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the engineered bronchiole and estimating the number of ASM cells to cover the surface with three layers of cells. Seeding densities of 0.5, 1, 2, and 3 million cells/bronchiole were investigated (n = 10, using three lots of ASM cells). ASM cells were dynamically seeded by suspending the cells in SmGM-2. After 48 h, the entire medium volume was removed and a cell count was done. For 0.5 × 106 cell-seeding density/bronchiole, no viable ASM cells were remaining in the medium, indicating that all of the cells adhered to the exterior surface of the bronchiole. For 1 × 106 ASM cells, there were >0.5 × 106 ASM cells remaining 9 ± 1% of the time. For 2 × 106 ASM cells/bronchiole seeding density, 31 ± 2% of the time, there were >0.5 × 106 ASM cells/bronchiole that did not adhere to the exterior of the engineered bronchiole. For 3 × 106 cells, there were >0.5 × 106 ASM cells remaining more than 83 ± 5% of the time. Therefore, 1 × 106 ASM cells/engineered bronchiole was considered optimal.

Epithelial cells were statically seeded into the lumen of the bronchiole. The cell density was determined by calculating the surface area of the inner diameter of the tissue construct. A monolayer of cells was deemed the fastest way to prepare the lumen, rather than waiting for the epithelial cells to proliferate and form a monolayer. The seeding time was determined by seeding the cells and then gently flushing the bronchiole after 6, 8, 12, 18 and 24 h (n = 6 for three lots of SAEC). Cell viability was checked by placing the flushed SAGM kit into a flask and observing the number of cells to adhere. At 6 h, 2 × 105 epithelial cells were flushed from the lumen 73 ± 5% of the time. At 8 h, 1 × 105 epithelial cells were flushed from the lumen 41 ± 4% of the time. At 12 h, 18 ± 2% of the time, <1 × 105 epithelial cells were removed from the bronchiole. At 18 and 24 h, <1 × 105 epithelial cells were flushed from the bronchiole 12 ± 2% of the time but cell viability decreased by 33 ± 5%. The best seeding time was 12 h. Therefore, 3.5 × 105 epithelial cells were suspended in 100 µl proliferative medium and injected into the lumen of the engineered bronchiole and incubated for 12 h.

3.2.3. Medium composition

Since the bronchiole wall diameter was 200 µm or less, the medium in the chamber likely affects all three cell types by diffusion of soluble factors. The initial fabrication step was conducted using FGM. The fibroblasts were embedding in collagen matrix and after the airway had solidified FGM was added to the chamber. The tissue contracted in the first 24 h and then the ASM cells were seeded. For ASM cell seeding, the chamber was filled with SmGM-2 for 48 h while attaching to the periphery of the bronchiole. After 48 h, the SmGM-2 was replaced with FGM. This was done because the fibroblast medium has a lower serum concentration (2%) and different growth factors (eliminates EGF), which allowed the tissue to stop contracting and stabilize within 10 days.

The epithelial cells were very sensitive to medium composition. It has been shown (Gray et al., 2001) that

Copyright 2010 John Wiley & Sons, Ltd.

C. Miller et al.

a high EGF concentration helps to establish an epithelial monolayer. After 3 days of exposure to 25 ng/ml EGF (proliferative medium), the concentration was decreased to 0.5 ng/ml EGF (quiescent medium) for 4–7 days (Figure 3). These manipulations allowed the epithelial cells to form a tight monolayer, produce a basement member and present proper morphology.

3.3. Tissue-engineered bronchiole fabrication

At the time of fabrication, the bronchiole wall thickness of the collagen–fibroblast construct is 1 mm. Over the first 5–7 days after fabrication, the fibroblasts branch (Figure 4b) and adhere to the collagen I fibres, causing the tissue wall to thin, thereby forming a ‘lamina propria’ of 100–200 µm thickness (Figure 4c). The thinness of the bronchiole wall is important for the diffusion of mediators between the different cell types (Figure 4d). It was determined by PCNA staining that the fibroblasts’ proliferation was negligible within the first 60 days (Figure 4e). Moreover, the fibroblasts remain viable in the collagen I matrix, with little to no evidence of apoptosis (Figure 4f). Since the engineered bronchiole contracted and then stabilized, and the fibroblasts exhibited quiescent behaviour (branching, negligible proliferation and apoptosis), the seeding density of 2 × 105 fibroblasts/ml tissue matrix was deemed adequate (Agarwal et al., 2003).

The ASM cells were dynamically seeded on the periphery of the engineered bronchiole, forming a multi-layer of cells. ASM cells were not seeded until the collagen–fibroblast construct contracted around the silicone rubber tubing (24 h) to prevent ASM contamination of the airway lumen.

The fabrication timeline for bronchiole contraction was integral to establish this model. Preliminary trials were conducted at 5, 7, 10, 14, 21 and 28 days to determine the fabrication timeline. The contraction period of 5–7 days for the bronchiole to decrease from 1000 to 100–200 µm in thickness was repeatable 98% ±1 (n = 10, three lots of ASM, one lot of NHLF). The wall diameter did not change from day 10 to day 14, indicating that the tissue construct had stabilized. The fibroblasts and ASM cells did not cause tissue contraction after day 10, which was crucial for the removal of the thin-walled silicone rubber tubing and seeding of the epithelial cells in the bronchiole lumen. When epithelial cells were seeded before the bronchiole wall was finished contracting (during days 2–4), the epithelial cells sloughed off of the wall. due to compression of the monolayer.

3.4. Tissue-engineered bronchiole analyses

Preliminary trials were conducted at 5, 7, 10, 14, 21 and 28 days to determine the fabrication timeline and to select sampling times (n = 6 bronchioles). Subsequently, tissue characteristics were determined at 7, 14, 28 and

J Tissue Eng Regen Med (2010). DOI: 10.1002/term

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