Long term stability of isolated human serum derived exosomes

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1 Long term stability of isolated human serum derived exosomes Candice de Boer (PhD student) Regenerative Medicine Laboratory Supervisor: Associate Professor Neil Davies

2 Exosomes First discovered during the growth of sheep reticulocytes Secreted membrane vesicles Found in body fluids (blood, urine, saliva and cerebrospinal fluid) Diameter of nm Paracrine effect: Shown to reduce myocardial infarction size Anti-apoptosis Anti-inflammatory effects (Kourembanas, 2015)

3 Exosome biogenesis Mammalian cells engulf a portion of their plasma membrane forming an endosome multivesicular bodies (MVBs) Once released into the extracellular space exosomes Lysosomes will degrade MVBs that do not release exosomes (Waldenström and Ronquist, 2014)

4 Intracellular uptake of exosomes Endocytosis Membrane fusion Receptor-mediated internalization (Kourembanas, 2015)

5 Absorbance (280 nm) 1. Exosome isolation 1.1 Size exclusion chromatography (SEC) 1.2 Ultracentrifugation (UC) Fraction Number Elution profile of size exclusion chromatography of pooled human serum UC isolated contaminating serum proteins SEC better purer isolation Sepharose CL-4B 1 ml Pooled human serum (2 donors) 26 Fractions collected (500 µl) Eluted with PBS SDS-PAGE (12%)

6 Absorbance 450 nm (percent of serum control) 2. Exosome characterisation (SEC exosomes) 2.1 TEM 2.2 Western blotting kda kda kda CD CD TSG Functional assay Fraction 9 Fraction 10 Exosomes nm in fraction 9 More smaller vesicles in fraction No serum * 10% SEC exosome fresh ** * 10% SEC exosome snap frozen 10% SEC exosome -80⁰C Significant increase in human dermal fibroblast proliferation * p < 0.05 ** p < (compared to no serum) Treatment

7 2. Exosome characterisation (SEC exosomes) 2.4 Exosome uptake (HT1080 cells + 3 hours incubation with exosomes) SYTO RNASelect labels the exosomal RNA green exosome Bodipy TR ceramide labels the exosomal membrane red exosome Actin red Actin green

8 Long term stability of isolated human serum derived exosomes 3. Exosomes incubated at 37⁰C for 3 weeks 3.1 TEM SEC-derived exosomes UC-derived exosomes -80⁰C 37⁰C 3 weeks -80⁰C 37⁰C 3 weeks

9 3. Exosomes incubated at 37⁰C for 3 weeks 3.2 Exosome Uptake (HT1080 cells) PBS control -80⁰C 37⁰C 3 weeks SEC-derived exosomes UC-derived exosomes

10 Absorbance 450 nm (percent of serum control) Absorbance 450 nm (percent of serum control) 3. Exosomes incubated at 37⁰C for 3 weeks 3.3 Functional assay SEC-derived exosomes UC-derived exosomes * * No serum 10% SEC exosome -80⁰C 10% SEC exosome 37⁰C 3 weeks 0 No serum 3.33% UC exosome -80⁰C 3.33% UC exosome 37⁰C 3 weeks Treatment Treatment Significant increase in human dermal fibroblast proliferation No difference in storage temperatures * p < 0.05 (compared to no serum) Trended towards increase in human dermal fibroblast proliferation High variability may be due to contaminating serum proteins

11 Exosomes delivered in a hydrogel Hydrogels have been used for controlled release of growth factors and gene delivery HyStem-HP hydrogel Advantages of hydrogels controlled release localized and sustained delivery In the last year exosomes have been actively explored for delivery from hydrogels (most not injectable) Deliver nucleic acids, growth factors, proteins Sodium alginate hydrogel Regenerative Bone regeneration Cutaneous healing in chronic wounds Hydroxyapatite/chitosan composite hydrogel Exosomes dispersed in a hydrogel Slow release of exosomes from the hydrogel (Qin et al., 2016)

12 4. Exosomes in a fibrin gel Fibrin hydrogel applications 4.1 Confocal Z-Stacked image of exosomes in a fibrin gel Function as Haemostatic plug Matrix for cell migration and wound healing Fibrin glue used instead of sutures or staples Enhances healing Minimizes scarring Good initial delivery vehicle Gels in situ Injectable Biocompatible Degrades naturally Controlled release 45 µl fibrin gel 30% Bodipy TR ceramide labelled exosomes (red) 3 mg/ml fibrinogen

13 4. Exosomes in a fibrin gel 4.2 Transwell assay using exosomes in a fibrin gel Green labelled exosomes in a fibrin gel HT1080 cells Cells uptake the RNA labelled exosomes Image Chemoattractant media

14 4. Exosomes in a fibrin gel 4.2 Transwell assay using exosomes in a fibrin gel PBS control 24 Hours of migration 63.6% exosomes in fibrin hydrogel 24 hours of migration HT1080 cells uptake exosomes embedded in a fibrin hydrogel

15 Conclusion Successful isolation of human serum exosomes using size exclusion chromatography Characterised using TEM and western blots Functional stability of exosomes stored in PBS at 37ºC for 3 weeks Physical properties retained Bioactivity (cellular uptake and stimulation of cell proliferation) Exosomes are stable enough to be embedded in a hydrogel 3D cell transfection Future possibility Long term stability of exosomes is an advantage for further therapeutic applications using exosomes Clinically approved fibrin hydrogel could potentially be used to achieve localized delivery of exosomes

16 Funding Acknowledgements

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