Developing a tissue-engineered bronchiole model
Figure 4. (a) The cylindrical bronchiole is 3 mm diameter and 3 cm long. (b) Tissue strength is provided by the fibroblasts in the collagen construct. (c, d) The ASM cells (m), fibroblasts and epithelial cells (e) are in close proximity to promote cell–cell communication. (e) The fibroblasts do not extensively proliferate or (f) undergo significant apoptosis. Bar = 10 µm unless indicated otherwise
Bright-field microscopy was performed for macroscopic observations. Haematoxylin and eosin (H&E) was used to view collagen fibres and the structure of the engineered bronchioles. Apoptosis and proliferating cell nuclear antigen (PCNA) were performed on the fibroblasts for cell viability and proliferation. For apoptosis, an ApopTAG kit (S7100, Chemicon) was used. Proliferating cells were identified with a primary antibody against PCNA (1 : 300; DAKO) and secondary antibody conjugated with horseradish peroxidase, using a VectaStain ABC Elite kit and DAB (PK-6102 and SK-4100, Vector Laboratories).
Bioreactor design and feature optimization
The biomaterials for construction of the bioreactor system were chosen to promote ease of sterilization and low cost, minimize the quantity of biological agents and provide long-term use without defects.
The current version of the bioreactor is very user- friendly (Figure 1). The bioreactor insert can be com- pletely disassembled for cleaning or part replacement and easily reassembled. The flint glass chamber is econom- ical and can also be replaced. The bioreactor insert is assembled with the PTFE moulds in place and then the entire reactor is sterilized. This minimizes the risk of contamination.
The pump system affords the most unique attribute of the bioreactor system (Figure 2a). It supplies the engineered bronchioles with pulsed air to mechanically stimulate the tissue during the contractile phase and dis- tributes humidified air to the epithelialized lumen during the differentiation phase (Figure 2b). The peristaltic pump was selected for its slow rotation (1–100 rpm) and pump
head selection for the number of rollers. The PharMed L/S 18 tubing has a 7.9 mm inner diameter (i.d.) that forces a large volume of air into the three-way splitter and through the L/S 13 tubing (0.8 mm i.d.). Pump speed was varied to pulse the thin-walled silicone rubber tubing 15 times/min. The roller positions in the pump head and the diameter of the tubing allowed for a 2% radial disten- sion, which increased the diameter of the bronchiole by 60 µm (from 3 mm to approximately 3.06 mm).
3.2. Determination of tissue fabrication parameters
Fabrication of the tissue-engineered airways involved parameter optimization of matrix concentration, fab- rication mechanics, cell density and seeding methods, medium composition and cell phenotype. Proper propor- tioning of these parameters was necessary for the creation of a stable engineered bronchiole (Figure 4a).
3.2.1. Matrix composition
The matrix concentration was determined by modifying a pre-existing protocol (Agarwal et al., 2003). Utilizing 5 mg/ml collagen for the cylindrical bronchiole main- tained a tubular shape after the PTFE mould was removed. Lower concentrations of collagen tended to deform or tear during mould removal.
3.2.2. Cell seeding density
Optimal seeding densities were determined to achieve the goal of a monolayer of epithelial cells and a multilayer of ASM cells. ASM cell-seeding density was initially estimated by calculating the exterior surface area of
Copyright 2010 John Wiley & Sons, Ltd.
J Tissue Eng Regen Med (2010). DOI: 10.1002/term