Exosome Isolation after in vitro Shock Wave Therapy

Summary

Exosomes are released upon in vitro application of shockwaves. Here, we describe how to apply shockwaves on cultured endothelial cells and subsequently isolate exosomes for further investigation.

Abstract

Shock wave therapy is routinely applied in orthopedic indications including tendinopathies such as lateral epicondylitis (tennis elbow) and Achilles tendinitis (heel spurs) as well as non-healing wounds and bones. Despite different pathologies, the combination of an angiogenic and an anti-inflammatory effect of shock wave therapy leads to regeneration in soft tissue and bones. In over 30 years of clinical application, no side effects were observed. Furthermore, basic research even revealed regenerative effects on ischemic myocardium.

In a previous work we could show that the mechanical stimulus of cultured cells is translated via an exosome release into a biological response. However, the exact mechanism remains to be elucidated. Mechanical coupling is crucial when applying shock wave therapy as even small air bubbles can absorb shock waves. The previously described water bath method is a valid method to guarantee adequate and reproducible shock wave application in vitro.

We were able to develop a feasible and replicable protocol to isolate exosomes from cultured cells after shock wave application. Thereby we demonstrate a possibility to study underlying mechanisms of mechanotransduction as well as the regenerative and angiogenic potential of shock wave released exosomes.

Introduction

Shock waves (SW) are sound pressure waves that appear in nature when a high amount of energy is released in a short period of time (e.g., thunder with lightning). In clinical routine, shock waves are used in lithotripsy to disintegrate kidney stones without relevant side effects for over 30 years^1,2. By incident, a thickening of the iliac bone was observed in X-rays after kidney stone disintegration. This observation provided the basis for research in bone healing disorders and lead to treatment in long-bone non unions^3,4,5....

Protocol

Human umbilical vein endothelial cells were obtained from caesarean sections at the Department of Gynecology. Therefore, written informed consent of patients was obtained. Permission was given from the ethics committee of Innsbruck Medical University (no. UN4435).

NOTE: Work under a sterile laminar flow hood to avoid contamination.

1. 24 h before the experiment

1. Prepare exosome free endothelial growth medium by ultra-centrifuging fetal bovine serum (FCS) .......

Discussion

In multiple basic research works, the regenerative effect of the shock wave therapy could be demonstrated and is routinely applied in orthopedic indications^3,4,5. In diverse animal models, the regenerative effect on the ischemic myocardium could be demonstrated and lead to the initiation of the CAST-HF trial^9,10. This randomized controlled trial aims to evaluate the bene.......

Materials

Name	Company	Catalog Number	Comments
Cell culture flasks	Cellstar	658170	75 cm2
Collection tubes – Falcon Tube	Corning	352070
Endothelial Cell Growth Medium-2 BulletKit	Lonza	CC-3162
Endothelial Cell Growth Basal Medium	Lonza	CC-3121
Fetal bovine serum	Sigma	F0804
Parafilm	Pechiney	PM996
Phosphat buffered saline	gibco	14190-904
Shockwave applicator	MTS	Orthowave 180c