Artificial Cells and Bioencapsulation in Bioartificial Organs

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<ul><li><p>Artificial Cells and Bioencapsulation in Bioartificial Organsa </p><p>THOMAS MING SWI CHANGh Artificial Cells &amp; Organs Research Centre </p><p>Departments of Physiology, Medicine &amp; Biomedical Engineering Faculty of Medicine McGill University </p><p>Montreal, Quebec, Canada H3G I Y6 </p><p>Modern research on bioartificial organs is based on molecular and cellular ap- proaches. Even before 1972 there were many ways of preparing bioartificial materi- als. 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. </p><p>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 materi- als. 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 </p><p> 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:; web:, artcell </p><p>249 </p></li><li><p>250 ANNALS NEW YORK ACADEMY OF SCIENCES </p><p>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.) </p><p>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-" </p><p>CELL ENCAPSULATION </p><p>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 </p><p>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.) </p></li><li><p>CHANG: ARTIFICIAL CELLS AND BlOENCAPSULATlON 251 </p><p>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 bioen- capsulation of islets."'-" These efforts will be reported on by others in this con- ference. </p><p>Bioencapsulation of Hepatocytes </p><p>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%." </p><p>Experimental Cell Therapy in Rats with Fulminant Hepatic Failure </p><p>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 &lt; 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.?" </p><p>Experimental Cell Therapy in Gunn Rats-An Animal Model for Human Non-Hemolytic Hyperbilirubinemia (Crigler-Najjar Type I) </p><p>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 </p></li><li><p>252 ANNALS NEW YORK ACADEMY OF SCIENCES </p><p>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 re- ceiving 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. </p><p>Immunoisolation of Bioencapsulated Rat Hepatocytes When Implanted into Mice </p><p>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 ana- lyzed since hepatocytes do not have good viability under this condition. </p><p>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 &lt; 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%. </p><p>In conclusion, rat hepatocytes in free floating microcapsules can be immunoiso- lated. 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. </p><p>Hepatocyte-secreted Hepatic Stimulatory Factor Is Retained Inside the Microcapsule Artificial Cells </p><p>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 micro- encapsulated hepatocyte suspension, helps to increase the viability and recovery of the membrane integrity of hepatocytes inside the artificial cells. </p></li><li><p>CHANG: ARTIFICIAL CELLS AND BIOENCAPSULATION 253 </p><p>The Single Major Obstacle to Clinical Use of Cell Encapsulation Is Long-Term Biocompatibility </p><p>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. </p><p>Procedure Specijic for Encapsulating High Concentrations of Smaller Cells Like Hepatocytes or Microorganisms </p><p>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. </p><p>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- </p><p>ALGINATE GEL SPHERE </p><p>FORM MEMBRANE DISSOLVE GEL </p><p>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 &amp; Chang.*&gt; Used with permission of Marcel Dekker Publisher.) </p></li><li><p>254 ANNALS NEW YORK ACADEMY OF SCIENCES </p><p>FIGURE 4. A two-step method has been designed to prevent this problem. (From Wong &amp; Chang?' Used with permission of Marcel Dekker Publisher.) </p><p>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. </p><p>BIOENCAPSULATION OF GENETICALLY ENGINEERED MICROORGANISMS </p><p>Bioencapsulation of Genetically Engineered E. coli Cells for Urea and Ammonia Removal </p><p>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 </p><p>Factors in the Bioencapsulation Process </p><p>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 </p><p>We find that an air flow rate of 2.00 L/min is suitable for preparing microcap- sules 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 microcapsul...</p></li></ul>


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