Chemistry: molecular biology and microbiology – Apparatus – Bioreactor
Reexamination Certificate
2001-09-19
2003-12-23
Beisner, William H. (Department: 1744)
Chemistry: molecular biology and microbiology
Apparatus
Bioreactor
C435S029000, C435S400000, C435S288200, C435S359000, C359S398000, C210S321800
Reexamination Certificate
active
06667172
ABSTRACT:
BACKGROUND OF THE INVENTION
Knowledge of the blood-brain barrier (BBB) has progressed rapidly over the past several years as new techniques (e.g. in vitro cell cultures) have become available. Improved technologies for the monitoring of barrier integrity in terms of electrical resistance and macromolecule permeability are readily accessible. Concomitant with the advancement of these techniques has come a wealth of knowledge regarding the relevant factors that promote the expression of a BBB phenotype particularly in endothelial cells.
The BBB maintains the homeostasis of the brain microenvironment, which is crucial for neuronal activity and function. Brain microvascular endothelial cells (EC) that constitute the BBB are responsible for the transport of metabolites, precursors and nutrients from the blood to the brain. The same cells are involved in the clearance of potassium and hydrogen ions from the brain. While blood-brain barrier EC retard the transcellular migration of most hydrophilic solutes, nutrients and sugars gain rapid access into the brain. In mammals and in higher vertebrates, the sites of the BBB are the complex tight junctions between EC that prevent the paracellular migration of hydrophilic molecules from blood to brain and vice versa. The perivascular glia process with encompass the basal lamina of the endothelial cells in the central nervous system influence the integrity of the tight junctions. Specialized transporters for sugars (e.g. glucose) and amino acids have been described in blood-brain barrier EC and account for the transendothelial permeability of otherwise membrane-impermeant substances.
As one may expect given the seemingly opposite properties of these BBB cells, two different subcellular mechanisms are responsible for the ‘barrier’ and ‘transport’ features of blood-brain barrier EC: tight junctions and specialized transcellular transporters, including micropinocytotic vesicles for macromolecules.
An often neglected aspect of the BBB relates to its capacity to act simultaneously as a barrier and as a transporter for any ion or molecule. For example, the BBB is virtually impermeant to intraluminal potassium (K
plasma
,). However, brain (i.e. abluminal) potassium is transported to the blood by a specialized and topographically segregated Na/K-ATPase. Thus, by combining the tight junction-mediated ‘tightness’ of the BBB with an asymmetric transporter, K
CSF
remains constant in spite of K
plasma
variations or parenchymal increases resulting from neuronal activity.
Most of these specialized properties (tight junctions, micropinocytotic vesicles, transporters, ion homeostasis mechanisms) are bestowed on endothelial cells by the brain tissue. Peripheral capillaries that vascularize brain tissue acquire BBB properties. However, isolated blood-brain barrier EC lose their properties after culturing in vitro.
Therefore, two key factors must be present in order for central nervous system endothelial cells to express a barrier phenotype in vitro which distinguishes them from their peripheral counterparts. First, the exposure of the apical membrane to shear stress, which is generated by the flow of blood across the apical surfaces of the endothelial cells, is vital to promote growth inhibition and differentiation of endothelial cells. Also, the exposure to shear stress serves to induce metabolic changes that limit the oxygen and substrate consumption of such cells and allow for trafficking of metabolic fuels to the brain. A by-product of the metabolic changes induced by flow is an improved capacity for endothelial cells to handle oxidative stress. The second vital factor for BBB formation by endothelial cells is exposure of these cells to as yet unidentified “permissive” or “promoting” factors presumably secreted by glia, specifically astrocytes. The basis for this comes from several series of experiments documenting close apposition of astrocyte foot processes to endothelial cells in instances of barrier expression and the absence of such expression when astrocytes or astrocytic factors are lacking. Astrocytic influences promote both a variety of changes in gene expression in endothelial cells as well as phenotypic changes including segregation of transporters and enzymes.
The goal of any study of BBB physiology or biology is to reproduce as many aspects as possible of the in vivo endothelial cells. The apposition of endothelial cell and astrocyte cell cultures in physically separate, but biochemically contiguous compartments and the exposure of the endothelial cells to apical shear stress are primary features of any dynamic model of the blood-brain barrier. Furthermore, an in vitro BBB model should simulate as many of the following properties as possible: (1) expression of tight junctions between ECs and the relative lack of pinocytotic vesicles (commonly assessed by measuring trans-endothelial electrical resistance (TEER) or permeability to radioactive molecules of poor or negligible permeation such as sucrose or mannitol); (2) selective (and asymmetric) permeability to physiologically relevant ions, such as Na
+
or K
+
; (3) selective permeability to molecules, based on their molecular weight and oil/water partition coefficient; (4) expression of BBB-specific transporters for metabolic substrates or building blocks necessary for neuronal and glial cell physiology; and (5) functional expression of mechanisms of active extrusion of otherwise permeable substances (such as antineoplastic agents).
A first attempt at an in vitro BBB model included the use of cone and plate viscometers as well as parallel plate apparatuses combined with semipermeable membranes that are able to generate shear stress and co-culture conditions. However, these models did not possess the three-dimensional architecture characteristic of brain tissue in situ and lacked the necessary glial factors. Another model design known in the art uses a hollow fiber apparatus to conduct BBB studies. This model results from a modification of a traditional cell culture system that is normally used for extensive culturing of non-EC cells. The general design of the hollow fiber apparatus is derived from attempts to develop a ‘cell factory’. U.S. Pat. No. 3,821,087 to Knazek et al. and U.S. Pat. No. 4,220,725 to Knazek et al. describe cell culturing devices using hollow fibers. Since then, these cell culturing devices have been extensively exploited for mass production of rare cell types, antibody production, and modeling of organ-like structures such as the BBB. Ott et al. used a hollow fiber cell culture apparatus for studies of flow-mediated effects on endothelial cell growth. A cell culturing device that is commercially available is CELLMAX® from Spectrum Laboratories.
Applicant has co-authored several publications describing attempts to simulate the blood brain barrier utilizing cell culture models by co-culturing endothelial cells intraluminally (i.e., intracapillary) and glia extraluminally (i.e., extracapillary). These publications include “A New Model of the Blood Brain Barrier: Co-Culture of Neuronal, Endothelial, and Glial Cells Under Dynamic Conditions,” NeuroReport, Vol. 10, No. 1816, December 1999; “Understanding the Physiology of the Blood Brain Barrier: In Vitro Models,” News in Physiological Sciences, Volume 13, December 1998; “Dynamic In Vitro Modeling of the Blood Brain Barrier: A Novel Tool for Studies of Drug Delivery to the Brain,” PSTT, Vol. 2, No. 1, January 1999; Morphological and Functional Characterization of an In Vitro Blood-Brain Barrier Model,” Brain Research, No. 771, 1997; and Mechanisms of Glucose Transport at the BBB: An In Vitro Study, Brain Research 409, 2001 which are all hereby incorporated by reference in their entireties to the extent they discuss the utilization of cell culturing models to simulate the BBB.
However, although cell culture models may be used to model the BBB, the cell culture models known in the art have proven to be of limited applicability. For example, these models provide poor visualization of the intracapillary or extracapillar
Janigro Damir
McAllister Mark S.
Beisner William H.
Miller Raymond A.
Pepper Hamilton LLP
The Cleveland Clinic Foundation
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