|Budget Amount *help
¥3,400,000 (Direct Cost : ¥3,400,000)
Fiscal Year 1999 : ¥400,000 (Direct Cost : ¥400,000)
Fiscal Year 1998 : ¥3,000,000 (Direct Cost : ¥3,000,000)
The periosteum of Femur, which prepared from chick embryo of 18ィイD1thィエD1 day after fertilization, was cryopeserved with a cryoprotectant (0.2M treharose, 50% egg yolk, 12% dimethy sulfoxide (DNSO) in huecm AH25). The following program of freezing was employed ; room temperature to -7℃ : -2.0s℃/min, -7.0℃ to -40℃ : -1.0℃/min, transfer in liquid nitrogen. The package of cryopreserved periosteum was thawed in tap water (37℃), and cultured on allantoic sac of chick embryo of 9ィイD1thィエD1 day after ferilization.
When compared the stepwise addition and removal of cryoprotectant with those direct procedures, there found no significant difference in the survival rate. The present study, therefore, employed direct procedure. The periosteum cryopreserved without DNSO gave no osteogenesis, and the optimum concentration of DMSO was found to be 6.0 to 18.0%, the study added 12% DMSO in the cryoprotectat. Addition of egg yolk improved the rate of osteogenesis, and similar effect was observed by using egg yolk lecithin. Even after long term preservation (1 week, 1 month and 3months), similar survival rates were observed. Electron microscopic observation suggested that surface cell layer were denatured after thawing, whereas those of deep layers were kept their cell structures such as plasma membrane and organelas.