Cryopreservation And Culture Of Chicken Primordial Germ Cells

This paper was focus on isolating primordial germ cells (PGCs) from gonads at stage 19 and stage 28 by Ficoll density-gradient centrifugation. Cryopreserved the PGCs at different freezing media, cryopretectant concentration and crypreservation procedure, then thawed after 7 days. PGCs was cultured in vitro in DMEM medium, let the PGCs differentiated spontaneously. The results showed:1. Under the condition of 10% of the cryopretectant concentration. At the same freezing media, the viability of the frozen-thawed PGCs showed significant difference (P<0.05 )or very significant difference(P<0.01) between the different cryopreservation procedures. At the same cryopreservation procedure, the viability of the frozen-thawed PGCs showed significant difference (P<0.05) or very significant difference(P<0.01) among freezing media I , freezing media II, freezing media III, freezing media IV, freezing media V, freezing media VI. At the same cryopreservation procedure and freezing media, the viability of the frozen-thawed PGCs showed no significant difference between stage 19 and stage28. Under the condition of 10% of the cryopretectant concentration. At the same cryopreservative condition, cryopreserved the PGCs after 24h cultured in vitro, the viability of the frozen-thawed PGCs showed very significant(P<0.01) lower than that of fresh PGCs cryoprederved .3. Among the cryoprotectant concentration, the results were: between the concentration of 10% and 15%, the viability of the frozen-thawed PGCs showed no significant difference (P>0.05) except freezing media IV. When the cryoprotectant concentration was 20%, the viability of the frozen-thawed PGCs was the lowest, and showed significant difference (P<0.05 ) or very significant difference(P<0.01) compared to other concentration.4. The thawed PGCs were cultured in DMEM media, supplemented with 10% fetal bovine serum, 2% chicken serum, 2 mmol glutamine, 1mmol sodium pyruvate, 5.5 x 10-5mol -mercaptoethanol, 100U/mL gentamycin sulfate. The thawed PGCs can adhere to the cell layer and form cell mass, but the PGCs can not proliferation. The PGCs gradually died when cultured continue.5. The thawed PGCs were cultured in DMEM media, supplemented with 10% fetal bovine serum, 2% chicken serum, 2 mmol glutamine, Immol sodium pyruvate, 5.5 x 10"5mol p-mercaptoethanol, 5ng/mL hSCF, 10UI/mL mLIF, 10ng/mL bFGF, 0.04ng/mL hIL-11, 10ng/mL IGF, 100u/mL gentamycin sulfate. The PGCs can adhere to the cell layer, form cell mass and the PGCs can proliferation. When subcultured, PGCs still could form cell mass and proliferation, it also could differentiated into other cells.