Instructions

Embryoid body formation

This protocol describes a defined, serum-free method for generating embryoid bodies (EBs) from human pluripotent stem cells cultured on Biolaminin 521. It supports reproducible EB formation from single cells or small aggregates and is compatible with animal-origin–free workflows.

What’s inside

  • Recommended pre-culture conditions for hPSCs on Biolaminin 521 before EB induction
  • Step-by-step EB formation using EDTA-based dissociation, ROCK inhibitor, and low-attachment plates
  • Guidance on seeding densities, centrifugation, and culture timelines to obtain one EB per well
  • Instructions for transferring EBs to low-attachment plates for 2–3 weeks of spontaneous differentiation, with tips to optimize EB size and efficiency
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