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BIOARTIFICIAL LIVER
Altan Tabanoğlu, 22 May 2000
cell culture reactor
INTRODUCTION
Liver is a vital organ, which has regenerative property. Liver functions include metabolism, storage, synthesis and release of vitamins, carbohydrates, proteins, lipids and cyclic tetrapyrroles. It also detoxifies and inactivates endogenous and exogenous substances including toxins and metals, and activates precursor molecules, such as proenzymes and coagulation factors. The liver also produces bile, which is involved in intestinal lipid plasma proteins.
The liver cells are called as hepatocytes, and they have the unique regenerative ability among other cells found in the body. They rarely replicate in healthy adults and are usually preoccupied with accomplishing a numerous tasks, which cannot be performed by any other tissue. Liver cells are highly specialized like heart or the brain. However, unlike the brain or the heart, when the liver is injured, virtually all of the surviving hepatocytes leave their normal, growth arrested state and replicate until the destroyed part of the liver is replaced. It is found that, as little as 10% of total liver mass is compatible with life. [1] Moreover, the liver has the ability to recognize when its functional mass has been normalized and to respond by terminating the compensatory growth response. By clarifying the regulation mechanisms we can be able to construct better designs for liver support systems. Still, it is unclear how different growth-regulatory factors interact with unique receptors on the surface of the hepatocytes and trigger a complex, yet orderly cascade of events within the cell, that, together, conclude in a “re-programming” of the hepatocyte’s gene expression which, in turn, permits the cell to escape growth arrest and to do things that are necessary for it to replicate. Emerging evidence indicates that, these different signaling pathways often intersect and, occasionally overlap, providing the cell ample opportunity to amplify or abort any given growth-regulatory signal. Much work will be required to diagram the cell's signaling network and to fully understand how it works. Also mysterious is how the hepatocyte is able to accomplish its specialized functions while it is shouldering the additional burdens required for replication. Clarification of this aspect of hepatocyte replication will be particularly important because the fact that mature hepatocytes replicate refutes previously accepted dogma that only undifferentiated (i.e., non-specialized) cells can replicate.
Due to multi-functions of liver, development of an artificial liver is a real challenge. Unlike the heart, lung, or kidney, which have one primary function, the liver has multiple functions. The complexity of these processes and our poor understanding of the pathogenesis of hepatic failure make what is essential to keep the liver failure patient alive, an unclear phenomenon. The major disease that arises by sudden impairment of liver function with hepatic encephalopathy, resulting from acute necrosis or dysfunction of a large portion of liver parenchyma is called as fulminant hepatic failure (FHF). An estimated 2000 to 2500 deaths per year in the United States are due to FHF. The recent advances in medical supportive therapy is not enough to heal patients completely, mortality rate approaches to 90% due to associated cerebral edema, sepsis, and multi-organ failure. Several attempts were made to improve survival by using various extracorporeal liver support systems loaded with sorbents and liver tissue preparations. However none of them succeeded in gaining clinical acceptance and orthotopic liver transplantation remains a primary therapeutic option for patients with FHF. But limited organ availability and high cost of transplantation ($150,000/transplant) enable only 10% of patients with FHF to receive transplantation. Furthermore, the donor organ deficit is increasing as the indications for liver transplantation are broadened. [2] It should also be noted that many patients with hepatic failure do not qualify for transplantation because of metastatic cancer, concomitant infection, active alcoholism, drug abuse, or concurrent medical problems. Still others do not recover after OLT because of irreversible brain damage caused by hepatic failure. [3] Hepatic regeneration can be increased by the use of short-term metabolic and physiologic support. Such an "artificial liver" can be used as a "bridge" to OLT and as a temporary support during the period of functional recovery of a transplanted liver.
What is expected from an artificial liver is that, it should stabilize the patient, improve his/her general conditions, and decrease the operative and perioperative risks of liver transplantation. An ideal artificial liver should be able to either arrest or reverse cerebral edema and the development of intracranial hypertension. Additional desirable beneficial effects should include improvement of hemodynamic parameters and correction of coagulopathy and other metabolic and physiologic derangements. Bilirubin conjugation, decrease in blood ammonia, maintenance of low lactic acid levels, and increase in the ratio between the branched chain and aromatic chain amino acids are the aimed during artificial liver support.
HISTORY
Scientists have investigated several liver support models and we can classify them as follows:
Blood and plasma exchange
Cross-circulation with healthy humans
Ex vivo whole liver perfusion
Plasma and hemofiltration utilizing various specific and nonspecific adsorptive columns
Hemodialysis
Hepatocyte transplantation
Artificial liver support systems utilizing various liver tissue preparations
None of these systems has achieved wide clinical use and none has demonstrated an ability to reverse the symptoms of hepatic encephalopathy. However, as stated before, artificial liver system can be used as a bridge to OLT.
Kimoto was the first scientist to use the term “artificial liver” in 1959. [4] In his study, he made a short-term hemodialysis between a comatous patient and a dog. Rapid recovery from hepatic coma was accompanied by a significant decrease in blood ammonia level. Then Nose et al, tried hemodialysis against various canine liver tissue preparations (homogenate, fresh liver slices, freeze-dried granules of liver tissue) and observed that it had beneficial effects on glucose homeostasis, hyperlactemia and hyperammonemia.
Later, in 1973 Eiseman et al, constructed a liver assist system by using liver cells.[5] They introduced two important concepts: (i) use of plasma separation; (ii) placement of liver cells within a high-flow plasma recirculation loop. However, this system was not tested clinically. During the past 20 years, artificial liver devices utilizing isolated hepatocytes were described several scientists. They were all tested in vitro and/or in animals with chemically or surgically induced hepatic insufficiency. However, clinical data are still limited. Also in Turkey, by the co-study of TUBITAK Mediterranean Research Center, Eczacıbaşı Medicine and Italy, artificial liver is trying to be constructed which will keep the patients alive until a donor organ is obtained.
DESIGN CONSIDERATIONS
1. Biological Component
The biological component is a collection of mammalian hepatocytes. As it is stated before, the enzyme regulation mechanism that would make us to manipulate the rate of the reactions to speed-up the healing process is still unavailable due to complex mechanism of the regeneration of liver cells. However, we can use readily available mammalian hepatocytes extracorporeally. These mammalians include, mouse, rat, rabbit, dog, porcine, and pig. Several attempts were made to use human hepatocyte cell lines, short population doubling time and contact inhibition is observed. But unfortunately these cells were not fitting biosafety requirements; a potential risk of leakage of tumor cells and their products into the patient’s circulation is present. Therefore, until a liver cell line which is safe and also proliferates and maintains differentiated liver functions is developed, the use of xnogeneic hepatocytes appears to be a more practical and safe alternative. [1] Pig hepatocytes are best to be used due to their morphological and functional homology with human hepatocytes. However, the cells should be immediately used in the Bioartificial Liver (BAL), because differentiation property is lost within 24h to 48h after isolation and there is no better storage method than cryopreservation. The advantages of using pig heaptocytes is as follows:
Pigs are widely used in medical research due to their similar physiology to man
Unlike smaller mammals, such as rabbits or rats, one adult porcine liver can provide enough hepatocytes for several patient treatments.
Isolated pig hepatocytes have a tendency toward formation of cell aggregates.
For an extracorporeal bioreactor system, the method of cell isolation, cell purification techniques, the type of matrix used to maintain the cell viability and expression of differential functions, functional assessment of cells and storage methods (cryopreservatation and cultivation) are important issues.
To anchor hepatocytes, solid supports in the form monolayer-type culture of microcarrier-attached hepatocytes and collagen-coated dextran microcarriers are used. In the first one, incubation of 1.5 x 108 hepatocytes with 1.6 g hydrated microcarriers in a variety of media and in the presence of 10% fetal bovine serum resulted in a monolayer-type culture of microcarrier-attached hepatocytes. Recently, by using only a small number of collagen-coated microcarriers (1.6g / 1.0 x 109 liver cells) cell clusters are obtained. These solid supports have three-dimensional structure, so that the total surface area is available for cell attachment is greatly increased. Also, after inoculation into the extrafiber compartment of the hollow-fiber bioreactor, the micro-carrier cell aggregates do not attach to the fiber wall thereby allowing free convection plasma. It is understood that when isolated hepatocytes are entrapped within gel or gel droplets, the transport of nutrients and metabolites across the fiber wall is less efficient compared with unrestricted convection of plasma.
For mathematically relating the changes observed, one can measure the amount of protein synthesis, ureagenesis, oxygen uptake, drug metabolic assays, clearance techniques, and others.
By cryopreservation of hepatocytes one can obtain:
High efficiency and low cost
Better availability of cells on demand if preservation methods are improved. (In fact, significant loss of cell viability (10% to 20%) and attachment (as much as 50%) is observed. So still fresh cultures are preferred although it takes too much time to isolate and grow them). [6]
Use of an adequate matrix and growth factors, coculture with other types of cells, entrapment of liver within three-dimensional gels, use of a sandwich configuration, and aggregation of hepatocytes into spheroids have also been tried to improve survival and/or function of cultured hepatocytes.
When we consider implantable systems, direct injection, microcarrier and encapsulation systems and finally biodegradable polymer devices are used to anchor hepatocytes. In the case of direct injection of hepatocytes into various organs, body cavities, or blood vessels, recepients’ stromal tissue is used as an extracellular matrix (ECM) required for hepatocyte growth and differentiation. Although corrections of metabolic defects and chemically induced liver failure for 2 weeks to 1 year were observed, significant necrosis and granuloma formation occurred. In the case of microcarriers, the best one is found to be type I collagen-coated dextran beads [7]; they did not induce inflammation or toxicity, intraperitoneal engraftment of hepatocytes were obtained, and cell organization is stimulated. Transplantation of hepatocytes encapsulated in biocompatible membranes (such as alginate-polylysine) also allows incorporation of ECM features into the system, and permits identification of transplanted hepatocytes and analyses of their survival and function. But still for a long period hepatic support, encapsulation is not efficient and remains under investigation. Use of biodegradable polymer can combine advantage of synthetic and natural materials. Physical properties of the synthetic polymers (mechanical strength, degradation rate) can be controlled, and synthetic scaffolds can be produced with fewer batch-to-batch variations made from natural materials. Also, regulation, function, and reorganization of the cell can be controlled by the use of signals. Simultaneous processes of polymer degradation and expansion of transplanted cells ideally lead to creation of the desired tissue. Scaffolds are usually polyglycolic acid (PGA) or polylactic acid (PLA) polymers or co-polymers. By this method, transfer of autologous hepatocytes can be made possible without the risk of immunosuppression. Also, as pancreas is an important source of hepatrotrophic stimuli, co transplantation of hepatocytes and islets of Langerhans as free grafts effectively delivers hepatrotrophic stimuli that improve the growth and function of the transplanted hepatocytes. However, it is difficult to assess and compare the efficacy of various liver assist systems in vivo, in part because most of the animal models with acute liver failure do not reproduce derangements seen clinically in FHF. Additionally, in the hepatoxin-induced (acetaminophen, CCl4, D- galactosamine) liver failure models and transient liver ischemia, the extent of the liver injury is difficult to standardize.
Liver Cell Mass
Optimal cell mass that is going to be used should be determined to provide adequate bioactive support. It should be noted that, even in the most severe form of liver failure there is always a certain amount of residual liver function and as little as 3% to 5% of additional liver mass in a liver support system could potentially make a difference. Kamlot et al, demonstrated that, rats transplanted intraperitoneally with only 1.0 x 107 microcarrier-attached hepatocytes (1% to 2% of the estimated liver mass) prior to subtotal (90%) hepatectomy, had significant higher blood glucose levels and markedly improved survival when compared to nontransplanted controls.
Bioreactor
Isolated hepatocytes have been tested in various devices, which in general can be divided into two groups: (i) those based on hemodialysis principle, (ii) those utilizing hollow fiber technology [8,9]. Hemodialysis has the advantage of protecting xenogenic cells against immunological attack and preventing sensitivity of the patient to proteins produced by the donor cells. However, liver support provided by such a device is restricted to detoxification, and to synthesis and bioprocessing of only small water-soluble molecules.
Wolf and Munkelt [10] were first to use hollow-fiber capillaries as an artificial liver. The bioreactor contains a bundle of small-diameter polysulfone of celluloseacetate tubes enclosed in a rigid plastic housing. The fibers are sealed at each end, so that the two compartments, intra- and extra-fiber, communicate through the pores in the fiber wall. The diameter of the pore can be as much as 0.45 mm. Except for Nyberg [8] et al, all investigators inoculate cells in the extra-fiber (shell) space, while culture medium, blood, or plasma is pumped through the fiber lumen. By this method, immunoisolation of cells placed in the extrafiber compartment can be achieved, although at the cost of impairing mass transport across the fiber wall. In Nyberg’s design, hepatocytes are suspended in a collagen gel, injected into the lumen of hollow-fibers, and then extrafiber space of the bioreactor is perfused for 24h with recirculating medium. The main aim was to construct a third space by the contraction of the gel with the fibers, and while blood passes through the extra-fiber compartment, the gel-entrapped cells are stimulated by the factors present in the medium flowing through a path adjacent to the contracted collagen. This design was original, however, it is difficult to scale it up to accommodate more cells.
Perfusion Systems
It has been discussed, whether a liver support system should be perfused with whole blood or plasma. Proponents of whole blood perfusion argue that plasma separation complicates liver support treatment, increases extracorporeal priming space, and increases the cost of the procedure. Other advocates of plasma perfusion states that with the availability of the modern state-of-art-technology (single venous access, minimal fluid volume removal, continuous high-yield collection of plasma, low extracorporeal volume) separation of plasma can be carried out rapidly and at low risk. As hollow-fiber bioreactors contain liver cells separated from the blood by a membrane similar to that used in the plasma membrane separator, one should also use plasma to obtain an efficient mass transport and oxygen support. Also, by placing activated charcoal, some of the scientists were able to depress the toxic effects of plasma to the donor hepatocytes.
An overall scheme of the extracorporeal process can be shown as:
The VITAGEN [11] company achieved using human hepatocytes at Extracorporeal Liver Assist Device (ELAD), and they were able to keep patients alive for 10 days with continuous changing of cartridges every few hours. It is being used at the University of Chicago Hospitals. Used cartridges will be returned to VITAGEN for further analysis.
REFERENCES
1. Kamlot, A., et al, “Review: Artificial Liver Support Systems”, Biotechnology and Bioengineering, Vol 50, pp 382-391 (1996)
2. Rozga, J., et al, “Isolated Hepatocytes in a Bioartificial Liver: A Single Group View and Experience”, Biotechnology and Bioengineering, Vol 43, pp 645-653 (1994)
3. Cohen, C., et al, “Alcoholics and liver transplantation”, JAMA 265: 1299-1301 (1991)
4. Kimoto, S., “The Artificial Liver Experiments and Clinical Application”, ASAIO Trans. 5: 102-110 (1959)
5. Eiseman, B., et al, “Hepatocyte Perfusion within a centrifuge”, Surg. Gynecol.Obstet., 142: 21-28 (1976)
6. Karlsson, J. O. M., et al, “Long-term Storage of tissues by cryopreservation: critical issues”, Biomaterails 17: 243-256 (1996)
7. Davis, W. M., et al, “Toward Development of an Implantable Tisuue Engineered Liver”, Biomaterails 17: 365-372 (1996)
8. Nyberg, S. L., et al, “Evaluation of a Hepatocyte-Entrapment Hollow Fiber Bioreactor: A Potential Bioartificial Liver”, Biotechnology and Bioengineering, Vol 41, pp 194-203 (1993)
9. Wu, F. J., et al, “Hollow Fiber Bioartificial Liver Utilizing Collagen-Entrapped Porcine Hepatocyte Spheroids”, Biotechnology and Bioengineering, Vol 52, pp 34-44 (1996)
10. Wolf, C. F. W., et al, “Bilirubin Conjugation by an Artificial Liver Composed of Cultured Cels and Synthetic Capillaries”, Trans. Am. Soc. Artif. Org. 21: 16-26 (1975)
11. http://www.vitagen.com
