Transmural flow bioreactor for vascular tissue engineering

Abstract

Nutrient transport limitation remains a fundamental issue for in vitro culture of engineered tissues. In this study, perfusion bioreactor configurations were investigated to provide uniform delivery of oxygen to media equivalents (MEs) being developed as the basis for tissue-engineered arteries. Bioreactor configurations were developed to evaluate oxygen delivery associated with complete transmural flow (through the wall of the ME), complete axial flow (through the lumen), and a combination of these flows. In addition, transport models of the different flow configurations were analyzed to determine the most uniform oxygen profile throughout the tissue, incorporating direct measurements of tissue hydraulic conductivity, cellular O(2) consumption kinetics, and cell density along with ME physical dimensions. Model results indicate that dissolved oxygen (DO) uniformity is improved when a combination of transmural and axial flow is implemented; however, detrimental effects could occur due to lumenal pressure exceeding the burst pressure or damaging interstitial shear stress imparted by excessive transmural flow rates or decreasing hydraulic conductivity due to ME compaction. The model was verified by comparing predicted with measured outlet DO concentrations. Based on these results, the combination of a controlled transmural flow coupled with axial flow presents an attractive means to increase the transport of nutrients to cells within the cultured tissue to improve growth (increased cell and extracellular matrix concentrations) as well as uniformity.

Figure 1

A: Image of flow chamber with ME mounted within. B: Image of flow chambers housed with reservoir inside incubator. All bioreactors had independent perfusion loops. C: Schematic of bioreactor configurations (from top to bottom): translumenal, lumenal, and combination.

Figure 2

Representative plots showing experimental determination of Michaelis–Menten parameters. A: The slope of the linear portion of the O₂ consumption curve is used to determine V_max. B: Oxygen consumption rate plotted versus P_O2 for determination of K_m. K_m is the oxygen concentration at which the OCR is one-half V_max. (–––), V_max constant; (●), K_m.

Figure 3

Static culture DO concentration profiles for cell densities of (A) 45, (B) 135, and (C) 225 million cells/mL within ME tissue. The white semi-circle represents the ME lumen, which is supported by a solid mandrel (impermeable to oxygen). Results in (A) indicate maximal (constant) oxygen consumption throughout the tissue, while (B) and (C) indicate sub-maximal (first-order) consumption since the concentrations quickly approach zero within the tissue.

Figure 4

Predicted DO concentration profiles along ablumenal wall for three different bioreactor configurations and three different cell densities. The legend in the upper left graph applies to each of the top six graphs (flow rates of 50, 200, and 400 µL/min, Pe_r = 0.53, 2.13, and 4.26, respectively, for translumenal flow). The legend in the bottom left graph applies to the entire bottom row (back pressures of 0, 10, and 15 mmHg), for which the inlet axial flow rate is 400 µL/min.

Figure 5

Relationship of shear stress and lumenal pressure to oxygen transport properties in the translumenal flow configuration. A: Interstitial shear stress on the vSMCs within the ME tissue. B: Intralumenal pressure. In each graph, the shaded region indicates a radial Peclet number <1 (diffusion-dominated transport) and the non-shaded region designates Pe_r greater than one (convection-dominated transport). Each line indicates varying L_p (in cm/s/Pa): (—), 10⁻⁵; (– –), 10⁻⁶; (–-), 10⁻⁷; (---), 10⁻⁸.

Figure 6

Predicted and measured DO concentration versus residence time (thickness divided by transmural velocity) for the translumenal bioreactor configuration. The shaded region indicates transmural flow velocities yielding Pe_r > 1, with flow rates ranging from 50 to 400 µL/min. (---), Predicted DO concentrations for (top to bottom) 45 × 10⁶, 135 × 10⁶, and 225 × 10⁶ cells/mL. Measured outlet DO for (●) low, (■) medium, and (▲) high cell densities (targeted cell densities to compare to predictions). Note that the measured DO inlet in these measurements was 165 nmol/mL and is also reflected in the predictions for consistency.

Figure 7

Error analysis for predicted outlet DO concentrations at varying residence times by varying (A) D_t between 1.06 × 10⁻⁵ ± 10%cm²/s, (B) V_max between 1.96 ± 10%fmol/min, and (C) K_m between 7.75 ± 10% nmol/mL. The three curves in each plot correspond to the same cell densities investigated in Figures 3, 4, and 6 (45, 135, and 225 × 10⁶ cells/mL).