article published yes Stefanie Rus author 4D9EC9B6-F248-11E8-B48F-1D18A9856A870000-0001-8703-1093 Jack Merrin author 4515C308-F248-11E8-B48F-1D18A9856A870000-0001-5145-4609 Monika Aleksandra Kulig author 3331f5ae-e896-11ec-af79-eeb79769bcb7 Thomas Minchington author 7d1648cb-19e9-11eb-8e7a-f8c037fb3e3f Anna Kicheva author 3959A2A0-F248-11E8-B48F-1D18A9856A870000-0003-4509-4998 AnKi department NanoFab department Mechanisms of tissue size regulation in spinal cord development project The regulatory logic of pattern formation in the vertebrate dorsal neural tube project Geometrically controlled stem cell differentiation promotes reproducible pattern formation. Here, we present a protocol to fabricate elastomeric stencils for patterned stem cell differentiation. We describe procedures for using photolithography to produce molds, followed by molding polydimethylsiloxane (PDMS) to obtain stencils with through holes. We then provide instructions for culturing cells on stencils and, finally, removing stencils to allow colony growth and cell migration. This approach yields reproducible two-dimensional organoids tailored for quantitative studies of growth and pattern formation. For complete details on the use and execution of this protocol, please refer to Lehr et al.1 https://research-explorer.ista.ac.at/download/18601/18610/2024_STARProtoc_Lehr.pdf application/pdfno Elsevier2024 eng 2666-1667 3960231010.1016/j.xpro.2024.103187 54 https://research-explorer.ista.ac.at/record/19763 S. Rus, J. Merrin, M. A. Kulig, T. Minchington, and A. Kicheva, “Protocol for fabricating elastomeric stencils for patterned stem cell differentiation,” <i>STAR Protocols</i>, vol. 5, no. 4. Elsevier, 2024. Rus, S., Merrin, J., Kulig, M. A., Minchington, T., &#38; Kicheva, A. (2024). Protocol for fabricating elastomeric stencils for patterned stem cell differentiation. <i>STAR Protocols</i>. Elsevier. <a href="https://doi.org/10.1016/j.xpro.2024.103187">https://doi.org/10.1016/j.xpro.2024.103187</a> Rus, Stefanie, Jack Merrin, Monika Aleksandra Kulig, Thomas Minchington, and Anna Kicheva. “Protocol for Fabricating Elastomeric Stencils for Patterned Stem Cell Differentiation.” <i>STAR Protocols</i>. Elsevier, 2024. <a href="https://doi.org/10.1016/j.xpro.2024.103187">https://doi.org/10.1016/j.xpro.2024.103187</a>. Rus S, Merrin J, Kulig MA, Minchington T, Kicheva A. 2024. Protocol for fabricating elastomeric stencils for patterned stem cell differentiation. STAR Protocols. 5(4), 103187. Rus S, Merrin J, Kulig MA, Minchington T, Kicheva A. Protocol for fabricating elastomeric stencils for patterned stem cell differentiation. <i>STAR Protocols</i>. 2024;5(4). doi:<a href="https://doi.org/10.1016/j.xpro.2024.103187">10.1016/j.xpro.2024.103187</a> S. Rus, J. Merrin, M.A. Kulig, T. Minchington, A. Kicheva, STAR Protocols 5 (2024). Rus, Stefanie, et al. “Protocol for Fabricating Elastomeric Stencils for Patterned Stem Cell Differentiation.” <i>STAR Protocols</i>, vol. 5, no. 4, 103187, Elsevier, 2024, doi:<a href="https://doi.org/10.1016/j.xpro.2024.103187">10.1016/j.xpro.2024.103187</a>. 186012024-12-01T23:01:53Z2026-08-06T22:30:36Z