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Blackwell Science, LtdOxford, UKAORArtificial Organs0160-564X2004 International Society for Artificial Organs282167172Original ArticleT.M.S. CHANG

Artificial Organs 28(2):167–172, Blackwell Publishing, Inc. © 2004 International Society for Artificial Organs

Artificial Cells for Cell and Organ Replacements

Thomas Ming Swi Chang

Artificial Cells and Organs Research Center, Faculty of Medicine, McGill University, Montreal, Quebec, Canada

Abstract: The artificial cell is a Canadian invention based on modified hemoglobin are already in Phase III (Chang, Science, 1964). This principle is being actively clinical trials in patients, with as much as 20 units being investigated for use in cell and organ replacements. The infused into each patient during trauma surgery. Artificial earliest routine clinical use of artificial cells is in the form cells containing are being developed for clinical of coated activated charcoal for hemoperfusion for use in trial in hereditary deficiency diseases and other the removal of drugs, and toxins and waste in uremia and diseases. The artificial cell is also being investigated for liver failure. Encapsulated cells are being studied for the and for other uses in biotechnology, chem- treatment of , liver failure and kidney failure, and ical engineering and medicine. Key Words: Artificial the use of encapsulated genetically-engineered cells is cells—Hybrid—Liver—Kidney—Gene therapy—Blood being investigated for gene therapy. Blood substitutes substitutes.

Artificial cells were first reported by Chang at After initial clinical trials for poisoning, kidney fail- McGill University a number of years ago (1–4) ure and liver failure (7), it is now in routine clinical (Fig. 1). Biologically-active materials inside the arti- use, especially for the treatment of suicidal or acci- ficial cells are prevented from coming into direct dental poisoning from medications (8). It is also contact with external materials like leukocytes, being used in combination with the hybrid artificial 3 or tryptic enzymes. Smaller molecules can liver in clinical trials. equilibrate rapidly across the ultrathin membrane, which has a large surface-to-volume relationship. A CELL ENCAPSULATION FOR HYBRID number of potential medical applications using arti- ARTIFICIAL ORGANS ficial cells have been proposed (2–6). The first of Chang first reported the encapsulation of biologi- these to be developed successfully for routine clinical cal cells in 1966 based on a drop method and pro- use is hemoperfusion (4). After initial clinical trials posed that “protected from immunological process, for poisoning, kidney failure and liver failure (5), it encapsulated endocrine cells might survive and is now in routine clinical use (7,8). Some exciting maintain an effective supply of hormone” (3,5). recent developments include their use for blood sub- stitutes and for the replacement of the metabolic Artificial pancreas, artificial liver and others functions of cells and organs (6). Chang approached the Conaught Laboratory to develop his crosslinking drop method for use in islet HEMOPERFUSION transplantation for diabetes. Sun from Conaught The first successful use of the artificial cell in rou- and his collaborators later developed this drop tine clinical applications is hemoperfusion (5–8). method by using milder physical crosslinking (9). This resulted in alginate-polylysine-alginate (APA) microcapsules containing cells. They showed that, Received November 2003. after implantation, the islets inside artificial cells Address correspondence and reprint requests to Thomas remained viable and continued to secrete insulin to Ming Swi Chang, Artificial Cells and Organs Research Center, control the levels of diabetic rats (9). Cell Faculty of Medicine, McGill University, 3655, Promenade Sir- William-Osler, Montreal, Quebec, Canada H3G 1H6. E-mail: encapsulation for has been extensively 2 [email protected] developed by many groups, especially using artificial

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168 T.M.S. CHANG

ARTIFICIAL CELLS IN BIOTECHNOLOGY & MEDICINE Klebsiella aerogenes urease gene to lower systemic

Chang (1964) SCIENCE urea in renal failure rats (27,28). However, these Chang et al (1966) Can J Physiol Pharm Chang & Poznansky (1968) NATURE genetically-engineered micro-organisms are not suf- Chang (1971) NATURE ficiently stable in their ability to remove urea. We are looking at the metabolic induction of , similar to those used in yogurt, in order not to intro- CELLS duce genetically-engineered cells into the body (29). HEMOGLOBIN ENZYMES ARTIFICIAL RED BLOOD CELLS oxygen, oxygen, Nutrients BIOREACTANTS Wastes metabolites Substrates ETC Products, drugs Toxins, drugs Hormones, Complete artificial red blood cells of micron dimensions The original complete artificial red blood cells (RBC) prepared here containing hemoglobin and 4 WBC TRYPTIC ENZYMES enzymes have all the properties of RBC when tested in vitro (1,2). However, they did not survive for a FIG. 1. The basic principle of artificial cells. (With permission sufficient length of time in the circulation after from Artificial Cells, Blood Substitutes and Immobilization Bio- technology, an international journal 2004;32:1–14.) infusion.

Polyhemoglobin as a blood substitute cells containing endocrine tissues, and As a result of the above, we used a simpler molec- other cells for cell therapy (9–15) (Fig. 1). ular version based on the use of bifunctional agents, We have been studying the use of the implanta- such as diacid (2,5) or later glutaraldehyde (30), to tion of encapsulated hepatocytes for liver support crosslink hemoglobin molecules into polyhemoglo- (16–24). We found that implantation increases the bin. Due to problems related to human immunodefi- survival of rats with acute liver failure (17), maintains ciency (HIV) in donor blood, there has been 5 a low bilirubin level in hyperbilirubinemic Gunn rats extensive development toward blood substitutes, (18), and prevents xenograft rejection (19). We starting in the early 1990s (31–34). At present, two of developed a two-step cell encapsulation method to these are in the final stages of clinical trials and are improve the APA method, resulting in the improved waiting for Food and Drug Administration (FDA) 6 survival of implanted cells (20,21). Using this two- approval. These have been developed independently step method together with the coencapsulation of by two groups based on our basic principle of gluat- stem cells and hepatocytes, we have further increased the viability of encapsulated hepatocytes both in cul- ture and also after implantation (22,24) (Fig. 2). One HEPATOCYTES COENCAPSULATED WITH STEM CELLS implantation of the coencapsulated hepatocytes and VIABILITY AFTER IMPLANTATION stem cells into Gunn rats lowered the systemic biliru- ( Liu & Chang, ACBSIB 2002) bin levels and maintained this low level for two 90 Encaphepatocytes with stem cells months (24). Implanted encapsulated hepatocytes 80 Encap hepatocytes only can only maintain a low level for one month. 70 60 Microencapsulated genetically-engineered cells 50 Microencapsulated genetically-engineered cells 40 have been studied by many groups for potential 30 HViability(%) applications in amyotrophic lateral sclerosis, dwarf- 20 ism, pain treatment, IgG1 plasmacytosis, hemophilia 10 B, Parkinsonism and axotomized septal cholinergic 0 neurons (25,26). One group uses hollow fibers to 0246810121416 macroencapsulate genetically-engineered cells. This Time(week) way, the fibers can be inserted and then retrieved after use, without being retained in the body (26). FIG. 2. An experiment showing that coencapsulation with stem To avoid the need for implantation, we studied the cells increases the viability of hepatocytes after implantation. (With permission from Artificial Cells, Blood Substitutes and oral use of microencapsulated genetically-engi- Immobilization Biotechnology, an international journal 2002; neered nonpathogenic E.coli DH5 cells containing 30:99–112.)

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aradehyde crosslinked polyhemoglobin (30). One is NANO DIMENSION ARTIFICIAL RBC pyridoxalated glutaraldehyde human polyhemoglo- NANOENCAPSULATED HB & ENZYMES bin (35,36). In a Phase III clinical trial it was shown that this can successfully replace extensive blood loss in trauma surgery by maintaining the hemoglobin GLUCOSE

level with no reported side-effects (36). Up to 20 ADENINE, INOSINE units have been infused into individual trauma GLUCOSE surgery patients (35). Another blood substitute is ATP glutaraldehyde crosslinked bovine polyhemoglobin, EMBDEN-MEYERHOF which has been extensively tested in Phase III clinical SYSTEM trials (37,38). This bovine polyhemoglobin has been HEMOGLOBIN NAD REDUCING approved for veterinary medicine in the U.S.A. and 2,3-DPG AGENT for routine clinical use in South Africa. Conjugated NADH hemoglobin development and Phase II clinical tri- METHB alsConjugated hemoglobin: development and Phase LACTATE 7 II clinical trials The above two polyhemoglobins CARBONCIC ANHYDREASE CO2 have been approved for compassionate use in SUPEROXIDE DISMUTASE SUPEROXIDE humans and they are waiting for regulatory approval LACTATE CATALASE H2O2 for routine clinical use in humans in North America. They have a number of advantages when compared FIG. 4. Nanodimension artificial red blood cells (RBC) with a to donor RBC and they are particularly useful in polyethylene-glyco-polylactide membrane. In addition to hemo- surgery. However, these are only oxygen carriers and globin, this contains the same enzymes that are normally present in RBC. Thus, it has the complete function of the RBC. (With do not have all of the functions of RBC that may be permission from Artificial Cells, Blood Substitutes and Immobili- needed for certain clinical conditions (39). zation Biotechnology, an international journal 2003;31:231–248.)

Polyhemoglobin crosslinked with RBC antioxidant enzymes tion of oxygen radicals and tissue injury (31,39). We Reperfusion using an oxygen carrier alone in are using a crosslinked polyhemoglobin-superoxide sustained severe hemorrhagic shock or sustained dismutase-catalase (PolyHb-SOD-CAT) (40–43). ischemic organs, as in stroke, myocardial Unlike PolyHb, PolyHb-SOD-CAT did not cause a or , may result in the produc- significant increase in oxygen radicals when it was used to reperfuse ischemic rat intestines (42). More recently (43), in a transient global cerebral

BRAIN EDEMA IN RATS AFTER ACUTE GLOBAL rat model, we found that, after 60 min of ischemia, CEREBRAL ISCHEMIA & REPERFUSION reperfusion with polyHb resulted in significant (Powanda & Chang ACBSIB 2002 ) increases in the blood–brain barrier and the break- down of the blood–brain barrier (Fig. 3). On the other hand, polyHb-SOD-CAT did not result in these adverse changes (43) (Fig. 3). PolyHb

Nanodimension artificial RBC Chang’s original idea of a complete artificial RBC (1,2) is now being developed as a third generation blood substitute (39). Hemoglobin lipid vesicles is PolyHb-SOD-CAT one of these approaches (44–46). We are using a dif- ferent approach based on a biodegradable and nanotechnology, resulting in nanoartificial RBC of 80–150 nm diameter (47–49). These nanoartificial RBC contain all of the RBC enzymes needed for the FIG. 3. This is a rat model of acute global cerebral ischemia long-term function of the nanoartificial RBC (49) followed by reperfusion with different oxygen-carrying solutions. (Fig. 4). Our recent studies show that, using a poly- Unlike polyhemoglobin, polyHb-CAT-SOD does not cause brain ethylene-glycol-polylactide copolymer membrane, edema when used in this situation. (With permission from Artifi- cial Cells, Blood Substitutes and Immobilization Biotechnology, we are able to increase the circulation time of these an international journal 2002;30:25–42.) nanoartificial RBC to double that of polyHb (49).

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ARTIFICIAL CELLS IN ENZYME THERAPY approaches based on nanodimension artificial cells in the form of liposomes, nanoparticles and nanocap- Enzyme therapy by implantation sules are being increasingly used by many groups for We have previously implanted artificial cells con- drug delivery, as reviewed in a recent book (62). taining catalase into acatalesemic mice, animals with a congenital deficiency in catalase (50). This replaced CONCLUSION 8 the deficient enzymes and prevented the animals The principle of artificial cells was a very novel from the damaging effects of oxidants. The artificial idea when it was first proposed (2). As a result, it cells protect the enclosed enzyme from immunolog- took some time before others started to actively ical reactions (51). It was also shown that artificial investigate and extend this principle for use in cell cells containing asparaginase implanted into mice and organ replacements. In the earliest routine clin- with lymphosarcoma delayed the onset and growth ical use of artificial cells, a very simple principle of of lymphosarcoma (52). The single problem prevent- artificial cells in the form of coated activated charcoal ing the clinical application of enzyme artificial cells for hemoperfusion was used in the removal of drugs, is the need to repeatedly inject these enzyme artifi- and toxins and waste in uremia and liver failure. cial cells. The successful clinical application of this simpler approach has resulted in the increasing development Oral administration to avoid the need of the more complicated approaches of artificial cells. for implantation For example, encapsulated cells are being studied for To solve this problem, artificial cells were given the treatment of diabetes, liver failure and kidney orally. As they travel through the intestine, they act failure, and the use of encapsulated genetically- as microscopic dialyzers. By encapsulating enzymes engineered cells is being investigated for gene and other material inside the microcapsules, they can therapy. Blood substitutes based on modified hemo- act as a combined dialyzer–bioreactor. For example, globin are already in Phase III clinical trials in artificial cells containing urease and ammonia adsor- patients, with as much as 20 units being infused into bent were used to lower the systemic urea level (5). each patient during trauma surgery. Artificial cells We found that microencapsulated phenylalanine- containing enzymes are being developed for clinical ammonialyase given orally can lower the elevated trial in hereditary enzyme deficiency diseases and phenylalanine levels in phenylketonuria (PKU) rats other diseases. The artificial cell is also being investi- (53). This is because of our more recent finding of an gated for drug delivery and for other uses in biotech- extensive recycling of amino acids between the body nology, chemical engineering and medicine. and the intestine (54). This is now being developed for clinical trial in PKU (55,56). In addition to PKU, other examples from our recent studies show that REFERENCES oral artificial cells containing tyrosinase are effective 1. Chang TMS. (1957) Hemoglobin Corpuscles. Report of a research project for Honours Physiology. Quebec: Medical in lowering systemic tyrosine levels in rats (57). We 9 have also used oral microencapsulated xanthine oxi- Library, McGill University. Also reprinted as part of “30 anni- versary in Artificial Red Blood Cells Research”. J Biomat Artif dase to lower the systemic hypoxanthine levels in a Cells Artif Org 1988;16:1–9. patient with Lesch–Nyhan disease (58). 2. Chang TMS. Semipermeable microcapsules. Science 1964;146: 524–5. 3. Chang TMS, MacIntosh FC, Mason SG. 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Artificial Organs Volume 28, 2004 BSA article no: 47343 AUTHOR QUERY FORM

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