Cell culture systems and methods for organ assist devices

Abstract

The invention features-modular cell culturing devices including one or more flat-plate modules, and is based on the discovery that if the flows of liquid medium and oxygenated fluid are separated by a gas-permeable, liquid-impermeable membrane, and the cells are grown attached to the liquid side of the membrane, the device can be used to culture cells with transport of oxygen through the membrane (i.e., direct oxygenation), without regard for the flow rate of the liquid medium 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, or add cell-synthesized products, or both.

What is claimed is:  
     
         1 . A method for culturing cells, the method comprising: 
 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.    
     
     
         2 . A method of  claim 1 , wherein the oxygen contained in the oxygenated fluid is at or above the critical partial pressure of oxygen.  
     
     
         3 . The method of  claim 1 , wherein the cells are hepatocytes.  
     
     
         4 . The method of  claim 3 , wherein the device is seeded with 2 to 20 billion hepatocytes.  
     
     
         5 . The method of  claim 3 , wherein the device is seeded with porcine, equine, ovine, bovine, rabbit, rat, canine, feline, or murine hepatocytes.  
     
     
         6 . The method of  claim 3 , wherein the device is seeded with human hepatocytes.  
     
     
         7 . The method of  claim 1 , wherein the cells are preserved.  
     
     
         8 . The method of  claim 7 , wherein the cells are preserved by cryopreservation, hypothermic storage, or lyophilization.  
     
     
         9 . The method of  claim 1 , wherein the gas-permeable, liquid-impermeable membrane material is selected from the group consisting of polystyrene, polyolefin, polyethylene, polypropylene, polyvinylidene fluoride, polycarbonate, hydrophobic-treated nylon, polyurethane, polyester, layered styrene-butadiene-styrene/ethyl vinyl acetate/styrene-butadiene-styrene, and layered styrene-butadiene-styrene/polyethylene.  
     
     
         10 . The method of  claim 1 , wherein the first surface of the gas-permeable, liquid-impermeable membrane is corona treated.  
     
     
         11 . The method of  claim 1 , wherein the first surface of the gas-permeable, liquid-impermeable membrane is collagen coated.  
     
     
         12 . The method of  claim 1 , wherein the concentration of oxygen in the oxygenated fluid is between about 0% to about 90% oxygen.  
     
     
         13 . The method of  claim 12 , wherein the concentration of oxygen in the oxygenated fluid is between about 19% to about 60% oxygen.  
     
     
         14 . The method of  claim 12 , wherein the concentration of oxygen in the oxygenated fluid is between about 40% to about 60% oxygen.  
     
     
         15 . The method of  claim 1 , wherein the concentration of oxygen in the oxygenated fluid is controlled to promote or downregulate cell function.  
     
     
         16 . The method of  claim 1 , wherein the nutrient-containing culture medium is perfused.  
     
     
         17 . The method of  claim 1 , wherein the method further comprises filtering blood plasma.  
     
     
         18 . The method of  claim 1 , wherein the cells are seeded across the entire membrane from above the membrane.  
     
     
         19 . The method of  claim 3 , wherein the hepatocytes are seeded directly onto the gas-permeable, liquid-impermeable membrane and then coated with collagen.  
     
     
         20 . The method of  claim 3 , wherein the gas-permeable, liquid-impermeable membrane is coated with collagen, and the hepatocytes are seeded directly onto the collagen-coated membrane.  
     
     
         21 . A flow-through cell culturing device comprising: 
 a housing with an oxygenated fluid inlet and an oxygenated fluid outlet, a liquid inlet and a liquid outlet, and first and second walls to form a chamber;    a gas-permeable, liquid-impermeable membrane arranged between the first and second walls to separate the chamber into an oxygenated fluid compartment comprising an oxygenated fluid entry and an oxygenated fluid exit, and a liquid compartment comprising a liquid entry and liquid exit; and    a liquid-permeable membrane arranged between a wall and the gas-permeable, liquid-impermeable membrane to separate the liquid compartment into a cell compartment and a liquid perfusion compartment,    wherein the oxygenated fluid inlet and oxygenated fluid outlet are arranged such that oxygenated fluid entering the oxygenated fluid inlet flows into the oxygenated fluid entry and through the oxygenated fluid compartment and exits the oxygenated fluid compartment through the oxygenated fluid exit and the housing through the oxygenated fluid outlet, and    wherein the liquid inlet and liquid outlet are arranged such that liquid entering the liquid inlet flows into the liquid entry and through the liquid-perfusion compartment and exits the liquid-perfusion compartment through the liquid exit and the housing through the liquid outlet.    
     
     
         22 . The device of  claim 21 , wherein in use, cells are seeded onto the gas-permeable, liquid-impermeable membrane, and the space between the gas-permeable, liquid-impermeable and liquid-permeable membranes is greater than the size of a cell.  
     
     
         23 . The device of  claim 21 , wherein in use, cells are seeded onto either of the gas-permeable, liquid-impermeable membrane or the liquid-permeable membrane, and the space between the gas-permeable, liquid-impermeable and liquid-permeable membranes is about equal to the size of one cell.  
     
     
         24 . The device of  claim 21 , wherein in use, cells are seeded onto the gas-permeable, liquid-impermeable membrane, and onto the liquid-permeable membrane, and the space between the gas-permeable, liquid-impermeable and liquid permeable membranes is about equal to the size of two adjacent cells.  
     
     
         25 . The device of  claim 21 , further comprising a liquid-permeable hollow fiber arranged in the liquid compartment.  
     
     
         26 . The device of  claim 21 , wherein the housing is arranged to enable stacking of one device on top of another device.  
     
     
         27 . A liver assist system comprising 
 a flow-through cell culturing device of claim  21 ;    a first conduit for conducting plasma from a patient to the housing inlet;    a second conduit for conducting plasma from the cell culturing device to the patient; and    a pump for moving plasma through the conduits and cell culturing device.    
     
     
         28 . The system of  claim 27 , further comprising a plasma separator to remove blood cells from whole blood to provide plasma that is passed through the cell culturing device.  
     
     
         29 . The system of  claim 27 , further comprising a bubble trap, to remove bubbles from the plasma in the first conduit prior to entering the cell culturing device.  
     
     
         30 . A liver assist system comprising 
 a flow-through cell culturing device of claim  21 ;    an immunoisolation device;    a first conduit for conducting plasma from a patient to an immunoisolation device;    a second conduit for conducting plasma from the immunoisolation device to the patient;    a third conduit for conducting liquid medium from the cell culturing device to the immunoisolation device; and,    a fourth conduit for conducting liquid medium from the immunoisolation device to the patient; and,    a pump for moving plasma through the conduits and cell culturing device.    
     
     
         31 . A method of filtering blood plasma, the method comprising 
 seeding a flow-through cell culturing device of  claim 21  with hepatocytes;    introducing blood plasma into the liquid inlet of the device;    supplying an oxygenated fluid into the oxygenated fluid inlet of the device;    allowing the oxygenated fluid to flow through the oxygenated fluid compartment and out of the device through the oxygenated fluid outlet; and    allowing the blood plasma to flow through the device and exit through the liquid outlet, thereby filtering the blood plasma.    
     
     
         32 . A method for treating a patient in need of liver assist, the method comprising attaching the liver assist system of  claim 27  to the blood flow of a patient and treating the patient.  
     
     
         33 . A flow-through cell culturing device comprising 
 a housing with a liquid inlet and a liquid outlet, an oxygenated fluid inlet and an oxygenated fluid outlet, and first and second walls to form a chamber; and    a gas-permeable, liquid-impermeable membrane arranged between the walls to separate the chamber into an oxygenated fluid compartment comprising an oxygenated fluid entry and an oxygenated fluid exit, and a liquid compartment comprising a liquid entry and liquid exit,    wherein the gas-permeable, liquid-impermeable membrane is seeded with cells,    wherein the liquid inlet and liquid outlet are arranged such that biological liquid entering the liquid inlet flows into the liquid entry and through the liquid compartment and exits the liquid compartment through the liquid exit and the housing through the liquid outlet, and wherein the oxygenated fluid inlet and oxygenated fluid outlet are arranged such that oxygenated fluid entering the oxygenated fluid inlet flows into the oxygenated fluid entry and through the oxygenated fluid compartment and exits the oxygenated fluid compartment through the oxygenated fluid exit and the housing through the oxygenated fluid outlet.    
     
     
         34 . The device of  claim 33 , wherein the gas-permeable, liquid-impermeable membrane is porous or non-porous.  
     
     
         35 . The device of  claim 33 , wherein the gas-permeable, liquid-impermeable membrane comprises polystyrene, a polyolefin, polyethylene, polypropylene, polyvinylidene fluoride, polyurethane, poly(styrene-butadiene-styrene), poly(ethyl vinylacetate), nylon, silicon rubber, poly(tetrafluoroethylene), or composites, mixtures, or copolymers thereof.  
     
     
         36 . The device of  claim 33 , wherein the gas-permeable, liquid-impermeable membrane is surface treated.  
     
     
         37 . The device of  claim 33 , wherein the gas-permeable, liquid-impermeable membrane is surface treated with a corona discharge.  
     
     
         38 . The device of  claim 33 , wherein the gas-permeable, liquid-impermeable membrane is surface treated with a coating of extracellular matrix.  
     
     
         39 . The device of  claim 33 , wherein a gel is disposed on the cells.  
     
     
         40 . The device of  claim 33 , wherein a gel is disposed on the gas-permeable, liquid-impermeable membrane.  
     
     
         41 . The device of  claim 39 , wherein the gel contains cells suspended within said gel.  
     
     
         42 . The device of  claim 40 , wherein the gel contains cells suspended within said gel.  
     
     
         43 . A liver assist system comprising 
 a flow-through cell culturing device of claim  33 ;    a first conduit for conducting plasma from a patient to the housing inlet;    a second conduit for conducting plasma from the cell culturing device to the patient; and    a pump for moving plasma through the conduits and cell culturing device.    
     
     
         44 . A method of filtering blood plasma, the method comprising 
 seeding a flow-through cell culturing device of  claim 33  with hepatocytes;    introducing blood plasma into the liquid inlet of the device;    supplying an oxygenated fluid into the oxygenated fluid inlet of the device;    allowing the oxygenated fluid to flow through the oxygenated fluid compartment and out of the device through the oxygenated fluid outlet; and    allowing the blood plasma to flow through the device and exit through the liquid outlet, thereby filtering the blood plasma.