Monday, December 29, 2003 6:06:56 PM
Principles of Liver Support Systems
I do not know how old this article is, but Dr. Hugo Jauregui (now deceased), Multicell, is mentioned in it. There are pictures at the link and I found the article interesting.
Erika Olson, Erin Bradley and Kedar Mate
Our thanks to Dr. Hugo Jauregui of MultiCell Co. for his assistance and guidance in our attempt to understand the bioengineering, cell biology, politics and economics of the bioartificial liver.
Table of Contents:
LIVER FUNCTION
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http://biomed.brown.edu/Courses/BI108/BI108_1999_Groups/Liver_Team/liver.gif
How does the liver work?
LIVER FAILURE
How does the liver fail?
FUNCTIONAL NEEDS AND HETEROGENEOUS SOLUTIONS
What are the needs for liver support?
What functional requirements must LADs perform/fulfill?
What device models have been proposed?
What is a Bio-Artificial/Bio-Hybrid LAD?
SYSTEMS IN CLINICAL TRIALS
What is the HepatAssist 2000" System?
What is the Hepatix/Vitagen ELAD( System?
How do both devices compare?
THE FUTURE
What major improvements are in the works?
How is the bioartificial liver research industry changing?
Liver Function
How does the liver work?
The biological complexity of the liver is immense. Many of the specific processes involved with this organ's multiple functions are not yet fully understood. It is commonly agreed, however, that the liver's main purpose is the maintenance of homeostasis.
The liver is central to the body's metabolism, or the process by which living matter is produced, destroyed, or maintained. This involves the breakdown, synthesis, modification, storage, and regulated release of carbohydrates, lipids, amino acids, proteins, and nucleic acids. The liver is capable of all of these tasks. Its production of bile, which it delivers to the intestine, aids in digestion and excretion of wastes. All the rest of the liver's metabolic processes are accomplished within the liver itself. Receiving two-thirds of its blood from the portal vein of the gastro-intestinal tract, the liver comes in direct contact with nutrient-rich blood after it has coursed through the gut, spleen and pancreas. It is capable of temporarily storing over half of the nutrients that are absorbed by it, thereby balancing elevated levels of carbohydrates, amino acids, and fats immediately after a meal's digestion. Five to eight percent of the liver's total weight is stored glycogen, which, like the rest of the liver's stored nutrients, is systematically released according to biological need. The liver is therefore also essential to the body's regulation of energy.
Due to its synthesis of certain plasma proteins, the liver plays a large role in regulating proper coagulation function, osmotic pressure, and enzymatic functions of the blood. Its synthesis of cholesterol, due to its fat and lipid metabolism, also effects the body's hormonal homeostasis.
The liver is also significant to maintaining immunological health. Phagocytic cells lining this organ, called Kuppfer cells, filter bacteria and other potentially harmful particulates out of the gastric blood passing over them. This prevents these antigens from coming in contact with the body's general bloodstream circulation. The liver's synthesis of globulin also enhances an organism's immune system.
One of the liver's most crucial functions is detoxification. The liver transforms potentially dangerous metabolites, toxins, and excess hormones into biologically harmless water-soluble compounds. Urea is one such compound - produced from ammonia, a toxic substance capable of creating acute negative effects on an organism. Ammonia is formed during the deamination of amino acids, which occurs any time a cell uses protein for energy. A method of ammonia breakdown is therefore imperative to an organism's survival. The liver provides this breakdown, thereby preventing hazardous ammonia accumulation, by transforming ammonia into urea, which can then be excreted by the body.
The function of the liver is therefore manifold, involving:
* Detoxification
* Carbohydrate Metabolism
- Glyconeogenesis and glycogenolysis
* Fat and Lipid Metabolism
* Synthesis of lipoproteins and cholesterol
* Protein Metabolism
* Synthesis of Plasma Proteins - albumin, fibrinogens, coagulation factors, transferrin, globulin
- Albumin accounts for colloid osmotic pressure in the plasma
- Fibrinogen is crucial to blood clotting
- Globulins have enzymatic function in the plasma and enhance immunity
* Conjugation of Bile Acids
* Storage of Essential Nutrients and Vitamins
* Biotransformation of Pharmaceuticals and Vitamins
- Causes transformations in drugs making them useful to the body
* Immunity
What is a hepatocyte and what does it do?
The liver is the largest organ of the body, weighing 1500 grams. Two percent of the liver's mass is formed by its phagocytic Kuppfer cells, which provide the organ with its filtrative immune function, and a comparable amount of the liver is made of the cells of Ito, which function as fat stores. The majority of the liver, however, is composed of hepatocytes. These are parenchymal cells of the liver, and make up 80 - 90% of its total mass. They are relatively large cells, measuring about 25 microns on a side. There exist a total of 250 billion to 500 billion such cells in the liver. They are arranged in plates, which are radially distributed around the central vein of the 4 or 5-lobed organ.
Hepatocytes possess an abundance of smooth and rough endoplasmic reticulum, and many ribosomes, lysosomes and mitochondria. This abundance is required for fat and lipid metabolism, synthesis of cholesterol and lipoproteins, protein metabolism, and the synthesis of complex proteins. The hepatocytes are also the primary cells involved in detoxification and bile secretion. They are the storehouse of many essential nutrients as well.
Only 40% of a liver's functioning hepatocytes are required for its adequate performance. This is due to their efficiency and their ability to regenerate. The liver is the only internal organ capable of regeneration.
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Liver Failure
How does the liver fail?
Hepatic failure is either acute or chronic, depending on the amount of time the liver takes to fail. Liver failure is caused either by traumatic, extensive injury or disease. There are over 100 different types of liver diseases, afflicting one in 10 individuals or 25 million Americans. Each year over 43,000 people die of liver disease and hospital costs exceed $8 billion annually.
According to the American Liver Foundation:
* 25,000 people die annually from chronic liver disease and cirrhosis
* 10,000 people die annually from Hepatitis C
* 5,000 people die annually from Hepatitis B with an estimated new infection rate of 250,000 people annually
* 1,000 people die annually from primary liver cancer
Most liver diseases result from a variety of build-ups within the liver including fats, sugars, and other metabolites. The build-ups within the liver cause cirrhosis. Cirrhosis is a degenerative process in the liver, which is identified by excess formation of connective tissue, destruction of functional cells, and, often, contraction of the organ.
As mentioned previously, the liver possesses the unique ability to regenerate itself with new healthy tissue. If the liver is damaged beyond its capacity to regenerate new cells, the resulting loss of liver function severely effects the entire body's metabolism.
Liver failure causes the following body-wide complications:
* metabolic instability
* disruption of energy supply
* disruption of acid-base balance
* disruption of thermoregulation
* uncontrolled bleeding and sepsis
* toxic by-product build-up stops dependant organ function
* cease of other organ functions because lack of liver-synthesized nutrients
The varieties of liver diseases cause either acute or chronic hepatic failure. Acute fulminant hepatic failure (FHF) is the result of extensive hepatocyte death over the course of days or weeks. This necrosis of hepatocytes is due to toxic substances or viral infections.
Body-wide symptoms include:
* jaundice
* mental confusion
* stupor or coma
The mortality rate is 80-90%, with rapid (a week or two) death. Though liver transplantation is the only effective form of treatment, 15-30% of FHF patients will regenerate their liver under proper medical treatment.
Chronic hepatic failure is the most common form of the disease. With chronic hepatic failure, the hepatocytes are damaged (usually by long-term exposure to toxins such as alcohol or by viral infections like hepatitis) and unable to detoxify. This build-up of toxic nitrogenous products (i.e., ammonia) within the body causes serious damage.
Body-wide complications include:
* deterioration of mental status
* cerebral edema
* impaired blood coagulation
* gastro-intestinal bleeding
* brain cell damage
* coma
Though there are management techniques for chronic hepatic failure, 60-90% of patients require an eventual transplantation.
These two different types of liver failure require different approaches to liver assist devices. While those suffering from FHF have a chance at liver regeneration, an extracorporeal liver assist device (LAD) is used to detoxify the blood, giving the damaged liver a chance to grow new cells. In this case, the LAD acts as a organ replacement for the time it takes to regenerate. In the case of chronic hepatic failure, regeneration of new cells is no longer viable, so the LAD acts as a bridge while waiting for the availability of a transplant.
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Functional Needs and Heterogeneous Solutions
What are the needs for liver support?
As suggested above, there are two principle needs/functions for liver-assist devices (LAD):
* As a Bridge-To-Transplant Device
* As a Bridge-To-Native Organ Regeneration
Bridge-To-Transplant Needs:
Patients affected by fulminant hepatic failure and hepatic encephalopathy have an extremely high mortality rate - published mortality rates varying from 86% to 93%. The present availability of liver transplants (the treatment of choice for liver failure) has stabilized according to the American Liver Foundation at approximately 4000 transplants a year in the US. The United Network for Organ Sharing (UNOS) schematic to the left charts the rapid growth of the liver transplant waitlist over the past 10 years. As of 4.21.99, according to UNOS there are 12,805 people awaiting liver donations. In 1998, UNOS reported a slight increase from the average in successful transplants as 4450 livers were transplanted.
For more information on liver and other organ donations, transplants and waitlists,
UNOS data can be accessed by going to the "Data" hyperlink at:
http://www.unos.org
These data demonstrate that a significant number of individuals are left waiting for biocompatible liver donations from cadaveric, or in rare cases, living donors. Furthermore, an unspecified number of liver transplants do not function properly in the post-operative period. Thus, a LAD is needed to bridge these primary non-function (PNF) patients to a functional liver status or to a secondary transplant. Finally, liver transplant is an extremely costly operation ranging between $150,000-$250,000 with the American Liver Foundation (ALF) reporting an average cost of $230,000 for the immediate operative costs (See ALF funding report at: http://gi.ucsf.edu/alf/ALF/fundrolo.html). Estimates of annual follow-up costs average between $15,000-$25,000. Clearly, there is a need for LADs that can effectively manage liver failure to bridge waitlist patients to transplant and perhaps to avoid costly transplant altogether by taking advantage of the liver's unique regenerative properties.
Bridge-To-Native Organ Regeneration Needs:
As has been stated earlier liver tissue has the unique ability to regenerate itself. Conditions and factors that encourage latent tissue to move into a regenerative cycle are currently under investigation. Though sources differ on exactly how much of the liver's bio-mass must be replaced to provide a minimum amount of liver function, the literature generally suggests that between 15-30% of the liver's total weight must be reconstituted. Thus, LADs must be created and tested to provide temporary relief of liver function while the native liver undergoes the hepatocyte-regenerative process discussed above. Thus, more livers will be made available, and costly transplant procedures can be avoided. This type of liver-assist is most clearly indicated when the patient's liver has undergone a massive liver resection (cancer removal) or has experienced some kind of trauma.
What functional requirements must LADs perform/fulfill?
LADs must attempt to imitate certain biochemical processes that the native liver routinely performs. In order to conduct these biochemical activities, a LAD will require mechanical support, thus the ideal LAD must fulfill two basic functional requirements:
* Biochemical Functions
* Mechanical Functions
Biochemical Functions:
In some professional estimates, the human liver performs upwards of 900 biochemical functions in the body. It is impossible for any artificial device to duplicate all of these activities; the sum total of these functions must be narrowed down to the essential biochemical functions that ensure patient survival. The acute indications of FHF and the variety of symptoms caused by CHF thus call for different LADs. Both illnesses, however, call for the substitution of the following principle biochemical functions:
* Ammonia control - Elevated levels of ammonia found in cerebrospinal fluid of FHF patients and elevated intestinal production of ammonia directly correlate with a progression of HE stages.
* Endogenous benzodiazepines, GABA and acetaminophen control.
* Articulation and careful regulation of the cytochrome P450 detoxification cycle.
Mechanical Functions:
The two main mechanical functions that the physical configuration of any LAD must allow and/or provide are:
* Immune Protection - Isolation of any foreign (xeno- or allo-) biological components found in the LAD from the body's natural immune response
* Cell Nourishment - Provision of oxygen and other nutrients to biological components that are housed within the LAD.
What device models have been proposed?
A plethora of liver support devices have reached varying stages of conceptualization, manufacture and experimental implementation. The history of liver-assist innovation is long and complex, exhibiting the typical slow-growth curve from 1950-1970 and the subsequent rapid acceleration due to the inception, incorporation (literal) and acceptance of biohybrid technologies. Though research interests in the bioartificial liver are constantly growing, the devices that have been produced have met with only marginal success. Some researchers claim, in a familiar metaphor, that the bioartificial liver remains the "Holy Grail" of organ replacement.
See Friedman's editorial article entitled,
"Why bioartificial liver support remains the Holy Grail"
ASAIO Jul-Aug of 1998.
Historically, a dividing line can be drawn among the devices that have been conceived and/or constructed:
* Those devices that primarily focus of mechanical replacement of basic liver function.
* Those devices that focus of biochemical or "global" replacement of liver function.
Though devices can be divided along these guidelines, it must be stipulated that the LADs that are presently in clinical trials often use a synthesis of devices from both categories.
Mechanical Models for basic replacement of liver function:
* Hemodialysis: Mechanism exactly parallel to kidney dialyzer that clears only ammonia and small molecules. Relies upon convective pressure or diffusion models
* Hemofiltration: Has larger molecular pores that allows for the convective clearance of larger toxins. Relies upon the same mechanics of hemodialysis.
* Hemoperfusion: Passing blood component over particular absorbent compounds that bind toxins. A hemoperfusion device using activated charcoal is often used in biohybrid LADs as an additional part of the blood pathway.
* Exchange Transfusion or Plasmapheresis: Operates on the principle that either whole blood or toxin-bearing plasma can be replaced entirely over time to reduce toxin-content. Though there are thoughts about combining plasma replacement with hemoperfusion, these procedures are not yet functional long-term solutions to liver insufficiency.
Several innovations upon these basic detoxification-based liver support mechanisms have been proposed including lipophilic membrane systems and cross-dialysis with limited or unexplored success. The principle problem with mechanical devices is they are metabolically inert, while the liver's principle functions are largely biochemical and excretory - functions that mechanical models cannot duplicate.
Biochemical models for "global" replacement of liver function:
* Cross-Circulation: Links a biocompatible individual's circulation to the circulation of the patient with liver failure in a prolonged exchange transfusion that allow the patient's blood to be processed "globally" by the healthy individual's hepatocytes.
* Hemoperfusion over Liver Slices: Similar to the mechanical model of hemoperfusion, except that the absorbent compound is active, biocompatible hepatocytes.
* Organ Transplant: The procedure of choice, though severely limited by donor organ supply. Recently segments of living-related donor liver tissue have proven effective in treatment of liver failure.
* Heterotopic Hepatocyte Transplant: Direct transplantation of hepatocytes suspended in a matrix to provide temporary relief to a failing liver. There is much disagreement on how and where to deliver the hepatocytes, how many to deliver and what kind of matrix to place them in.
* Bio-Artificial/Bio-Hybrid Replacement: A device that fixes and sustains functional, harvested hepatocytes within a matrix that allows for biochemical interactions with patient's blood or plasma. These mechanisms are the models for LADs that are currently in clinical trials.
What is a Bio-Artificial/Bio-Hybrid LAD?
Bioartificial LADs apply mechanical principles to the biologically active models for the "global" replacement of primary liver functions. A variety of culture systems and bioreactors have been developed to perform as bioartificial liver substitutes including: static cultures, stirred suspensions, packed beads, matrix cultures, fluidized beds and hollow-fiber mechanisms. Among these various configurations, cell suspensions, packed bead chambers and hollow-fiber bioreactors have been actually used in human patients, with hollow-fiber devices being the principle device design that has gone on to human clinical trials.
The Principles of Hollow-Fiber Bioreactors as LADs
Hollow-fiber bioreactors, similar to hemodialysis devices, contain a number of hollow fibers of a semipermeable material. In a design that is quite familiar by now, this device allows for two compartments, an intracapillary space (ICS) and an extracapillary space (ECS). Most bioreactors in clinical trials, culture or seed hepatocytes in the ECS and perfuse patient's blood or plasma through the capillaries.
There are a number of disagreements among scientists who are currently investigating hollow-fiber bioreactors as a replacement for primary liver functions. These disagreements are currently posited as research questions, some of which have been answered and some of which are currently under investigation.
What kind of hepatocytes to use?
Jauregui et al. describe the ideal hepatocyte for LADs as, "human in origin, rapidly and easily grown in culture at high densities...of normal (non-malignant) phenotype...remain in a well-differentiated state for days or weeks...[and] must display synthetic and detoxifying features of mature hepatocytes." The bioactive, hepatocyte component of the hollow-fiber bioreactors is perhaps the most hotly debated aspect of LADs - questions raised generally revolve around the following concerns:
* Human or Porcine? Human hepatocytes are preferable because the biochemicals they manufacture are same products that native hepatocytes produce. Also, they are less immunogenic and there is no risk of porcine endogenous retroviruses (PERV). However, human hepatocytes grow poorly in culture media and there is a problem with accumulating enough human hepatocytes considering the enormous existing liver support burden. Furthermore, it appears that human C3A cells (the most common human hepatoma cell line) have less ammonia clearing ability than porcine cells.
Relative performance of Porcine Hepatocytes (PPH) in decomplemented human serum medium (DHS) versus Human Hepatomas (C3A) in human serum (HS). Each of the 3 measurements has its own scale - here they are shown together and relative to one another. Synthesized from Wang-Lischeng et al.; Cell-Transplantation 1998: 7(5): 459-468.
* Tumorigenic (malignant) or Not? Tumorigenic cell lines provide rapidly proliferating, non-differentiated cells that allow for long-term hepatocyte replacement and regeneration. These cells require a minimal seeding dose and minimal anchorage in comparison to normal cells. However, they may not respond to physiologic controls and thus there is a danger of bioreactor escape and subsequent tumor formation.
* Immortalized or Not? These cells may not be tumorigenic and can be subjected to physiological controls. Simultaneously, immortalized cells will provide long-term functional hepatocytes. However, there is a question of whether multiple generations will contribute to long-term degradation of metabolic capabilities.
What should be the particular geometry of the device?
* Should hepatocytes be cultured or seeded in the ECS or in the ICS? As stated above hepatocytes are generally being cultured/seeded in the ECS.
* How can the hepatocytes be evenly distributed and how can this distribution be maintained with time? This question is one of the biggest problems with hollow-fiber bioreactors today and is the subject of a number of investigations. One of the latest innovations addresses this issue in a very interesting way.
Should the perfusate be blood or plasma?
* Blood - Easy uncomplicated circuit design, sufficient oxygen and nutrient support for hepatocytes, coagulation problems.
* Plasma - Less coagulation problems, can be filtered before returning it to the blood stream, circuit is complicated and plasma has a lower oxygen-carrying capacity.
What should the nature of the membrane be?
* Open-membrane - Allows toxin-bearing albumin to cross and interact with hepatocytes allowing for increased overall effectiveness of the LAD. However, this also increases the immunogenicity of the device.
* Closed-membrane - Allows for greater immunoisolation, prevents toxin-bearing albumin from interacting with hepatocytes and therefore decreases the effectiveness of the LAD.
Most devices have switched over to an open-membrane LAD and have generated other solutions to the immune comprise that this concession creates.
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Systems in Clinical Trials
What is the HepatAssist 2000" System?
Circe Biomedical
http://www.circebio.com
Circe Biomedical is a bio-tech company devoted to developing bioartificial organs based on its exclusive technologies involving mammalian cells and semi-permeable membranes. These technologies include the ability to:
* Fabricate semipermeable and biocompatible membranes
* Isolate, purify and preserve mammalian cells
* Design, produce and commercialize biomedical systems incorporating these membranes and cells, providing for essential organ function
Its liver support system, HepatAssist(r), is currently Circe Biomedical's leading product. It is the most clinically advanced system of its kind.
HepatAssist(r) is an extracorporeal cell-based bioartificial liver device, based on the use of an open membrane hollow fiber bioreactor. This membrane is microporous, with a pore size of .2_m. This size is just small enough to halt the passage of whole cells, such as hepatocytes, through it, but large enough to allow for free exchange of toxins (soluble and protein-bound) and large molecular weight proteins between the hepatocytes housed outside the hollow fibers and the plasma traveling on the inside of the fibers. The cells used in this device are microcarrier-attached primary porcine hepatocytes.
Basic Steps Involved in HepatAssist(r) Treatment:
Circe's bioartificial liver device (BAL) consists of four parts:
* A hollow fiber bioreactor containing primary porcine hepatocytes
* Two charcoal filters
* A membrane oxygenator
* A pump
BAL is used in conjunction with a commercially available plasma separation machine, heater, and temperature and oxygen monitors.
To begin a treatment, which generally lasts around 6 hours, a patient's blood is separated into blood and plasma in a plasma separation machine. The blood portion is then kept in the plasmapheresis device until reconstituted with the plasma after processing in the bioreactor. Once separated from the blood, a person's plasma, by use of the device's pumping system, is moved via the system's tubing through two charcoal filters. These filters essentially act as Kuppfer cells, filtering the plasma from massive bacteria and particulate matter which could overwhelm the system's hepatocytes. Thus, they provide the system with its first detoxificative action. The rest of the detoxification occurs within the hepatocyte-lined hollow fiber column, through which the plasma then flows. After this, the "clean" plasma is reconstituted with the blood which was stored in the plasmapheresis system, and the whole blood is reinfused into the patient.
A membrane oxygenator and heater are placed between the charcoal filters and the hepatocyte bioreactor. The heater keeps the plasma (and therefore the hepatocytes over which they flow) at body temperature. The membrane oxygenator provides the housed hepatocytes with the oxygen they require for proper function.
The Use of Porcine Cells:
There is great concern involving the use of xenogeneic tissue in this BAL, especially since the large size of HepatAssist's membrane pores doesn't provide an immunoisolated environment for its porcine hepatocytes. Great effort has therefore been devoted to the issue of cell supply through the use of intensive animal sourcing programs.
The main problem involved with the use of porcine cells in this device is their inability to be sterilized. Once hepatocytes are isolated from porcine livers, they cannot undergo any sort of sterilization process and still remain functional. A comprehensive herd and animal bioburden screening program was therefore instituted to provide animals free from zoonotic viruses which could be harvested for HepatAssist(r). Under such screening, the desired porcine cells are cryopreserved and quarantined until the results of bioburden assays performed on them are acquired. Once deemed acceptable, the cells remain cryopreserved for shipping and storage, and are thawed just prior to HepatAssist(r) treatment. So far zoonotic viruses, including PERV (porcine endogenous retroviruses), have not been a problem in clinical trials of this system. The isolation, processing, and cryopreservation involved with porcine hepatocytes is in compliance with Good Manufacturing Practices (cGMP).
Phase I Clinical Trials:
Circe's HepatAssist(r) is meant to function simply as a bridge to orthotopic liver transplantation (OLT) or natural liver recovery. So far, FHF patients have received one 6 hour long treatment per day for an average bridge time of 39.3 hrs (21 - 96 hour range). Individual treatments have been shown to induce intracranial pressure improvement in patients after 2 - 2.5 hours of use.
Most of HepatAssist's phase I clinical testing occurred at the General Clinical Research Center at Cedars-Sinai Medical Center, where many of the creators of the device are from. HepatAssist(r) was used in combination with an effective support group of doctors, nurses, nutritionists, physical and respiratory therapists and pharmacists during its trial. The trial was finished early in 1997. Thirty-six patients, total, were treated with the device; 23 of whom had FHF, 10 acute worsening of chronic liver disease, and 3 nonfunctioning transplants. Each patient was treated 1 - 5 times with the device.
The Results of Phase I Clinical Testing:
* 19 of the FHF and nonfunctioning transplant patients were bridged to OLT
* one of these died 7 days after implantation
* 6 recovered spontaneously - with no need for transplantation
* 2 patients with chronic liver disease recovered to the point of receiving an OLT
* 9 patients died
* eight of the chronic liver disease patients were denied OLT
* one died of pancreatitis
* All survivors reached full neurologic recovery
These results demonstrated safety and tolerance to the point that the FDA granted Circe permission to proceed with a phase II/III clinical trial testing the device's efficacy on the 30 day survival of patients with acute liver failure. These trials are ongoing in 12 different centers in the US and Europe.
What is the Hepatix/Vitagen ELAD( System?
VITAGEN, INC
(formerly Hepatix, Inc.)
http://www.hepatix.com
Vitagen Incorporated is a biotechnology and medical products company that describes itself as having "developed the world's first artificial liver [with the goal] of creating products to help reduce the needs for transplants of this vital organ." Vitagen has created the first medical device to incorporate "immortalized" human liver cells (C3A).
Basic Steps Involved in ELAD( Treatment:
VitaGen's ELAD is plasma perfused, meaning the patient's plasma flows through the cartridge where it interacts with the metabolically active C3A cells. The treated plasma is then filtered and returned to the patient. The basic steps involved in treatment are comparable to those described above in the HepatAssist(r) device.
The Use of Human Hepatoma C3A Cells:
VitaGen's ELAD (Extracorporeal Liver Assist Device) artificial liver consists of a hollow fiber cartridge populated with an immortalized liver cell line. These C3A liver cells, located in the extra-capillary space, can be reproducibly manufactured in culture and express normal liver-specific metabolic pathways such as ureogenesis, gluconeogenesis, and P-450 activity. They also secrete clotting factors and other liver-specific proteins. The metabolic capacity of each cartridge is equal to that of about 200 g of normal liver.
Phase I Clinical Trials:
VitaGen conducted two trials to assess the role of an artificial liver in the treatment of acute liver failure (ALF). The trials were held at the University of Chicago Hospital and at King's College in London. The initial trial involved 11 patients, all with advanced ALF, while the second trial (12 patients) was divided into two groups. Group 1 suffered from moderate liver disease, group 2 from advanced. The causes of ALF in the patients breaks down in the following manner:
* 7 due to acetaminophen
* 5 due to NANB hepatitis
* 3 due to fialuridine
* 2 due to anti-TB drugs
* 1 idiopathic
* 1 due to syncytila giant cell hepatitis
* 1 due to liver resection for primary graft non-function
Patients received ELAD therapy continuously for 3-168 hrs (7 days).
The Results of Phase I Clinical Testing:
Results showed satisfactory biocompatability, improved hemodynamic parameters and improvement in encephalopathy, which indicated that patients could safely be removed from ELAD treatment.
However, the survival outcomes of the two trials differed greatly:
Trial 1
* 1 out of the 11 survived
Trial 2
* 8 out of 12 survived
The discrepancy is described as being due to the medical conditions of those in each trial.
These results demonstrate that ELAD treatment appears to increase the rate of recovery from sublethal ALF, and has the capacity to support patients for several days (even in the presence of severe liver failure), serving as an effective bridge to transplantation.
How do both devices compare?
SYSTEM
Device Configuration
Perfusion Medium
Cell Selection
Phase I Clinical Trial Survival Rate
Hepatix ELAD(
Hollow-fiber ELAD
Open-Membrane
Plasma
Human Hepatoma (C3A)
9/23 = 39.1%
HepatAssist(r)
Hollow-fiber BAL
Open-Membrane
Plasma
Porcine Hepatocytes
(PPH)
27/36 = 75.0%
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The Future
So, what major improvements are in the works?
There are a number of improvements to the present models that are in various phases of development. Two systems, in particular, should be mentioned - one which has been used under extreme circumstances in a single patient and another which is very close to being finalized and put into human trials.
Gerlach et al.
This device pioneered at the University of Berlin by Dr. JC Gerlach uses a three-dimensional design for a LAD that is very different from the hollow-fiber models that are currently being tested. His model essentially involves four different capillary membrane systems, each serving a different purpose, woven into a 3-D lattice. These capillaries independently and locally provide oxygen, nutrients and plasma perfusate inflow and outflow to hepatocytes. This allows for, "decentralized cell perfusion with low metabolite gradients and decentralized oxygenation and CO2 removal," and has contributed to increased hepatocyte performance in long-term ex vivo assessment. Data for this technique is encouraging, though as of 1997, only 1 patient had been bridged-to-transplant using this device. The duration of liver-assist for this patient was 40 hours.
Chamuleau et al.
At the University of Amsterdam, Dr. Chamuleau et al. have come up with a novel and exciting design technique that will hopefully resolve the matrix support and hepatocyte anchorage problems that have thus far plagued non-tumorigenic cell bioreactors. In order to fix these cells more firmly and to preserve their distribution, Dr. Chamuleau has taken a flat hepatocyte-seeded polyethylene sheet and rolled each hollow-fiber one after another to create a tight multiple sushi-roll-style bioreactor. The hollow-fibers provide oxygen and nutrient support to the hepatocytes while toxin-bearing plasma is perfused directly through the extrafiber space allowing for direct hepatocyte contact. This device has been tested in pigs and will soon enter human trials supervised by Dr. Calise of the Cardarelli Hospital in Naples, Italy.
How is the bioartificial liver research industry changing?
Our conclusions, based upon trends we have observed in the existing literature and our conversations with Dr. Jauregui, indicate that as the bioartificial liver research industry grows it is becoming increasingly more and more specialized. What this means is that, although there are many companies working on complete LADs, companies now entering the LAD market tend to chose very specific aspects of such devices as their specialty. MultiCell Co., and other bio-tech companies like it, have intelligently narrowed their research focus to just the cell lines that will eventually be used to populate the devices that are engineered by other companies whose primary focus is on the device itself. Thus, the process of forming a complete LAD has become a cooperative effort of highly specialized companies that share their expertise (at a price) in a highly competitive market.
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http://biomed.brown.edu/Courses/BI108/BI108_1999_Groups/Liver_Team/Liver.html
BACK TO BI108 HOMEPAGE
I do not know how old this article is, but Dr. Hugo Jauregui (now deceased), Multicell, is mentioned in it. There are pictures at the link and I found the article interesting.
Erika Olson, Erin Bradley and Kedar Mate
Our thanks to Dr. Hugo Jauregui of MultiCell Co. for his assistance and guidance in our attempt to understand the bioengineering, cell biology, politics and economics of the bioartificial liver.
Table of Contents:
LIVER FUNCTION
How does the liver work?
LIVER FAILURE
How does the liver fail?
FUNCTIONAL NEEDS AND HETEROGENEOUS SOLUTIONS
What are the needs for liver support?
What functional requirements must LADs perform/fulfill?
What device models have been proposed?
What is a Bio-Artificial/Bio-Hybrid LAD?
SYSTEMS IN CLINICAL TRIALS
What is the HepatAssist 2000" System?
What is the Hepatix/Vitagen ELAD( System?
How do both devices compare?
THE FUTURE
What major improvements are in the works?
How is the bioartificial liver research industry changing?
Liver Function
How does the liver work?
The biological complexity of the liver is immense. Many of the specific processes involved with this organ's multiple functions are not yet fully understood. It is commonly agreed, however, that the liver's main purpose is the maintenance of homeostasis.
The liver is central to the body's metabolism, or the process by which living matter is produced, destroyed, or maintained. This involves the breakdown, synthesis, modification, storage, and regulated release of carbohydrates, lipids, amino acids, proteins, and nucleic acids. The liver is capable of all of these tasks. Its production of bile, which it delivers to the intestine, aids in digestion and excretion of wastes. All the rest of the liver's metabolic processes are accomplished within the liver itself. Receiving two-thirds of its blood from the portal vein of the gastro-intestinal tract, the liver comes in direct contact with nutrient-rich blood after it has coursed through the gut, spleen and pancreas. It is capable of temporarily storing over half of the nutrients that are absorbed by it, thereby balancing elevated levels of carbohydrates, amino acids, and fats immediately after a meal's digestion. Five to eight percent of the liver's total weight is stored glycogen, which, like the rest of the liver's stored nutrients, is systematically released according to biological need. The liver is therefore also essential to the body's regulation of energy.
Due to its synthesis of certain plasma proteins, the liver plays a large role in regulating proper coagulation function, osmotic pressure, and enzymatic functions of the blood. Its synthesis of cholesterol, due to its fat and lipid metabolism, also effects the body's hormonal homeostasis.
The liver is also significant to maintaining immunological health. Phagocytic cells lining this organ, called Kuppfer cells, filter bacteria and other potentially harmful particulates out of the gastric blood passing over them. This prevents these antigens from coming in contact with the body's general bloodstream circulation. The liver's synthesis of globulin also enhances an organism's immune system.
One of the liver's most crucial functions is detoxification. The liver transforms potentially dangerous metabolites, toxins, and excess hormones into biologically harmless water-soluble compounds. Urea is one such compound - produced from ammonia, a toxic substance capable of creating acute negative effects on an organism. Ammonia is formed during the deamination of amino acids, which occurs any time a cell uses protein for energy. A method of ammonia breakdown is therefore imperative to an organism's survival. The liver provides this breakdown, thereby preventing hazardous ammonia accumulation, by transforming ammonia into urea, which can then be excreted by the body.
The function of the liver is therefore manifold, involving:
* Detoxification
* Carbohydrate Metabolism
- Glyconeogenesis and glycogenolysis
* Fat and Lipid Metabolism
* Synthesis of lipoproteins and cholesterol
* Protein Metabolism
* Synthesis of Plasma Proteins - albumin, fibrinogens, coagulation factors, transferrin, globulin
- Albumin accounts for colloid osmotic pressure in the plasma
- Fibrinogen is crucial to blood clotting
- Globulins have enzymatic function in the plasma and enhance immunity
* Conjugation of Bile Acids
* Storage of Essential Nutrients and Vitamins
* Biotransformation of Pharmaceuticals and Vitamins
- Causes transformations in drugs making them useful to the body
* Immunity
What is a hepatocyte and what does it do?
The liver is the largest organ of the body, weighing 1500 grams. Two percent of the liver's mass is formed by its phagocytic Kuppfer cells, which provide the organ with its filtrative immune function, and a comparable amount of the liver is made of the cells of Ito, which function as fat stores. The majority of the liver, however, is composed of hepatocytes. These are parenchymal cells of the liver, and make up 80 - 90% of its total mass. They are relatively large cells, measuring about 25 microns on a side. There exist a total of 250 billion to 500 billion such cells in the liver. They are arranged in plates, which are radially distributed around the central vein of the 4 or 5-lobed organ.
Hepatocytes possess an abundance of smooth and rough endoplasmic reticulum, and many ribosomes, lysosomes and mitochondria. This abundance is required for fat and lipid metabolism, synthesis of cholesterol and lipoproteins, protein metabolism, and the synthesis of complex proteins. The hepatocytes are also the primary cells involved in detoxification and bile secretion. They are the storehouse of many essential nutrients as well.
Only 40% of a liver's functioning hepatocytes are required for its adequate performance. This is due to their efficiency and their ability to regenerate. The liver is the only internal organ capable of regeneration.
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Liver Failure
How does the liver fail?
Hepatic failure is either acute or chronic, depending on the amount of time the liver takes to fail. Liver failure is caused either by traumatic, extensive injury or disease. There are over 100 different types of liver diseases, afflicting one in 10 individuals or 25 million Americans. Each year over 43,000 people die of liver disease and hospital costs exceed $8 billion annually.
According to the American Liver Foundation:
* 25,000 people die annually from chronic liver disease and cirrhosis
* 10,000 people die annually from Hepatitis C
* 5,000 people die annually from Hepatitis B with an estimated new infection rate of 250,000 people annually
* 1,000 people die annually from primary liver cancer
Most liver diseases result from a variety of build-ups within the liver including fats, sugars, and other metabolites. The build-ups within the liver cause cirrhosis. Cirrhosis is a degenerative process in the liver, which is identified by excess formation of connective tissue, destruction of functional cells, and, often, contraction of the organ.
As mentioned previously, the liver possesses the unique ability to regenerate itself with new healthy tissue. If the liver is damaged beyond its capacity to regenerate new cells, the resulting loss of liver function severely effects the entire body's metabolism.
Liver failure causes the following body-wide complications:
* metabolic instability
* disruption of energy supply
* disruption of acid-base balance
* disruption of thermoregulation
* uncontrolled bleeding and sepsis
* toxic by-product build-up stops dependant organ function
* cease of other organ functions because lack of liver-synthesized nutrients
The varieties of liver diseases cause either acute or chronic hepatic failure. Acute fulminant hepatic failure (FHF) is the result of extensive hepatocyte death over the course of days or weeks. This necrosis of hepatocytes is due to toxic substances or viral infections.
Body-wide symptoms include:
* jaundice
* mental confusion
* stupor or coma
The mortality rate is 80-90%, with rapid (a week or two) death. Though liver transplantation is the only effective form of treatment, 15-30% of FHF patients will regenerate their liver under proper medical treatment.
Chronic hepatic failure is the most common form of the disease. With chronic hepatic failure, the hepatocytes are damaged (usually by long-term exposure to toxins such as alcohol or by viral infections like hepatitis) and unable to detoxify. This build-up of toxic nitrogenous products (i.e., ammonia) within the body causes serious damage.
Body-wide complications include:
* deterioration of mental status
* cerebral edema
* impaired blood coagulation
* gastro-intestinal bleeding
* brain cell damage
* coma
Though there are management techniques for chronic hepatic failure, 60-90% of patients require an eventual transplantation.
These two different types of liver failure require different approaches to liver assist devices. While those suffering from FHF have a chance at liver regeneration, an extracorporeal liver assist device (LAD) is used to detoxify the blood, giving the damaged liver a chance to grow new cells. In this case, the LAD acts as a organ replacement for the time it takes to regenerate. In the case of chronic hepatic failure, regeneration of new cells is no longer viable, so the LAD acts as a bridge while waiting for the availability of a transplant.
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Functional Needs and Heterogeneous Solutions
What are the needs for liver support?
As suggested above, there are two principle needs/functions for liver-assist devices (LAD):
* As a Bridge-To-Transplant Device
* As a Bridge-To-Native Organ Regeneration
Bridge-To-Transplant Needs:
Patients affected by fulminant hepatic failure and hepatic encephalopathy have an extremely high mortality rate - published mortality rates varying from 86% to 93%. The present availability of liver transplants (the treatment of choice for liver failure) has stabilized according to the American Liver Foundation at approximately 4000 transplants a year in the US. The United Network for Organ Sharing (UNOS) schematic to the left charts the rapid growth of the liver transplant waitlist over the past 10 years. As of 4.21.99, according to UNOS there are 12,805 people awaiting liver donations. In 1998, UNOS reported a slight increase from the average in successful transplants as 4450 livers were transplanted.
For more information on liver and other organ donations, transplants and waitlists,
UNOS data can be accessed by going to the "Data" hyperlink at:
http://www.unos.org
These data demonstrate that a significant number of individuals are left waiting for biocompatible liver donations from cadaveric, or in rare cases, living donors. Furthermore, an unspecified number of liver transplants do not function properly in the post-operative period. Thus, a LAD is needed to bridge these primary non-function (PNF) patients to a functional liver status or to a secondary transplant. Finally, liver transplant is an extremely costly operation ranging between $150,000-$250,000 with the American Liver Foundation (ALF) reporting an average cost of $230,000 for the immediate operative costs (See ALF funding report at: http://gi.ucsf.edu/alf/ALF/fundrolo.html). Estimates of annual follow-up costs average between $15,000-$25,000. Clearly, there is a need for LADs that can effectively manage liver failure to bridge waitlist patients to transplant and perhaps to avoid costly transplant altogether by taking advantage of the liver's unique regenerative properties.
Bridge-To-Native Organ Regeneration Needs:
As has been stated earlier liver tissue has the unique ability to regenerate itself. Conditions and factors that encourage latent tissue to move into a regenerative cycle are currently under investigation. Though sources differ on exactly how much of the liver's bio-mass must be replaced to provide a minimum amount of liver function, the literature generally suggests that between 15-30% of the liver's total weight must be reconstituted. Thus, LADs must be created and tested to provide temporary relief of liver function while the native liver undergoes the hepatocyte-regenerative process discussed above. Thus, more livers will be made available, and costly transplant procedures can be avoided. This type of liver-assist is most clearly indicated when the patient's liver has undergone a massive liver resection (cancer removal) or has experienced some kind of trauma.
What functional requirements must LADs perform/fulfill?
LADs must attempt to imitate certain biochemical processes that the native liver routinely performs. In order to conduct these biochemical activities, a LAD will require mechanical support, thus the ideal LAD must fulfill two basic functional requirements:
* Biochemical Functions
* Mechanical Functions
Biochemical Functions:
In some professional estimates, the human liver performs upwards of 900 biochemical functions in the body. It is impossible for any artificial device to duplicate all of these activities; the sum total of these functions must be narrowed down to the essential biochemical functions that ensure patient survival. The acute indications of FHF and the variety of symptoms caused by CHF thus call for different LADs. Both illnesses, however, call for the substitution of the following principle biochemical functions:
* Ammonia control - Elevated levels of ammonia found in cerebrospinal fluid of FHF patients and elevated intestinal production of ammonia directly correlate with a progression of HE stages.
* Endogenous benzodiazepines, GABA and acetaminophen control.
* Articulation and careful regulation of the cytochrome P450 detoxification cycle.
Mechanical Functions:
The two main mechanical functions that the physical configuration of any LAD must allow and/or provide are:
* Immune Protection - Isolation of any foreign (xeno- or allo-) biological components found in the LAD from the body's natural immune response
* Cell Nourishment - Provision of oxygen and other nutrients to biological components that are housed within the LAD.
What device models have been proposed?
A plethora of liver support devices have reached varying stages of conceptualization, manufacture and experimental implementation. The history of liver-assist innovation is long and complex, exhibiting the typical slow-growth curve from 1950-1970 and the subsequent rapid acceleration due to the inception, incorporation (literal) and acceptance of biohybrid technologies. Though research interests in the bioartificial liver are constantly growing, the devices that have been produced have met with only marginal success. Some researchers claim, in a familiar metaphor, that the bioartificial liver remains the "Holy Grail" of organ replacement.
See Friedman's editorial article entitled,
"Why bioartificial liver support remains the Holy Grail"
ASAIO Jul-Aug of 1998.
Historically, a dividing line can be drawn among the devices that have been conceived and/or constructed:
* Those devices that primarily focus of mechanical replacement of basic liver function.
* Those devices that focus of biochemical or "global" replacement of liver function.
Though devices can be divided along these guidelines, it must be stipulated that the LADs that are presently in clinical trials often use a synthesis of devices from both categories.
Mechanical Models for basic replacement of liver function:
* Hemodialysis: Mechanism exactly parallel to kidney dialyzer that clears only ammonia and small molecules. Relies upon convective pressure or diffusion models
* Hemofiltration: Has larger molecular pores that allows for the convective clearance of larger toxins. Relies upon the same mechanics of hemodialysis.
* Hemoperfusion: Passing blood component over particular absorbent compounds that bind toxins. A hemoperfusion device using activated charcoal is often used in biohybrid LADs as an additional part of the blood pathway.
* Exchange Transfusion or Plasmapheresis: Operates on the principle that either whole blood or toxin-bearing plasma can be replaced entirely over time to reduce toxin-content. Though there are thoughts about combining plasma replacement with hemoperfusion, these procedures are not yet functional long-term solutions to liver insufficiency.
Several innovations upon these basic detoxification-based liver support mechanisms have been proposed including lipophilic membrane systems and cross-dialysis with limited or unexplored success. The principle problem with mechanical devices is they are metabolically inert, while the liver's principle functions are largely biochemical and excretory - functions that mechanical models cannot duplicate.
Biochemical models for "global" replacement of liver function:
* Cross-Circulation: Links a biocompatible individual's circulation to the circulation of the patient with liver failure in a prolonged exchange transfusion that allow the patient's blood to be processed "globally" by the healthy individual's hepatocytes.
* Hemoperfusion over Liver Slices: Similar to the mechanical model of hemoperfusion, except that the absorbent compound is active, biocompatible hepatocytes.
* Organ Transplant: The procedure of choice, though severely limited by donor organ supply. Recently segments of living-related donor liver tissue have proven effective in treatment of liver failure.
* Heterotopic Hepatocyte Transplant: Direct transplantation of hepatocytes suspended in a matrix to provide temporary relief to a failing liver. There is much disagreement on how and where to deliver the hepatocytes, how many to deliver and what kind of matrix to place them in.
* Bio-Artificial/Bio-Hybrid Replacement: A device that fixes and sustains functional, harvested hepatocytes within a matrix that allows for biochemical interactions with patient's blood or plasma. These mechanisms are the models for LADs that are currently in clinical trials.
What is a Bio-Artificial/Bio-Hybrid LAD?
Bioartificial LADs apply mechanical principles to the biologically active models for the "global" replacement of primary liver functions. A variety of culture systems and bioreactors have been developed to perform as bioartificial liver substitutes including: static cultures, stirred suspensions, packed beads, matrix cultures, fluidized beds and hollow-fiber mechanisms. Among these various configurations, cell suspensions, packed bead chambers and hollow-fiber bioreactors have been actually used in human patients, with hollow-fiber devices being the principle device design that has gone on to human clinical trials.
The Principles of Hollow-Fiber Bioreactors as LADs
Hollow-fiber bioreactors, similar to hemodialysis devices, contain a number of hollow fibers of a semipermeable material. In a design that is quite familiar by now, this device allows for two compartments, an intracapillary space (ICS) and an extracapillary space (ECS). Most bioreactors in clinical trials, culture or seed hepatocytes in the ECS and perfuse patient's blood or plasma through the capillaries.
There are a number of disagreements among scientists who are currently investigating hollow-fiber bioreactors as a replacement for primary liver functions. These disagreements are currently posited as research questions, some of which have been answered and some of which are currently under investigation.
What kind of hepatocytes to use?
Jauregui et al. describe the ideal hepatocyte for LADs as, "human in origin, rapidly and easily grown in culture at high densities...of normal (non-malignant) phenotype...remain in a well-differentiated state for days or weeks...[and] must display synthetic and detoxifying features of mature hepatocytes." The bioactive, hepatocyte component of the hollow-fiber bioreactors is perhaps the most hotly debated aspect of LADs - questions raised generally revolve around the following concerns:
* Human or Porcine? Human hepatocytes are preferable because the biochemicals they manufacture are same products that native hepatocytes produce. Also, they are less immunogenic and there is no risk of porcine endogenous retroviruses (PERV). However, human hepatocytes grow poorly in culture media and there is a problem with accumulating enough human hepatocytes considering the enormous existing liver support burden. Furthermore, it appears that human C3A cells (the most common human hepatoma cell line) have less ammonia clearing ability than porcine cells.
Relative performance of Porcine Hepatocytes (PPH) in decomplemented human serum medium (DHS) versus Human Hepatomas (C3A) in human serum (HS). Each of the 3 measurements has its own scale - here they are shown together and relative to one another. Synthesized from Wang-Lischeng et al.; Cell-Transplantation 1998: 7(5): 459-468.
* Tumorigenic (malignant) or Not? Tumorigenic cell lines provide rapidly proliferating, non-differentiated cells that allow for long-term hepatocyte replacement and regeneration. These cells require a minimal seeding dose and minimal anchorage in comparison to normal cells. However, they may not respond to physiologic controls and thus there is a danger of bioreactor escape and subsequent tumor formation.
* Immortalized or Not? These cells may not be tumorigenic and can be subjected to physiological controls. Simultaneously, immortalized cells will provide long-term functional hepatocytes. However, there is a question of whether multiple generations will contribute to long-term degradation of metabolic capabilities.
What should be the particular geometry of the device?
* Should hepatocytes be cultured or seeded in the ECS or in the ICS? As stated above hepatocytes are generally being cultured/seeded in the ECS.
* How can the hepatocytes be evenly distributed and how can this distribution be maintained with time? This question is one of the biggest problems with hollow-fiber bioreactors today and is the subject of a number of investigations. One of the latest innovations addresses this issue in a very interesting way.
Should the perfusate be blood or plasma?
* Blood - Easy uncomplicated circuit design, sufficient oxygen and nutrient support for hepatocytes, coagulation problems.
* Plasma - Less coagulation problems, can be filtered before returning it to the blood stream, circuit is complicated and plasma has a lower oxygen-carrying capacity.
What should the nature of the membrane be?
* Open-membrane - Allows toxin-bearing albumin to cross and interact with hepatocytes allowing for increased overall effectiveness of the LAD. However, this also increases the immunogenicity of the device.
* Closed-membrane - Allows for greater immunoisolation, prevents toxin-bearing albumin from interacting with hepatocytes and therefore decreases the effectiveness of the LAD.
Most devices have switched over to an open-membrane LAD and have generated other solutions to the immune comprise that this concession creates.
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Systems in Clinical Trials
What is the HepatAssist 2000" System?
Circe Biomedical
http://www.circebio.com
Circe Biomedical is a bio-tech company devoted to developing bioartificial organs based on its exclusive technologies involving mammalian cells and semi-permeable membranes. These technologies include the ability to:
* Fabricate semipermeable and biocompatible membranes
* Isolate, purify and preserve mammalian cells
* Design, produce and commercialize biomedical systems incorporating these membranes and cells, providing for essential organ function
Its liver support system, HepatAssist(r), is currently Circe Biomedical's leading product. It is the most clinically advanced system of its kind.
HepatAssist(r) is an extracorporeal cell-based bioartificial liver device, based on the use of an open membrane hollow fiber bioreactor. This membrane is microporous, with a pore size of .2_m. This size is just small enough to halt the passage of whole cells, such as hepatocytes, through it, but large enough to allow for free exchange of toxins (soluble and protein-bound) and large molecular weight proteins between the hepatocytes housed outside the hollow fibers and the plasma traveling on the inside of the fibers. The cells used in this device are microcarrier-attached primary porcine hepatocytes.
Basic Steps Involved in HepatAssist(r) Treatment:
Circe's bioartificial liver device (BAL) consists of four parts:
* A hollow fiber bioreactor containing primary porcine hepatocytes
* Two charcoal filters
* A membrane oxygenator
* A pump
BAL is used in conjunction with a commercially available plasma separation machine, heater, and temperature and oxygen monitors.
To begin a treatment, which generally lasts around 6 hours, a patient's blood is separated into blood and plasma in a plasma separation machine. The blood portion is then kept in the plasmapheresis device until reconstituted with the plasma after processing in the bioreactor. Once separated from the blood, a person's plasma, by use of the device's pumping system, is moved via the system's tubing through two charcoal filters. These filters essentially act as Kuppfer cells, filtering the plasma from massive bacteria and particulate matter which could overwhelm the system's hepatocytes. Thus, they provide the system with its first detoxificative action. The rest of the detoxification occurs within the hepatocyte-lined hollow fiber column, through which the plasma then flows. After this, the "clean" plasma is reconstituted with the blood which was stored in the plasmapheresis system, and the whole blood is reinfused into the patient.
A membrane oxygenator and heater are placed between the charcoal filters and the hepatocyte bioreactor. The heater keeps the plasma (and therefore the hepatocytes over which they flow) at body temperature. The membrane oxygenator provides the housed hepatocytes with the oxygen they require for proper function.
The Use of Porcine Cells:
There is great concern involving the use of xenogeneic tissue in this BAL, especially since the large size of HepatAssist's membrane pores doesn't provide an immunoisolated environment for its porcine hepatocytes. Great effort has therefore been devoted to the issue of cell supply through the use of intensive animal sourcing programs.
The main problem involved with the use of porcine cells in this device is their inability to be sterilized. Once hepatocytes are isolated from porcine livers, they cannot undergo any sort of sterilization process and still remain functional. A comprehensive herd and animal bioburden screening program was therefore instituted to provide animals free from zoonotic viruses which could be harvested for HepatAssist(r). Under such screening, the desired porcine cells are cryopreserved and quarantined until the results of bioburden assays performed on them are acquired. Once deemed acceptable, the cells remain cryopreserved for shipping and storage, and are thawed just prior to HepatAssist(r) treatment. So far zoonotic viruses, including PERV (porcine endogenous retroviruses), have not been a problem in clinical trials of this system. The isolation, processing, and cryopreservation involved with porcine hepatocytes is in compliance with Good Manufacturing Practices (cGMP).
Phase I Clinical Trials:
Circe's HepatAssist(r) is meant to function simply as a bridge to orthotopic liver transplantation (OLT) or natural liver recovery. So far, FHF patients have received one 6 hour long treatment per day for an average bridge time of 39.3 hrs (21 - 96 hour range). Individual treatments have been shown to induce intracranial pressure improvement in patients after 2 - 2.5 hours of use.
Most of HepatAssist's phase I clinical testing occurred at the General Clinical Research Center at Cedars-Sinai Medical Center, where many of the creators of the device are from. HepatAssist(r) was used in combination with an effective support group of doctors, nurses, nutritionists, physical and respiratory therapists and pharmacists during its trial. The trial was finished early in 1997. Thirty-six patients, total, were treated with the device; 23 of whom had FHF, 10 acute worsening of chronic liver disease, and 3 nonfunctioning transplants. Each patient was treated 1 - 5 times with the device.
The Results of Phase I Clinical Testing:
* 19 of the FHF and nonfunctioning transplant patients were bridged to OLT
* one of these died 7 days after implantation
* 6 recovered spontaneously - with no need for transplantation
* 2 patients with chronic liver disease recovered to the point of receiving an OLT
* 9 patients died
* eight of the chronic liver disease patients were denied OLT
* one died of pancreatitis
* All survivors reached full neurologic recovery
These results demonstrated safety and tolerance to the point that the FDA granted Circe permission to proceed with a phase II/III clinical trial testing the device's efficacy on the 30 day survival of patients with acute liver failure. These trials are ongoing in 12 different centers in the US and Europe.
What is the Hepatix/Vitagen ELAD( System?
VITAGEN, INC
(formerly Hepatix, Inc.)
http://www.hepatix.com
Vitagen Incorporated is a biotechnology and medical products company that describes itself as having "developed the world's first artificial liver [with the goal] of creating products to help reduce the needs for transplants of this vital organ." Vitagen has created the first medical device to incorporate "immortalized" human liver cells (C3A).
Basic Steps Involved in ELAD( Treatment:
VitaGen's ELAD is plasma perfused, meaning the patient's plasma flows through the cartridge where it interacts with the metabolically active C3A cells. The treated plasma is then filtered and returned to the patient. The basic steps involved in treatment are comparable to those described above in the HepatAssist(r) device.
The Use of Human Hepatoma C3A Cells:
VitaGen's ELAD (Extracorporeal Liver Assist Device) artificial liver consists of a hollow fiber cartridge populated with an immortalized liver cell line. These C3A liver cells, located in the extra-capillary space, can be reproducibly manufactured in culture and express normal liver-specific metabolic pathways such as ureogenesis, gluconeogenesis, and P-450 activity. They also secrete clotting factors and other liver-specific proteins. The metabolic capacity of each cartridge is equal to that of about 200 g of normal liver.
Phase I Clinical Trials:
VitaGen conducted two trials to assess the role of an artificial liver in the treatment of acute liver failure (ALF). The trials were held at the University of Chicago Hospital and at King's College in London. The initial trial involved 11 patients, all with advanced ALF, while the second trial (12 patients) was divided into two groups. Group 1 suffered from moderate liver disease, group 2 from advanced. The causes of ALF in the patients breaks down in the following manner:
* 7 due to acetaminophen
* 5 due to NANB hepatitis
* 3 due to fialuridine
* 2 due to anti-TB drugs
* 1 idiopathic
* 1 due to syncytila giant cell hepatitis
* 1 due to liver resection for primary graft non-function
Patients received ELAD therapy continuously for 3-168 hrs (7 days).
The Results of Phase I Clinical Testing:
Results showed satisfactory biocompatability, improved hemodynamic parameters and improvement in encephalopathy, which indicated that patients could safely be removed from ELAD treatment.
However, the survival outcomes of the two trials differed greatly:
Trial 1
* 1 out of the 11 survived
Trial 2
* 8 out of 12 survived
The discrepancy is described as being due to the medical conditions of those in each trial.
These results demonstrate that ELAD treatment appears to increase the rate of recovery from sublethal ALF, and has the capacity to support patients for several days (even in the presence of severe liver failure), serving as an effective bridge to transplantation.
How do both devices compare?
SYSTEM
Device Configuration
Perfusion Medium
Cell Selection
Phase I Clinical Trial Survival Rate
Hepatix ELAD(
Hollow-fiber ELAD
Open-Membrane
Plasma
Human Hepatoma (C3A)
9/23 = 39.1%
HepatAssist(r)
Hollow-fiber BAL
Open-Membrane
Plasma
Porcine Hepatocytes
(PPH)
27/36 = 75.0%
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The Future
So, what major improvements are in the works?
There are a number of improvements to the present models that are in various phases of development. Two systems, in particular, should be mentioned - one which has been used under extreme circumstances in a single patient and another which is very close to being finalized and put into human trials.
Gerlach et al.
This device pioneered at the University of Berlin by Dr. JC Gerlach uses a three-dimensional design for a LAD that is very different from the hollow-fiber models that are currently being tested. His model essentially involves four different capillary membrane systems, each serving a different purpose, woven into a 3-D lattice. These capillaries independently and locally provide oxygen, nutrients and plasma perfusate inflow and outflow to hepatocytes. This allows for, "decentralized cell perfusion with low metabolite gradients and decentralized oxygenation and CO2 removal," and has contributed to increased hepatocyte performance in long-term ex vivo assessment. Data for this technique is encouraging, though as of 1997, only 1 patient had been bridged-to-transplant using this device. The duration of liver-assist for this patient was 40 hours.
Chamuleau et al.
At the University of Amsterdam, Dr. Chamuleau et al. have come up with a novel and exciting design technique that will hopefully resolve the matrix support and hepatocyte anchorage problems that have thus far plagued non-tumorigenic cell bioreactors. In order to fix these cells more firmly and to preserve their distribution, Dr. Chamuleau has taken a flat hepatocyte-seeded polyethylene sheet and rolled each hollow-fiber one after another to create a tight multiple sushi-roll-style bioreactor. The hollow-fibers provide oxygen and nutrient support to the hepatocytes while toxin-bearing plasma is perfused directly through the extrafiber space allowing for direct hepatocyte contact. This device has been tested in pigs and will soon enter human trials supervised by Dr. Calise of the Cardarelli Hospital in Naples, Italy.
How is the bioartificial liver research industry changing?
Our conclusions, based upon trends we have observed in the existing literature and our conversations with Dr. Jauregui, indicate that as the bioartificial liver research industry grows it is becoming increasingly more and more specialized. What this means is that, although there are many companies working on complete LADs, companies now entering the LAD market tend to chose very specific aspects of such devices as their specialty. MultiCell Co., and other bio-tech companies like it, have intelligently narrowed their research focus to just the cell lines that will eventually be used to populate the devices that are engineered by other companies whose primary focus is on the device itself. Thus, the process of forming a complete LAD has become a cooperative effort of highly specialized companies that share their expertise (at a price) in a highly competitive market.
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http://biomed.brown.edu/Courses/BI108/BI108_1999_Groups/Liver_Team/Liver.html
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