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Artificial Cells and Bioencapsulation in Bioartificial Organsa

THOMAS MING SWI CHANGh Artificial Cells & Organs Research Centre

Departments of Physiology, Medicine & Biomedical Engineering Faculty of Medicine McGill University

Montreal, Quebec, Canada H3G I Y6

Modern research on bioartificial organs is based on molecular and cellular approaches. Even before 1972 there were many ways of preparing bioartificial materials. In 1972 an ad hoc committee formulated the term Immobilization to cover all the different approaches.’ This blanket term is subdivided into four main classes: 1) Adsorption; 2) Covalent linkage, 3) Matrix entrapment, and 4) Encapsulation. Since Immobilization Biotechnology is a very large area, this paper discusses only one of the four approaches-encapsulation.

The first reports on artificial cells bioencapsulating biologically active material were published by this author as early as 19572 and 1964.’ He showed that it is possible to bioencapsulate hemoglobin, enzymes, proteins, cells, microorganisms, adsorbents, magnetic materials and other biologically active materials (FIG. 1).1-6 The artificial cells protect the encapsulated biological materials from the extracellu- lar environment. At the same time the enclosed materials continue to act in the intracellular environment. Like biological cells, artificial cells contain biologically active materials. However, artificial cells can contain both biological and synthetic materials. The membranes of artificial cells can also be extensively varied using many different types of synthetic or biological materials. The permeability can be controlled over a wide range. For example, selectively permeable ultrathin synthetic membranes can retain macromolecules like proteins and enzymes. At the same time it allows the rapid diffusion of peptide and smaller molecules. This way, the enclosed material can be retained and separated from undesirable external materials. At the same time, the large surface area and the ultrathin membrane allows permeant substrate and products to diffuse randomly. Ten ml of 20 micron diameter artificial cells have a total surface area of about 20,000 cm’. The membrane thickness is 200 ingstroms. As a results, mass transfer of permeant molecules across 10 ml of artificial cells can be 100 times higher than that for a standard hemodialysis machine. Variations in dimensions are also possible. Dimensions depend on the type of use and contents. Microcapsules in the microns diameter range are used for bioencapsulation of enzymes, protein, peptide, microorganisms, organelles, cells and others. Nanocapsules in the nanometer range are best used for enzymes, protein, peptide, antibiotics, and hemoglobin. Artificial red blood cells have been prepared

“ T.M.S.C. gratefully acknowledges the operating grants and career investigatorship from the Medical Research Council of Canada and the Quebec MESST Virage “Centre of Excellence in Biotechnology” award. ’ Fax: (514) 398-4983; E-mail: [email protected]; web: http://www.physio,mcgill.ca/ artcell

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250 ANNALS NEW YORK ACADEMY OF SCIENCES

FIGURE 1. Artificial cells for bioencapsulation of a larger number of biologically active materials for use in biotechnology and medicine. (From Chang." Used with permission of Marcel Dek- ker Publisher.)

by crosslinking hemoglobin molecules to form polyhemoglobin consisting of 4 to 10 molecules. These possible variations in contents and membrane materials allow for one to have unlimited variations in the properties of artificial cells.2-"

CELL ENCAPSULATION

Chang first developed a drop method for the bioencapsulation of cells and proposed its use in cell therapy (FIG. 2).5.6 "Microencapsulation of intact

FIGURE 2. Artificial cells for bioencapsulation in bioartificial organs. The enclosed materials are separated from external antibodies and leukocytes and prevented from immunological rejection. Smaller permeant materials can equilibrate rapidly across the membrane to be acted on by the enclosed material. (From Chang?n Used with permission of Marcel Dekker Pub- lisher.)

CHANG: ARTIFICIAL CELLS AND BlOENCAPSULATlON 251

cells. . . . . . . . . . the enclosed material might be protected from destruction and from participation in immunological processes, while the enclosing membrane would be permeable to small molecules of specific cellular product which could then enter the general extracelluar compartment of the recipient.. . . . . . . The situation is comparable to that of a graft placed in an immunologically favourable site."j With increasing interests in biotechnology, many groups are now actively exploring this approach?"' A large amount of work has been reported on using this for bioencapsulation of islets."'-" These efforts will be reported on by others in this con- ference.

Bioencapsulation of Hepatocytes

We have been using bioencapsulated hepatocytes in basic studies to study the feasibilities of encapsulated cells in cell and gene therapy. Typically, hepatocytes were enclosed within alginate-polylysine-alginate (APA) microcapsules of 300 pm mean diameters. Different concentrations can be encapsulated, for example 1.1 mL of microcapsules can contain 15 X 10' hepatocytes.lS-l7 The 300 pm diameter microcapsules are flexible. They can be injected using syringes with 18 gauge needles. Permeability of the membrane can be adjusted. Detailed analysis has been carried out using HPLC analysis of a large spectrum of molecular weight dextran.In The permeability can be adjusted to have different cut off molecular weight depending on the applications. Thus, for hepatocytes, it can be adjusted to allow albumin to pass through but not immunoglobulin. After isolation from the liver, the percentage of viable hepatocytes as determined by trypan blue stain exclusion was about 80%. After bioencapsulation of hepatocytes the percent of viable cells was 63.40%."

Experimental Cell Therapy in Rats with Fulminant Hepatic Failure

Our earlier studies showed that rats with galactosamine-induced fulminant hepatic failure which received control artificial cells died 66.1 2 18.6 hours after galactosamine indu~ti0n.l~ The survival time of the group which received one peri- toneal injection of 4.00 ml of microcapsules containing 7.40 X lo6 hepatocytes was 117.3 5 52.7 hours S.D. Paired analysis showed that this is significantly ( p < 0.025) higher than that of the control group. The total number of hepatocytes injected in this initial study was very small. Later study by another group using higher concentrations of hepatocytes resulted in increase in long-term survival rates.?"

Experimental Cell Therapy in Gunn Rats-An Animal Model for Human Non-Hemolytic Hyperbilirubinemia (Crigler-Najjar Type I)

We have investigated the use of artificial cells containing hepatocytes as cell therapy to lower bilirubin levels in Gunn In the first experiment, 3.5 months old Gunn rats weighing 258 -C 12 grams were used. During the 16-day control period, the serum bilirubin increased at a rate of 0.32 2 0.07 mg/100 ml per day. This reached 14.00 ? 1.00 mgll00 ml at the end of the control period. Each animal then received an intraperitoneal injection of 1.10 ml of microcapsules containing 15 X 10' viable Wistar rat hepatocytes. Twenty days after implantation of the


encapsulated hepatocytes, the serum bilirubin decreased to a level of 6.00 2 1.00 mg/100 ml. The level remained low 90 days after the implantation. In the second experiment, control groups of Gunn rats were compared to those receiving cell therapy. The bilirubin levels did not decrease in the control group and the group which received control microcapsules contained no hepatocytes. In the group receiving encapsulated hepatocytes there was significant decreases in the plasma bilirubin level. Analysis showed that implanted encapsulated hepatocytes lowered bilirubin by carrying out the function of the liver in the conjugation of bilirubin?' Dixit's groups has also carried out extensive studies using these Gunn rats and their results supports our findings. They will be reporting in details on their research.

Immunoisolation of Bioencapsulated Rat Hepatocytes When Implanted into Mice

We also studied the implantation of free or bioencapsulated rat hepatocytes intraperitoneally into 20-22 g male normal CD-1 Swiss mice or CD-1 Swiss mice with galactosamine-induced fulminant hepatic failure (FHF).I9 This is a basic study to see if rat hepatocytes can remain viable and be immunoisolated inside freely floating artificial cells in mice. Therefore, aggregated microcapsules were not analyzed since hepatocytes do not have good viability under this condition.

As expected, rat hepatocytes implanted into normal CD-1 Swiss mice were rapidly rejected. By the 14Lh day, there were no intact hepatocytes detected in the mice. Rat hepatocytes after implantation into CD-1 Swiss mice with galactosamine- induced FHF were rejected completely after 4-5 days. In the case of bioencapsulated hepatocytes, not only did they stay viable, there was also a significant increase (p < 0.001) in the percentage of viable hepatocytes within the microcapsules after two days of implantation. The percentage of viable cells increased with time so that 29 days after implantation, the viability increased from the original 62% to nearly 100%. There was no significant changes in the total number of hepatocytes in the microcapsules. The viability of encapsulated rat hepatocytes implanted into galactosamine induced FHF mice also increased to nearly 100%.

In conclusion, rat hepatocytes in free floating microcapsules can be immunoisolated. As a result, xenograft of rat hepatocytes are not immunologically rejected in mice. Instead, we have the unexpected findings of improvement in cell viability when followed for up to 29 days.

Hepatocyte-secreted Hepatic Stimulatory Factor Is Retained Inside the Microcapsule Artificial Cells

We found that hepatocytes in the microcapsule secrete factor(s) capable of stimulating liver regeneration?* This factor is retained inside the microcapsules after secretion. Sephacryl gel chromatography shows that this factor has a molecular weight of over 110,000 D. The hepatic-stimulating factor accumulating in the microencapsulated hepatocyte suspension, helps to increase the viability and recovery of the membrane integrity of hepatocytes inside the artificial cells.

CHANG: ARTIFICIAL CELLS AND BIOENCAPSULATION 253

The Single Major Obstacle to Clinical Use of Cell Encapsulation Is Long-Term Biocompatibility

Basic research using bioencapsulated hepatocytes, islets and other types of cells, shows the feasibility of using bioencapsulation for cell therapy. Further improve- ments in biocompatibility may allow this approach to be used for cell and gene therapy in human. This is becoming increasingly feasible because of the increasing progress in genetic engineering and molecular biology. The one single major obstacle is to have microcapsules which are so biocompatible that they can function for a sufficient length of time after implantation. Much research on this very important issue is ongoing by many excellent research groups, whose progress is being reported in this volume. We have examined one area in the biocompatibility of microcapsules containing cells as follows.

Procedure Specijic for Encapsulating High Concentrations of Smaller Cells Like Hepatocytes or Microorganisms

The general procedure used for alginate-polylysine-alginate cell encapsulation was originally designed for the bioencapsulation of a few When this procedure is used to bioencapsulate a high concentration of dispersed cells like hepatocytes or microorganisms, the following problems can occur.23 Some cells are incorporated into the membrane matrix and some of these cells are also exposed on the surface of the membrane (FIG. 3). When these microcapsules are implanted, the hosts immediately recognize the protruding cells on the surface resulting in acute cell-mediated host immune response and rejection. Even when the cells were integrated into the membrane matrix without protrusion, this resulted in a weak and poorly formed area in the membrane. When these were implanted into mice, macrophage and lymphocytes perforated the capsular membrane and infiltrated the microcapsule at these sites.

To prevent the above problem, we worked out the following approach (FIG. 4), the details of which have been p~blished.*~.*~ Very briefly, small calcium alginate gel microspheres containing entrapped cells were first formed. These gel micro-

ALGINATE GEL SPHERE

FORM MEMBRANE DISSOLVE GEL

FIGURE 3. Standard method of cell encapsulation when used for high concentrations of small cells can result in some cells being entrap or exposed on the surface of the membrane. When implanted this can result in membrane weakness and immunorejection. (From Wong & Chang.*> Used with permission of Marcel Dekker Publisher.)


FIGURE 4. A two-step method has been designed to prevent this problem. (From Wong & Chang?' Used with permission of Marcel Dekker Publisher.)

spheres are then resuspended in alginate solution to form larger microspheres. When these smaller microspheres are entrapped within the large microspheres, the larger ones do not have cell extruding on the surface. Thus during membrane formation, there was no cell entrapment in the membrane. Microscopic studies show that the encapsulated cells are not embedded into the membrane matrix.242s Implantation resulted in better immunoisolation.

BIOENCAPSULATION OF GENETICALLY ENGINEERED MICROORGANISMS

Bioencapsulation of Genetically Engineered E. coli Cells for Urea and Ammonia Removal

Urea and ammonia removal are needed in kidney failure, liver failure, environ- mental decontamination, and regeneration of the water supply in space travel. Standard dialysis is effective for terminal renal failure patients, but it is expensive and many countries cannot afford to use dialysis to treat their patients. Many years ago, extensive effort to find an oral approach was not successful. The one major obstacle was the inability to remove the large amount of urea. With the availability of genetically engineered microorganisms, we are studying the use of bioencapsulated genetically engineered E. coli DH5 cells containing K. aerogens urease gene.26,27

Factors in the Bioencapsulation Process

The concentration of alginate is very crucial in the microencapsulation of this genetically microorganism. Our analysis shows that 2.00% (WN) alginate resulted in well formed microcapsules with the maximum number of encapsulated bacterial

We find that an air flow rate of 2.00 L/min is suitable for preparing microcapsules with an average diameter of 500 ? 45 pm diameter. We also analyzed the optimal liquid flow rate.26 The mechanical strength of the alginate microbeads and APA microcapsules as a function of cell leakage was studied. Results showed that

CHANG: ARTIFICIAL CELLS AND BIOENCAPSULATION 255

the microencapsulated microorganisms were stable when agitated up to 210 rpm for 7 hours.”

Analysis of in Vitro Efficiency and Stability in the Removal of Urea and Ammonia

Log phase microencapsulated bacteria lowered 87.89 ? 2.25% of the plasma urea within 20 minutes and 99.99% of urea in 30 minutes. Furthermore, the encapsulated bacteria decrease plasma ammonia concentration from 975 -C 70.15 to 81.15 -C 7.37 p M in 30 minutes.” This ammonia removal efficiency of encapsulated bacteria in plasma is not significantly different from that in the aqueous media. One can use encapsulated bacteria prepared by the general procedure for up to three cycles. The urea removal rate is greater in the second and third cycles than in the first. This is probably due to increase in total biomass inside microcapsules with time. There is no leakage of encapsulated bacteria in the first, second and third cycles. We used a single pool model to analyze urea removal efficiency by the encapsulated bacteria. The total fluid compartment was 40 liters with an urea concentration of 100 mg/d1(26). 40.00 5 8.60 g of APA encapsulated bacteria can remove 87.89 ? 2.25% of the total body urea (40 grams) within 20 minutes and 99.99% in 30 minutes. It requires 388.34 g of oxystarch to remove the same amount of urea under the same conditions. It requires 1212.12 g of microcapsule containing urease-zirconium- phosphate to remove 40 g urea from the total body water. Overall, urea removal efficiency of microencapsulated genetically engineered bacteria is therefore much higher than the best available urea removal systems.

Effectiveness in the Lowering of Systemic Urea Levels in Uremic Rats

Our detailed study has just been reported.?’ The uremic rats are prepared by the surgical removal of one kidney and partial ligation of the other. Blood urea levels in these animals reached uremic levels. Oral administration of microencapsulated E. coli DH5 cells once a day resulted in the lowering of the systemic urea level to normal level. This normal level is maintained during the 21 days of daily oral administration. On discontinuation of the oral administration, systemic urea level rapidly returned to its high uremic levels showing that there is no retention of microorganisms in the intestine. Unlike these treated animals, 50% of the control uremic rats receiving control microcapsules died during the 21-day period. Based on this study, calculation shows in a 70 kg patient we only need to give 4 grams of the biomass each day. This is the first time that this is possible. This has therefore solved the major single obstacle of urea removal, the single major problem which has prevented the investigation of oral therapy for terminal renal failure.

Encapsulation of Other Microorganisms

We have also studied the encapsulation of other microorganisms.2R,2Y For example we have studied the use of microencapsulated Pseudomonas pictorum (ATCC #23328) as another model system because of its ability to d$grade In order for lipoprotein-cholesterol macromolecules (50-1000 A) in plasma to enter the microcapsules we have designed a new method’ based on open-pore agar to form the microcapsules. These high porosity agar beads stored at 4°C did not show


any sign of deterioration. The beads retained its activity even after 9 months of storage. There was no evidence of leakage of the enclosed bacteria. Open pore agar beads were incubated in serum and their cholesterol depletion activity was compared to controls and non-immobilized bacteria. The bacterial action was not significantly different between the immobilized and non-immobilized forms. Bacte- rial reaction was found to be the limiting step in the overall reaction of immobilized bacteria. Other methods to remove cholesterol (e.g., LDL immunosorbents) are capacity limited. The immobilized microorganism as shown here, has an almost unlimited capacity to deplete cholesterol levels. However, for practical applications, a suitable bacterium with higher rates of cholesterol removal is needed. No doubt this will become available in the future with the help of genetic engineering. Another example is the bioencapsulation of Erwinia herbicola for the production of t y r o ~ i n e . ~ ~ This also has implications for converting phenylalanine to tyrosine in phenylketonuria. Detailed in uitro kinetics have been carried o ~ t . 2 ~

OTHER AREAS OF RESEARCH ON ARTIFICIAL CELLS

Bioencapsulation of Bioactive Sorbents

Microencapsulation of bioacitve sorbents is the simplest form of artificial cells which has already been used in routine clinical applications in human for many years. Sorbents like activated charcoal, resins and immunosorbents cannot be used in direct blood perfusion. This is because of particulate embolism and blood cells removal. Sorbents like activated charcoal inside artificial cells no longer caused particulate embolism and blood cells remova1.4~8~9.30~3’ Artificial cells with polymer membrane containing adsorbents have been used in hemoperfusion for the routine treatment of patients for many years.”.31 This includes acute poisoning, high blood aluminum and iron, and supplement to dialysis in kidney failure and in liver failure.3031

Bioencapsulation of Enzymes

Our earlier studies of artificial cells for hereditary enzyme defects includes its successful use for replacement of catalase in acatalasemic mice? It has also been studied for asparagine removal in the treatment of leukemia in animals8 The major obstacle was the problem of long-term biocompatibility in implantation. More recently, we found an extensive enterorecirculation of amino acids in the This allows the use of orally administered artificial enzyme cells to selectively remove specific amino acids from the body, as in phenylketonuria. We also studied the oral administration of artificial cells containing xanthine 0xidase.3~ This resulted in decrease in systemic hypoxanthine in a pediatric patient with hypoxanthinuria (Lesch-Nyhan Disease).

Red Blood Cell Substitutes

We reported the use of artificial cells containing hemoglobin as artificial red blood cell substitutes as early as 1957.2 We extended this to crosslinked hemoglobin in 1964.3 We then carried out detailed There was no major interest until

CHANG: ARTIFICIAL CELLS AND BlOENCAPSULATlON 257

the 1980s when problems of HIV in donor blood has resulted in extensive research and developments by many g r o ~ p s . ~ - ‘ ~ Many new developments resulted in crosslinking of hemoglobin from different sources: human hemoglobin, bovine hemoglobin and recombinant hem~globin,‘~-” A number of these are now in Phase I, Phase I1 and Phase I11 clinical The second generation of hemoglobin-based blood substitutes based on microencapsulated and nanoencapsulated hemoglobin is being investigated as a more complete red blood cell s~bstitute.”-~’

SUMMARY

The most common use of artificial cells is for bioencapsulation of biologically active materials. Many combination of materials can be bioencapsulated. The permeability, composition and configurations of artificial cell membrane can be varied using different types of synthetic or biological materials. These possible variations in contents and membranes allow for large variations in the properties and functions of artificial cells.

REFERENCES

1. SCJNDARIIM, P. V., E. K. PYE, T. M. S. CHANG, V. H. EDWARDS, A. E. HUMPHREY, 0. KAPLAN, E. KATCHALSKI, Y . LEVIN, M. D. LILLY, G . MANECKE, K. MOSBACH, A. PATCHORNIK, J . PORATH, H. H. WEETALL & I. B. WINGARD, JR. 1972. Recommenda- tions for standardization of nomenclature in enzyme technology. Biotechnol. Bioeng. Id. 15-18.

CHANG, T. M. S. 1957. Hemoglobin corpuscles. Research Report for Honours Physiology, Medical Library, McGill University. Also reprinted as part of “30th anniversary in Artificial Red Blood Cells Research.” J. Biomat. Artificial Cells & Artificial Organs

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5. CHANG. T. M. S. 1965. Semipermeable Aqueous Microcapsules. Ph.D. Thesis, McGill Uni- versity.

6. CHANG, T. M. S., F. C. MACINTOSH & S. G . MASON. 1966. Semipermeable aqueous microcapsules: I. Preparation and properties. Can. J. Physiol. Pharmacol. 44: 115-128.

7. CtiANci, T. M. S., F. C. MACIN’rOSlI & s. G . MASON. 1971. Encapsulated hydrophilic compositions and methods of making them. Canadian Patent, 873, 815, 1971.

8. CHANG, T. M. S . 1972. Artificial Cells. C. C. Thomas Publisher. Springfield, IL. 9. CtiANG. T. M. S. 1991. Artificial Cells. In Encyclopedia of Human Biology. R. Dulbecco,

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MURPIIY, M. K. MOLONEY, M. SCHMEHLL, M. HARRIS, R. MENDEZ, R. MENDEZ & P. A. SANDFORD. 1994. Insulin independence in a type 1 diabetic patient after encapsulated islet transplantation. Lancet 343: 950-951.


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BRUNI, S. & T. M. S. CHANG. 1989. Hepatocytes immobilized by microencapsulation in artificial cells: Effects on hyperbilirubinemia in Gunn Rats. Biomater. Artif. Cells & Artif. Organs 17: 403-412.

BRUNI, S. & T. M. S. CHANG. 1991. Encapsulated hepatocytes for controlling hyperbilirub- inernia in Gunn Rats. Int. J. Artificial Organs 14: 239-241.

CoRoMiLi, V. & T. M. S. CHANG. 1993. Polydisperse dextran as a diffusing test solute to study the membrane permeability of alginate polylysine microcapsules. Biornater. Artif. Cells Immobilization Biotechnol. 21: 323-335.

WONG, H. & T. M. S. CHANG. 1988. The viability and regeneration of artificial cell microencapsulated rat hepatocyte xenograft transplants in mice. Biomater. Artif. Cells Artif. Organs 16: 731-740.

DIXIT, V., V. P. GORDON, S. C. PAPPAS & M. M. FISHER. 1989. Increased survival in galactosamine induced fulminant hepatic failure in rats following intraperitoneal transplantation of isolated encapsulated hepatocytes. In Hybrid Artificial Organs. C. Baquey & B. Dupuy, Eds. Colloque ISERM ln. 257-264. Paris, France.

BRUNI, S. & T. M. S. CHANG. 1995. Kinetics of UDP-glucuronosyltransferase in bilirubin conjugation by encapsulated hepatocytes for transplantation into Gunn rats. J. Artifi- cial Organs 19 449-457.

KASHANI, S. & T. M. S . CHANG. 1991. Physical chemical characteristics of hepatic stimulatory factor prepared from cell supernatant of hepatocyte cultures. Biomater. Artif. Cells Immobilization Biotechnol. 19: 565-578.

WONG, H. & T. M. S. CHANG. 1991. Microencapsulation of cells within alginate poly-L- lysine microcapsules prepared with standard single step drop technique: Histologically identified membrane imperfections and the associated graft rejection. J. Biomater. Artif. Cells Immobilization Biotechnol. 182: 675-686.

WONG, H. & T. M. S. CHANG. 1991. A novel two step procedure for immobilizing living cells in microcapsules for improving xenograft survival. Biomater. Artif. Cells Immobilization Biotechnol. 19: 687-698.

CHANG, T. M. S. & H. WONG. 1992. A novel method for cell encapsulation in artificial cells. United States Patent No. 5,084,350.

PRAKASH, S. & T. M. S. CHANG. 1995. Preparation and in-vitro analysis of microencapsulated genetically engineered E. coli DH5 for urea and ammonia removal. Biotechnol. Bioeng. 46: 621-626.

PRAKASH, S. & T. M. S. CHANG. 1996. Microencapsulated genetically engineered live E. coli DH5 cells administered orally to maintain normal plasma urea level in uremic rats. Nature Medicine 2: 883-887.

GAROFALO, F. & T. M. S. CHANG. 1991. Effects of mass transfer and reaction kinetics on serum cholesterol depletion rates of free and immobilized Pseudomonm pictorum. Appl. Biochem. Biotechnol. 27: 75-91.

LLOYD-GEORGE, I. & T. M. S. CHANG. 1995. Characterization of free and alginate- polylysine-alginate microencapsulated Erwiniu herbicolu for the conversion of ammonia, pyruvate and phenol into L-tyrosine and L-DOPA J. Bioeng. Biotechnol. 48:

CHANG, T. M. S. 1975. Microencapsulated adsorbent hemoperfusion for uremia, intoxica- tion and hepatic failure. Kidney Int. 7: S387-S392.

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