C. Miller et al.
Figure 1. (a) Bioreactor insert in place. (b) The bioreactor insert shows a complete PTFE mould unit and a mould unit with half of the mould removed. The moulds are held in place by grooves in the parallel PTFE plates. The thin-walled silicone rubber tubing is inserted through the needle tubing, through the PharMed L/S 16 tubing, and connected to a 3/32 inch connector. (c) Once the tissue matrix solidifies, the moulds are removed and the chamber is filled with medium
air flow, and allows us to manipulate the growth environment. The overall intent is to develop a tissue- engineered bronchiole model of airway remodelling that approximates the behaviour of native tissue. This model system may advance understanding of the cumulative effects of individual factors associated with remodelling of human bronchioles.
Native bronchioles are affected by many factors. Lung fibroblasts, smooth muscle cells and epithelial cells are influenced by cell–cell signalling and interactions with the extracellular matrix. Stimulation of one cell type has been found to influence the behaviour of other cells types that are in close proximity (Zhang et al., 1999). The extracellular matrices bind soluble regulatory molecules that also mediate cell behaviour. Mechanical forces exerted on the matrix and cells during respiration influence pathophysiological conditions (Hirst et al., 2000; Swartz et al., 2001; Black et al., 2003). Cytoskeletal-mediated contraction of the airway is equilibrated dynamically, affecting the adaptability of the airway smooth muscle in response to mechanical changes (An and Fredberg, 2007). Shear stress and pressure exerted by air flow through the lumen also influence cell behaviour (Liu et al., 1999).
As part of a novel approach to model the human bronchiole, we have developed an in vitro model that mimics bronchiole wall physiology. The engineered bron- chioles are composed of a collagen scaffold containing embedded lung fibroblasts. The exterior surface is sur- rounded by multiple layers of airway smooth muscle (ASM) cells, and the inner surface (lumen) is lined with bronchial epithelial cells with an air interface. The human airway cells are in close proximity to one another to promote cell–cell communications. The bioreactor envi- ronment can be manipulated to focus on various aspects of airway remodelling, such as subepithelial fibrosis (Woodruff and Fahy, 2002), smooth muscle hyperpla- sia and hypertrophy (Hirst, 1996) and epithelial cell metaplasia (Woodruff and Fahy, 2001; Doherty and Broide, 2007), all of which are key components of airway remodelling.
Materials and methods
Bioreactor design and features
The bioreactor is constructed using polytetrafluoroethy- lene (PTFE), stainless steel, and glass, based on their biocompatibility (Figure 1a). The tissue-engineered bron- chioles are vertically supported by a unit constructed of two plates separated by stainless steel rods (Figure 1b). Scored stainless steel needle tubing is inserted into the parallel PTFE plates to act as grips for the contracting tissue (Figure 1c). Thin-walled silicone rubber tubing is threaded through the needle tubing (Figure 1c). Bisected, cylindrical PTFE tissue moulds insert into grooves on the top and bottom PTFE plate. The PTFE mould produces the tube-like shape of the airway (Figure 1c), while the thin-walled silicone rubber tubing creates the lumen of the bronchiole and also exerts dilatory forces on the engi- neered bronchiole when air is pulsed through the system. The glass outer housing (W216 904, Wheaton) of the bioreactor has a maximum volume of 120 ml.
One of the unique features of the bioreactor system is the ability to mechanically stimulate the engineered bronchiole through radial distension. The bioreactor applies mechanical stimulation by pulsing air through thin-walled silicone tubing. The thin-walled silicone rubber tubing (2.4 mm i.d. and 3 mm o.d.; Saint Gobain Performance Plastics, Beaverton, MI, USA) has a 0.6 mm wall thickness. which allows for radial distension. PharMed L/S 18 tubing is loaded into the head of a peristaltic pump (Masterflex L/S model 7553-80, Cole- Parmer; Figure 2a). Six PharMed L/S 13 tubes (06 485-13, Cole-Parmer) connect to six ports in the lid of the bioreactor (Figure 2a). The lid also has a vent port with 0.2 µm filter (02 915-08, Cole-Parmer) for pH stability and an injection port (2N3399, Baxter; Figures 1a, 2a). The injection port is used for medium exchange, sample withdrawal and supplement input.
Radial distension of the bronchioles is controlled by connecting the pump-tubing network to the six ports in the lid (Figure 1a). Once connected, the system is air-tight
Copyright 2010 John Wiley & Sons, Ltd.
J Tissue Eng Regen Med (2010). DOI: 10.1002/term