Studies on development of serum-free conditioned media using Vero cells and DMEM with controlled concentration of glucose and pyruvate

Kim, Ju Hwan; Seo, Young Seok; Song, Hai Bum; Yang, Jung Bo; Lee, Kyung En; Lee, Ki Hwan

Abstract

OBJECTIVE
The purpose of this study was to examine in vitro development of early preimplantation mouse embryos in various kind of serum-free conditioned media (SF-VCM) manufactured from DMEM cultured with Vero Cells.
METHODS
A total of 846 two-cell mouse embryos were cultured in different kind of SF-VCM. SF-VCMs were divided into SF-VCM-10, -30 and -50 by media volume using DMEM #1 media, and divided into SF-VCM #1, #2 and #3 by controlled concentration of glucose and pyruvate (manufactured by DMEM #1: mixed three volume of DMEM-G (DMEM with glutamine without glucose and pyruvate) and one volume of DMEM-GGP (DMEM with glutamine, glucose, pyruvate), #2: mixed same volume of DMEM-G and DMEM-GGP and #3: mixed one volume of DMEM-G and three volume of DMEM-GGP, respectively). Experimental groups were mainly added 10% SSS, and 20% hFF was added to only Control group co-cultured with Vero cells. Development of embryos was observed every 24 hours. Results between different groups were analyzed using Chi-square test, and considered statistically significant when P-value was less than 0.05.
RESULTS
In vitro developmental rate by each cleavage stages of mouse embryos cultured in SF-VCMs with a various volumes were significantly (P<0.05) higher in SF-VCM-30 (morula< or =: 97.2%, Blastocyst (BL)< or =: 97.2%, Hatching BL< or =: 82.2%) than other groups. In the rate of development on in vitro co-culture vs. a various SF-VCMs manufactured by DMEM controlled concentration of glucose and pyruvate, Group I (SF-VCM #1) was higher than other groups in each cleavage stages (morula< or =: 98.1%, Blastocyst (BL)< or =: 97.1%, hatching BL< or =: 81.7%, respectively). Moreover, specially, in the developmental rate into the hatching blastocyst < or = after 96 hours in vitro culture, Group I (81.7%) was significantly higher than control group (67.6%, P<0.05).
CONCLUSION
SF-VCM #1 manufactured by volume of 30 mL DMEM #1 media cultured in vitro for 48 hours in 250 mL flask was the most effective on in vitro developmental rate of mouse preimplantation embryos. Therefore, it is expected that SF-VCM #1 has application to human IVF-ET.