including bioencapsulation, microencapsulation, cell encapsulation, gene therapy, oral therapy for uremia, enzyme therapy, tissue engineering, hemoperfusion, sorbents, nanocapsules and drug delivery
Author: Professor Thomas Ming Swi Chang, O.C.,M.D.,C.M.,Ph.D.,FRCP(C) , Director, Artificial Cells & Organs Research Centre,Departments of Physiology,Medicine & Biomedical Engineering, Faculty of Medicine, McGill University, 3655 Drummond Street, Montreal, P.Q., Canada, H3G 1Y6
This is a detailed scientific review on artificial cell except blood substitutes.
Please click here for Blood substitutes
For more general
readings on artificial cells please click here.
I. Introduction click
II. Methods of preparation click
III. Artificial cells containing sorbents and immunosorbents including use in acute poisoning click
IV. Bioencapsulation of cells and microorganisms including use in diabetes, liver failure, etc click
V. Artificial cells containing genetically engineered cells including studies on the use of this approach in oral therapy for uremia click
VI. Artificial cells containing enzymes and multienzyme systemes and use in enzyme therapy including phenylketonuria click
VII. Blood Substitutes click
VIII. Biodegradable membrane artificial cells including use as drug delivery systems click
IX. Discussions and future perspectives click
X. General selected references click
XI. Publications on Artificial Cells by Chang's group(1957-2002) click
i. General:
Artificial Cells (1) prepared from microencapsulation of biologically active materials were first reported by Chang in 1957 (2) and 1964 (3). These were first prepared as ultrathin polymer membrane of cellular dimensions microencapsulating the proteins and enzymes extracted from biological cells (2,3). This was followed by the encapsulation of biological cells, adsorbents, magnetic materials, drugs, vaccine, hormones and many other biologically active materials for applications in biotechnology and medicine (3,4,5,6) (Fig.1). The first major interest shown by other groups was after its successful use for treating poisoning, kidney failure and liver failure in patients (4,7). The second impetus in the research on artificial cells was in the 1980's. This was the time of increasing international interests in all areas of biotechnology. Many groups have therefore investigated extensively microencapsulation of cells, microorganisms, enzymes and other biotechnological materials (8-11). The third impetus in this area was in the late 1980's. AIDS due to H.I.V. virus in transfusion blood has led to extensive research into artificial red blood cell substitutes.
ii. Basic Principles.
Like biological cells, artificial cells contain biologically active
materials. However, the content of artificial cells can be more varied than biological
cells (Fig.1). The membranes of artificial cells can also be extensively varied using
synthetic or biological materials. The permeability can be controlled over a wide range.
This way, the enclosed material can be retained and separated from undesirable external
materials (Fig. 2). While the large surface area and the ultrathin membrane allow selected
substrates and products to permeate rapidly (Fig.2). Mass transfer across 100 ml of
artificial cells can be 100 times higher than that for a standard hemodialysis machine.
i. Principles of methods of preparations.
Many methods are now available for the preparation of artificial cells. This brief overview is not the place to describe these methods. The most commonly used approaches are based on the following principles. Small artificial cells in the micron dimensions are prepared by emulsification procedures that are usually modifications of the original basic procedures (26). Here materials for microencapsulation are dissolved or suspended in an aqueous solution. An emulsionis then form. Membranes are then formed on the surface of each microdroplets. The microcapsules formed are then resuspended in an aqueous medium. Smaller artificial cells of nanometer dimensions are formed based on the same principles except that the initial emulsion formed are of much smaller dimensions. Larger artificial cells especially those in the millimeter dimensions are prepared based on modifications of the original drop method (2,5-7). These are generally used to microencapsulate cells or microorganisms in tissue engineering. Spray technic can also be used to encapsulated particulate matters (2). This approach has now been developed into techniques for large scale production. Artificial cells containing sorbents are usually prepared based on the original method of ultrathin membrane coating of sorbent granules (7,12).
ii. Membranes used for encapsulation.
Different types of synthetic polymers can be used (10). Variations in configuration are also possible. A single ultrathin polymer membrane is the most common one. The unlimited vairations in polymers available allow for possible variations in permeability, biocompatibility and other characteristics (211). Artificial cells can also be made to contain smaller "intracellular compartments " (57). Others can be prepared to form solid polymer microspheres containing microdrolets of biologically active materials (5). Liquid hydrocarbons form microdroplets containing biologically active materials which are useful in biotechnology and other applications (13). Membranes formed from biodegradable or biological materials are useful for artificial cells which need to be degraded. Different materials have been used. Protein membrane artificial cells and polyhemoglobin are two examples (3,57). The use of lipid is another common approach. This includes the use of lipidprotein membrane (7), concentric lipid membranes and submicron ultrathin lipid membrane (14). Another approach is biodegradable synthetic polymer. The first one used is polylactide (15). Many types of polylactides and polyglycolic acids are being used for artificial cells at present (16). Other synthetic biodegradable polymers can also be used. Polyanhydride is one example (17). Biodegradable artificial cells is now a very active field.
iii. Variations in contents.
Extensive variation in contents is also possible. The following are
examples of microencapsulation of biologically active materials for use in biotechnology
and medicine.
III. MICROENCAPSULATION OF
BIOACTIVE SORBENTS
i. General:
This 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 removal (4,7,12). This was developed and used successfully in patients (7). The hemoperfusion device being used in patients contains 70 gm of artificial cells. Each artificial cell is formed by applying an ultrathin coating of collodion membrane or other polymer membranes on each of the 100 micron diameter activated charcoal microspheres. The mass transfer for this small device is many times higher than that for a standard dialysis machine.
ii. Routine clinical uses for treating patients.
This is now a routine method of treatment for both adult and pediatric patients for poisoning (19). This is applicable to the many cases where the medication of toxin can be adsorbed by activated charcoal. In kidney failure, this is more effective than hemodialysis in removing organic waste metabolites (7,12,20). This is being used in 2 ways. (1) In series with dialysis, to shorten dialysis time and improved dialysis resistant symptoms (7,20). (2) In series with a small ultrafiltrator (20) it can replace the dialysis machine (20). Here oral adsorbents can be used to control potassium and phosphates. An urea removal system is being developed to complete the hemoperfusionultrafiltrator approach. The detoxifying functions of hemoperfusion resulted in temporary recoveries of coma in grade IV hepatic coma patient(26). This was supported by other groups(19,22,23,24,25,27). Hemoperfusion is very effective in detoxication. It is being studied as part of an artificial liver system. Hemoperfusion when used with a chelating agent, desferroxaimine, is effective in lower high aluminum levels in patients (29,19,30).
iii. Microencapsulation of immunosorbents:
Immunosorbents, like other sorbents described above also have problems
when in direct contact with blood. This includes the same embolisms of particulate and
adverse effects on blood cells. The same ultrathin coating has been applied to
immunosorbents (21) to prevent these problems. This has been tested clinically in patients
(10).
IV.
MICROENCAPSULATION OF CELLS OR MICROORGANISMS
i. General:
The first encapsulation of biological cells was reported in 1965 based on a drop method (5). It was proposed that (5) "..protected from..immunological process... encapsulated endocrine cells might survive and maintain an effective supply of hormone " "For organ deficiency ... cultures of liver cells...in artificial cells " This original drop-method for cell encapsulation involves chemically crosslinking the surface of aqueous droplets which contains cells (5,6). This was modified into the following drop technique using milder physical crosslinking (22,23). This resulted in alginate-polylysine-alginate (APA) microcapsules containing cells . Alginates are hetropolymer carboxylic acids, coupled by 1-E4 glycosidic bonds of $-D-mannuramic (M) and "-L-gluronic acid unit (G). Alkali and magnesium alginate are soluble in water, whereas alginic acids and the salts of polyvalent metal cations are insoluble. Thus, gel spheres can be formed by drops of sodium alginate solution entering a calcium chloride solution.
ii. General method is based on the following procedure:
1. Cells or microorganisms are suspended in the sodium alginate solution. The suspension is pumped through a 23 G stainless steel needle. Sterile compressed air, through a 16 G coaxial stainless steel needle, was used to shear the droplets coming out of the tip of the 23 G needle. Each droplet falls into the sterile ice cold solution of calcium chloride (1.40 %, pH 7.20 heat sterilized). Upon contact with the calcium chloride buffer, alginate gelation is immediate. The droplets were allowed to gel for 15 minutes in the ice cold sterile calcium chloride solution (1.40 %).
2. After gelation in the calcium chloride solution, alginate gel beads were suspended for 10 minutes in a 0.05 % polylysine solution. The positively charged polylysine forms a complex with surface alginate to form a semipermeable membrane.
3. The beads were then washed and placed in an alginate solution (0.10 %) for 4 minutes. The alginate neutralizes any excess polylysine on the surface. The alginate-poly-L-lysine-alginate capsules were then washed in a 3.00 % citrate bath (3.00 % in 1:1 HEPES-buffer saline, pH 7.20) to liquefy the gel in the microcapsules. The APA microcapsules formed, which contains a suspension of hepatocytes or genetically engineered bacteria E. coli, are stored at 4 0 C for use in experiments.
This is the most commonly used method cell encapsulation (23). This method is the result of extensive research (23) to improve the original drop method (5,6). This works well for larger cell aggregates like islets. However,it is not as suitable for encapsulating high concentrations of smaller cells like hepatocytes and microorganisms. Some cells or microorganisms remained on the surface of the microcapsule membrane (24). This would result in the rejection of the whole artificial cell.
iii. Method specific for high concentrations of smaller cells like hepatocytes and microorganisms.
We have therefore devised a new method to allow for more complete encapsulation of higher concentrations of smaller cells in artificial cells (25,26) as follows:
1. Small calcium alginate gel microspheres containing entrapped cells were first formed. Like the general procedure, there are cells protruding out of the surface of the smaller calcium alginate gel microspheres. These gel microspheres are then resuspended in alginate solution to go through the same droplet formation as before.
2. This way the small microspheres were entrapped within larger calcium alginate gel microspheres. This way there are no cells protruding out of the larger alginate gel microspheres.
3. The next two steps are the same as before. In the last step the entire content of the microcapsule was liquified by citrate. This also liquified the small calcium alginate gel microspheres inside the microcapsule. This way the hepatocytes in the smaller gel microsphere are released to float freely inside the microcapsule.
iv. Microencapsulated islets for Diabetes Mellitus
This author persuaded Connaught Laboratory of insulin fame to develop this for diabetes. This was finally carried out there (22,23) and later in other centers (27). They showed that islets inside artificial cells are indeed prevented from immunorejection after implantation into animals. Islets can indeed remain viable and continued to secrete insulin to control the glucose levels of diabetic rats. They improved the biocompatibility by the use of a alaginatepolylysinealginate membrane improved biocompatibility (23). One group used a special alginate to further improve the biocompatibility (27).
v. Microencapsulated hepatocytes for Liver failure:
We found that artificial cells containing hepatocytes increased the survival time of fulminant hepatic failure rats (28). Xenografts of rat hepatocytes in artificial cells were not immunorejected in mice (29). Instead of rejection the viability of the enclosed liver cells increased after intraperitoneal implantation(29). This was because the hepatotrophic factor secreted by the encapsulated hepatocytes accumulate in the artificial cells (30). After implantation, hepatocytes in artificial cells can lower the high bilirubin level in the Gunn rats (31,32). Recent reports by another group (33) also support this finding.
vi. Microencapsulation of cholesterol removing microorganisms.
We (36,37) selected Pseudomonas pictorum (ATCC #23328) as another model system because of its ability to degrade cholesterol. The standard encapsulation method does not result in high porosity membrane to allow lipoprotein-cholesterol to cross. We therefore devised a modified method to prepare high porosity agar microspheres. There was no evidence of leakage of the enclosed bacteria. Open pore agar beads were incubated in serum. The bacterial action was not significantly different between the encapsulated and free bacterial. Bacterial action was found to be the limiting step in the overall reaction. For practical applications, a suitable bacteria with higher rates of cholesterol removal is needed. No doubt this become available in the future with the help of genetic engineering.
vii. Microencapsulation of other types of cells.
Many other groups are now seriously studying the microencapsulation of
cells and microorganisms (38,39).
V. MICROENCAPSULATION OF
GENETICALLY ENGINEERED CELLS:
There is increasing research on using artificial cells to microencapsulate genetically engineered cells for gene therapy.
i. One appraoch is to encapsulate genetically engineered cells and implant this into the body. this includes the use of beta-endorphin secreting cells for pain treatment, NGF secreting cells for parkinsonism, human nerve growth factor secreting cells as trophic factors for striatal neurons, recombinant ciliary neurotrophic factor secreting cells for neurodegenerative diseases, CNTF secreting cells for amyotrophic lateral sclerosis, A number of problems related to implantation are being studied - this includes (a) potential safety problems related to the introduction of genetically engineered material into the body and (b) Although protected from rejection by leucocytes and antibodies, there is potential rejection by complements and cytokines. This approach of implantation of microencapsulated genetically engineered cells for gene therapy is being actively studied by many groups - for example Cell Transplant 1997 6:527-530, Art Cells Blood Sub Imm Biot 1996 24:219-255, Exp neurol 1997 147:10-17, Neuroscience 72:63-77, Human gene ther 1966 10:7:2135-2146,
ii. We have recently reported a new approach to avoid the need for implantation. This has much implications in an oral therapy for uremia (Nature Medicine 1996 2:883-887 and 1997 3: 2-4; Art Cells Blood Sub Imm Biot 1998 26: 35-51. For other more recent references and more details please see Appendix I at the end of this section).
At present only 15% of the world's uremic patients can afford dialysis treatment. Furthermore, dialysis is inconvenient for the patients. Our study shows that oral administration of microencapsulated genetically engineered E.coli DH5 cells can remove urea and increase the survival of uremic rats. Recent in-vitro studies here show that this also remove from uremic rat plasma other uremic metabolites like potassium, phosphate, uric acid, creatinine etc.
Microencapsulated genetically engineered cells are given once a day orally. The microcapsules with its genetically engineered cells passes through the intestine and is excreted in the stool. There is therefore no retention in the body. On their passage through the intestinal tract, small molecules (e.g. urea) from the body enters the microcapsules. The genetically engineered cells in the microcapsules then act on these molecules. Our first feasibility test is to study kidney failure rats using microencapsulated genetically engineered microorganisms (E.coli DH5 cells). Oral administration once a day allows the return of the elevated systemic urea to normal level. Unlike the control untreated group receiving controlled microcapsules, the treated group survived throughout the 21 days of study and continued to grow. More details are available in appendix I below.
APPENDIX I: Does orally administered artificial cells containing genetically engineered cells have a role in uremia therapy?
Advances in molecular biology have resulted in the availability of nonpathogenic genetically engineered microorganisms that can effectively used uremic metabolites for cell growth. We therefore studied the oral use of microencapsulated genetically engineered nonpathogenic E.coli DH5 cells containing Klebsiella aerogenes urease gene in renal failure rats [1-8].
In-vitro removal of urea, ammonium, electrolytes and other metabolites: Our in-vitro results show that 40.00 ± 8.60 g of encapsulated E. coli DH5 can remove 40 grams of urea (100mg/dl in 40 litres). There was no increase in ammonia level. This is compared to the much higher amount needed when we used oral microencapsulated urease and ammonia adsorbent in rats and the 1,212 g of microcapsulated urease-zirconium-phosphate in Kjellstran et al’s clinical trial . E. coli DH5 cells in culture grow to many times its original number and reaches a plateau after about 12 hours . This rapid growth will require ions and other metabolites in addition to nitrogen source. Indeed, our recent in-vitro studies using uremic rat plasma show significant decreases in the followings . When 10 microgram of E. coli DH5 cells were added to 1.0 ml of the uremic plasma [except for creatinine where 100 microgram of the cells were used] the decreases in plasma levels after 24 hours are as follows. Potassium decreased from 5.80+ 0.40 mEq/l to 3.50 + 0.03 mEq/l (p<0.001) in 24 hours; phosphate from 2.20+ 0.9 mg/dl to 1.49 + 0.03 mg/dl (p<0.005); sodium from 172 + 11.00 mEq/l to 129 + 6.12 mEq/l in 24 hours (p<0.001);. chloride from 137 + 6.60 mEq/l to 107 + 2.00 mEq/l (p<0.005) and cholesterol from 1.86 + 0.10 mmol/l to 1.37 + 0.06 mmol/l (p<0.005). Removal of uric acid was so high that we had to add a higher concentration of uric acid into the uremic plasma to 84.80 + 3.40 mg/ dl that was lowered to 8.80 + 3.12 mg/ dl (p<0.001) in 24 hours. Removal of creatinine from the uremic plasma was 83. 31 + 2.40% remaining after 24 hours of incubation using 100 microgram of the E.coli DH5 cells.
Daily oral administration to partially nephrectomized rats including survival studies: We prepared renal failure rats by the surgical removal of one kidney and the partial ligation of the other kidney. The degree of ligation was adjusted to result in an elevation of urea and other waste metabolites without severe disturbances in water and electrolytes. All the rats were placed on a standard rat diet containing 22.5% protein. We gave daily oral log phase microencapsulated genetically engineered E. coli DH5 cells daily for 21 days to a group of renal failure rats. Their original plasma urea level of 52.08 ± 2.06 % mg was lowered and maintained at the normal range of 9.10 ± 0.71 % mg during the treatment period [23]. The blood ammonia level also decreased significantly[23]. A return to high urea levels was observed when the treatment was stopped, showing that there was no significant retention of E.coli DH5 cells in the intestine. The microcapsules containing the rapidly growing E.coli DH5 cells with the urea-nitrogen used for growth are excreted all together in the stool. Calculations based on the result of this study showed that in a 70kg patient with the same degree of renal failure as in the partially nephrectomized rats, we would only need to give 4 grams of E.coli DH5 cells orally each day. This would not be an excessive amount for use in patients. Daily oral administration for 21 days was started 31 days after partial nephrectomy. In the group of uremic rat that received control microcapsules containing no E. coli DH 5 cells, 25 % died in the first 46 days after partial nephrectomy, 50 % died after 54 days, and 75 % died after 67 days. Surprising, the uremic rats that received daily oral microcapsulated E. coli DH 5 cells, all survived during the treatment period . After stopping oral administration 50% of the animals died by day 81, equivalent to 29 days after stopping treatment . In addition, we carried out a very severe test of the potential toxicity of what happens if some encapsulated cells leak out during their passage through the intestinal tract. We gave another group of renal failure rats the same daily oral doses of E .coli DH5 cells but all in the free form for the same 21 days period. The results showed that even if all the E. coli DH5 cells were to have leaked out there was no adverse effect on the growth or survival of the renal failure rats.
Clinical potentials of oral encapsulated E.coli DH5 cells:
Even though free E.coli DH5 cells are not toxic when given orally, it does not mean that
they should be given orally in the free form [8]. When free E.coli DH5 cells were used,
some of them were retained in the intestine. Repeated large doses of these
bacteria in the free form would result in their taking over the normal intestinal flora.
Now, urea removal is based on the use of urea as a nitrogen source by the encapsulated
E.coli DH5 cells which are then excreted in the stool with its nitrogen content [8]. If
the E.coli DH5 cells stay in the intestine, the urea-nitrogen is not excreted and is
recycled in the body. Indeed, attempts in the past to lower urea levels by manipulating
the intestinal flora [9,10] would involve the retention of bacteria in the intestine. With
the retention of the microorganisms in the intestine the nitrogen source from the urea
also stays with the microorganisms and is therefore retained in the body and recycled [8].
The oral administration of artificial cells containing nonpathogenic genetically
engineered cells may contribute to the possibility for oral therapy in uremia [8]. Removal
of fluid can be carried out using oral fluid removal system [11] especially for the 85% of
the uremic patients who cannot receive dialysis. In addition, the recent potential
benefits of daily dialysis to prevent large fluctuations in the systemic waste metabolites
come with the unwelcome need for daily treatment. This oral approach could be used with
the regular three times a week dialysis to prevent large fluctuations in the systemic
waste metabolites.
Prakash S, Chang TMS: Microencapsulated genetically Engineered
live E. coli DH5 cells administered orally to maintain normal plasma urea level in uremic
rats. Nature Medicine 1996; 2 [8]:883-887.
As discussed above we have studied the use of microencapsulated genetically engineered microorganisms using as a model E. coli DH5 cells containing K. aerogens urease gene. Overall, urea removal efficiency of microencapsulated genetically engineered bacteria is 10-30 times higher than the best available urea removal systems available at present. In addition to its potential use in uremia, the removal of urea ,ammonia, uric acid, bilirubin and other metabolites are needed in liver failure, environmental decontamination, regeneration of water supply in space travel and other conditions.
VI. MICROENCAPSULATION OF
ENZYMES, AND MULTIENZYME SYSTEMS
Artificial cell protects the enclosed enzyme for immunological rejection or tryptic enzymes (37). However, substrates can equilibrate rapidly into the artificial cells for conversion into products which can diffuse out. This is now being developed for use in the treatment of an hereditary disease, phenylketonuria (PKU) and in other conditions.
i. Urea removal:
We showed that artificial cells containing urease can convert urea to ammonia which is then removed by ammonia adsorbent (37). This approach has since been developed further by us and other groups (10). Ammonia adsorbents with better adsorbing capacity for ammonia are required to improve this approach.
ii. Enzyme therapy in hereditary enzyme defects and other conditions:
Artificial cells have been used in hereditary enzyme defects. This includes our earliest use for replacement of catalase in acatalasemic mice (40). It has also been studied for asparagine removal in the treatment of leukaemia in animals (41). We used phenylalanine ammonia lyase artificial cells in phenylketonuria rats (42). More recently, we found an extensive enterorecirculation of amino acids in the intestine. This allows the use of orally enzyme artificial cells to selectively remove specific amino acids from the body, as in Phenylketonuria (43). We also studied the oral administration of artificial cells containing xanthine oxidase(44). This resulted in decrease in systemic hypoxanthine in a pediatric patient with hypoxanthinuria (LeschNyhan Disease).
iii. Multienzyme system:
Most enzymes in biological cells function as complex enzyme systems. We
have prepared artificial cells to contain multienzyme systems with cofactor recycling
(45). This approach can convert metabolic wastes like urea and ammonia into essential
amino acids like leucine, isoleucine and valine which are required by the body(46). We
have also prepared artificial cells containing hemoglobin with pseudoperoxidase activity
and glucose oxidase to remove bilirubin(47,48) .
VII. RED BLOOD CELL SUBSTITUTES
The 2 major approaches are (i) Modified hemoglobin and (ii) Perfluorochemicals.
Detailed reviews in the field are available (49,50). This is a very large area which will be discuss under a separate title
of Artificial Blood.
VIII. BIODEGRADABLE
ARTIFICIAL CELLS
Another area is the use of biodegradable artificial cells especially
for drug delivery. This was discussed under the section on preparation. We have used
crosslinked protein (3,57) and biodegradable polylactide artificial cells (15). Many
groups are extending these approaches for use in drug delivery (medications, hormones,
peptides and proteins)(10,16,7). We prepared lipidprotein and lipidpolymer artificial
cells to encapsulate biologically active materials(51). Later, Gregoriadis prepared
concentric lipid membrane liposomes containing enzymes (14). Liposomes are multiple lipid
layers onionskinlike microspheres originally used by Bangham for basic membrane research.
Workers in liposomes more recently turned to preparing small submicron artificial cells
with a single bilayer lipid membrane(14). These lipid membrane artificial cells are no
longer concentric lipid membrane liposomes. Some still continue to call these
"liposomes " (14). This has created some confusions in the field. The most
extensive research is in its use for drug delivery (14).
i. Present status:
The present uses of artificial cells in biotechnology and medicine includes the following:
1. Hemoperfusion for acute poisoning routine treatment in patients
2. Hemoperfusion for aluminium and iron overloadroutine treatment in patients
3. Supplement to hemodialysis in endstage renal failure routine treatment in patients
4. Artificial liver support: hemoperfusion and hybrid systems experimental
5. Red blood cell substitutes for transfusion Phase I and Phase II clinical trials
6. Blood group antibodies removal (immunosorbents) clinical trial
7. Hereditary enzyme deficiency clinical trial
8. Clinical laboratory analysis clinical application
9. Production of monoclonal antibodies
10. Diabetic mellitus and other endocrine diseases animal experiment, clinical trial started
11. Drug delivery systems clinical application and experimental
12. Conversion of cholesterol into carbon dioxide experimental
13. Bilirubin removal experimental
14. Production of fine biochemicals
15. Food and aquatic culture
16. Conversion of wastes into useful products experimental
17. Other biotechnological and medical applications
Artificial cells can contain an unlimited number of biologically active materials (Fig 1). There are therefore many other areas of applications and research. For example, the author has enclosed magnetic and biological materials together inside artificial cells (4). This allows for localization with external magnetic fields (4). Kato applied magnetic field applied outside the body of animals(11). This can direct magnetic artificial cells containing radioactive materials and chemotherapeutic agents to specific sites of bladder cancer. Magnetic artificial cells are also used in bioreactors. Others have used artificial cells in laboratory analysis of free and proteinbound hormones in patients (11). We have studied its use for 1shot vaccine (10) and for removing large lipohyllic molecules from small hydrophyllic molecules (10). Still others have used artificial cells for industrial aquatic culture for shrimps, lobsters, cannot cover all areas of of artificial cells(10). We have also studied artificial cells containing hepatic microsome and cytosol (10).
ii. Future perspectives:
The author wrote in his 1972 book on Artificial Cells (7):
"Artificial cell " is a concept; the examples described....are but physical
examples for demonstrating this idea. In addition to extending and modifying the present
physical examples, completely different systems could be made available to further
demonstrate the clinical implications of the idea of "artificial cells. "
imagination. An entirely new horizon is waiting impatiently to be explored. This future
perspective is even more valid now.
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14. Gregoriadis F. Liposomes as drug carriers: Recent trends and progress. New York: John Wiley & Sons, 1989.
15. Chang TMS. Biodegradable semipermeable microcapsules containing enzymes, hormones, vaccines, and other biologicals. J Bioengineering 1976;1:2532.
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24. Wong H, Chang TMS. Microencapsulation of cells within alginate polyLlysine microcapsules prepared with standard single step drop technique: Histologically identified membrane imperfections and the associated graft rejection. J. Biomaterials, Artificial Cells and Immobilization Biotechnology 1991;675686.
25. Wong H, Chang TMS. A novel two step procedure for immobilizing living cells in microcapsules for improving xenograft survival. Biomaterials, Artificial Cells and Immobilizing Biotechnology 1991;19:687698.
26. Chang TMS, Wong H. A novel method for cell encapsulation. Patent U.S.A. 1991.
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29. Wong H, Chang TMS. The viability and regeneration of artificial cell microencapsulated rat hepatocyte xenograft transplants in mice. J Biomat Artif Cells and Artif Organs 1988;16:73140.
30. Kashani S, Chang TMS. Effects of hepatic stimulatory factor released from free or microencapsulated hepatocytes on galactosamine induced fulminant hepatic failure animal model. J. Biomaterials, Artificial Cells and Immobilization Biotechnology 1991;19:579598.
31. Bruni S, Chang TMS. Hepatocytes immobilized by microencapsulation in artificial cells: Effects on hyperbilirubinemia in Gunn Rats. J. Biomat Artif Cells and Artif Organs 1989;17:40312.
32. Bruni S, Chang TMS. Encapsulated hepatocytes for controlling hyperbilirubinemia in Gunn Rats. Int. J. Artificial Organs 1991;14:239241.
33. Dixit V, Darvasi R, Arthur M, Brezina M, Lewin K, Gitnick G. Restoration of liver function in Gunn rats without immunosuppression using transplanted microencapsulated hepatocytes. Hepatol. 1990;12:134249.
34. Prakash S and Chang TMS Genetically engineered E. coli cells containing K. aerogenes gene, microencapsulated in artificial cells for urea and ammonia removal. Biomaterials, Artificial Cells and Immobilization Biotechnology, an international journal. 21:629-636, 1993.
35. Prakash S , Chang TMS. Kinetic studies of microecnapsulated genetically engineered E.coli cells containing K. aerogenes gene for urea and ammonia removal. Biotechnology & Bioengineering 1995 (in press)
36. Garofalo F, Chang TMS. Immobilization of P. pictorum in open pore agar, alginate polylysinealginate microcapsules for serum cholesterol depletion. J Biomat, Artif Cells and Artif Organs 1989;17:27190.
37. Garofalo F, Chang, TMS. Effects of mass transfer and reaction kinetics on serum cholesterol depletion rates of free and immobilizedPseudomonas pictorum. Applied Biochemistry and Biotechnology 1991;27:7591.
38. Goosen MFA, King GA, McKnight CA, Marcotte, N. Animal cell culture engineering using alginate polycation microcpasules of controlled membrane molecular weight cutoff. Journal of Membrane Science 1989;40:233243.
39 Goosen, M (editor) Fundamentals of animal cell encapsulation and immobilization CRC Press, 1992.
40. Chang TMS, Poznansky MJ. Semipermeable microcapsules containing catalase for enzyme replacement in acatalsaemic mice. Nature, 1968;218(5138):242245.
41. Chang TMS. The in vivo effects of semipermeable microcapsules containing
Lasparaginase on 6C3HED lymphosarcoma. Nature, 1971;229(528):117118.
42. Bourget L, Chang TMS. Phenylalanine ammonialyase immobilized in microcapsules for the depleture of phenylalanine in plasma in phenylketonuric rat model. Biochim. Biophys. Acta, 1986;883:432438.
43. Chang TMS, Bourget L & Lister C . new theory of enterorecirculation of amino acids and its use for depleting unwanted amino acids using oral enzyme-artificial cells, as in removing phenylalanine in phenylketonuria. Artificial Cells, Blood Substitutes & Immobilization Biotechnology,25: 1-23.1995.
44. Palmour RM, Goodyer P, Reade T, Chang TMS. Microencapsulated xanthine oxidase as experimental therapy in LeschNyhan Disease. Lancet 1989;2(8664):6878.
45. Chang TMS. Recycling of NAD(P) by multienzyme systems immobilized by microencapsulation in artificial cells. Methods in Enzymology 1987;136:6782.
46. Gu KF, Chang TMS. Production of essential L-branched chained amino acids, in bioreactors containing artificial cells immobilized multienzyme systems and dextranNAD+. Applied Biochemistry and Biotechnology 1990;26:263269.
47. Daka JN, Chang TMS. Bilirubin removal by the pseudoperoxidase activity of free and immobilized hemoglobin and hemoglobin coimmobilized with glucose oxidase. J Biomat, Artif Cells and Artif Organs 1989;17:55362.
48. Chang TMS, Daka JN. Removal of bilirubin by the pseudoperoxidase activity of immobilized hemoglobin. U.S. Patent No. 4820416, 1989.
49. Chang TMS, ed. Blood Substitutes and Oxygen Carriers New York: Marcel Dekker Publisher, 1992.
50. Chang TMS, J Reiss and R Winslow (eds) Blood substitutes: general. Special issue, Artificial Cells, Blood Substitutes and Immbolization Biotechnology, An International Jounral, 22:123-360, 1994.
51. Chang TMS. Lipid coated spherical ultrathin membranes of polymer or crosslinked protein as possible cell membrane
52. CHANG TMS and PRAKASH, S (1996).
Artificial cells for bioencapsulation of cells and genetically engineered e. Coli :
for cell therapy, gene therapy and removal of urea and ammonia. Chapter 75 in book on
"Expression and Detection of Recombinant Genes" in Methods in Molecular Biology
series
53. PRAKASH, S and CHANG TMS (1996)
Microencapsulated genetically engineered E.coli DH5 cells for plasma urea and aommonia
removal based on: 1. column bioreactor and 2. oral administration in uremic rats.
Artificial Cells, Blood Substitutes & Immobilization Biotechnology, an international
journal. 24:201-218.
54. PRAKASH, S. and CHANG, T.M.S.(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, August
1996.
55. CHANG TMS (1997) Live E. coli cells to treatment uremia: replies to letters to the editor, Nature Medicine. 3:2-3, 1997
56. CHANG TMS and PRAKASH, S (1998) Therapeutic uses of microencapsulated genetically engineered cells. Molecular Medicine Today. 4:221-227
57. Abstract book (1998): Engineering Foundation Conference on Bioartificial Organs August, 1998
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