Chemistry: molecular biology and microbiology – Carrier-bound or immobilized enzyme or microbial cell;... – Enzyme or microbial cell is immobilized on or in an...
Reexamination Certificate
2001-10-12
2004-05-11
Naff, David M. (Department: 1651)
Chemistry: molecular biology and microbiology
Carrier-bound or immobilized enzyme or microbial cell;...
Enzyme or microbial cell is immobilized on or in an...
C435S029000, C435S071100, C435S071200, C435S283100, C435S395000
Reexamination Certificate
active
06734000
ABSTRACT:
FIELD OF THE INVENTION
The invention relates to the field of bioreactors. More specifically, it relates to nanoporous silicon bioreactors for the maintenance of cells in culture for use in development and testing of bioactive compounds, protein and metabolite production, and simulated organ function.
BACKGROUND OF THE INVENTION
Silicon has begun to receive increasing attention for use in biomedical applications. In particular crystalline silicon has been utilized as a textured surface to guide cell alignment, to encapsulate cells for implantation, and as an electroactive substrate to stimulate excitable cells. Several properties of silicon have led to its use in these diverse applications: (1) well-described silane chemistries for immobilization of adhesive ligands, (2) wet and dry micromachining capability to form 3-dimensional structures on biologically relevant length scales, and (3) semiconductor properties that allow incorporation of microelectronic elements. In comparison, porous silicon, a nanocrystalline material generated by etching of crystalline silicon in hydrofluoric acid, has been less extensively utilized for biomedical applications. Its open pore structure and large surface area, combined with unique properties such as photo and electroluminescence have provided a platform for sensors for non-biological species (e.g. solvents, gases, and explosives) as well as biological species (DNA, proteins). Indeed, the range of tunable pore sizes (5 to 1200 nm) in porous silicon spans a range of sizes important in biology; a small DNA fragment is on the order of a few tens of nm, proteins are generally in the 100 nm range, and bacteria and cells can be a few microns in diameter.
Previously it has been shown that manipulations of cellular microenvironment by “micropatterning” on inorganic surfaces can alter the behavior of cells in culture (Chen et al, 1997; Bhatia et al, 1999). Methods to alter the support for cell growth to allow for distinct localized cell adhesion involved the manipulation of glass, gold or polymer supports such that cell adhesion molecules were differentially deposited on the support (e.g. U.S. Pat. Nos. 6,004,444; 6,103,479 and 6,133,030; all incorporated herein by reference). A common method involves the use of photoresist, a UV-sensitive polymer. Borisilicate substrates (e.g. coverslips) are coated with photoresist and exposed to light through a mask, creating a photoresist pattern. Patterned substrates are used to control subsequent immobilization of extracellular matrix components (ECM) (e.g. collagen I). The localization of a specific ECM component allows for the adhesion of cells to specific regions of the substrate (e.g. primary hepatocytes adhere to collagen, but not to glass). In some cases, co-cultures of two cell types are achieved by subsequent addition of a second cell type to attach to the periphery. Thus, micropatterned arrays have been used to generate defined co-cultures of hepatocytes and fibroblasts for the study of the maintenance of cell fate and function (Bhatia et al, 1999). Similar arrays have also been used for use in an apparatus for cell based screening. The method may be used for the establishment of any of a number of patterns, including non-uniform arrays (U.S. Pat. No. 6,103,479). However, such a method requires that the cell types of interest have different adhesive properties that are well known. Thus the system is limited to the use of cell types with well defined, and distinct, characteristics.
Some researchers have begun to explore the use of porous silicon as a biodegradable material for the slow release of drugs or essential trace elements to cells or as an in vivo diagnostic
[10-12]
. Promising findings by Canham et al. have shown hydroxyapatite nucleation on porous silicon in vitro, suggesting that porous silicon, in contrast to crystalline silicon, could be a bioactive surface (Canham, 1995; Canham et al, 1997). Nonetheless, porous silicon has not been extensively characterized as a material for implantation or the formation of hybrid (biological
on-biological) devices in vitro (Rosengren et al, 2000). Studies on the compatibility of this material with mammalian tissues have been performed in immortalized cell lines, that are known to be relatively robust. Cells proliferated in vitro in the presence of silicon and “bulk” metabolic assays revealed no toxicity.
There have recently been a number of papers demonstrating the feasibility of interfacing crystalline silicon and mammalian cells (Mayne et al, 2000; Thomas et al, 1999; Curtis and Wilkinson, 1997). The motivation for such studies includes the fact that silicon is easily manipulated into a variety of structures due to developments in the optoelectronics industry and the production of micro electromechanical (MEMS) devices (Steiner and Lang, 1995; Meyer and Biehl, 1995). Starting with crystalline silicon as a substrate, photolithography and etching techniques allow the facile construction of micron- and submicron-sized structures. Silicon surface chemistries targeting the reactivity of silicon oxide via Si—OH groups and silicon hydride (Si—H) have been well explored allowing a variety of surface modifications (Bhatia et al, 1997; Stewart and Buriak, 2000). Other methods for the modification of silicon included electrodeposition machining, laser ablation, laser drilling, micromachining, lithographic galvanic fabrication (LIGA) and embossing. Furthermore, silicon-based cellular arrays can be easily integrated with other silicon-based components such as sensors, heaters, microfluidics arrays, and the like. Porous silicon has recently received considerable interest in applications as a biomaterial due to its solubility in physiologic environments. The primary dissolution product is silicic acid [Si(OH)
4
] a naturally occurring form of silicon that can be processed and excreted by the body. The rate of dissolution can be controlled by chemical derivatization by methods well known to those skilled in the art (Canham et al, 2000).
The use of crystalline silicon chips as a scaffold for the growth of vascularized perfused microtissue and micro-organ arrays has been taught by Griffith et al. (U.S. Pat. No. 6,197,575, incorporated herein by reference). The apparatus consists of a micromatrix and a perfusion assembly suitable for seeding and attachment of cells on and throughout the matrix and for morphogenesis of seeded cells into complex, hierarchical tissue or organ structures, wherein the matrix includes channels or vessels through which culture medium, blood, gases or other nutrients or body fluids can be perfused. The functional unit in these micromatricies is the channel containing cells and their exudates (such as extracellular matrix molecules) in the desired morphological structure. The channel refers to a hole with defined dimensions, typically 75-1000 micron across, that goes through a sheet of scaffold material approximately 50-500 micron thick. Each channel is sufficiently large to contain a microscale tissue which is a synthetically formed mass of cells forming a tissue structure or a structure that carries out tissue functions. Griffith suggests that such bioreactors would be ideal to simulate liver. One could seed the micromatricies with endothelial cells, followed by the addition of hepatocytes. Alternatively stem cells may be plated directly onto the scaffold and treated with appropriate growth factors to induce differentiation. Such microtissues can be used in the context of an artificial liver apparatus or in drug toxicity and screening assays.
It would be desirable to develop an artificial liver apparatus, similar to a kidney dialysis apparatus, for hepatic support in individuals waiting for liver transplant. However, the liver is a more complex organ than the kidney which is predominantly responsible for salt balance and filtering of molecules based on size. The liver is responsible for detoxification of xenobiotics and hormones, energy metabolism, production of plasma proteins, and production of bile, rather than the simple
Bhatia Sangeeta N.
Chin Vicki I.
Collins Boyce E.
Sailor Michael J.
Gordon & Rees LLP
Naff David M.
Regents of the University of California
LandOfFree
Nanoporous silicon support containing macropores for use as... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Nanoporous silicon support containing macropores for use as..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Nanoporous silicon support containing macropores for use as... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-3270699