. The exosomes isolated using these methods were characterized for their size, morphology, concentration, and surface marker protein expression.

Result: It was observed that the recovery of exosomes with cup-shaped morphology from one-step sucrose cushion ultracentrifugation was comparatively high as estimated by nanoparticle tracking analysis and electron microscopy. These results were confirmed by Western blotting and flow cytometry.

Conclusion: We conclude that this one-step sucrose cushion ultracentrifugation method provides an effective and reproducible potential standard method which could be used for various starting materials for isolating exosomes. We believe that this method will have a wide application in the field of extracellular vesicle research where exosome isolation with high yield and purity is an imperative step. Schematic representation of comparison of UC and SUC exosome isolation methods for tissue-specific human mesenchymal stem cells. The SUC isolation method yields a greater number of cup-shaped exosomes with a relatively homogenous population for mass-scale production of exosomes for downstream analysis.

Abbreviations: SUC One-step sucrose cushion ultracentrifugation, UC Direct ultracentrifugation.

Keywords: Exosomes; Mesenchymal stem cells; Sucrose cushion ultracentrifugation.

Fig. 1

Schematic representation of exosome isolation using standard method (direct ultracentrifugation) and modified method (one-step sucrose cushion ultracentrifugation). Abbreviations: MSC mesenchymal stem cell, NTA Nanoparticle Tracking Analysis, PBS phosphate-buffered saline, TEM transmission electron microscopy

Fig. 2

Characterization of BMSCs and ADSCs using (a) flow cytometry for positive markers such as CD29, CD73, CD90, CD105, and HLA-I and negative markers such as HLA-II, CD34, and CD45. b Tri-lineage differentiation potential. (i) Osteogenesis was determined by Alizarin red S staining of the extracellular mineralized matrix. (ii) Adipogenesis was determined by using Oil red O staining of lipid droplets. (iii) Chondrogenesis was determined by Alcian blue staining of proteoglycan. All images were taken at 20× magnification. Abbreviations: ADSC adipose tissue–derived mesenchymal stem cell, BMSC bone marrow–derived mesenchymal stem cell

Fig. 3

Size distribution and concentration of hMSC-derived exosomes. a As measured by Nanoparticle Tracking Analyses (NTA), particle size for hMSC exosomes isolated by both methods was within the range of 30 to 120 nm. Size distribution and concentration of hMSC-derived exosomes as measured by NTA. b Representative graph plots depicting the significant increase in the yield of exosomes isolated by using the modified method (SUC). Results are mean ± standard error of the mean of three independent experiments. *Significant with P value of less than 0.05. Abbreviations: ADSC adipose tissue–derived mesenchymal stem cell, BMSC bone marrow–derived mesenchymal stem cell, hMSC human mesenchymal stem cell, ns non-significant, SUC one-step sucrose cushion ultracentrifugation, UC direct ultracentrifugation

Fig. 4

Transmission electron microscopic (TEM) pictures of exosomes isolated by hMSCs by using the UC and SUC methods. a Representative images depicting a higher number of exosomes in the SUC method for both tissuespecific hMSCs. b & c Morphometric evaluation of exosome by further diluting the samples for shape, size, and number. Exosomes isolated by the SUC method were cup-shaped and greater in number compared with those isolated by the UC method. Also, small-sized vesicles (<30 nm) were observed to be in clusters of four to five vesicles each. Red circle depicts grouped or coupled vesicles Yellow circle depicts individual vesicles. All images were taken at 15,000× magnification and for morphological analyses images were magnified at 20,000× magnification. Abbreviations: hADSC human adipose tissue–derived mesenchymal stem cell, hBMSC human bone marrow–derived mesenchymal stem cell, hMSC human mesenchymal stem cell, ns non-significant, SUC one-step sucrose cushion ultracentrifugation, UC direct ultracentrifugation

Fig. 5

Surface marker profiling of exosomes by Western blotting. a Total protein concentration of exosomes by bicinchoninic acid assay (BCA): Representative graph plots depict the comparison of total protein concentration in exosomes which showed that the concentration of exosomal protein was significantly higher for the SUC isolation method. b Western blot for CD63 and Alix protein expression in exosomes (50 μL of total exosomal protein was loaded). GAPDH was used as a housekeeping gene. Unlike CD63, Alix was expressed specifically in exosomes. c Densitometric analysis of CD63 and Alix expression in exosomes showed a higher protein expression for the SUC isolation method. Results are mean ± standard error of the mean of three independent experiments. *Significant with P value of less than 0.05. Abbreviations: AD adipose tissue, ADSC adipose tissue–derived mesenchymal stem cell, AU arbitrary unit, BM bone marrow, BMSC bone marrow–derived mesenchymal stem cell, ns non-significant, SUC one-step sucrose cushion ultracentrifugation, UC direct ultracentrifugation

Fig. 6

a Representative flow cytometry histogram plots for CD63-positive exosomes isolated by both methods and (b) its quantification. c Confocal microscopy (Leica, Wetzlar, Germany) was used to visualize the PKH26-labeled exosomes bound to magnetic beads (magnification 63×). d Representative flow cytometry histogram plots for BMSC- and ADSC-derived exosomes to analyze the expression of parent cell surface markers (CD73and CD29). Results are mean ± standard error of the mean of three independent experiments. *Significant with P value of less than 0.05. Abbreviations: ADSC adipose tissue–derived mesenchymal stem cell, BMSC bone marrow–derived mesenchymal stem cell, ns non-significant, SUC one-step sucrose cushion ultracentrifugation, UC direct ultracentrifugation