Rebecca H. Li

Stability of hydrogels used in cell encapsulation : An in vitro comparison of alginate and agarose

The present studies were undertaken to evaluate the in vitro gel stability of the hydrogels alginate and agarose. Gel strength (of alginate and agarose) and protein diffusion (of alginate only) were shown to correlate with gel stability and to be useful techniques to monitor gel stability over time. The gel strengths of alginate and agarose were followed for a 90‐day period using gel strength as a measure of gel stability. The gel strength of agarose diminished in the presence of cells because the cells likely interfered with the hydrogen bond formation required for agarose gelation. In the presence of cells, the gel strength of agarose decreased by an average of 25% from time 0 to 60 days, thereafter maintaining that value to 90 days. The gel strength of calcium‐ or barium‐crosslinked alginate decreased over 90 days, with an equilibrium gel strength being achieved after 30 days. The presence of cells did not further decrease alginate gel strength. The gel strengths of calcium‐ and barium‐crosslinked alginates were similar at 60 days—350 ± 20 g and 300 ± 60 g, respectively—indicating equivalence in their stability. The stability of calcium‐crosslinked sodium alginate gels over a 60‐day time period was monitored by diffusion of proteins ranging in molecular weight from 14.5 to 155 kD. From these diffusion measurements, the average pore size of the calcium‐crosslinked alginate gels was estimated, using a semi‐empirical model, to increase from ∼176 to 289 Å over a period of 60 days.

Transport characterization of hydrogel matrices for cell encapsulation

Current membrane‐based bioartificial organs consist of three basic components: (1) a synthetic membrane, (2) cells that secrete the product of interest, and (3) an encapsulated matrix material. Alginate and agarose have been widely used to encapsulate cells for artificial organ applications. It is important to understand the degree of transport resistance imparted by these matrices in cell encapsulation to determine if adequate nutrient and product fluxes can be obtained. For artificial organs in xenogeneic applications, it may also be important to determine the extent of immunoprotection offered by the matrix material. In this study, diffusion coefficients were measured for relevant solutes [ranging in size from oxygen to immunoglobulin G (IgG)] into and out of agarose and alginate gels. Alginate gels were produced by an extrusion/ionic crosslinking process using calcium while agarose gels were thermally gelled. The effect of varying crosslinking condition, polymer concentration, and direction of diffusion on transport was investigated. In general, 2–4% agarose gels offered little transport resistance for solutes up to 150 kD, while 1.5–3% alginate gels offered significant transport resistance for solutes in the molecular weight range 44–155 kD—lowering their diffusion rates from 10‐ to 100‐fold as compared to their diffusion in water. Doubling the alginate concentration had a more significant effect on hindering diffusion of larger molecular weight species than did doubling the agarose concentration. Average pore diameters of approximately 170 and 147 Å for 1.5 and 3% alginate gels, respectively, and 480 and 360 Å for 2 and 4% agarose gels, respectively, were estimated using a semiempirical correlation based on diffusional transport of different‐size solutes. The method developed for measuring diffusion in these gels is highly reproducible and useful for gels crosslinked in the cylindrical geometry, relevant for studying transport through matrices used in cell immobilization in the hollow fiber configuration.

Transport characterization of membranes for immunoisolation

This study relates to the diffusive transport characterization of hollow fibre membranes used in implantable bio-hybrid organs and other immunoisolatory devices. Techniques were developed to accurately determine the mass transfer coefficients for diffusing species in the 10(2)-10(5) MW range, validated and then used to study one membrane type known to effectively immunoisolate both allografts and xenografts in vivo. Low-molecular-weight diffusing markers included glucose, vitamin B12 and cytochrome C; higher-molecular-weight molecules were bovine serum albumin, immunoglobulin G, apoferritin and a range of fluorescein-tagged dextrans. Overall and fractional mass transfer coefficients through the hollow fibres were determined using a resistance-in-series model for transport. A flowing dialysis-type apparatus was used for the small-molecular-weight diffusants, whereas a static diffusion chamber was used for large-molecular-weight markers. For diffusion measurements of small-molecular-weight solutes, convective artefacts were minimized and the effect of boundary layers on both sides of the membrane were accounted for in the model. In measuring diffusion coefficients of large-molecular-weight species, boundary layer effects were shown to be negligible. Results showed that for small-molecular-weight species (< 13,000 MW) the diffusion coefficient in the membrane was reduced relative to diffusion in water by two to four times. The diffusion rate of large-molecular-weight species was hindered by several thousand-fold over their rate of diffusion in water.

Poly(vinyl alcohol) synthetic polymer foams as scaffolds for cell encapsulation.

Poly(vinyl alcohol) (PVA) foams were used as scaffolds in hollow fiber membrane-based cell encapsulation devices. The surrounding permselective membrane serves as an immunoisolation barrier while allowing metabolites and other small molecules to be freely transported. The internal matrix defines the microenvironment for the encapsulated cells. PC12 cell-containing devices represent one possible strategy for safe transplantation of dopamine-secreting cells for the treatment of dopamine-deficient diseases such as Parkinsons disease. PC12 cells--a dopamine-secreting cell line--were encapsulated with PVA foam as a matrix material in the lumen of these hollow fibers. In this work, we demonstrate the presence of the PVA matrix increased the catecholamine secretion efficiency of the cells as compared to devices containing a chitosan matrix. Devices were implanted in vivo into rodent striatum and device output of catecholamines was measured preimplant and post-explant. Evoked stores of dopamine remained constant (preimplant vs explant) for devices encapsulated with the foam matrix and increased with devices encapsulated with chitosan matrix. Cell proliferation within devices was inhibited in the presence of the foam matrix. Cell viability and distribution was significantly improved with the inclusion of the foam matrix in both in vitro and in vivo studies. In comparison to chitosan--a typical matrix material for PC12 cells--addition of a foam-type matrix altered the encapsulated cell microenvironment and resulted in more efficient secretion of catecholamines and improved distribution within the device resulting in smaller necrotic regions and a lower rate of cell proliferation.

Encapsulation Matrices for Neurotrophic Factor-Secreting Myoblast Cells

Encapsulated-cell therapy is an emerging technology that entails implantation of cell-containing devices that secrete therapeutic factors. One potential application of this technology is the delivery of neurotrophic factors to treat neurodegenerative disease. These devices typically use an internal matrix to serve as a cell scaffold. This study compares collagen-coated polyethylene terephthalate (PET) yarn scaffold versus collagen as a matrix for engineered C2C12 myoblasts. C2C12 cells transfected to secrete ciliary neurotrophic factor (CNTF) were immobilized in matrices and encapsulated into hollow fiber membrane devices. Encapsulated cells were monitored in vitro for viability, morphology, and factor secretion. Two independent methods (histology assessment and metabolic assay) were used to estimate viable cell density; a high correlation between the methods was found. After 4 weeks, encapsulated devices with PET scaffold had an almost nine-fold greater number of viable cells compared to collagen. PET matrix devices contained a thick annulus of compact, highly oriented cells. Collagen matrix devices contained sparse viable cells in a thin rim. Secretion assays showed cells in PET matrix released approximately four-fold the amount of CNTF versus cells in collagen (averaging 542 and 129 ng/day per device for PET and collagen matrix, respectively). The choice of encapsulation matrix was found to have a profound effect on cell morphology, level of secreted factor, and viability of encapsulated C2C12 cells.

DESIGN OF MEMBRANE-BASED BIOARTIFICIAL ORGANS

The goal of encapsulated cell therapy research is to develop implants containing living xenogeneic cells to treat serious and disabling human conditions. The concept is straightforward: cells or small clusters of tissue are surrounded by a selective membrane barrier which admits oxygen and required metabolites, releases bioactive cell secretions but restricts the transport of the larger cytotoxic agents of the body’s immune defense system. Use of a selective membrane both eliminates the need for chronic immuno-suppression in the host and allows cells to be obtained from non-human sources, thus avoiding the cell-sourcing constraints which have limited the clinical application of generally successful investigative trials of unencapsulated cell transplantation for chronic pain1, Parkinson’s disease2, and type I diabetes.3–5 Cross-species immunoisolated cell therapy has been validated in small and large animal models of chronic pain6,7, Parkinson’s disease8,9, and Type 1 diabetes10–12, and is under active investigation by several groups in animal models of Huntington’s13, ALS14 and Alzheimer’s15–19. In addition, the first encapsulated therapy using xenografts in humans has been performed in chronic nain20 and ALS21.

Transplantation of Encapsulated Cells into the Central Nervous System

Targeted Delivery to the CNS Targeted delivery of active biomolecules to the central nervous system (CNS) has been applied as a potential therapy to treat a variety of disorders, including chronic pain and neurodegenerative diseases. This approach is advantageous for delivery of neuroactive agents that do not readily cross the blood-brain barrier. The implantable systems available for the targeted delivery of these substances to the CNS are either cell-based or non-cell-based. The non-cell-based systems include controlled release polymers and infusion via pumps and catheters. These systems involve relatively simple surgical procedures and allow high control over dose over the short term. The main disadvantages involve poor long-term release kinetics for the polymer delivery systems, high risk of infection for the catheters and pumps, and the need for highly purified and stable factors for both approaches. Cell-based systems involve the encapsulation of living secretory cells in either microencapsulation or macroencapsulation devices. The main advantage of cell-based delivery systems is the continuous production and secretion of the neuroactive agents from the encapsulated cells, thereby eliminating requirements for purified and stable therapeutic factors and the need for frequent refilling or replacement. Macroencapsulation devices have the additional advantage of high mechanical strength, ease of retrievability, and increased control over the manufacturing. This chapter will focus on the delivery of therapeutic factors to the CNS using macroencapsulation devices.