Developing a tissue-engineered bronchiole model
Figure 2. (a) The bioreactor is connected to the pump system via PharMed tubing to dilate the bronchioles during the contraction phase. (b) The air humidifier is connected to flowing air through the engineered bronchioles, creating an air interface. This system provides essential mechanical stimulation to the tissue
and pulses the thin-walled tubing. The expansion and relaxation of the tubing mimics adult quiet breathing. The resistance to air flow and viscous resistance to movement are nearly negligible. The engineered bronchiole is pulsed at a rate of 15 pulses/min, with a radial distension of approximately 2% and distension velocity of 0.015 mm/s.
The bioreactor system can also supply humidified air flow to the epithelialized lumen of the tissues (Figure 2b), once the luminal silicone tubing is removed. Room air is filtered with a 0.2 µm PTFE filter and then passes through a porous stone submerged in a bottle of sterile normal saline for humidification. A dew loop in the L/S 18 and a port in the three-way splitter traps extra moisture and allows for the removal of condensation.
2.2. Tissue-engineered bronchiole fabrication
The tissue-engineered bronchioles in this study are composed of three primary human lung cell types and a collagen matrix scaffold. The human primary cells (Lonza, formerly Clonetics) include normal human lung fibroblasts (CC2512), airway smooth muscle cells (CC2576) and small airway epithelial cells (CC2547). The cells are expanded in their respective medium: FGM Bullet kit (CC3131), SmGM-2 Bullet kit (CC3182) and SAGM Bullet kit (CC3118). The fabrication of the bronchioles occurs as a step-wise process. The complete process takes 28 days to create a phenotypically stable bronchiole.
First, the cylindrical-shaped bronchioles are fabricated by embedding fibroblasts (passages 1–6, 2 × 105 cells/ml matrix) in a collagen I matrix. The collagen matrix (BD Biosciences, Bedford, MA, USA) is prepared on ice by mixing 5× Dulbecco’s modified Eagle’s medium (DMEM; Sigma D5523, pH adjusted to 7.4) and 10× reconstitution buffer (2.2 g sodium bicarbonate, 4.77 g HEPES, 95 ml nanopure de-ionized water, and 5 ml 1 N sodium hydroxide) and then adding the type I collagen (Agarwal et al., 2003). The ratio of components is 20 : 10:65 w/v, respectively, with the remaining 5% consisting of fibroblasts and FGM.
Fibroblasts are suspended in a small volume of FGM and pipetted into the chilled collagen matrix and then mixed. The chilled fibroblast–collagen solution is pipetted
into a PTFE mould, and the tissue solidifies during a 2 h incubation at 37 ◦C, after which the moulds are removed. The reactor is filled with FGM [2% fetal bovine serum (FBS)].
During the ensuing 24 h, the tissue contracts around the silicone rubber tubing. ASM cells (passages 1–5, ∼6 × 106 cells/reactor) are dynamically seeded onto the periphery of the collagen bronchiole constructs by stirring the SmGM-2 at 150 rpm (Figure 3). After 48 h, the medium is changed to remove any unattached ASM cells. Once the ASM cells are seeded, the airways are pulsed to mechanically stimulate circumferential ASM alignment. The tissue is maintained in FGM and stirred at 50 rpm to facilitate oxygen, pH and nutrient distribution. One-half of the FGM is refreshed every third day in the bioreactor. Large-volume exchange is necessary due to the high density of ASM cells and fibroblasts (≥7 × 106 cells/reactor). ASM death occurred when the FGM was refreshed at longer intervals.
In the final cell seeding step, the lumen of the bronchiole is epithelialized. The epithelial cells (passages 1–3, 3.5 × 105 cells/tissue) are suspended in 100 µl proliferative medium (bronchiole epithelial medium with single quots, 25 ng/ml epithelial growth factor (EGF), retinoic acid 5 × 10−8 M) (Gray et al., 2001) and injected into the lumen of the bronchiole. Twelve hours after static seeding of the epithelial cells, the medium is gently circulated through the lumen to remove the cells that did not adhere. The system is not pulsed during epithelial cell seeding. Proliferative medium is injected (200 µl) into the lumen twice daily for 3 days and then substituted with quiescent medium (50% LHC basal, 50% DMEM, 0.5 ng/ml EGF) for 4–7 days (Gray et al., 2001). After 7–10 days post-epithelial seeding, the epithelial cells form a monolayer with an established basement membrane and the quiescent medium is removed (Figure 3).
After epithelial seeding and culture, the epithelial cells are exposed to air. The air humidifier is connected to the pump system, allowing humidified air flow through the epithelialized lumen. During epithelial cell seeding, the periphery of the tissue is maintained in FGM because a small amount of serum is necessary to support the ASM and fibroblasts. Figure 3 expresses the fabrication steps
Copyright 2010 John Wiley & Sons, Ltd.
J Tissue Eng Regen Med (2010). DOI: 10.1002/term