Studies On In Vitro Cell Culture Of Pheretima Aspergillum

Objective:The aim of the study was to explore a rapid and efficacious approach for the successful isolation of intestinal epithelium cells (IEC) from Pheretima aspergillum (E. Perrier). The culturing condition was optimized and the stable cell line of P. aspergillum was thus obtained. The work present here in had established a homogenous and stable sterile cell line of P. aspergillum, which might contribute to illuminate the heavy metal enrichment of P. aspergillum and its underlying mechanism at both celluar and molecular levels.Methods:1. In vitro cell viability assay of eight anatomical origins from P. aspergillum was carried out. Enzymatic digestion regime and tissue culturing were employed according to respective organizational characteristics. The integument, intestinal tract, oncus and prostate gland were enzymatically digested, and seminal vesicle and seminal receptacle tissues were cultured conventionally. Based on the morphological changes of cells and proliferation capacity, the optimal anatomical origin was attained.2. On the basis of previous work, primary cultural system was optimized according to the biological property of IEC from P. aspergillum. The cultural varieties, serum concentration as well as different types of digestive enzymes were assessed. And a steady cultivation system for P. aspergillum cell was thus established. 3. MTT assay was exploited to curve plot the growth of IEC from P. aspergillum. The steady exponential phase for IEC growth was investigated. Culturing and PCR protocol were applied to ensure the celluar authentication.4. Species original, tissue derivatives and immune enzyme assay were initiated for the identification and confirmation of the specific cell line. The genomes of IEC cells of1st,5th,7th generetions were extracted, and PCR and DNA sequencing were employed to determine the mitochondrial COI fragment. The results obtained were comparatively analysized with identified P. aspergillum COI standard sequence for the origin verification and the possible cytodifferentiation during passage. Isoenzymatic profiles of subcultured cells were compared with8different tissues from P. aspergillum for its origin confirmation-whether of intestinal tissue origin. IEC-specific enzymatic staining was performed to authenticate the specific culture from IEC and its purification.ResultsA stable cultivation system for P. aspergillum was established and the cultural varieties, serum concentration as well as different types of digestive enzymes were optimized. The results were summarized as follows:the optimal culture were intestinal epithelium cells (IEC) and seminal vesicle(SV); the optimum cultural medium was mix Schneider’s Drosophila Medium and F12DMEM, adding serum with the concentration of10%,adding Benzyl penicillin Sodium200U/mL+Streptomycin Sulfate400μg/ml+Gentamycin Sulfate300U/ml+Amphotericin B5μg/ml,adding20ng/ml EGF,2μg/ml insulin.Within this system, the IEC and SV cells grow vigorously and wall-adhere smoothly. The IEC cells have now been of the eighth generation and SV cells of the ninth. The confirmation of IEC of P. aspergillum was undertaken by means of species original, tissue derivatives and specified enzyme assay.(Presumptively nominated as IEC-6-P.A.)ConclusionsIn the investigation, a rapid and efficacious approach to isolate the intestinal epithelium cells from P. aspergillum was attained and a stable cultivation system for P. aspergillum was achieved. The findings outlined have not only paved the way for the further exploration into the underlying mechanism of heavy metal acculmulation of P. aspergillum and possible physiological involvement, but also represented a feasible experience for the research of other varieties of earthworms and other coelenterates. Moreover, the specific cell line of P. aspergillum was further envisaged to facilitate the relevant scientific researches with indicative methodological inplications.