TY - JOUR

T1 - In Vitro Development of Human iPSC-Derived Functional Neuronal Networks on Laser-Fabricated 3D Scaffolds

AU - Koroleva, Anastasia

AU - Deiwick, Andrea

AU - El-Tamer, Ayman

AU - Koch, Lothar

AU - Shi, Yichen

AU - Estévez-Priego, Estefanía

AU - Ludl, Adriaan Alexander

AU - Soriano, Jordi

AU - Guseva, Daria

AU - Ponimaskin, Evgeni

AU - Chichkov, Boris

N1 - Funding Information: This work was funded by European Union’s Horizon 2020 projects MESO-BRAIN, Grant 713140, and‘Scaffold Needs’, Grant 851734. This work was supported by Deutsche Forschungsgemeinschaft through Grant PO732 and Excellence Cluster REBIRTH (E.P.), and through Grant GU1521/4-1 (D.G.). A.K. acknowledges Grant 19-75-10008 (2PP scaffold characterization) from Russian Science Foundation. J.S. also acknowledges the support from the Ministerio de Ciencia e Innovación (Spain), grants FIS2016-78507-C2-2-P and PID2019-108842GB-C21, and to the Generalitat de Catalunya, grant 2017SGR1061.

PY - 2021/2/24

Y1 - 2021/2/24

N2 - Neural progenitor cells generated from human induced pluripotent stem cells (hiPSCs) are the forefront of ″brain-on-chip″ investigations. Viable and functional hiPSC-derived neuronal networks are shaping powerful in vitro models for evaluating the normal and abnormal formation of cortical circuits, understanding the underlying disease mechanisms, and investigating the response to drugs. They therefore represent a desirable instrument for both the scientific community and the pharmacological industry. However, culture conditions required for the full functional maturation of individual neurons and networks are still unidentified. It has been recognized that three-dimensional (3D) culture conditions can better emulate in vivo neuronal tissue development compared to 2D cultures and thus provide a more desirable in vitro approach. In this paper, we present the design and implementation of a 3D scaffold platform that supports and promotes intricate neuronal network development. 3D scaffolds were produced through direct laser writing by two-photon polymerization (2PP), a high-resolution 3D laser microstructuring technology, using the biocompatible and nondegradable photoreactive resin Dental LT Clear (DClear). Neurons developed and interconnected on a 3D environment shaped by vertically stacked scaffold layers. The developed networks could support different cell types. Starting at the day 50 of 3D culture, neuronal progenitor cells could develop into cortical projection neurons (CNPs) of all six layers, different types of inhibitory neurons, and glia. Additionally and in contrast to 2D conditions, 3D scaffolds supported the long-term culturing of neuronal networks over the course of 120 days. Network health and functionality were probed through calcium imaging, which revealed a strong spontaneous neuronal activity that combined individual and collective events. Taken together, our results highlight advanced microstructured 3D scaffolds as a reliable platform for the 3D in vitro modeling of neuronal functions.

AB - Neural progenitor cells generated from human induced pluripotent stem cells (hiPSCs) are the forefront of ″brain-on-chip″ investigations. Viable and functional hiPSC-derived neuronal networks are shaping powerful in vitro models for evaluating the normal and abnormal formation of cortical circuits, understanding the underlying disease mechanisms, and investigating the response to drugs. They therefore represent a desirable instrument for both the scientific community and the pharmacological industry. However, culture conditions required for the full functional maturation of individual neurons and networks are still unidentified. It has been recognized that three-dimensional (3D) culture conditions can better emulate in vivo neuronal tissue development compared to 2D cultures and thus provide a more desirable in vitro approach. In this paper, we present the design and implementation of a 3D scaffold platform that supports and promotes intricate neuronal network development. 3D scaffolds were produced through direct laser writing by two-photon polymerization (2PP), a high-resolution 3D laser microstructuring technology, using the biocompatible and nondegradable photoreactive resin Dental LT Clear (DClear). Neurons developed and interconnected on a 3D environment shaped by vertically stacked scaffold layers. The developed networks could support different cell types. Starting at the day 50 of 3D culture, neuronal progenitor cells could develop into cortical projection neurons (CNPs) of all six layers, different types of inhibitory neurons, and glia. Additionally and in contrast to 2D conditions, 3D scaffolds supported the long-term culturing of neuronal networks over the course of 120 days. Network health and functionality were probed through calcium imaging, which revealed a strong spontaneous neuronal activity that combined individual and collective events. Taken together, our results highlight advanced microstructured 3D scaffolds as a reliable platform for the 3D in vitro modeling of neuronal functions.

KW - 3D neuronal culture

KW - Ca imaging

KW - Human neural stem cells

KW - NETCAL analysis

KW - Scaffold

KW - Two-photon polymerization

UR - http://www.scopus.com/inward/record.url?scp=85101527575&partnerID=8YFLogxK

U2 - 10.1021/acsami.0c16616

DO - 10.1021/acsami.0c16616

M3 - Article

C2 - 33559469

AN - SCOPUS:85101527575

VL - 13

SP - 7839

EP - 7853

JO - ACS Applied Materials and Interfaces

JF - ACS Applied Materials and Interfaces

SN - 1944-8244

IS - 7

ER -