Chemistry: molecular biology and microbiology – Animal cell – per se ; composition thereof; process of... – Solid support and method of culturing cells on said solid...
Reexamination Certificate
2000-06-21
2004-07-06
Lankford, Jr., Leon B. (Department: 1651)
Chemistry: molecular biology and microbiology
Animal cell, per se ; composition thereof; process of...
Solid support and method of culturing cells on said solid...
C435S284100, C435S297100, C435S297200, C435S402000, C435S395000, C435S399000
Reexamination Certificate
active
06759245
ABSTRACT:
FIELD OF THE INVENTION
The invention relates to systems and methods of culturing cells in organ assist devices.
BACKGROUND OF THE INVENTION
Over 43,000 Americans die each year from liver disease, making it the tenth leading disease-related cause of death in the U.S. When liver disease progresses to liver failure, the mortality is 80% unless a compatible donor organ is found. As with other organs, there is a critical shortage of donor livers. Over 12,000 patients are currently listed as transplant candidates, but fewer than half that number of donor livers become available each year. Treatment with a liver assist device (LAD) would decrease the mortality associated with liver failure by stabilizing patients so that they are suitable candidates for a transplant, by supporting them until a suitable donor liver becomes available, and/or by preventing deterioration to the point where a liver transplant is required. Improving the pre-operative health of these patients would also increase transplant success, thereby decreasing the frequency of retransplantation and easing the demand for donor organs.
In cases of sudden or hepatic failure, which often occurs as a result of viral infection or toxicity, treatment with a LAD would eliminate the need for a transplant by supporting these individuals until their own livers regenerate. Liver transplantation is currently the most expensive organ transplant procedure. Successful development of a LAD would consequently provide major benefits to the US in reduced deaths and health-care costs.
Extracorporeal devices for temporary liver support have been investigated since the 1960s. Two strategies have been explored in the development of liver assist devices: (1) non-biological devices based on hemoperfusion on sorbents, hemodialysis across selectively-permeable membranes, and plasma exchange (Malchesky, “Non-biological liver support: historic overview,” Artif. Organs, 18:342-347, 1994); and (2) biological devices that incorporate cells or cellular components (Yarmush et al., “Assessment of artificial liver support technology,” Cell Trans., 1:323-341, 1992).
Non-biological devices have shown only limited efficacy, confirming that synthetic materials cannot replace the range and level of complex metabolic functions normally performed by the liver. On the other hand, a biological LAD in which hepatocytes are seeded on the outer surface of hollow fibers and blood or plasma circulates through the lumen of these fibers was proposed almost 25 years ago by Wolf and colleagues (Wolf et al., “Bilirubin conjugation by an artificial liver composed of cultured cells and synthetic capillaries,” Tran. Amer. Soc. Artif. Int. Organs, 21:16-23, 1975).
Current biological LAD designs use the inverse of this concept today. Modern designs are often based on providing critical liver function by supporting high-density hepatocyte suspensions in hollow fibers, with circulation of blood or plasma outside the fibers. In this design, intermittent extracorporeal liver function is to be provided until the patient recovers through liver regeneration or until a transplant becomes available. However, the hollow fiber design is limited by several factors, including: a) inadequate mass transport, particularly of oxygen, b) lack of understanding of hepatocyte function in an in vitro environment, c) randomized tissue architecture for support of cell viability and function, and d) constraints of void volume on the perfusion circuit for the device.
Hollow fibers have been chosen for LADs on the basis of ready availability rather than demonstrated ability to support hepatocyte function. Perfusion of high-density hepatocyte cultures in hollow fibers has shown a lack of convincing benefit due to, among other reasons, transport limitations that undermine their support of high-density cultures. Such limitations are particularly acute for oxygen, which is required for both basic metabolic function as well as for initial steps in detoxification. Perfusion of oxygenated plasma or medium through or around a network of hollow fibers fails to address this problem because these aqueous liquids are poor carriers for oxygen and the associated distances for transport are relatively large. Modifications to the core hollow-fiber design (e.g., the use of a woven network of three independent sets of capillaries providing integral oxygenation) significantly complicate fabrication and incompletely address underlying transport limitations. They also lack the ability to orient hepatocytes in a more organotypic laminar configuration.
SUMMARY OF THE INVENTION
The invention features modular cell culturing devices comprised of one or more flat-plate modules. The invention is based on the discovery that if the flows of liquid medium and an oxygenated fluid are separated by a gas-permeable, liquid-impermeable membrane, and the cells are grown cultured on the liquid side of the membrane, the device can be used to culture cells with transport of oxygen through the membrane to the cells with independent control of the flow rate of the liquid passing through the device. The new flow-through cell culturing devices can thus be used to culture cells, e.g., hepatocytes, with high levels of cell function in organ, e.g., liver, assist systems, for production of cells, for production of cell-derived products, such as proteins or viruses, or for systems to treat biological liquids to remove toxins, such as ammonia, add cell-synthesized products, or both.
In general, the invention features methods and devices for the culture of cells that provide direct oxygenation of cells through planar, gas-permeable membranes. When the apparatus is seeded with the appropriate cells and is incorporated into a device, the device can be used to treat a patient with an organ, such as the liver, in need of functional assistance.
The invention features methods for culturing cells including: providing a gas-permeable, liquid-impermeable membrane having a first surface and a second surface; seeding cells on the first surface of the gas-permeable, liquid-impermeable membrane; contacting the cells with a nutrient-containing culture medium; providing an oxygenated fluid to the second surface of the gas-permeable, liquid-impermeable membrane at a pressure sufficient to provide transmembrane oxygenation to the cells seeded on the first surface; and culturing the cells under conditions that promote viability and function of the cells.
The device can be seeded with hepatocytes, e.g., porcine, equine, ovine, bovine, rabbit, rat, canine, feline, or murine hepatocytes. Additionally, the device can be seeded with human hepatocytes. The device can be seeded with 2 to 20 billion hepatocytes. The hepatocytes can be seeded directly onto the gas-permeable, liquid-impermeable membrane and then coated with collagen. Alternatively, the gas-permeable, liquid-impermeable membrane can be coated with collagen, and the hepatocytes can be seeded directly onto the collagen-coated membrane. Cells can seeded across the entire membrane from above the membrane.
In one embodiment, the oxygen contained in the oxygenated fluid is at or above the critical partial pressure of oxygen.
In one embodiment, the cells are preserved. The cells can be preserved by cryopreservation, hypothermic storage, or lyophilization.
The gas-permeable, liquid-impermeable membrane material can be made of, e.g., polystyrene, polyolefin, polyethylene, polypropylene, polyvinylidene fluoride, polycarbonate, hydrophobic-treated nylon, polyurethane, polyester, layered styrene-butadiene-styrene/ethyl vinyl acetate/styrene-butadiene-styrene, or layered styrene-butadiene-styrene/polyethylene.
The first surface of the gas-permeable, liquid-impermeable membrane can be treated, e.g., corona treated. In another embodiment, the first surface of the gas-permeable, liquid-impermeable membrane is collagen coated.
In one embodiment, the concentration of oxygen in the oxygenated fluid is between about 0% to about 90% oxygen. Additionally, the concentration of oxygen in the oxygenated fluid can be between about 19% to a
Balis Ulysses J.
Cosman Maury D.
Dimilla Paul A.
Tilles Arno W.
Toner Mehmet
Fish & Richardson P.C.
Lankford , Jr. Leon B.
The General Hospital Corporation
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