自2014年以来,已经描述了在卵内容物首次在壳中孵育55-70小时后从无壳培养物中孵化鸡胚的方法。本报告首次描述了从胚盘阶段到孵化的鸡胚的无壳培养系统。对于第一个69-70小时,鸡蛋内容物悬浮在聚甲基戊烯厨房包裹物中(F.O.R.包裹,RikenFabro,东京,日本)支撑在内径6.35或6.67厘米的三脚架中,并用固定的Milli-Wrap圆盘覆盖,以每分钟16或22个循环(CPM)来回旋转90°。随后,取出Milli-Wrap圆盘,将培养三脚架转移到环境室,在孵育第8.5天(E8.5)期间摇摆±20°。从E9开始,环境室保持在水平位置,直到用受控的O2和CO2孵化。提供补充钙,在E9(2.5mL)和E13(1.0mL)或E15(1.0mL)处,通过保鲜膜将含有100mg/mL1-乳酸钙水合物的水溶液注入蛋白中。在16或22CPM下孵育69-70小时后,80%-83%的先前未孵育的卵内容物产生了明显的正常胚胎。在16或22CPM的可转盘孵育产生的正常胚胎的孵化率约为43%。值得注意的是,从胚盘阶段到孵化,卵含量保持在同一培养三脚架中。该技术可能会用作教育工具和早期胚胎发生的基础研究,致畸,和基因转移实验。
Since 2014, methods have been described to hatch chick embryos from shell-less culture after egg contents are first incubated within shells for 55-70 h. The present report describes for the first time a shell-less culture system for chick embryos from the
blastoderm stage to hatching. For the first 69-70 h, egg contents suspended in polymethylpentene kitchen wrap (F.O.R. Wrap, Riken Fabro, Tokyo, Japan) supported in 6.35 or 6.67 cm inside diameter tripods and covered with a disc of immobilized Milli-Wrap, were rotated back and forth through 90° at 16 or 22 cycles per minute (CPM). Subsequently, the Milli-Wrap disc was removed and culture tripods were transferred to environmental chambers, which were rocked ±20° through incubation day 8.5 (E8.5). From E9, environmental chambers were maintained in the horizontal position through to hatching with controlled O2 and CO2 . To provide supplemental calcium, an aqueous solution containing 100 mg/mL of calcium l-lactate hydrate was injected through the plastic wrap into the albumen at E9 (2.5 mL) and at E13 (1.0 mL) or E15 (1.0 mL). After incubation for 69-70 h at 16 or 22 CPM, 80%-83% of previously unincubated egg contents yielded apparently normal embryos. Hatch rate of normal embryos resulting from turntable incubation at 16 or 22 CPM was approximately 43%. Of note, egg contents remained in the same culture tripod from
blastoderm stage to hatching. This technique may find use as an educational tool and in basic investigations of early embryogenesis, teratogenesis, and gene transfer experiments.