Chitosan Materials As Scafford For Lymphoid Tissue And Cell In Vitro Culture From Penaeus Chinensis

With the wide and rapid explosion of shrimp viral diseases, in vitro culture ofshrimp tissue and cell has therefore attracted attention recently. Although the cellmonolayer of many species of shrimp was acquired, to date, no continuous cell lineshave been successfully established. This phonomenon mainly lie in the lack ofappropriate culture conditions and the loss of the ability to proliferate in succession ofcultured cells. Shrimp shell is one of the raw materials of chitosan (CS), so to someextent, homology might exist between shrimp cells and CS. It’s pioneering andmeaningful to use chitosan for shrimp cell culture.Chitosan/α, β-glycerophosphate (CS/α, β-GP) thermo-sensitive hydrogel wassuccessfully prepared by physical cross-linking method. Rheological analysis foundgel temperature (Tgel) of CS/α, β-GP aqueous solution slightly increased with theincrease of CS molecular weight. Measured Tgel were as follows: CS1/α, β-GP,25.2°C; CS2/α, β-GP,25.9°C; CS3/α, β-G,26.2°C; CS4/α, β-GP,27°C; CS5/α,β-GP,27.5°C. Frequency sweep experiments showed changes of storage modulus G’and loss modulus G’’ of aqueous solutions were diverse with temperature. Whenambient temperature was much lower than Tgel, the system was characteristic of stableviscous liquid. When temperature was close or equal to Tgel, the solution began totransform to hydrogel with weak mechanical strength. When the ambient temperaturewas obviously higher than Tgel, the system was characteristic of strong gel. Theseresults were consistent with inverted tube experiment for sol-gel transforming. SEMobservations revealed that the inner of the hydrogel was three dimensional networkstructures composed of loose and irregular macroporous, while the surface wascompact hydration film. Elements Na and P reflected the existence of α, β-GP. Thesignificant decrease of Na and P content in thermo-sensitive hydrogels afterequilibrium by double distilled water indicated that no new chemical bonds generated between CS and α, β-GP during the process of gelation.Primary lymphocytes from Penaeus chinensis were acquired by traditional tissueexplants method. Cell monolayer was attained with7days’ culture and cellmorphology changed from roundness to fusiform. After25days, a part of primarycells were still alive with adhesive cellularstroma around. When lymphoid tissueswere seeded on the surface of CS/α, β-GP thermo-sensitive hydrogel, primary cellswere rounded and covered the hydrogel after7days. By analyzing the whole level ofperoxidase (POD) and alkaline phosphatase (ALP) activities and relative activity inprimary lymphocytes, it could be concluded that CS/α, β-GP thermo-sensitivehydrogel could simulate the extra cellular matrix, reduce the oxidized stress andpromote cell proliferation compared with traditional culture method. Considering theeffect of CS molecular weight on lymphocyte growth comprehensively, CS3/α, β-GPperformed the best culture outcome.It was reported that shrimp cells were sensitive to trypsin, and traditional trypsindigestion method in subculture did harm to shrimp cell. In the present study cells wereably to be dispersed with the broken of thermo-sensitive hydrogel for futhersubculture. Living cell detection by trypan blue proved that this method was able tomaintain cell viability and little adhesion was observed. Lymphocytes had beensubcultured for4passages but the cell mumber decreased obviously underfluorescence microscope. This might not only be related with cells unable tobreakthrough M2limit but also caused by the loss of cell number on broken hydrogels.Hydrolysis of hydrogel by chitosanase could overcome the cell loss but appropriatetime was important. Results found hydrolysis by chitosanase in HBSS for4hourscould not only make cells dispersed but also maintain good cell activity. Afteradherence for12hours, subcultured cells began to multiplicate.Chitosan microspheres (CMs) were successfully prepared by emulsificationcrosslinking method. The resulting CMs were compact and complete in spherical withclean cut and good uniformity. When primary lymphocytes was blended with CMsand cultured for certain time, the cells began to migrate to microsphere surface and the contours of CMs became blurred. Considering all these results, it could beconcluded that it is feasible to adopt CMs for lymphocyte culture.In vitro cytotoxicity of α, β-GP、CS/α, β-GP thermo-sensitive hydrogel and CMswere evaluated. Under the experimental concentration, α, β-GP showed toxic effect onmouse embryonic fibroblast (MEF). Cytotoxicity of hydrogels and microspheres werereflected indirectly by extraction. Compared with the control, the relative growth ofcells with extracted solution exceeded100%, which indicated that the extraction couldpromote cell proliferation and the hydrogels had good biocompatibility. Theextractions of hydrogels balanced with culture medium showed better promotingeffects. The toxicity of CMs extracts was lower with longer incubation time, whichindicated CMs complied with the requirements of biological safety.