Protocol for 3D Bioprinting Mesenchymal Stem Cell-derived Neural Tissues Using a Fibrin-based Bioink

Milena Restan Perez; Nadia Z Masri; Jonathan Walters-Shumka; Sarah Kahale; Stephanie M Willerth

doi:10.21769/BioProtoc.4663

Protocol for 3D Bioprinting Mesenchymal Stem Cell-derived Neural Tissues Using a Fibrin-based Bioink

Bio Protoc. 2023 May 5;13(9):e4663. doi: 10.21769/BioProtoc.4663.

Authors

Milena Restan Perez¹, Nadia Z Masri², Jonathan Walters-Shumka², Sarah Kahale³, Stephanie M Willerth^{1

2

4

5

6}

Affiliations

¹ Axolotl Biosciences, 3800 Finnerty Road, Victoria, BC, V8W 2Y2, Canada.
² Division of Medical Sciences, University of Victoria, 3800 Finnerty Road, Victoria, BC, V8W 2Y2, Canada.
³ Cloud Nine Editing and Design.
⁴ Department of Mechanical Engineering, University of Victoria, 3800 Finnerty Road, Victoria, BC, V8W 2Y2, Canada.
⁵ Centre for Advanced Materials and Technology, University of Victoria, 3800 Finnerty Road, Victoria, BC, V8W 2Y2, Canada.
⁶ School of Biomedical Engineering, University of British Columbia, 2222 Health Sciences Mall, Vancouver, BC, V6T 1Z3, Canada.

Abstract

Three-dimensional bioprinting utilizes additive manufacturing processes that combine cells and a bioink to create living tissue models that mimic tissues found in vivo. Stem cells can regenerate and differentiate into specialized cell types, making them valuable for research concerning degenerative diseases and their potential treatments. 3D bioprinting stem cell-derived tissues have an advantage over other cell types because they can be expanded in large quantities and then differentiated to multiple cell types. Using patient-derived stem cells also enables a personalized medicine approach to the study of disease progression. In particular, mesenchymal stem cells (MSC) are an attractive cell type for bioprinting because they are easier to obtain from patients in comparison to pluripotent stem cells, and their robust characteristics make them desirable for bioprinting. Currently, both MSC bioprinting protocols and cell culturing protocols exist separately, but there is a lack of literature that combines the culturing of the cells with the bioprinting process. This protocol aims to bridge that gap by describing the bioprinting process in detail, starting with how to culture cells pre-printing, to 3D bioprinting the cells, and finally to the culturing process post-printing. Here, we outline the process of culturing MSCs to produce cells for 3D bioprinting. We also describe the process of preparing Axolotl Biosciences TissuePrint - High Viscosity (HV) and Low Viscosity (LV) bioink, the incorporation of MSCs to the bioink, setting up the BIO X and the Aspect RX1 bioprinters, and necessary computer-aided design (CAD) files. We also detail the differentiation of 2D and 3D cell cultures of MSC to dopaminergic neurons, including media preparation. We have also included the protocols for viability, immunocytochemistry, electrophysiology, and performing a dopamine enzyme-linked immunosorbent assay (ELISA), along with the statistical analysis. Graphical overview.

Keywords: 3D bioprinting; Bioink; Dopaminergic neurons; Mesenchymal stem cells; Stem cells; Tissue engineering.