578 JOURNAL OF BIOSCIENCE AND BIOENGINEERING © 2007, The Society for Biotechnology, Japan Vol. 103, No. 6, 578–581. 2007 DOI: 10.1263/jbb.103.578 A Novel Apparatus Applying Long Term Intermittent Cyclic Hydrostatic Pressure to In Vitro Cell Cultures Kenneth A. Myers, 1 Nigel G. Shrive, 2 and David A. Hart 1 * McCaig Centre for Joint Injury and Arthritis Research, Faculty of Medicine, University of Calgary, 3330 Hospital Dr. NW, Calgary, AB, T2N 4N1, Canada 1 and Department of Civil Engineering, University of Calgary, 2500 University Dr., Calgary, AB, T2N 1N4, Canada 2 Received 2 November 2006/Accepted 8 March 2007 A novel apparatus has been designed that allows for application of long term intermittent cyclic hydrostatic pressure to in vitro cell cultures, while maintaining cell viability on par with a stan- dard incubator. The system has been tested with monolayer cultures, but could also accommodate three dimensional constructs. [Key words: hydrostatic pressure, bioreactor, in vitro cell culture, drug delivery, mechanobiology, mechanotransduction] The issue of mechanical stimuli is important when con- sidering in vitro methods of cell culture. The influence of mechanical loading on the cellular microenvironment is cru- cial, stimulating changes in proliferation, differentiation, and matrix synthesis/degradation. In order to ensure normal cell function, the chemistry, temperature, and mechanics of the environment must be carefully controlled. Achieving this goal requires the development of bioreactors with substan- tial flexibility; a major challenge in the field of tissue engi- neering today. One area currently being explored is the application of hydrostatic pressure (HP) for in vitro culture protocols. HP is an important stimulus for many types of cells; in animal tissues, the functional behaviour of cells is often directed by this type of loading (1–4). There exist at present, many specialized experimental ap- paratuses for the application of HP to cellular cultures, how- ever in order to represent physiological conditions accu- rately, a cyclic, rather than static, loading regime is desir- able. Several such systems have been developed (5–8) and while they are useful for certain purposes, most have spe- cific limitations restricting their flexibility in application. Long-term (>12 h) testing experiments require replacement of culture media during loading in order to restore levels of nutrients and dissolved gases, as well as remove metabolites which could modify cell function and behaviour. Jannasch et al. (9) and Carver and Heath (10) have designed HP ex- perimentation systems that allow for media exchange, how- ever both are only capable of static HP, which significantly restricts applications. More recently, Watanabe et al. (7) de- scribed a bioreactor capable of cyclic HP and media ex- change. This apparatus appears to have potential, however the system is designed for a specific type of three dimen- sional (3D) construct which again, constrains application. Other designs involve compression of the gas phase, rather than direct compression of the liquid culture medium (11, 12). This approach is not viable for relatively high ampli- tude cyclic HP (i.e., levels experienced by connective tissues in vivo), as significant piston displacement is required. Fur- thermore, the varying partial pressures of O 2 and CO 2 in the gaseous phase would lead to variations in the dissolved levels of these gases in the medium. While there have been a number of recent advances, the limitations of existing devices continue to restrict our ability to learn about mechanical adaptations of cells to loading, disease progression, and long term drug effectiveness on specific cell types. The types of cellular adaptations that occur over days and weeks, rather than hours, continue to be a mystery because many loading experiments cannot be con- ducted over these longer time scales with existing systems. In this paper, we describe a novel apparatus designed to apply intermittent cyclic HP for longer term experiments. This system was developed to be compatible with standard cell culture materials in order to facilitate integration into existing culture protocols. This device will allow for the conduction of previously limited experiments, studies which will hopefully shed light on the nature of cellular mechan- otransduction occurring over longer time scales in multiple cell configurations (e.g., 2D vs 3D cultures). Figure 1A shows the overall set up of the system. Cyclic pressure is generated by the QX-1500 pump, a computer- controlled, belt-driven pump (Quizix, Broken Arrow, OK, USA). Through a coordinated effort with Quizix, special- ized software was designed to add an oscillating mode op- tion to the existing capabilities of the machine. The output pressure of the Quizix pump was amplified with a pressure multiplier: an aluminium piston with a 2:1 surface area dif- ferential. The consequence of this design is that the output pressure is twice the input. An aluminium chamber was designed and manufactured * Corresponding author. e-mail: hartd@ucalgary.ca phone: +1-403-220-4571 fax: +1-403-283-7742