Mouse embryo culture and implantation by Type I Collagen

Kim, Chung Hoon; Chae, Hee Dong; Kang, Byung Moon; Chang, Yoon Seok; Kang, Eun Hee; Cheon, Yong Pil; Park, Kyun

Abstract

OBJECTIVES
To examine the in vitro interactions of blastocyst attachment using type I collagen.
MATERIALS AND METHODS
ICR mice were used and follicular growth was stimulated by pregnant mare serum gonadotropin and human chorionic gonadotropin. On day 4 of pregnancy, the uteri were removed and blastocysts were flushed. Mixtures of 1mL sterile water, 0.5mL DMEM, 2mL type collagen solution and 0.5mL 0.1M NaOH were prepared and transferred to an incubator where the collagen solution polymerized. Blastocysts were transferred to dishes previously coated with type I collagen. CMRL 1066 was used as the basic culture medium. It was supplemented with 1mM glutamine and 1mM sodium pyruvate plus 50 IU/ml penicillin and 50 mg/ml streptomycin. During the first 4 days the culture medium was supplemented with 20% fetal calf serum and thereafter with 20% heat inactivated human cord serum. All blastocysts were initially cultured for 2 days without media change. After 2 days, fresh medium was renewed daily. The stages of embryo growth were examined and recorded everyday under a dissecting microscope and classified according to the standard in vivo criteria set forth by Witschi.
RESULTS
By 48h, nearly all blastocysts had attached to the surface of collagen pad. Following adhesion to the collagen pad, the blastocysts maintained their 3-dimensional integrity in contrast to control. The embryos in collagen pad were not flattening and kept polarity and spherical shape during culture. The polar trophoblast invaded the type I collagen downward unlike the horizontal growth in control. In the developmental stage of mouse blastocyst, there were significant differences between control and type I collagen group during day 4 and 5 culture.
CONCLUSION
Blastocyst development was better in type I collagen group than control. Therefore, in vitro culture study using type I collagen could provide improved model for the establishment of blastocyst implantation study.