Shock Wave Application to Cell Cultures

Summary

Shock waves nowadays are well known for their regenerative effects. Therefore in vitro experiments are of increasing interest. We therefore developed a model for in vitro shock wave trials (IVSWT) that enables us to mimic in vivo conditions thereby avoiding distracting physical effects.

Abstract

Shock waves nowadays are well known for their regenerative effects. Basic research findings showed that shock waves do cause a biological stimulus to target cells or tissue without any subsequent damage. Therefore, in vitro experiments are of increasing interest. Various methods of applying shock waves onto cell cultures have been described. In general, all existing models focus on how to best apply shock waves onto cells.

However, this question remains: What happens to the waves after passing the cell culture? The difference of the acoustic impedance of the cell culture medium and the ambient air is that high, that more than 99% of shock waves get reflected! We therefore developed a model that mainly consists of a Plexiglas built container that allows the waves to propagate in water after passing the cell culture. This avoids cavitation effects as well as reflection of the waves that would otherwise disturb upcoming ones. With this model we are able to mimic in vivo conditions and thereby gain more and more knowledge about how the physical stimulus of shock waves gets translated into a biological cell signal (“mechanotransduction").

Introduction

Shock waves are sound pressure waves arising from a sudden release of energy, e.g. as thunder when lightning. In medicine shock waves have been used for over 30 years in Lithotripsy for the disintegration of kidney stones. Since the incidental finding of iliac bone thickening in Lithotripsy patients in the early 1980s, first studies were conducted to evaluate the effect of shock wave treatment (SWT) on bone healing¹. Impressive results of improved healing of long-bone nonunions could be observed². Subsequently, indications were expanded to soft tissue wounds³. Basic research findings showed that shock waves do cause a bio....

Protocol

Ethical permission

After obtaining written informed consent of patients, umbilical cords were obtained from Caesarean section at the Department of Gynaecology for isolation of human umbilical vein endothelial cells (HUVECs). Permission was given from the ethics committee of Innsbruck Medical University (no. UN4435).

1. Prepare the IVSWT Water Bath

1. Prepare 3.5 L of tap water in an appropriate tank. Water needs to be heated to 37 °C (see Protocol 6).
2. Fill the water into the water bath until the water level is approximately 3 cm below the edge. Mind to completely cover the membrane with ....

Results

Using the described method we applied shock waves to human umbilical vein endothelial cells (HUVECs) that we afore isolated from umbilical cords. Umbilical cords were obtained from elective caesarian sections.

HUVECs were treated at a confluence of 90% in a T25 cell culture flask with an electrohydraulic shock wave therapy system. Treatment parameters were an energy flux density of 0.1 mJ/mm² and a frequency of 5 Hz. 300 impulses were applied from a distance of 5 cm from the shock w.......

Discussion

The significance of the proposed model for in vitro shock wave treatment is the fact that waves can propagate after passing the cell culture in contrast to existing models. Thereby, disturbing physical effects such as tensile forces can be avoided. The model more closely resembles in vivo conditions than that by others applying waves to their cell culture flasks directly.

An additional advantage is the possibility of varying the distance between shock wave source and cells. T.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Orthogold shock wave device	Tissue Regeneration Technologies, Woodstock, GA – manufactured by MTS-Europe GmbH, Konstanz, Germany
IVSWT Water Bath V2.0	Johann Hohenegger - Technical Products
EBM-2 Basal Medium 500 m +EGM-2 SingleQuot Suppl. & Growth Factors	Lonza	CC-3156 & CC-4176	This medium was used for the shown experiments with HUVECs to fill the cell culture flask. For other cell types, use the recommended medium.
Pechiney Parafilm M PM996	Pechiney Plastic Packaging	PH-LF-PM996-EA at labplanet.com	for sealing flasks
Falcon Serological pipettes 25 ml	Becton Dickinson Labware	357525
CellMate II Serological Pipette	Matrix Technologies
Skintact Ultrasonic Gel	Skintact	UL-01 250 ml
T25 Cell culture flasks	COSTAR	3056
Mikrozid disinfectant	Schülke
3.5 L Degassed water
Paper towels