Probabilistic cell seeding and non-autofluorescent 3D-printed structures as scalable approach for multi-level co-culture modeling

Sebastian Buchmann; Alessandro Enrico; Muriel Alexandra Holzreuter; Michael Reid; Erica Zeglio; Frank Niklaus; Göran Stemme; Anna Herland

doi:10.1016/j.mtbio.2023.100706

Probabilistic cell seeding and non-autofluorescent 3D-printed structures as scalable approach for multi-level co-culture modeling

Mater Today Bio. 2023 Jun 16:21:100706. doi: 10.1016/j.mtbio.2023.100706. eCollection 2023 Aug.

Authors

Sebastian Buchmann^{1

2}, Alessandro Enrico^{3

4}, Muriel Alexandra Holzreuter^{2

3}, Michael Reid⁵, Erica Zeglio^{1

2}, Frank Niklaus³, Göran Stemme³, Anna Herland^{1

2}

Affiliations

¹ Division of Nanobiotechnology, KTH Royal Institute of Technology, Tomtebodavägen 23a, 171 65, Solna, Sweden.
² AIMES - Center for the Advancement of Integrated Medical and Engineering Sciences, Department of Neuroscience, Karolinska Institute, 17177, Stockholm, Sweden.
³ Division of Micro and Nanosystems, KTH Royal Institute of Technology, Malvinas väg 10, 100 44, Stockholm, Sweden.
⁴ Synthetic Physiology lab, Department of Civil Engineering and Architecture, University of Pavia, Via Ferrata 3, 27100, Pavia, Italy.
⁵ Department of Fiber and Polymer Technology, Wallenberg Wood Science Centre, KTH Royal Institute of Technology, Teknikringen 56-58, 100 44, Stockholm, Sweden.

Abstract

To model complex biological tissue in vitro, a specific layout for the position and numbers of each cell type is necessary. Establishing such a layout requires manual cell placement in three dimensions (3D) with micrometric precision, which is complicated and time-consuming. Moreover, 3D printed materials used in compartmentalized microfluidic models are opaque or autofluorescent, hindering parallel optical readout and forcing serial characterization methods, such as patch-clamp probing. To address these limitations, we introduce a multi-level co-culture model realized using a parallel cell seeding strategy of human neurons and astrocytes on 3D structures printed with a commercially available non-autofluorescent resin at micrometer resolution. Using a two-step strategy based on probabilistic cell seeding, we demonstrate a human neuronal monoculture that forms networks on the 3D printed structure and can establish cell-projection contacts with an astrocytic-neuronal co-culture seeded on the glass substrate. The transparent and non-autofluorescent printed platform allows fluorescence-based immunocytochemistry and calcium imaging. This approach provides facile multi-level compartmentalization of different cell types and routes for pre-designed cell projection contacts, instrumental in studying complex tissue, such as the human brain.

Keywords: Astrocytes; Calcium imaging; Co-culture models; IP-Visio; Neurons; Two-photon polymerization.