Bioengineered Artificial Lateral Liver (BALL) or Bioengineered Artificial Ectopic Liver (BAEL)

Abstract

The embodiments provide a bioengineered artificial functional liver which is connected to a patient suffering from acute liver failure and would functional like an ectopic liver. The device uses the cells derived from the patient's own body thereby nullifying the chances of self/non-self-recognition and related immune activation and rejection. The extracted liver cells are grown on a customized 3D matrix called as 3D cell cartridge and these cell cartridges individually function as miniature liver assemblies. Multiple such assemblies when working in parallel would rescue the condition of liver failure. A microfluidic chamber is built with the similar network as found in the liver and the chamber has flow circuits for plasma/de-cellularised blood and the flow circuits are lined by a coculture of hepatocytes, endothelial cells and fibroblasts. The array of cells in the chamber serve as a miniature liver and multiple such arrays will be stacked to achieve a significant hepatic function.

Claims

1. A bioartificial liver device for removing lipophilic albumin-bound substances such as bilirubin, bile acids, medium chain fatty acids, metabolites of aromatic amino acids, and cytokines from the patient blood, the bioartificial liver device comprising: an inline plasma separator; and one or more 3D cell cartridges with co-cultured liver cells—biologically active hepatocytes cells and endothelial cells on either side of a 3D construct. wherein, the blood from the patient is drawn using a centrifugal pump and subjected to separation of plasma or the de-cellularised blood and the separated plasma or the de-cellularised blood is pumped into the 3D cell cartridge for removing lipophilic albumin-bound substances from the plasma or the de-cellularised blood thereby allowing the damaged liver to regenerate by reducing the metabolic burden.

2. The bioartificial liver device as claimed in claim 1, wherein the 3D cell cartridge comprises of microfluidic chamber with the similar network as found in the human liver.

3. The bioartificial liver device as claimed in claim 1, wherein the chamber in the 3D cell cartridge has flow circuits for plasma/de-cellularised blood where the flow circuits are lined by a coculture of hepatocytes, endothelial cells and fibroblasts.

4. The bioartificial liver device as claimed in claim 1, wherein the array of cells in the chamber serve as a miniature liver and which multiple such arrays will be stacked to achieve a significant hepatic function.

5. The bioartificial liver device as claimed in claim 3, wherein the flow circuits are lined by a coculture of hepatocytes, endothelial cells and fibroblasts.

6. The bioartificial liver device as claimed in claim 1, wherein the 3D cell cartridge comprises of patient derived hepatocytes/somatic cells.

7. The bioartificial liver device as claimed in claim 1, wherein the ratio of the cell number cultured in the 3D cell matrix is at least 2:1 (Hepatocyte:Endothelial cells)

8. The bioartificial liver device as claimed in claim 1, wherein the extracted tissues that is the Hepatocyte and Endothelial cells are cultured on a single membrane.

9. A method of manufacturing a 3D cell cartridge for use in bioartificial liver device, the method comprising of steps: a. 6 trasnwell membrane for the cell culture is customized as per the need and is made of Polycarbonate material with a pore size of at least 0.4 microns b. The 6 transwell which is commonly used in culturing of cells on a membrane is used by removing the existing membrane applying a coating of collagen over the new membrane. c. the removed membrane is replaced by combining 6 transwells membrane with a parafilm. d. Once the 6 transwells are combined with the parafilm, the biologically active hepatocytes cells are cultured on one surface of the 6 trasnwell membrane. e. one of the 6 transwell is removed and 6 transwell reservoir is created using a support ring. The 6 transwell reservoirs is filled with tissue culture medium which support the growth of the biologically active hepatocytes cells f. The endothelial cells which are biologically active cells derived from the patients plasma is seeded and cultured over this vacant membrane collagen and filled with the tissue culture medium

10. The method as claimed in claim 1, wherein the extracted tissues that is the Hepatocyte and Endothelial cells are cultured on a single membrane.

Description

F) BRIEF DESCRIPTION OF THE DRAWINGS

[0035] The other objects, features and advantages will occur to those skilled in the art from the following description of the preferred embodiment and the accompanying drawings in which:

[0036] FIG. 1 illustrates a schematic of the 3D culture set-up for cell culture according to an embodiment of the present invention.

[0037] FIG. 2 illustrates the establishment of co-culture wherein MRP2 and ZO-1 are markers for cell polarization and DAPI is a nuclear marker according to an embodiment of the present invention.

[0038] FIG. 3 illustrates the status of cell co-culture after 14 days wherein the staining reflects that the cells are viable according to an embodiment of the present invention.

[0039] FIG. 4 illustrates the functional validation of the 3D cell culture setup under conditions of continuous flow by albumin measurements according to an embodiment of the present invention.

[0040] FIG. 5 illustrates the Schematic of the working principle of the device according to an embodiment of the present invention.

[0041] Although specific features of the present invention are shown in some drawings and not in others. This is done for convenience only as each feature may be combined with any or all of the other features in accordance with the present invention.

G) DETAILED DESCRIPTION OF THE INVENTION

[0042] In the following detailed description, reference is made to the accompanying drawings that form a part hereof, and in which the specific embodiments that may be practiced is shown by way of illustration. These embodiments are described in sufficient detail to enable those skilled in the art to practice the embodiments and it is to be understood that the logical, mechanical and other changes may be made without departing from the scope of the embodiments. The following detailed description is therefore not to be taken in a limiting sense.

[0043] The present invention discloses an engineered implantable device, which would functional like an ectopic liver. The device uses immunologically neutral cells thereby nullifying the chances of self/non-self-recognition and related immune activation and rejection. Here the cells are grown on a customized 3D matrix called as 3D cell cartridge. These cell cartridges individually function as miniature liver assemblies. Multiple such assemblies when working in parallel would rescue the condition of liver failure. As opposed to transplanting liver, which have serious disadvantages, the ectopic liver would be an alternative strategy to provide hepatic functioning in cases of ALF. This device can be used as an external attachment to the patient. This device will function like a live native liver. This external device will allow the damaged liver to regenerate by reducing the metabolic burden.

[0044] Accordingly, a microfluidic chamber is built with the similar network as found in the liver. This chamber has flow circuits for plasma/de-cellularised blood. The flow circuits are lined by a coculture of cells. This co-culture combination has been developed by us in the preliminary stages of the project. The array of cells in the chamber serve as a miniature liver. Multiple such arrays will be stacked to achieve a significant hepatic function. We have also co-cultured these cells of relevance on either side of a 3D construct and maintained them using complete medium resembling plasma for about 30 days. The material used for growing the 3D culture was uniquely identified by us. The culture was monitored for viability and functioning for this period. Systematic analysis of the cell proliferation, albumin production and cell polarization has been done. The data in support of this is included in the later part of this report. It was found that, during this time they functioned well and did all the functions of liver better than the cells maintained in a 2D culture set-up. We chose plasma to blood to maintain the cells, because WBC form part of the blood which identify foreign cells and destroy them. We believe that by using plasma, we will be avoiding this problem of destruction and sensitization.

[0045] In our proposed device, there are 2 innovative concepts. 1st is the use of a 3D cell cartridge which will use metabolically active cells which are capable of performing detoxification processes. 2nd innovative concept is the development of an in-line plasma separator. Both the 2 concepts when put together, make up a functional liver-like device which can be connected to a patient suffering from acute liver failure. Hence, we claim the design of a bioengineered artificial liver. This invention is the first of its own kind which has a potential to disrupt the existing treatment modalities related to liver failure and transplant.

[0046] FIG. 1 illustrates a schematic of the 3D culture set-up for culturing cells on a single membrane according to an embodiment of the present invention. Accordingly, the membrane (101) for the cell culture is customized as per the need and is made of Polycarbonate material with a pore size of at least 0.4 microns. The membrane (101) has two surfaces on which one side biologically active cells A and on the other surface biologically active cells B are cultured (which is a 3D cell cartridge). Precisely, the 3D cell culture setup comprises of a customized membrane with appropriate coating. Cell A and Cell B are layered on the membrane sequentially and the setup is incubated in 37-degree Celsius incubator for attachment. Upon successful attachment of the cells to the membrane, the reservoirs is filled with tissue culture medium which support the growth of the biologically active cells. Further, the ratio of the cell number is kept at optimal (Cell A:Cell B) which is cultured herein. Hence, this new process allows the growth of two biologically active cells on a single 6 membrane which are used in a 3D cartridge in the bioartificial liver device for removing lipophilic albumin-bound substances such as bilirubin, bile acids, medium chain fatty acids, metabolites of aromatic amino acids, and cytokines from the patient's blood.

[0047] FIG. 2 illustrates the establishment of co-culture wherein markers for cell viability and nucleus are used according to an embodiment of the present invention. Accordingly, once the cell culturing process has started on the membrane we need examine and track the state (alive or dead) and growth progress of these biological active cells. The validation of the cell growth is shown in the FIG. 2 at the zero hour.

[0048] FIG. 3 illustrates the status of cellular co-culture after 24 hours wherein the staining reflects that the cells are viable according to an embodiment of the present invention. Accordingly, the validation of the cell growth is shown in the FIG. 3 at the 24.sup.th hour. For the cell to be cultured and used in the 3D cartridge it requires 24 complete hours for culturing the biologically active cells.

[0049] FIG. 4 illustrates the functional validation of the 3D cell culture setup by albumin measurements according to an embodiment of the present invention. The effectiveness of the cultured 3D cell are measured by albumin measurements. The legend marked with “well 6” (the last purple bar) indicates its effectiveness of being fully functional even at day 14. Hence, this measurement proves that cultured cell in the 3D cell cartridge is healthy and active even on day 14.

[0050] FIG. 5 illustrates the Schematic of the working principle of the device according to an embodiment of the present invention. Accordingly, the device comprises of an inline plasma separator (600) and one or more 3D cell cartridges (602) with co-cultured cells on either side of a 3D construct. The blood from the patient is drawn using a centrifugal pump (601) and subjected to separation of plasma or the de-cellularised blood and the separated plasma or the de-cellularised blood is pumped (603) into the 3D cell cartridge (602) for removing lipophilic albumin-bound substances from the plasma or the de-cellularised blood thereby allowing the damaged liver to regenerate by reducing the metabolic burden. The 3D cell cartridge (602) herein comprises of microfluidic chamber with the similar network as found in the human liver and the 3D cell cartridge (602) has flow circuits for plasma/de-cellularised blood where the flow circuits are lined by a coculture of cells. The array of cells in the chamber serves as a miniature liver and which multiple such arrays will be stacked to achieve a significant hepatic function.

[0051] The foregoing description of the specific embodiments will so fully reveal the general nature of the embodiments herein that others can, by applying current knowledge, readily modify and/or adapt for various applications such specific embodiments without departing from the generic concept, and, therefore, such adaptations and modifications should and are intended to be comprehended within the meaning and range of equivalents of the disclosed embodiments.

H) ADVANTAGES OF THE INVENTION

[0052] The proposed invention provides bioengineered artificial functional liver which can be connected to a patient suffering from acute liver failure.

[0053] The proposed invention uses a 3D cell cartridge which will use biologically and metabolically active cells.

[0054] The proposed invention provides the development of an in-line plasma separator.

[0055] The proposed invention provides a method of tissue culture wherein the multiple type of cells are cultured on a single membrane in a particular arrangement.

[0056] The proposed invention will provide necessary support to the patient suffering from acute liver failure and will facilitate the regeneration of the native liver.