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.
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.
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 years1,2. 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 unions3,4,5.
Indications for shock wave therapy were expanded and today the method is in routine clinical use in orthopedic indications including tendinopathies such as lateral epicondylitis (tennis elbow) and Achilles tendinitis (heel spurs)6,7. Furthermore, basic research demonstrated a high angiogenic potential of shock wave therapy (SWT). Therein, a study showed an increase in angiogenic growth factors such as VEGF (vascular endothelial growth factor), PIGF (placental growth factor) and FGF (vascular endothelial growth factor) upon SWT followed by angiogenesis8.
To investigate a possible beneficial role of shock wave induced angiogenesis in other pathologies, we applied SW therapy in an animal model of ischemic heart disease9,10. After a regenerative effect in the ischemic myocardium could be demonstrated, we could identify the indispensable role of the innate immune receptor TLR 3 (Toll like receptor 3) upon shock wave therapy11,12. Further studies showed that the mechanical stimulus of SW therapy is translated into a biological signal via exosome release. Compared to exosomes physiologically released from endothelial cells, exosomes released upon SWT contain an increased cargo of angiogenic microRNA. Injected into ischemic myocardium, SWT released exosomes induced regeneration13.
Since air absorbs SW, perfect coupling between the applicator and the cell culture flasks is crucial. A standardized water bath represents a feasible method to apply SWT in vitro and a reproducible experimental setup. In order to avoid a reflection and consequently interference of waves, a wedge-shaped absorber destructs the primary waves running to the back of the water bath. For this reason, we recommend applying shock waves in vitro using the described water bath only.
In this protocol we describe the application of shock waves in vitro to release angiogenic exosomes into the supernatant. This protocol provides the possibility to investigate the role of exosomes in mechanotransduction and is the basis for further investigation of exosome release upon SWT.
In multiple basic research works, the regenerative effect of the shock wave therapy could be demonstrated and is routinely applied in orthopedic indications3,4,5. In diverse animal models, the regenerative effect on the ischemic myocardium could be demonstrated and lead to the initiation of the CAST-HF trial9,10. This randomized controlled trial aims to evaluate the bene…
The authors have nothing to disclose.
This study was supported by an unrestricted AUVA research grant to JH and CGT.
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 |
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