Study On Cytomedicine Of Alginate-Poly(L)lysine-Alginate Microcapsules

AIginate-poly(L)lysine-alginate (APA) membrane has been developed for the microencapsulation of living cells which protects these cells from the immune system. It was preparaed for the first time by Lim and Sun in 1980's. APA microcapsules have semipermeable membranes which permit passage of low molecular weight substance, such as nutrients and oxygen, but not of cells and high molecular weight substance. APA microencapsulated biological active molecules, tissue, and cells for allo- or xenograft transplanting have been studied by many researchers. Recently, someone has proposed an term "cytomedicine" for cell therapy. This novel medical treatments against diseases using living cells makes the best use of cell functions which naturally act as a drug delivery system (DDS) in vivo. APA microcapsules acting as the vehicle of the cytomedicine will be potential for clinical use.Multiple myeloma (MM) is a disease of immunoglobulin structural abnormality. B-cell malignancies produce myeloma protein (M protein). In patients of MM with the IgG type kappa light chain subclass, kappa is dominantly produced, resulting in a marked change of light chain/heavy chain ratio in the early phase of the disease. If the excessive light chain is suppressed, it will be useful for clinical therapy.In this study, we optimized the parameters during the process of APA microencapsulation. Firstly, the morphologic study, the mean diameter, the suface finish, the uniformity, the mechanical strength and the chemical intensity of APA microcapsules were established as standards. Then, we performed the APAmicrocapsules with a high-voltage electrostatic system. Briefly, with the syringe pump, alginate solution was extruded at a controlled rate through a needle. When the suspension was forced out of the end of the needle, spherical droplets were pulled off in the electrical field by the high-voltage electrostatic generator. The spherical droplets were collected and transformed to alginate microbeads by gelling in 100 mmol -L CaCl2 solution for 10 minutes. After washing three times with 0.9 % saline, the microbeads suspended with poly-L-lysine (PLL) solution. Before and after treatment with 0.15 % alginate, the microbeads were washed three times with 0.9 % saline. In order to liquefy their inner cores, the microcapsules were allowed to react with 55 mmol L sodium citrate, and then washed three times with 0.9 % saline to remove excess citrate and liquefied alginate.After optimized the parameters individially, we conformed the best concentration of first alginate solution 1.5 %, the CaCh solution 100 mmol L, and the suitable needle 7. Also we found that different concentration of PLL solution reacted with different diameter of alginate microbeads led to varied surface finish. After orthogonal experiment design, the other parameters were optimized, such as push speed of the syringe pump, the voltage of high-voltage electrostatic generator, the distance between needle and the CaCl2 solution. It was confirmed that qualityl (the alginate microbeads diameter) and quality 2 (roundness) were affected by the push speed mostly, and quality 3 (homogeneity) was decided by the distance. Thus, the microbeads of 150m can be prepared with the parameters of the push speed 4.5 ml min, the voltage 12.5 kv, the distance 2.0 cm, followed with the reaction of 0.02 % PLL solution. With optimum parameters, the APA microcapsules of mean diameter 250 um can be made which were found round shape, homogeneous and had the best membrane strength.To get the cell line we need in this study, the hybridoma cell line secreting anti-human IgG1 type monoclonal antibodies was established. The spleen cells taken from BALB/C mice immunized repeatedly by purified human IgG1 type were fused with mouse myeloma cells SP2/0. After culture and screened by ELISA, thehybridoma cell lines secreting monoclonal antibodies (mAb) against human IgG1. type named JY-A1 was established. The subclass of mAb secreted by JY-A1 is belonged to IgGl.By the high-voltage electrostatic system with the op...