Dual-Purpose Bioreactors to Monitor Noninvasive Physical and Biochemical Markers of Kidney and Liver Scaffold Recellularization

Abstract

Analysis of perfusion-based bioreactors for organ engineering and a detailed evaluation of physical and biochemical parameters that measure dynamic changes within maturing cell-laden scaffolds are critical components of ex vivo tissue development that remain understudied topics in the tissue and organ engineering literature. Intricately designed bioreactors that house developing tissue are critical to properly recapitulate the in vivo environment, deliver nutrients within perfused media, and monitor physiological parameters of tissue development. Herein, we provide an in-depth description and analysis of two dual-purpose perfusion bioreactors that improve upon current bioreactor designs and enable comparative analyses of ex vivo scaffold recellularization strategies and cell growth performance during long-term maintenance culture of engineered kidney or liver tissues. Both bioreactors are effective at maximizing cell seeding of small-animal organ scaffolds and maintaining cell survival in extended culture. We further demonstrate noninvasive monitoring capabilities for tracking dynamic changes within scaffolds as the native cellular component is removed during decellularization and model human cells are introduced into the scaffold during recellularization and proliferate in maintenance culture. We found that hydrodynamic pressure drop (ΔP) across the retained scaffold vasculature is a noninvasive measurement of scaffold integrity. We further show that ΔP, and thus resistance to fluid flow through the scaffold, decreases with cell loss during decellularization and correspondingly increases to near normal values for whole organs following recellularization of the kidney or liver scaffolds. Perfused media may be further sampled in real time to measure soluble biomarkers (e.g., resazurin, albumin, or kidney injury molecule-1) that indicate degree of cellular metabolic activity, synthetic function, or engraftment into the scaffold. Cell growth within bioreactors is validated for primary and immortalized cells, and the design of each bioreactor is scalable to accommodate any three-dimensional scaffold (e.g., synthetic or naturally derived matrix) that contains conduits for nutrient perfusion to deliver media to growing cells and monitor noninvasive parameters during scaffold repopulation, broadening the applicability of these bioreactor systems.

FIG. 1.

Detailed schematic representation of kidney recellularization bioreactor. Photographs of the kidney bioreactor (A, C–E) and cross-sectional schematics (B) illustrate the geometry and components of the bioreactor vessel: (a) flange, metallic clamp, and O-ring to seal the head and body of the bioreactor, (b) adjustable vacuum valve, (c) adjustable vent connected to sterile filter for gas exchange and pressure equilibration with the surrounding environment, (d) cap for media addition/removal and sampling in a biological safety cabinet, (e) inlet Luer acceptor to a cannula within the renal artery to perfuse the vasculature, (f) ureter Luer acceptor, (g) media perfusate outlet, (h) kidney scaffold, and (i) silicone rubber tubing to withdraw media into the bioreactor outlet and back to the recirculation pump. The reactor dimensions are shown in (B). Up to four replicate perfusion circuits can be run in parallel for each pump, as shown in (D). High-magnification image (E) shows where the medium flows into the kidney scaffold through the renal artery and may freely flow out from the scaffold (h) through the open renal vein (data not shown). Color images available online at www.liebertpub.com/tec

FIG. 2.

Detailed schematic representation of liver recellularization bioreactor. Photographs of the liver bioreactor (A, C–E) and cross-sectional schematics (B) illustrate the cylindrical geometry of the vessel: (a) flange, metallic clamp, and O-ring to seal the bioreactor, (b) adjustable vent connected to sterile filter for gas exchange and pressure equilibration with the environment, (c) adjustable valve for draining media, (d) cap for media addition/removal and sampling in a biological safety cabinet, (e) inlet Luer acceptor connected to a cannula in the portal vein used to perfuse hepatic vasculature, (f) media perfusate outlet, (g) liver scaffold, and (h) restraint to hold the liver scaffold below the liquid level. The reactor dimensions are shown in (B). High-magnification image (C) shows the catheterized hepatic portal vein where media flows into the liver scaffold (g); media may then freely flow out from the scaffold through the open vena cava where it mixes with the surrounding medium. The liver scaffold is submerged by the restraint (h), as shown in (D). Perfusion into the liver scaffold is controlled by a peristaltic pump, as shown in (E). Color images available online at www.liebertpub.com/tec

FIG. 3.

Pressure drop (ΔP) measurement system. The perfusion circuit used to measure ΔP in kidneys or livers is shown in a photograph (A) and diagram (B). Various components of the perfusion flow circuit and data acquisition apparatus are shown: (a) Fluid Metering, Inc. controllable step pump, (b) Honeywell pressure transducer (0–260 mmHg), (c) National Instruments chassis used to collect data from the amplifier, (d) custom glass vessel to contain the organ/scaffold within a reservoir containing physiologically equilibrated fluid phosphate-buffered saline that is perfused during evaluation, (e) Honeywell amplifier to convert transducer signal and transport data to chassis.

FIG. 4.

Comparison of pressure drop and morphological characteristics of kidney scaffolds during decellularization and recellularization. (A) The mean ΔP across the organ (mean±standard deviation) at a constant normalized flow rate of 1.35 mL·g⁻¹·min⁻¹ for native (n=17), decellularized (Decell; n=18), decellularized process control kidneys perfused with medium for 7 days without cells (Process; n=4), kidney scaffolds recellularized through the ureter after 3 days (day 3; n=8) or 7 days (day 7; n=3), or kidneys scaffolds recellularized through the renal artery after 3 days (day 3; n=6) or 7 days (day 7; n=4). ^#Over each bar indicates a significant difference (p<0.05) as determined by the Tukey HSD compared to Decell. *Indicates a significant difference (p<0.05) as determined by the Tukey HSD between the groups specified. (B) Macroscopic images of native and decellularized kidneys, and representative images of hematoxylin and eosin (H&E)-stained histological sections from each condition. Macroscopic image scale bars represent 5 mm, while H&E scale bars represent 100 μm. (C) The mean metabolic activity in artery-seeded (n=3) or ureter-seeded (n=3) scaffolds at days 1, 3, 5, or 7 after seeding. Indicates a significant difference in metabolic activity in artery-seeded scaffolds compared to all other time points evaluated. Calculated number of cells, based on a standard curve as described in the Supplemental Materials and Methods section, is shown in the secondary vertical axis. HSD, honestly significant difference. Color images available online at www.liebertpub.com/tec

FIG. 5.

Characterization of morphological adaptation, sustained viability, and decrease in injury biomarker expression by CD133/1⁺ cell-repopulated kidney scaffolds. (A) CD133/1⁺ cells repopulated renal cortical tubules but were not found in glomeruli (g) 3 days after seeding. After 7 days of culture, CD133/1⁺ cells aggregated to form tubule-like structures (arrows). (B) One hour resazurin perfusion assay performed every 2 days indicates that CD133/1⁺ cells remained viable within the kidney scaffold over 7 days with no statistically significant deviation during this time course (p>0.05 by ANOVA). (C) Injury biomarker kidney injury molecule-1 (KIM-1) release from CD133/1⁺ cells decreases over 7 days suggesting adaptation and engraftment into renal scaffolds. ^#Denotes a significant difference as determined by the Tukey-HSD relative to all other time points (p<0.05). Data are presented as mean±standard deviation (n=3–4 replicates per time point). Color images available online at www.liebertpub.com/tec

FIG. 6.

Comparison of pressure drop and morphological characteristics of liver scaffolds during decellularization and recellularization. (A) The mean ΔP across the organ (mean±standard deviation) for native whole (n=16), decellularized whole (n=11), decellularized right lobe (n=14), and recellularized right lobe livers at 3 days (day 3, n=4) and 7 days (day 7, n=4) at a constant normalized flow rate of 0.72 mL·g⁻¹·min⁻¹. Asterisk indicates a significant difference between native and decellularized whole livers as determined by the Student's t-test (p<0.05). Indicates a significant difference as determined by one-way ANOVA (p=0.05). (B) Macroscopic images of native and decellularized whole livers, and representative images of H&E-stained histological sections from each condition. Macroscopic image scale bars represent 15 mm, while H&E scale bars represent 100 μm. (C) The mean metabolic activity in recellularized scaffolds at days 1, 3, 5, or 7 after seeding. Indicates a significant difference in metabolic activity between time points as indicated. Calculated number of cells, based on a standard curve as described in the Supplemental Materials and Methods section, is shown in the secondary vertical axis. (D) Cumulative albumin production by HepG2 cells within recellularized scaffolds during 1 week of perfusion culture. ^#Indicates a significant difference relative to all other time points as determined by Tukey-HSD (p<0.05). Color images available online at www.liebertpub.com/tec