Applications

3.3.1. Bioencapsulated Cells in Cell Therapy

The present example is to use bioencapsulated hepatocytes in basic investigations to study the feasibility of encapsulated cells in cell and gene therapy. Hepatocytes or hepatocytes with genetic induction can be used. One can immunoisolate the cells by using the bioencapsulation concept discovered by Chang (2). Hepatocytes and other types of cells can be obtained from humans or animals for implantation.

3.3.1.1. Bioencapsulation of Hepatocytes

Typically, hepatocytes were enclosed within aiginate-polylysine-alginate (APA) microcapsules of 300 pm mean diameters (18). Different concentrations of hepatocytes can be microencapsulated into the artificial cells. In a typical case, each 300-pm diameter artificial cell contains 120 ±20 SD hepatocytes, Thus, the 4 mL of 62,000 artificial cells injected contained a total of about 7.40

x 106 hepatocytes. Higher concentrations of hepatocytes can also be enclosed. Thus, 1.1 mL of microcapsules can contain 15 x 106 hepatocytes (19,20). The 300-pm diameter microcapsules are flexible. They can be easily injected using syringes with 20-gage needles. Permeability of the membrane can be adjusted. Detailed analysis has been carried out using HPLC of a large spectrum of mol wt dextran (21). The permeability can be adjusted to have different cutoff mol wt depending on the applications. Thus, for hepatocytes, it can be adjusted to allow albumin to pass through but not immunoglobulins.

3.3.1 2. The Viability of Rat Hepatocytes After Encapsulation

Wong and Chang (22) noted that after isolation from the liver, the percentage of viable hepatocytes as determined by trypan blue stain exclusion was about 80%. After bioencapsulation of hepatocyes within algmate-polylysme microcapsules, the percent of viable cells was 63.40%. This is mainly due to the effect of the procedure on the hepatocyte membrane integrity. As will be shown later, the membrane can recover in certain m vivo conditions.

3.3 1 3. Experimental Cell Therapy in Fulminant Hepatic Failure Rat

Wong and Chang showed that galactosamine-induced fulminant hepatic failure rats that received control artificial cells died 66.1 ± 18.6 h after galac-tosamine induction (18). The survival time of the group that received one peritoneal injection of 4 mL of microcapsules containing 7.40 x 106 hepatocytes was 117 3 + 52.7 h SD. 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 m long-term survival rates (23).

3.3.1.4 Experimental Cell Therapy in Gunn rats—An Animal Model for Human Nonhemolytic Hyperbilirubinemia (Crigler-Najjar Type I)

Brum and Chang investigated the use of artificial cells containing hepatocytes as cell therapy to lower bilirubin levels in Gunn rats (Fig. 4) (19,20). In the first experiment, 3.5-mo-old Gunn rats weighing 258 ± 12 g were used. During the 16 d control period, the serum bilirubin increased at a rate of 0.32 ± 0.07 mg/100 mL/d. This reached 14.00 ± 1 mg/100 mL at the end of the control period. Each animal then received an ip injection of 1.10 mL of microcapsules containing 15 x 106 viable Wistar rat hepatocytes. Twenty days after implantation of the encapsulated hepatocytes, the serum bilirubin decreased to a level of 6.00 ± 1 mg/100 mL. The level remained low 90 d 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

EFFECT OF ENCAPSULATED WISTAR HEPATOCYTES ON SERUM BILIRUBIN IN GUNN RATS

EFFECT OF ENCAPSULATED WISTAR HEPATOCYTES ON SERUM BILIRUBIN IN GUNN RATS

time (days)

Fig. 4. Implantation of artificial cells containing hepatocytes. Effects on hyperbilirubinemia m Gunn Rats. Reprinted with permission from ref 19

time (days)

Fig. 4. Implantation of artificial cells containing hepatocytes. Effects on hyperbilirubinemia m Gunn Rats. Reprinted with permission from ref 19

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. Significant decreases in the plasma bilirubin level were observed in the rat group receiving encapsulated hepatocytes. Analysis showed that implanted encapsulated hepatocytes lowered bilirubin by carrying out the function of the liver in the conjugation of bilirubin.

3.3 1.5. Immunoisolation of Bioencapsulated Rat Hepatocytes When Implanted into Mice

Wong and Chang reported studies on the lp implantation of free or bioencapsulated rat hepatocytes into 20-22 g male normal CD-1 Swiss mice or CD-I Swiss mice with galactosamine-induced fulminant hepatic failure (FHF) (22). 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, free rat hepatocytes implanted into normal CD-I Swiss mice were rapidly rejected. By day 14, there were no mtact hepatocytes detected in the mice (Fig. 5). Rat hepatocytes after implantation into CD-I Swiss mice with galactosamine-induced FHF (FHF) were rejected completely after 4—5 d. 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 hepato-

RAT XENOGRAFT IN NORMAL MICE

* VIABILITY

* VIABILITY

DAYS IMPLANTED

Fig. 5. Rat hepatocytes transplanted into mice. The viability and regeneration of artificial cell microencapsulated rat hepatocyte xenograft transplants in mice This is compared to rat hepatocytes without bioencapsulation. Reprinted with permission from ref 22

DAYS IMPLANTED

Fig. 5. Rat hepatocytes transplanted into mice. The viability and regeneration of artificial cell microencapsulated rat hepatocyte xenograft transplants in mice This is compared to rat hepatocytes without bioencapsulation. Reprinted with permission from ref 22

cytes within the microcapsules after 2 d of implantation (Fig. 5). The percentage of viable cells increased with time so that 29 d 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 had have the unexpected findings of improvement in cell viability when followed for up to 29 d.

3.3.1.6. Hepatocytes-Secreted Hepatic Stimulatory Factor is Retained Inside the Microcapsule Artificial Cells

Studies by Kashani and Chang show that hepatocytes in the microcapsule secrete factor(s) capable of stimulating liver regeneration (25). This factor is retained inside the microcapsules after secretion. Using Sephacryl gel chroma-

tography, they showed that this factor has a mol wt >110,000. 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.

3.3 1.7 Summary

Basic research using bioencapsulated hepatocytes shows the feasibility of this technology for cell therapy. Further improvements in biocompatibility may allow this approach to be used for cell and gene therapy in humans. This is becoming increasingly feasible because of the increasing progress in genetic engineering and molecular biology.

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