Fabrication of elastomeric stencils for patterned stem cell differentiation

Stem cell differentiation with controlled geometry results in reproducible pattern formation. In contrast to constraining differentiating cells on micropatterned surfaces, we initialise colony formation using elastomeric stencils that adhere to culture dishes and create microwells with defined sizes and shapes. After colony formation, stencils are removed to allow colony growth and cell migration. Stencil fabrication involves mould production by photolithography followed by replica-moulding polydimethylsiloxane (PDMS). This approach produces reproducible two-dimensional organoids tailored for quantitative studies of growth control and pattern formation.