Effect of culture medium volume and embryo density on early mouse embryonic development: Tracking the development of the individual embryo
Effect of culture medium volume and embryo density on early mouse embryonic development: Tracking the development of the individual embryo
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DOI：
10.1007/s10815-012-9744-8
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发表时间：
2012-07-01
影响因子：
3.1
通讯作者：
Chian, Ri-Cheng
中科院分区：
文献类型：
--
作者：
Dai, Shan-Jun;Xu, Chang-Long;Chian, Ri-Cheng
确定井中井（WOW）系统的最佳胚胎体积或密度，以跟踪单个胚胎的发育，并确定WOW系统是否可以逆转单独培养胚胎的负面影响。(1)小鼠胚胎（2）在石蜡油下，在6.25 μ l、12.50 μ l、25.00 μ l和50.00 μ l的培养基液滴中培养2细胞期的3、6、9和12个胚胎组;（3）在石蜡油下，在培养皿底部有或没有九个微孔的情况下，在50 μ l液滴中培养9个处于2-细胞期的胚胎的组，在每个微孔中放置9个胚胎中的一个（WOW系统）。此外，在石蜡油下在5.5 μ l培养基液滴中单独培养单个2-细胞期胚胎，在培养皿底部有或没有单个微孔（用于单培养的WOW系统）。培养结束时，比较各组囊胚发育率、孵化率和孵化囊胚率。将囊胚固定后进行差异染色，结果表明，50 μ l培养液中培养的9枚胚胎的囊胚发育率显著高于其它体积培养液中培养的胚胎（P < 0.05）。单胚培养的囊胚发育率显著低于单胚培养组（P < 0.05）。将单个胚胎在微孔中单独培养与不使用微孔单独培养相比，囊胚发育没有改善。无论是否使用WOW系统，在组胚胎培养中囊胚的总细胞数均显著高于单胚胎培养。此外，单胚培养中囊胚细胞总数显著高于单胚培养（P < 0.05），说明群体培养的囊胚发育效果优于单胚培养（上级）。具有50 μ l培养液液滴的WOW系统可用于跟踪成组培养的胚胎的个体发育，同时保持良好的胚胎发育。WOW系统不能改善单胚胎培养降低的胚胎发育。
To determine the optimal volume or density of embryos for the well-of-the-well (WOW) system in order to track the development of individual embryos and to determine whether the WOW system can reverse the negative impact of culturing embryos singly.(1) Mouse embryos (groups of nine at the 2-cell stage) were cultured in 6.25 mu l, 12.50 mu l, 25.00 mu l and 50.00 mu l of droplets of culture medium under paraffin oil; (2) Groups of three, six, nine and twelve embryos at the 2-cell stage were cultured in 50 mu l of droplet of culture medium under paraffin oil; (3) Groups of nine embryos at the 2-cell stage were cultured in 50 mu l of droplet under paraffin oil with or without nine micro-wells made on the bottom of the Petri dish into each of which were placed one of the nine embryos (WOW system). Also single 2-cell stage embryos was cultured individually in 5.5 mu l of droplet of culture medium under paraffin oil with or without a single micro-well made on the bottom of the Petri dish (WOW system for single culture). At the end of culture, the percentages of blastocyst development, hatching and hatched blastocysts were compared in each group. The blastocysts were fixed for differential staining.The blastocyst development was significantly higher (P < 0.05) when nine embryos were cultured in 50 mu l of droplet of culture medium compared with other volumes. The blastocyst development was significantly reduced (P < 0.05) in single embryo culture compared to group embryo culture with or without the WOW system. The blastocyst development was not improved when single embryo cultured individually in a micro-well was compared to single embryo cultured individually without micro-well. The total cell numbers of blastocysts were significantly higher in group embryo culture than single embryo culture regardless of whether the WOW system was used. In addition, the total cell numbers of blastocysts were significantly higher (P < 0.05) in single embryo culture with the WOW system than without.Group embryo culture is superior to single embryo culture for blastocyst development. The WOW system with 50 mu l of droplet of culture medium can be used to track the individual development of embryo cultured in groups while preserving good embryonic development. The reduced embryonic development with single embryo culture cannot be ameliorated by the WOW system.