TeSR-AOF 3D

An animal origin-free medium from STEMCELL Technologies, TeSR-AOF 3D is formulated for the expansion of human pluripotent stem cells (hPSCs), including embryonic stem (ES) and induced pluripotent stem (iPS) cells, as aggregates in three-dimensional (3D) suspension culture. The low-protein system is recommended for downstream applications that require cGMP, animal origin-free, stabilized, feeder-free conditions.

The medium uses a fed-batch strategy in which nutrients and growth factors are added daily, with a single medium change per week of culture. The kit consists of a Seed Medium (Seed Basal Medium plus Seed Supplement) for initiating cultures and performing the weekly medium change, and a Feed Supplement for daily addition. Both media contain recombinant human basic fibroblast growth factor (rh bFGF) and recombinant human transforming growth factor β (rh TGFβ); no further growth factors are required. The Seed Medium requires supplementation with 10 µM Y-27632 for optimal aggregate formation when initiating and passaging cultures.

In this system, cells grow as spherical aggregates in suspension without matrices or microcarriers. Compared with 2D adherent culture, the format eliminates the need to replace medium daily between passages, reduces pH and nutrient/growth-factor fluctuations, removes the need for manual removal of differentiated cells, and is amenable to automation with liquid-handling robots. The medium supports efficient transition from 2D cultures, robust expansion across multiple hPSC lines (including H1, H7, WLS-1C, and STiPS-F016) over 5 passages, expression of OCT4 and TRA-1-60 at levels equivalent to 2D TeSR cultures, maintenance of more consistent glucose and lower lactate levels than 2D cultures, and differentiation capacity to all three germ layers. Spherical aggregates are typically less than 400 µm in size, with clear edges and a dimpled or pockmarked appearance. Suitable vessels range from 2 mL cultures in non-tissue-culture-treated 6-well plates on an orbital shaker through to 500 mL cultures in PBS-MINI Bioreactors, with passaging on an alternating 3-day/4-day schedule that allows weekend-free workflows. Cultures are incubated at 37 °C with 5% CO₂.