Mariah S. Hout

Mouse Fetal Liver Cells in Artificial Capillary Beds in Three-Dimensional Four-Compartment Bioreactors

Bioreactors containing porcine or adult human hepatocytes have been used to sustain acute liver failure patients until liver transplantation. However, prolonged function of adult hepatocytes has not been achieved due to compromised proliferation and viability of adult cells in vitro. We investigated the use of fetal hepatocytes as an alternative cell source in bioreactors. Mouse fetal liver cells from gestational day 17 possessed intermediate differentiation and function based on their molecular profile. When cultured in a three-dimensional four-compartment hollow fiber-based bioreactor for 3 to 5 weeks these cells formed neo-tissues that were characterized comprehensively. Albumin liberation, testosterone metabolism, and P450 induction were demonstrated. Histology showed predominant ribbon-like three-dimensional structures composed of hepatocytes between hollow fibers. High positivity for proliferating cell nuclear antigen and Ki-67 and low positivity for terminal dUTP nick-end labeling indicated robust cell proliferation and survival. Most cells within these ribbon arrangements were albumin-positive. In addition, cells in peripheral zones were simultaneously positive for alpha-fetoprotein, cytokeratin-19, and c-kit, indicating their progenitor phenotype. Mesenchymal components including endothelial, stellate, and smooth muscle cells were also observed. Thus, fetal liver cells can survive, proliferate, differentiate, and function in a three-dimensional perfusion culture system while maintaining a progenitor pool, reflecting an important advance in hepatic tissue engineering.

Development of a low flow resistance intravenous oxygenator.

A potentially attractive support device for patients with acute respiratory failure is an intravenous membrane oxygenator. One problem, however, is that the membrane surface area required for sufficient gas exchange can unduly increase vena cavai pressure drop and impede venous return. The purpose of this study was to design and develop an intravenous oxygenator that would offer minimal venous flow resistance in situ. The device uses a constrained fiber bundle of smaller cross sectional size than the vena cava so as to effect an intentional shunt flow of venous blood around the fiber bundle and reduce the venous pressure drop caused by the device. A pulsating balloon within the fiber bundle redirects part of this shunt flow into reciprocating flow in and out of the fiber bundle. This offers dual advantages: 1) Blood flow through the fiber bundle is mainly perpendicular to the fibers; and 2) the requisite energy for driving flow comes largely

Recent progress in engineering the Pittsburgh intravenous membrane oxygenator.

The University of Pittsburgh intravenous membrane oxygenator (IMO) is undergoing additional engineering development and characterization. The focus of these efforts is an IMO device that can supply as much as one-half basal O2 consumption and CO2 elimination rates while residing within the inferior and superior vena cavae after peripheral venous insertion. The current IMO design consists of a bundle of hollow fiber membranes potted to manifolds at each end, with an intra-aortic type balloon integrally situated within the fiber bundle. Pulsation of the balloon using helium gas and a balloon pump console promotes fluid and fiber motion and enhances gas exchange. During the past year, more than 15 IMO prototypes have been fabricated and extensively bench tested to characterize O2 gas exchange capacity, balloon inflation/deflation over relevant frequency ranges, and the pneumatics of the sweep gas pathway through the device. The testing has led to several engineering changes, including redesign of the helium and sweep gas pathways within the IMO device. As a result, the maximum rate of balloon pulsation has increased substantially above the previous 70 bpm to 160 bpm, and the vacuum pressure required for sufficient sweep gas flow has been reduced. The recent IMO prototypes have demonstrated an O2 exchange capacity of as much as 90 ml/min/m2 in water, which appears within 70% of our design goal when extrapolated to scaled up devices in blood.

Interwoven Four-Compartment Capillary Membrane Technology for Three-Dimensional Perfusion with Decentralized Mass Exchange to Scale Up Embryonic Stem Cell Culture

We describe hollow fiber-based three-dimensional (3D) dynamic perfusion bioreactor technology for embryonic stem cells (ESC) which is scalable for laboratory and potentially clinical translation applications. We added 2 more compartments to the typical 2-compartment devices, namely an additional media capillary compartment for countercurrent ‘arteriovenous’ flow and an oxygenation capillary compartment. Each capillary membrane compartment can be perfused independently. Interweaving the 3 capillary systems to form repetitive units allows bioreactor scalability by multiplying the capillary units and provides decentralized media perfusion while enhancing mass exchange and reducing gradient distances from decimeters to more physiologic lengths of <1 mm. The exterior of the resulting membrane network, the cell compartment, is used as a physically active scaffold for cell aggregation; adjusting intercapillary distances enables control of the size of cell aggregates. To demonstrate the technology, mouse ESC (mESC) were cultured in 8- or 800-ml cell compartment bioreactors. We were able to confirm the hypothesis that this bioreactor enables mESC expansion qualitatively comparable to that obtained with Petri dishes, but on a larger scale. To test this, we compared the growth of 129/SVEV mESC in static two-dimensional Petri dishes with that in 3D perfusion bioreactors. We then tested the feasibility of scaling up the culture. In an 800-ml prototype, we cultured approximately 5 × 109 cells, replacing up to 800 conventional 100-mm Petri dishes. Teratoma formation studies in mice confirmed protein expression and gene expression results with regard to maintaining ‘stemness’ markers during cell expansion.

Specific removal of anti-A and anti-B antibodies by using modified dialysis filters

Removal of anti-A and anti-B blood group antibodies from human blood has been shown to facilitate cross-matched kidney transplantation by preventing hyperacute rejection. Patients in these studies had anti-A and anti-B antibodies removed by using plasmapheresis, followed by immunoadsorption onto packed bead columns. We conducted a study to assess the feasibility of selectively removing anti-A and anti-B antibodies directly from blood by using modified dialysis filters. An anti-A and anti-B specific antigen was covalently attached to the lumenal surfaces of hollow fibers within selected commercial dialysis modules. The filters were able to reduce the anti-A and anti-B titers of 300 ml of blood to 2 or below. A low molecular weight fraction of our antigen system was found to have no antibody binding capacity. The standard antigen was purified by removal of the low molecular weight fraction and a dialysis filter was modified by using the purified antigen. This filter displayed a six-fold higher capacity than a dialysis filter modified with the same mass of standard antigen. We conclude that selective blood group antibody removal by antigen modified dialysis filters is feasible and may be a simpler system than plasmapheresis followed by immunoadsorption.

Mathematical and experimental analyses of antibody transport in hollow-fiber-based specific antibody filters.

We are developing hollow fiber‐based specific antibody filters (SAFs) that selectively remove antibodies of a given specificity directly from whole blood, without separation of the plasma and cellular blood components and with minimal removal of plasma proteins other than the targeted pathogenic antibodies. A principal goal of our research is to identify the primary mechanisms that control antibody transport within the SAF and to use this information to guide the choice of design and operational parameters that maximize the SAF‐based antibody removal rate. In this study, we formulated a simple mathematical model of SAF‐based antibody removal and performed in vitro antibody removal experiments to test key predictions of the model. Our model revealed three antibody transport regimes, defined by the magnitude of the Damköhler number Da (characteristic antibody‐binding rate/characteristic antibody diffusion rate): reaction‐limited (Da≤ 0.1), intermediate (0.1 < Da < 10), and diffusion‐limited (Da ≥ 10). For a given SAF geometry, blood flow rate, and antibody diffusivity, the highest antibody removal rate was predicted for diffusion‐limited antibody transport. Additionally, for diffusion‐limited antibody transport the predicted antibody removal rate was independent of the antibody‐binding rate and hence was the same for any antibody‐antigen system and for any patient within one antibody‐antigen system. Using SAF prototypes containing immobilized bovine serum albumin (BSA), we measured anti‐BSA removal rates consistent with transport in the intermediate regime (Da ∼3). We concluded that initial SAF development work should focus on achieving diffusion‐limited antibody transport by maximizing the SAF antibody‐binding capacity (hence maximizing the characteristic antibody‐binding rate). If diffusion‐limited antibody transport is achieved, the antibody removal rate may be raised further by increasing the number and length of the SAF fibers and by increasing the blood flow rate through the SAF.