Extracapillary Space Research Articles

Optimal metabolite transport in hollow fibre membrane bioreactors

Hollow fibre membrane bioreactors provide a fast and efficient method for engineering functional tissue for use in medical treatments. Flow is utilised to overcome mass transport limitations by perfusing a nutrient-rich culture medium through the fibre lumen, which can then transport along the fibre lumen or across the porous membrane wall. Cells seeded at the outer membrane wall consume the nutrient and subsequently produce waste metabolites, which are transported away through an external extra-capillary space (ECS) along with excess nutrient. We present and investigate a two-dimensional axisymmetric model for fluid flow and solute transport through a single-fibre bioreactor configuration, with cells seeded to the external fibre wall. Fluid flow is modelled by steady lubrication and Darcy equations, which are coupled to the solute transport problem modelled by a system of advection–diffusion equations, supplemented with a reaction term to model the cell layer. Our model analysis reveals how spatially varying wall permeability distributions can be utilised to provide uniform nutrient delivery to a spatially uniform, homogeneous cell population. We also reveal how maximising the transmural pressure drop across the membrane wall is the dominant mechanism for waste removal rather than traditional experimental methods of flushing the ECS.

Biomechanical determinants of endothelial permeability assessed in standard and modified hollow-fibre bioreactors.

Effects of mechanical stress on the permeability of vascular endothelium are important to normal physiology and in the development of atherosclerosis. Here we elucidate novel effects using commercially available and modified hollow-fibre bioreactors, in which endothelial cells form confluent monolayers lining plastic capillaries with porous walls, contained in a cartridge. The capillaries were perfused with a near-aortic waveform, and permeability was assessed by the movement of rhodamine-labelled albumin from the intracapillary to the extracapillary space. Permeability was increased by acute application of shear stress and decreased by chronic shear stress compared with a static control: this has previously been shown only for multidirectional flows. Increasing viscosity reduced permeability under both acute and chronic shear; since shear rate remained unchanged, these effects resulted from altered shear stress. Reducing pulsatility increased permeability, contrary to the widely held assumption that flow which is highly oscillatory causes endothelial dysfunction. Chronic convection across the monolayer increased effective permeability more than could be explained by the addition of advective transport, contrary to results from previous acute experiments. The off-the-shelf and modified bioreactors provide an excellent tool for investigating the biomechanics of endothelial permeability and have revealed novel effects of flow duration, viscosity, pulsatility and transmural flow.

Hollow fiber membrane supported metal organic framework-based packed bed for gas/vapor adsorption

Crystalline metal–organic frameworks (MOFs) with high porosity have high sorption capacities for various gases. Their fragile and pulverulent characteristics have prompted significant efforts to prepare shaped bodies e.g., pellets, granules for use in adsorbers. A hollow fiber membrane-based strategy is adopted since hollow fiber membrane (HFM) modules are highly preferred for industrial separation processes due to very high surface area provided per unit device volume and their easy scalability. We report herein a solvothermal synthesis method whereby nanocrystals of the MOF, UiO-66-NH2, are synthesized directly inside submicron pores of hydrophilic hollow fiber membranes of Nylon 6 as well as in the bores of the HFMs. Nanocrystals of around 100 nm populate HFM pores. Cylindrical modules containing such HFMs and MOF nanocrystals and microcrystals in membrane pores, HFM bores and the extra capillary space were studied for adsorption of ammonia from a dilute gas stream. High values of ammonia breakthrough time were achieved. The corresponding behaviors of three MOF configurations namely, MOF in membrane pores, MOF in membrane pores and the HFM bores and MOF present in membrane pores, HFM bores and in extra capillary space were studied. The values of time/MOF weight achieved were very high. The MOFs synthesized were characterized by Scanning electron microscopy (SEM), Fourier-transform infrared spectroscopy (FTIR), powder X-ray diffractometer (PXRD), Brunauer-Emmett-Teller (BET) adsorption isotherms, surface area, and pore size distribution. High performance of HFM-supported MOF-based scalable devices for gas/vapor adsorption has been demonstrated with values of 20,000 min/g of MOF for trace ammonia breakthrough from humid ammonia feed gas stream employed. Other potential uses of such devices for adsorbing 2 to 3 gases and liquid phase adsorption have also been discussed.

Diametrical Effects of Glucose Levels on Microvascular Permeability of Peripheral Nerves in Patients With Type 2 Diabetes With and Without Diabetic Neuropathy.

Clinical studies investigating the benefit of glucose control on the progression of diabetic neuropathy (DN) have come to controversial results in patients with type 2 diabetes (T2D). This study aimed to assess associations of HbA1c levels with parameters of nerve perfusion in patients with T2D with and without DN using dynamic contrast-enhanced magnetic resonance neurography (DCE-MRN) at 3 Tesla. A total of 58 patients with T2D (20 with DN and 38 without DN) took part in this cross-sectional single-center study. Groups were matched for age, BMI, HbA1c, duration of T2D, and renal function. All patients underwent DCE-MRN with subsequent electrophysiologic and serologic testing. The extended Tofts model was used to quantify the sciatic nerve's microvascular permeability (Ktrans), volume fraction of the extracapillary extracellular space, and volume fraction of the plasma space. As a main result, we found that Ktrans correlated positively with HbA1c in patients with DN, while a negative correlation between the two parameters was found in patients without DN. Our results indicate that the effect of glucose control on the capillary permeability of peripheral nerves differs between patients with T2D with and without DN.

Towards the biomimetic design of hollow fiber membrane bioreactors for bioartificial organs and tissue engineering: A micro-computed tomography (μCT) approach

Hollow fiber membrane bioreactors (HFMBs) with cells cultured in the extracapillary space (ECS) have been proposed for bioartificial organs, to assist patients with failing organs, or to produce in vitro engineered biological substitutes of tissues and organs. They have not gained clinical acceptance yet. One factor limiting therapeutic application is the irregular membrane distribution in the HFMB shell, often considered a typical feature of clinical-scale HFMBs. Such distribution does not permit good control of shell spaces, prevents from offering cells a template structure mimicking the tissue-specific extracellular matrix (ECM) and an adequate supply of oxygen and nutrients, and limits control over cell migration, organization, and differentiation in the ECS. In this study, micro-computed tomography and image analysis techniques were used to characterize the space distribution in the shell of HFMBs varying for membrane packing density and bundling technique, and to investigate whether and how it is possible to manufacture HFMBs in which the distribution of intermembrane spaces in the ECS is uniform and biomimetic. Results suggest that the arrangement in HFMBs of hollow fiber membranes bundled in rolled cross-woven mats at high packing density permits to obtain a uniform shell-side membrane distribution with pore size distribution favoring cells migration around the membranes, and mimicking the ECM structure of bone tissue. • HFMBs were equipped with membranes in loose bundles or in cross-woven mats at varying packing density in the ECS. • Voids distribution in ECS was characterized by μCT and image analysis and was compared to natural bone and a synthetic bone substitute. • Results show that in HFMBs with densely packed membranes in cross-woven mats voids distribution is uniform and mimics bone ECM.

Drug Sensitivity Testing of Mycobacterium tuberculosis Growing in a Hollow Fiber Bioreactor.

Hollow fiber systems (HFSs) have been widely applied to study pharmacokinetic-pharmacodynamic (PK-PD) relationships in antibiotic research and development. The system comprises a bundle of high-density hollow capillary fibers that conduct a flow of medium with or without drug and an extra-capillary space (ECS) inoculated with the pathogen of interest. The semipermeable membrane of the hollow fibers allows for rapid exchange of small molecule drugs and solutes, while the pathogen is restricted to the ECS. The unique properties of the HFS are (1) the ability to simulate any PK profile within the fibers and ECS, including plasma or site-of-disease PK profiles, (2) the ability to simultaneously input several drugs with different half-lives, (3) the ability to manipulate growth conditions such as medium composition, carbon source, and pH, and (4) the ability to sample in both compartments in order to monitor drug concentrations and bacterial growth kinetics over time. The system is particularly suited for Mycobacterium tuberculosis research in a biosafety level 3 (BSL3) environment since pathogenic bacteria are sequestered in an isolated compartment. The HFS was qualified by the European Medicines Agency for antituberculosis drug development in 2015. Here, we describe the standard procedures used to study the growth kinetics of M. tuberculosis in the HFS and the killing effect of first-line antituberculous drugs applied under simulated human PK conditions. This animal-sparing and economical tool can be applied to optimize dosing schedules that minimize emergence of resistance and to prioritize drug regimens that accelerate sterilization.

On modelling of glucose transport in hollow fibre membrane bioreactor for growing three‐dimensional tissue

AbstractHollow fibre membrane bioreactors (HFMBs) have been shown to overcome the diffusion limitation of nutrients (e.g., glucose) from the hollow fibres (lumens) to the porous regions of a scaffold (extracapillary space). However, direct monitoring of glucose diffusion inside the HFMBs is almost impossible because of their small size; thus, various computational modelling frameworks have been developed in the past. These models have defined that the glucose diffusivity in the cell culture medium used in the HFMBs was similar to the diffusivity in water. Similarly, other assumptions have been made that do not represent the nutrient transport processes in the HFMB accurately. In addressing these issues, a mathematical model is presented in this paper, where we employ experimentally deduced effective glucose diffusivities of tissue engineering membranes and scaffolds with and without cells along with glucose diffusivity in cell culture medium. The governing equations are non‐dimensionalized, simplified and solved numerically. The results demonstrate the roles of various dimensionless numbers (e.g., Péclet and Damköhler numbers) and non‐dimensional groups of variables on determining the glucose concentration especially in the scaffold region. The result of this study is expected to help optimize designs of HFMB as well as carry out more accurate scaling analyses.

Next-generation polymerized human hemoglobins in hepatic bioreactor simulations.

Hepatic hollow fiber (HF) bioreactors can be used to provide temporary support to patients experiencing liver failure. Before being connected to the patient's circulation, cells in the bioreactor must be exposed to a range of physiological O2 concentrations as observed in the liver sinusoid to ensure proper performance. This zonation in cellular oxygenation promotes differences in hepatocyte phenotype and may better approximate the performance of a real liver within the bioreactor. Polymerized human hemoglobin (PolyhHb) locked in the tense quaternary state (T‐state) has the potential to both supply and regulate O2 transport to cultured hepatocytes in the bioreactor due to its low O2 affinity. In this study, T‐state PolyhHb production and purification processes were optimized to minimize the concentration of low‐molecular‐weight PolyhHb species in solution. Deconvolution of size‐exclusion chromatography spectra was performed to calculate the distribution of polymeric Hb species in the final product. Fluid flow and mass transport within a single fiber of a hepatic HF bioreactor was computationally modeled with finite element methods to simulate the effects of employing T‐state PolyhHb to facilitate O2 transport in a hepatic bioreactor system. Optimal bioreactor performance was defined as having a combined hypoxic and hyperoxic volume fraction in the extracapillary space of less than 0.05 where multiple zones were observed. The Damköhler number and Sherwood number had strong inverse relationships at each cell density and fiber thickness combination. These results suggest that targeting a specific Damköhler number may be beneficial for optimal hepatic HF bioreactor operation.

Modelling of hollow fiber membrane bioreactor for mammalian cell cultivation using computational hydrodynamics.

The hollow fiber membrane bioreactor (HFMB) has been investigated for the cultivation of mammalian Chinese hamster ovary cell expansion. The experiments were carried out in Petri's dishes and in the hollow fiber membrane bioreactor having 20 fibers (S2025 from FiberCell Systems). The approach to HFMB modelling which combines the model of cell growth kinetics and hydrodynamics has been proposed. The hydrodynamic model is made using ANSYS Fluent software. The mathematical model of HFMB was developed, allowing the study of the hydrodynamics into the lumen and the extracapillary spaces, the filtration through the membrane fiber with the cell expansion on outer membrane surface. The direct nutrient medium flow variant into the extracapillary space was suggested. Based on the numerical simulations, the optimal parameters were selected for daily changes in the medium flow-rate into the lumen space. The HFMB scaling up was performed for the larger size HFMB (60 fibers).

Membrane bioreactor for investigation of neurodegeneration.

To gain a better understanding of neurodegeneration mechanisms and for preclinical evaluation of new therapeutics more accurate models of neuronal tissue are required. Our strategy was based on the implementation of advanced engineered system, like membrane bioreactor, in which neurons were cultured in the extracapillary space of poly(l-lactic acid) (PLLA) microtube array (MTA) membranes within a dynamic device designed to recapitulate specific microenvironment of living neuronal tissue. The high membrane permeability and the optimized fluid dynamic conditions created by PLLA-MTA membrane bioreactor provide a 3D low-shear stress environment fully controlled at molecular level with enhanced diffusion of nutrients and waste removal that successfully develops neuronal-like tissue. This neuronal membrane bioreactor was employed as in vitro model of β-amyloid -induced toxicity associated to Alzheimer's disease, to test for the first time the potential neuroprotective effect of the isoflavone glycitein. Glycitein protected neurons from the events induced by β-amyloid aggregation, such as the production of ROS, the activation of apoptotic markers and ensuring the viability and maintenance of cellular metabolic activity. PLLA-MTA membrane bioreactor has great potential as investigational tool in preclinical research, contributing to expand the available in vitro devices for drug screening.

Restoring balance to the immune system via Ex Vivo MSC therapy

Background & Aim Mesenchymal stromal cells (MSCs) are shown to secrete potent immunomodulatory molecules and extracellular vesicles. However, delivery of these cells has yielded limited success in clinical applications, likely due to ineffective dosing and exposure times. To overcome these hurdles, Sentien Biotechnologies developed SBI-101, a continuous flow bioreactor with MSCs immobilized on the extraluminal side of a semi-permeable hollow-fiber membrane. Patient's blood flows through the lumen of the hollow fibers and is conditioned by MSC-secreted factors, allowing for the blood cells and MSCs to sense their environment and react to it. To study the effect of MSC-secreted factors delivered in this way, Sentien developed a scaled-down bioreactor system and explored its therapeutic efficacy through treatment of immune cell populations in vitro. Methods, Results & Conclusion MSCs were seeded into the extracapillary space of bioreactors at doses ranging from 0 (acellular control) to 9 million cells per device. Bioreactors were then perfused with media up to 8 days, with metabolite and cytokine analyses performed longitudinally. Data showed consumption of nutrients and production of cellular byproducts in a dose-dependent manner, indicating the maintenance of metabolically active MSCs over the perfusion time-course. Furthermore, MSCs were found to secrete a range of factors, including immunomodulatory and angiogenic, when immobilized in this way. Upon introduction of inflammatory stimuli to the in vitro system, MSCs modulated their secretion of factors and extracellular vesicles in response. Vesicle size and production of immunomodulatory factors, including IL-6 and PGE2, increased with stimuli. Activated PBMCs were then added to the perfusion medium to probe the effect of MSCs on inflamed blood cells. MSC treatment proved to regulate inflammatory milieu through modulation of cytokines and extracellular vesicles, suppressing T cell proliferation as well as decreasing cytotoxic and activated T cell populations. Conditioned medium and cells harvested from the perfusion circuits were further challenged via post-perfusion static assay. Treatment strategy was shown to influence lymphocyte and macrophage phenotype, proving a durable reprogramming effect. Thus, we report the ability of a hollow-fiber bioreactor to effectively maintain MSC function as well as impart an immunomodulatory effect on inflamed immune cell populations, laying the framework for a new generation of ex vivo cellular therapeutics.

Histological Image Analysis of Scaffold‐Based Cell Products to Estimate Percent Viability

IntroductionRegulatory agencies have an expectation that cell‐based products in clinical development be characterized for their percent (%) viability. However, many new cell‐based products do not readily lend themselves to determining this metric through classical methods such as enzymatic dissociation and cell enumeration. Vital Therapies, Inc. (VTL) uses four metabolically active cartridges containing VTL C3A cells in its investigational (phase 3) ELAD® System, an extracorporeal human allogeneic cellular treatment being evaluated for severe alcoholic hepatitis. This study objective was to develop and verify new image processing methods for automatically characterizing the % viability of ELAD C3A cell cartridge histological samples.MethodsELAD C3A cell cartridges were clinically manufactured by introducing VTL C3A cells into the extracapillary space and perfusing growth medium through the lumens of the porous polysulfone hollow fibers. At maturity, cartridges were fixed for histology by recirculating 10% neutral buffered formalin through the hollow fiber lumens for approximately 24 hr. Cross‐sectional discs (~2‐cm thick) were cut using a saw and further cut into 16‐grid samples prior to paraffin embedding. Thin (4‐mm) sections were sliced from the grid blocks, stained with hematoxylin and eosin, and imaged using a high‐resolution scanner (Olympus VS120 or Hamamatsu NanoZoomer 2.0). MATLAB software was utilized to develop a custom algorithm to automatically isolate, count, and mask the hollow fiber cross‐sections through image processing techniques (e.g., color thresholding and object feature segmentation). Grayscale histogram analysis of the differently stained cells in the remaining space was utilized to separate viable from non‐viable areas. The % viability for an entire histology grid image could then be reported as the integrated ratio of viable tissue area to total tissue area. This method was compared to blinded, independent mass measurements of printed and hand‐cut images to separate hollow fiber, viable tissue, and non‐viable tissue areas. Additional comparisons were made against blinded visual assessment.ResultsThis methodology demonstrated that reliable % viability estimates could be estimated from histological images despite fixation and/or sectioning artifacts (e.g., tissue separating from the hollow fibers). The automated image‐processing method was able to complete the analysis very rapidly (e.g., an 8000×8000 pixel image containing over 600 fibers is analyzed in a couple minutes), such that a large library of histological samples could be completed quickly and the results tallied for inter‐grid, intra‐ and inter‐cartridge, and intra‐ and inter‐lot comparisons.ConclusionsNew cell‐based products require creative approaches to address regulatory expectations. The image processing algorithm developed herein was shown to be in agreement with, and more reproducible and rapid than, visual or manual assessments. It was further recognized that this method could be extended to other types of histological stains (e.g., trichrome) or to other types of biomaterial/scaffold‐based tissue‐engineered products.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Mixtures of tense and relaxed state polymerized human hemoglobin regulate oxygen affinity and tissue construct oxygenation.

Pure tense (T) and relaxed (R) quaternary state polymerized human hemoglobins (PolyhHbs) were synthesized and their biophysical properties characterized, along with mixtures of T- and R-state PolyhHbs. It was observed that the oxygen affinity of PolyhHb mixtures varied linearly with T-state mole fraction. Computational analysis of PolyhHb facilitated oxygenation of a single fiber in a hepatic hollow fiber (HF) bioreactor was performed to evaluate the oxygenation potential of T- and R-state PolyhHb mixtures. PolyhHb mixtures with T-state mole fractions greater than 50% resulted in hypoxic and hyperoxic zones occupying less than 5% of the total extra capillary space (ECS). Under these conditions, the ratio of the pericentral volume to the perivenous volume in the ECS doubled as the T-state mole fraction increased from 50 to 100%. These results show the effect of varying the T/R-state PolyhHb mole fraction on oxygenation of tissue-engineered constructs and their potential to oxygenate tissues.

Anisotropic diffusion phantoms based on microcapillaries

Diffusion MRI is an efficient and widely used technique for the investigation of tissue structure and organisation in vivo. Multiple phenomenological and biophysical diffusion models are intensively exploited for the analysis of the diffusion experiments. However, the verification of the applied diffusion models remains challenging. In order to provide a “gold standard” and to assess the accuracy of the derived parameters and the limitations of the diffusion models, anisotropic diffusion phantoms with well known architecture are demanded. In the present work we built four anisotropic diffusion phantoms consisting of hollow microcapillaries with very small inner diameters of 5, 10 and 20μm and outer diameters of 90 and 150μm. For testing the suitability of these phantoms, we performed diffusion measurements on all of them and evaluated the resulting data with a set of popular diffusion models, such as diffusion tensor and diffusion kurtosis imaging, a two compartment model with intra- and extra-capillary water spaces using bi-exponential fitting, and time-dependent diffusion coefficients in Mitra’s limit. The perspectives and limitations of these diffusion phantoms are presented and discussed.

Mathematical modelling of cell layer growth in a hollow fibre bioreactor

Generating autologous tissue grafts of a clinically useful volume requires efficient and controlled expansion of cell populations harvested from patients. Hollow fibre bioreactors show promise as cell expansion devices, owing to their potential for scale-up. However, further research is required to establish how to specify appropriate hollow fibre bioreactor operating conditions for expanding different cell types. In this study we develop a simple model for the growth of a cell layer seeded on the outer surface of a single fibre in a perfused hollow fibre bioreactor. Nutrient-rich culture medium is pumped through the fibre lumen and leaves the bioreactor via the lumen outlet or passes through the porous fibre walls and cell layer, and out via ports on the outer wall of the extra-capillary space. Stokes and Darcy equations for fluid flow in the fibre lumen, fibre wall, cell layer and extra-capillary space are coupled to reaction–advection–diffusion equations for oxygen and lactate transport through the bioreactor, and to a simple growth law for the evolution of the free boundary of the cell layer. Cells at the free boundary are assumed to proliferate at a rate that increases with the local oxygen concentration, and to die and detach from the layer if the local fluid shear stress or lactate concentration exceed critical thresholds. We use the model to predict operating conditions that maximise the cell layer growth for different cell types. In particular, we predict the optimal flow rate of culture medium into the fibre lumen and fluid pressure imposed at the lumen outlet for cell types with different oxygen demands and fluid shear stress tolerances, and compare the growth of the cell layer when the exit ports on the outside of the bioreactor are open with that when they are closed. Model simulations reveal that increasing the inlet flow rate and outlet fluid pressure increases oxygen delivery to the cell layer and, therefore, the growth rate of cells that are tolerant to high shear stresses, but may be detrimental for shear-sensitive cells. The cell layer growth rate is predicted to increase, and be less sensitive to the lactate tolerance of the cells, when the exit ports are opened, as the radial flow through the bioreactor is enhanced and the lactate produced by the cells cleared more rapidly from the cell layer.

English

The performance of a hollow-fiber membrane bioreactor (HFBR) (molecular weight cut-off 30 kD, fiber surface area 2050 cm2) containing a culture of hybridoma cells has been investigated. Experimental data were used as basis to develop a model of general application. Concentrations of fundamental nutrients (glucose and glutamine), inhibitory products (ammonium and lactate), and monoclonal antibodies (MAb) against bovine lactoferrin (IgG1) were monitored over time. Exchange of nutrients and products occurred across the capillary surface, whereas cells and MAb remained in the extra-capillary space (ECS). A protein-free culture medium (Hybrimax) with and without antibiotics was used. In both cases, the final MAb concentration was the same; however, antibiotic presence slowed down the time to achieve this concentration. Diffusion assays have been carried out in order to support the development of a mathematical model that describes the performance of the HFBR, including mass transfer and reaction terms. Inhibition by ammonium and lactate has been considered in the kinetics, providing model results consistent with experimental data. Further research with other cell lines and/or culture media will allow to broaden the field of application of this model for general use in HFBR systems.

Bioinspired hollow fiber membrane bioreactor using human liver microtissue spheroids

Event Abstract Back to Event Bioinspired hollow fiber membrane bioreactor using human liver microtissue spheroids Loredana De Bartolo1, Haysam M. Ahmed1, 2, Simona Salerno1, Shervin Khakpour1, 2, Sabrina Morelli1 and Lidietta Giorno1 1 National Research Council of Italy - CNR, Institute on Membrane Technology, Italy 2 University of Calabria, Department of Environmental and Chemical Engineering, Italy Introduction: The development of bioartificial devices that are able to favour the liver reconstruction and to modulate cell behaviour is an important challenge in liver tissue engineering. As strategy we realized liver microtissue spheroids in a crossed hollow fiber membrane bioreactor, which was developed for the in vitro long-term perfusion. The bioreactor consists of two bundles of hollow fiber (HF) polyethersulfone (PES) membranes cross-assembled in alternating manner. One bundle of HF membranes provides cells nutrients and metabolites whereas the other HF bundle removes catabolites from cell compartment mimicking in this way the in vivo arterious and venous blood vessels. The combination of these two fiber set creates three compartments: two intraluminal compartments of PES HF in which the medium flows and one extraluminal compartment represented by extracapillary network formed by the fibers in which spheroids are cultured. Materials and Methods: The bioreactor consists of two bundles of PES HF membranes cross-assembled in alternating manner and potted with polyurethane adhesive within glass housing. The membranes were previously characterised in order to establish the physico-chemical, structural and transport properties[1]. The oxygenated medium enters to the membrane bioreactor with a flow rate Qf of 1.5 mL/min that was set on the basis of average retention time. Its fluid dynamics were characterized in terms of cumulative residence time distribution (RTD). Spheroids of primary human hepatocytes were realized in agarose mold and then cultured in the extralumen compartment of the bioreactor. The oxygen uptake, urea synthesis and albumin production rates as well as biotransformation functions were evaluated with time by using specific assays[2]. Numerical analysis of the mass transfer model for oxygen, urea and albumin concentration was performed using COMSOL Multiphysics. Results and Discussion: The geometry of the bioreactor ensured optimal perfusion conditions, which plaid a crucial role together with the surface and transport properties of the HF membranes in guiding the self-assembling of liver microtissue spheroids. Indeed, adjacent spheroids fused in the extracapillary space forming liver microtissues (Fig. 1). The liver microtissue spheroids expressed liver specific functions in terms of urea synthesis, albumin production and diazepam biotransformation at high levels up to 2 weeks. The liver specific functions of the bioreactor were significantly higher with respect to those of the batch system. Furthermore, the bioreactor provided an adequate oxygenation of the liver microtissue spheroids maintaining oxygen concentration levels well above the in vivo liver periportal zone concentration. Conclusion: The bioreactor thanks to its fluid dynamics and mass transfer properties of the fibers allowed to promote the formation liver microtissue spheroids with high specific functionality.

A multiphase model for chemically- and mechanically- induced cell differentiation in a hollow fibre membrane bioreactor: minimising growth factor consumption.

We present a simplified two-dimensional model of fluid flow, solute transport, and cell distribution in a hollow fibre membrane bioreactor. We consider two cell populations, one undifferentiated and one differentiated, with differentiation stimulated either by growth factor alone, or by both growth factor and fluid shear stress. Two experimental configurations are considered, a 3-layer model in which the cells are seeded in a scaffold throughout the extracapillary space (ECS), and a 4-layer model in which the cell-scaffold construct occupies a layer surrounding the outside of the hollow fibre, only partially filling the ECS. Above this is a region of free-flowing fluid, referred to as the upper fluid layer. Following previous models by the authors (Pearson et al. in Math Med Biol, 2013, Biomech Model Mechanbiol 1-16, 2014a, we employ porous mixture theory to model the dynamics of, and interactions between, the cells, scaffold, and fluid in the cell-scaffold construct. We use this model to determine operating conditions (experiment end time, growth factor inlet concentration, and inlet fluid fluxes) which result in a required percentage of differentiated cells, as well as maximising the differentiated cell yield and minimising the consumption of expensive growth factor.

Bioartificial Liver Device Based on Induced Pluripotent Stem Cell-Derived Hepatocytes

Decompensated liver disorders require liver transplantation. However, the donor organ shortage is a limiting factor. Harnessing the power of human induced pluripotent stem cell (iPSC) technology in combination with hollow fiber-based bioartificial liver (BAL) device can be beneficial to patients with liver failure. Our goal is to develop a BAL module comprised of iPSC-derived hepatocytes (iHeps) arrayed on the extracapillary space (ECS) of hollow fiber membranous capillaries that allow the flow of blood through the intracapillary space (ICS), thus mimicking the tissue microarchitecture. For the proof-of-concept in vitro study, a cartridge having semipermeable polysulfone membrane fibers was used as an artificial liver device. As a source for human liver cells, we derived metabolically active hepatocytes from iPSCs. The iHeps on microcarrier beads were loaded into the ECS of a hollow fiber bioreactor cartridge and cultured using a closed-circuit continuous flow system. The iHeps secreted human albumin, prothrombin, and apolipoprotein B into the hollow fiber ICS media, and the continuous flow system also improved maturation of iHeps. In conclusion, the iPSC-hepatocytes in the bioartificial liver device maintained the secretory function and exhibited cell maturation. The iPSC-hepatocyte BAL has the potential to be further developed as a liver support device for the treatment of decompensated liver diseases.

Multiphase modelling of the effect of fluid shear stress on cell yield and distribution in a hollow fibre membrane bioreactor.

We present a simplified two-dimensional model of fluid flow, nutrient transport and cell distribution in a hollow fibre membrane bioreactor, with the aim of exploring how fluid flow can be used to control the distribution and yield of a cell population which is sensitive to both fluid shear stress and nutrient concentration. The cells are seeded in a scaffold in a layer on top of the hollow fibre, only partially occupying the extracapillary space. Above this layer is a region of free-flowing fluid which we refer to as the upper fluid layer. The flow in the lumen and upper fluid layer is described by the Stokes equations, whilst the flow in the porous fibre membrane is assumed to follow Darcy’s law. Porous mixture theory is used to model the dynamics of and interactions between the cells, scaffold and fluid in the cell–scaffold construct. The concentration of a limiting nutrient (e.g. oxygen) is governed by an advection–reaction–diffusion equation in each region. Through exploitation of the small aspect ratio of each region and asymptotic analysis, we derive a coupled system of partial differential equations for the cell volume fraction and nutrient concentration. We use this model to investigate the effect of mechanotransduction on the distribution and yield of the cell population, by considering cases in which cell proliferation is either enhanced or limited by fluid shear stress and by varying experimentally controllable parameters such as flow rate and cell–scaffold construct thickness.