Patent Publication Number: US-2013236972-A1

Title: Liver Sinusoid Model

Description:
STATEMENT REGARDING GOVERNMENT SPONSORED RESEARCH OR DEVELOPMENT 
     The present invention was developed under grant number 0747752, awarded by the National Science Foundation and grant number R21AA017458, awarded by the National Institutes of Health. The U.S. Government has certain rights in this invention. 
    
    
     FIELD OF THE INVENTION 
     The present invention relates to a miniature liver tissue model that represents the architecture and functions of human liver tissue. 
     BACKGROUND OF THE INVENTION 
     The liver is the largest solid organ in the body and is involved in a myriad of metabolic processes required for body homeostasis, as well as the detoxification of harmful chemicals. Hepatocytes are the major cells within the liver and are responsible for many activities that are attributed to the liver. Liver biology studies predominantly rely on hepatocyte culture models. When hepatocytes are isolated and cultured in vitro, however, they lose their normal structure and functions because of a lack of cell-to-cell and cell-to-extracurricular matrix interactions that are essential for maintaining normal liver functions. While much progress has been made in the past in prolonging hepatocyte viability and maintaining liver functions in vitro, there are still no authentic liver models that accurately represent the architecture and functions of human liver tissue. 
     BRIEF SUMMARY OF THE INVENTION 
     Briefly, the present invention provides a liver sinusoid model comprising a generally planar substrate having first and second generally parallel microchannels formed therein. A microporous membrane is disposed between and separating the first and second generally parallel microchannels. A first layer of cells lines one side of the membrane in the first microchannel. The first layer of cells are all a first common cell type. A second layer of cells extends parallel to the first layer of cells in one of the first microchannel and the second microchannel. The second layer of cells is all of a second common cell type. 
     Further, the present invention provides a liver sinusoid functional unit comprising a generally planar substrate having first and second generally parallel microchannels formed therein. The first microchannel is disposed above the second microchannel. A microporous membrane is disposed between the first microchannel and the second microchannel. A layer of hepatocyte cells is disposed in the first microchannel and extends directly along the microporous membrane. A layer of liver sinusoidal endothelial cells is disposed in the first microchannel such that the layer of hepatocyte cells are sandwiched between the layer of liver sinusoidal endothelial cells (LSECs) and the microporous membrane 
     Additionally, the present invention provides bioreactor functions that include continuous perfusion of culture media and introduction of drugs. A first microchannel represents a sinusoid (blood vessel) and thus a fluid flow that simulates blood with proper oxygen and nutrient compositions is introduced. A second microchannel represents a duct of bile that is secreted from hepatocytes and transferred to the intestines, and thus a fluid flow will be introduced to collect the bile component. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
       The foregoing summary, as well as the following detailed description of the invention, will be better understood when read in conjunction with the appended drawings. For the purpose of illustrating the invention, there is shown in the drawing certain embodiments of the present invention. It should be understood, however, that the invention is not limited to the precise arrangements shown. In the drawings: 
         FIG. 1  is a side elevational view of a liver sinusoid model according to a first exemplary embodiment of the present invention; 
         FIG. 2  is a side elevational view of an enlarged portion of the liver sinusoid model of  FIG. 1 , showing flow through the first microchannel; 
         FIG. 3  is a schematic view of a mini liver bioreactor flow circuit incorporating the liver sinusoid model of  FIG. 1 ; and 
         FIG. 4  is a side elevational view of a liver sinusoid model according to a second exemplary embodiment of the present invention. 
     
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     In the drawings, like numerals indicate like elements throughout. Certain terminology is used herein for convenience only and is not to be taken as a limitation on the present invention. The terminology includes the words specifically mentioned, derivatives thereof and words of similar import. The embodiments illustrated below are not intended to be exhaustive or to limit the invention to the precise form disclosed. These embodiments are chosen and described to best explain the principle of the invention and its application and practical use and to enable others skilled in the art to best utilize the invention. 
     Referring to  FIG. 1 , a first exemplary embodiment of a liver sinusoid model  100  according to the present invention is shown. Liver sinusoid model  100  is an in vitro microfluidic model of a model of the liver tissue, a liver sinusoid. Liver sinusoid model  100  is formed using a generally planar substrate assembly  102  as a base. Substrate assembly  102  may be constructed from planar polymer sheets constructed from a material such as, for example, polymethyl methacrylate (“PMMA”) or polydimethylsiloxane (“PDMS”). PMMA and PDMS can be used because of the ease in forming microchannels therein. The processes of manufacturing microchannels in PMMA and PDMS substrates are well known by those skilled in the art. Substrate assembly  102  includes a top substrate  104  and a bottom substrate  106 . A microchannel assembly  110  is formed in the substrates  104 ,  106 . A first, or top, microchannel  112  is formed in top substrate  104  and a second, or bottom, microchannel  114  is formed in bottom substrate  106 . First microchannel  112  is used to simulate a capillary that provides blood to liver sinusoid model  100 . Second microchannel  114  is used to simulate a bile duct that removes toxins and other waste products from liver sinusoid model  100 . 
     In an exemplary embodiment, planar substrate assembly  102  has dimensions of approximately 10-20 millimeters wide, 20-40 millimeters long, and 5-10 millimeters thick. Top and bottom microchannels,  112  and  114  have a length of about 10-20 millimeters, a width of about 1-2 millimeters, and a depth of about 50-200 microns. Top microchannel  112  also includes a top inlet passage  115  at a first end  112   a  of top microchannel  112  and a top outlet passage  116  and a second end  112   b  of top microchannel  112 . Top inlet passage  115  and top outlet passage  116  each extend generally transverse to the length of top microchannel  112 . 
     Bottom microchannel  112  also includes a bottom inlet passage  117  at a first end  114   a  of bottom microchannel  114  and a bottom outlet passage  118  at a second end  114   b  of bottom microchannel  114 . A microporous membrane  120  is placed over the top of bottom substrate  106  so that membrane  120  covers microchannel  114 . In a first exemplary embodiment, membrane  120  may be constructed from a Transwell membrane (polyester) or parylene polymer (polyparaxylylene) that is about 10 microns thick. Pores in membrane  120  may be between about 0.3 and about 1 micron in diameter. It is desired that the pores are sufficiently large enough to allow liquids and proteins to pass through from one side of membrane  120  to opposing side of membrane  120 , yet small enough to prevent cells from passing through membrane  120 . Top substrate  104  is placed on top of bottom substrate  106  and substrates  104 ,  106  are secured to each other so that second microchannel  114  is generally parallel to first microchannel  112 , with microchannels  112 ,  114  being separated from each other by membrane  120 . In an exemplary embodiment, top substrate  104  includes a first groove  104   a  and a second groove  104   b  that are sized to accept and retain membrane  120  between top substrate  104  and bottom substrate  106 . Top substrate  104  is fixedly coupled to bottom substrate  106  via thermal fusion bonding or adhesive bonding or oxygen plasma, which welds top substrate  104  to bottom substrate  106 . 
     In order to prepare first microchannel  112  to receive liver cells, a collagen solution is flushed through first microchannel  112  via top inlet passage  115  and out of top outlet passage  116 . The collagen solution allows liver cells to adhere to membrane  120 . A plurality of liver cells  130  are disposed on membrane  120  in first microchannel  112 . In an exemplary embodiment, liver cells  130  may be rat liver cells. In an alternative exemplary embodiment, liver cells  130  may be human liver cells. Liver cells  130  include, extending outwardly from membrane  120 , a layer of hepatocyte cells  132  directly on membrane  120 , a collagen layer  134  directly on the layer of hepatocyte cells  132 , and a layer of liver sinusoidal endothelial cells (LSEC)  136  directly on collagen layer  134 . Included with the LSEC  136  are minority cells, such as stellate cells and Kupffer cells, which are liver-specific micro phages. In an exemplary embodiment, liver cells  130  are disposed on membrane  120  in the absence of any fibroblast cells. Those skilled in the art, however, will recognize that fibroblast cells may also be used to assist in culturing liver cells  130 . 
     Liver cells  130  are primary cells, meaning that they are freshly removed from a recently deceased body, and are viable within liver sinusoid model  100  for a timeframe greater than at least one month. Collagen layer  134  simulates the Space of Disse, which separates hepatocyte cells from the LSEC in a biological liver. In this embodiment, all liver cells are located in first microchannel  112 . Optionally, established cell lines such as rat adrenal medulla endothelial cells (RAMEC) can also be used to replace the LSECs. 
     Liver sinusoid model  100  can be used in a static mode. A first fluid, representing blood, may be inserted into first microchannel  112  via either top inlet passage  115  or top outlet passage  116 . Additionally, a second fluid, representing bile fluid, may be inserted into second microchannel  114  via either bottom inlet passage  117  or bottom outlet passage  118 . LSEC  136  and hepatocyte cells  132  act upon the fluid in first microchannel  112 . 
       FIG. 2  illustrates an enlarged version of top microchannel  112  having a height “H” and showing fluid flow having a velocity u x  along x and y axes. Oxygen diffuses from the first fluid, through liver cells  130  and membrane  120 , while cell uptake/secretion from liver cells  130  is absorbed by the first fluid. 
     Alternatively, in an exemplary embodiment, illustrated schematically in  FIG. 3 , a continuous perfusion system can be provided to liver sinusoid model  100  to simulate the flow of blood to liver sinusoid model  100  as well as the discharge of bile from liver sinusoid model  100 . The perfusion system provides bioreactor functions that include continuous perfusion of culture media and introduction of drugs. First microchannel  112  represents a sinusoid (blood vessel) and thus a fluid flow that simulates blood with proper oxygen and nutrient compositions is introduced. Second microchannel  114  represents a duct of bile that is secreted from hepatocytes and transferred to the intestines (not shown), and thus a fluid flow will be introduced to collect the bile component. 
     First microchannel  112  of liver sinusoid model  100  is in fluid communication with a first fluid circuit  150  that is used to provide continuous perfusion to simulate blood being pumped through first microchannel  112 . First fluid circuit  150  represents a sinusoid (blood vessel) and includes a medium reservoir  152  that includes a fluid medium  154  that simulates blood. Fluid medium  154  is a formulation that includes growth factors, hormones, nutrients, and oxygen. 
     A peristaltic pump  156  includes a suction end  158  in fluid communication with fluid medium  154  and is used to pump fluid medium  154  from medium reservoir  152 . In an exemplary embodiment, peristaltic pump  156  is a compact digital pump, manufactured by Ismatic. A discharge end  160  of peristaltic pump  156  is in fluid communication with a medium oxygenator  162  and pump fluid medium  154  into medium oxygenator  162 . In an exemplary embodiment, medium oxygenator  162  is realized by passing the fluid through a PDMS tube in an oxygen-rich bottle. 
     A discharge end of medium oxygenator  162  is in fluid communication with a bubble trap  164 , which is used to remove any air bubbles from fluid medium  154 . In exemplary embodiment, bubble trap  164  consists of a micro-porous, hydrophobic membrane where an aqueous fluid is retained and able to flow from inlet to outlet while air bubbles are forced through to a vent. 
     A discharge end of bubble trap  164  is in fluid communication with top inlet passage  115  of first microchannel  112 . Top outlet passage  116  of first microchannel  112  is in fluid communication with medium reservoir  152  such that fluid that flows into top inlet passage  115  of first microchannel  112  flows through first microchannel  112  and out of top outlet passage  116  of first microchannel  112  and back to medium reservoir  152 . The flow rate of fluid medium  154  through first microchannel  112  can be controlled by adjusting the operational speed of peristaltic pump  156 . Adjustment of the flow rate of fluid medium  154  through first microchannel  112  results in an adjustment of the oxygen concentration within fluid medium  154 , as well as the shear stress imparted upon LSEC  136  by fluid medium  154  flowing across LSEC  136 . 
     Additionally, second microchannel  114  is in fluid communication with a second fluid circuit  170  that is used to provide continuous perfusion to simulate bile fluid that is being pumped through second microchannel  114 . Second fluid circuit  170  is adapted to collect waste product generated by liver cells  130  and includes a bile collection reservoir  172  that includes a fluid medium  174  that simulates bile fluid. 
     A peristaltic pump  176  includes a suction end  178  in fluid communication with fluid medium  174  and is used to pump fluid medium  174  from bile collection reservoir  172 . A discharge end  180  of peristaltic pump  176  is in fluid communication with bottom inlet passage  117  of second microchannel  114 . Bottom outlet passage  118  of second microchannel  114  is in fluid communication with bile collection reservoir  172  such that fluid that flows into bottom inlet passage  117  of second microchannel  114  flows through second microchannel  114  and out of bottom outlet passage  118  of second microchannel  114  and back to bile collection reservoir  172 . The flow rate of fluid medium  174  through second microchannel  114  can be controlled by adjusting the operational speed of peristaltic pump  176 . As shown in  FIG. 2 , the flow direction of fluid medium  154  through first microchannel  112  is in a left-to-right direction, such that fluid flow in first fluid circuit  150  is counterclockwise. 
     Referring now to  FIG. 4 , a second exemplary embodiment of a liver sinusoid model  200  according to the present invention is shown. Liver sinusoid model  200  may be formed using a generally planar substrate assembly  202  similar to generally planar substrate assembly  102  disclosed above. Structural elements in liver sinusoid model  200  are similar to structural elements in liver sinusoid model  100  but are identified with a “2” as the first digit in the reference number instead of a “1” as the first digit in the reference number. 
     Liver sinusoid model  200  is similar to liver sinusoid model  100  disclosed above, but with a structural modification. A difference between liver sinusoid model  100  and liver sinusoid model  200  is that, while, in liver sinusoid model  100 , hepatocyte cells  132  and LSEC  136  are on the same side of membrane  120  in first microchannel  112 , in liver sinusoid model  200 , hepatocyte cells  232  are on an opposing side of a microporous membrane  220  in a second microchannel  214 . In liver sinusoid model  200 , LSEC  236  are attached directly to a first side  220   a  of membrane  220  while hepatocyte cells  232  are attached directly to a second side  220   b  of membrane  220 . Similar to liver sinusoid model  100 , a collagen solution is flushed through first microchannel  212  to assist in the adhesion of LSEC  236  to first side  220   a  of membrane  220  and a collagen solution is flushed through second microchannel  214  to assist in the adhesion of hepatocyte cell  232  to second side  220   b  of membrane  220 . In liver sinusoid model  200 , collagen layer  134  may be omitted. Similar to liver sinusoid model  100 , liver sinusoid model  200  can be used in a static or a dynamic environment. 
     The liver functional model according to the present invention provides an accurate liver tissue model for performing liver biology studies, liver cancer research, and viral infections, as well as toxicology studies, drug metabolism studies, the effects of alcohol and viral infection on the liver, as well as performing drug screening tests. 
     It will be appreciated by those skilled in the art that changes could be made to the embodiments described above without departing from the broad inventive concept thereof. It is understood, therefore, that this invention is not limited to the particular embodiments disclosed, but it is intended to cover modifications within the spirit and scope of the present invention as defined by the appended claims.