Study On NaCS Preparation And Immobilized Cultivation Of Dictyostelium Discoideum In NaCS-PDMDAAC Microcapsules

In this work,NaCS(sodium cellulose sulphate)-PDMDAAC(ploy dimethyl diallyal ammonium chlorid) microcapsule immobilization system was applied to cultivate recombinant Dictyostelium discoideum(D.discoideum),in order to raise its cell density and increase the product concentration.However,the NaCS has not been commercialized,thus its preparation process, characterization of NaCS-PDMDAAC microcapsules and application of the microcapsules in cell culture were investigated.In NaCS preparation,sulfate acid and n-propyl alcohol were used as the reaction solution according to the report at first.Then the n-propyl alcohol was replaced by alcohol to reduce the preparation cost.The response surface methodology(RSM) was employed to optimize the NaCS preparation condition.Two factors,i.e.the proportion of sulfate acid to alcohol in reaction solution and the reaction time were considered with the response of mechanical strength of the NaCS-PDMDAAC microcapsules.In general, higher mechanical strength of the NaCS-PDMDAAC microcapsules indicates better quality of the prepared NaCS.The optimized results were as follows:the ratio of acid to alcohol 1.51:1,and the reaction time 60.9 min.After the optimization of the preparation conditons,the reuse of the reaction solution was studied in order to reduce the preparation cost as much as possible,and it was found that the reaction solution could be reused for three times.The reaction system was scaled up to 1000 mL.After high quality NaCS has been prepared successfully,its substitution degree and viscosity were measured, which was 0.29 and 489 mPa.s(with 4%NaCS solution),respectively.The NaCS-PDMDAAC microcapsules were characterized.The diameter of the microcapsules and the membrane thickness were measured.Scan electron microscopy was employed to investigate the membrane structure. The factors influencing the microcapsule mechanical strength were investigated,and the results show that the mechanical strength could be obtain as high as 2010 g while the capsules remained sphere shape,which was much higher than that ever reported.In order to investigate their resistivity to shearing force,the microcapsues were shaken in a shake flask at 150 rpm on a rotating shaker,and the results indicate that they were not broken within 44 days.Subsequently,the NaCS-PDMDAAC microcapsule system was applied in immobilized cultivation of D.discoideum for production of soluble human Fas ligand(shFasL).The biocompatibility of the microcapsules with D.discoideum cells was studied at first.It was found that the NaCS could promote the cell growth,and the cells grew very well in the microcapsules.Then the microcapsule preparation conditions for the immobilized cultivation of D. discoideum were optimized as follows:NaCS 3.59%,PDMDAAC 6.18%,and reaction time 15.4 min.The encapsulated D.discoideum cells were cultivated on complex HL-5C and synthetic SIH medium,and cell densities up to 1.61×10⁸ mL^-1 and 1.92×10⁸ mL^-1 were reached,respectively,which were 6～8 times as much as those could be obtained in suspension culture.In the meanwhile,shFasL concentration of 1057μg L^-1and 1216μg L^-1 were accumulated on these two medium,respectively,which were 4～7 times higher than those could be achieved in suspension culture.Repeated-batch cultivation of immobilized D.discoideum in microcapsules was also studied. The repeated-batch cultivations were carried out by replacing the spent medium with fresh one when cell density reached a maximum stationary level in the microcapsules.It appeared diauxic growth during a cultivation period of 20～30 days.Finally,The cultivation of immobilized D.discoideum cells was scaled up by in a bubble column reactor.The results show that a considerable high cell density of 1.73×10⁸ mL^-1 could be achieved,although the cells grew a bit slower in the bubble column than in the shake flask culture.