1Homogeneous EBs generated from microwell-cultured
hESC aggregates hESCs cultured in microwells were typically constrained to the well boundaries. However, if care was taken to minimize shear during medium exchange, colony growth expanded into the medium above the microwell. These hESC aggregates
growing above the surface could be easily sheared into the medium by gentle pipetting, leaving behind a confluent layer of hESCs within the microwells.This base layer of cells then replicated to fill the microwell and form a new aggregate.hESC aggregates cultured for 11 days in 50 mm deep_ 100 mm lateral microwells grew as relatively monodisperse hESC colonies while attached to microwells (Fig. 6A) and were harvested by gentle pipetting to release them into the medium.To assess viability and differentiation state,aggregates were plated on Matrigel-coated TCPS plates and cultured for 6 days in CMF+. All aggregates attached to the Matrigel substrate and began replication within 1 day (Fig. 6B). Rates of hESC growth were consistent with standard hESC cultures and colonies reached passaging confluence within 6 days. To verify hESCs were undifferentiated, colonies were fixed on the sixth day and analyzed via immunocytochemistry for Oct-4 expression. Very little differentiation occurred around colony perimeter, with no visible differentiation in colony interior, similar to results obtained for enzymatic harvest of entire hESC colonies from microwells.Fig. 7 quantifies the size distributions of EBs generated from microwell-cultured hESC colonies and TCPS-harvested hESC colonies.
- 1 decade agoFavorite Answer
hESCs引起的EBs開化在microwells典型地被壓抑了到好的界限。 然而， 在中等交換期間，如果保重使剪減到最小， 殖民地成長擴展了入媒介在microwell之上。 這些hESC聚集體
hESC殖民地，當附有microwells時(。 6A) 并且通過發布他們的柔和吸取收穫 入媒介。估計生活能力和分化狀態，聚集體在Matrigel上漆的TCPS板材在CMF+被鍍了并且被開化了6天。 所有聚集體附有Matrigel基體和在1天之內開始了複製 (。 6B)。 hESC成長的率與標準hESC文化是一致的，并且殖民地在6天之內到達了通過的合流。 要核實hESCs是無差別， 殖民地是固定的在第六天并且通過immunocytochemistry被分析了為10月4表示。 很少分化在殖民地周長附近發生了，沒有可看見的分化在殖民地內部， 相似於為整個hESC殖民地酶收穫得到的結果從microwells。。 7定量從microwell被開化的hESC殖民地和TCPS被收穫的hESC殖民地引起的EBs的大小分佈。
- Anonymous1 decade ago