Assembling Spheroids of Rat Primary Neurons Using a Stress-Free 3D Culture System
Neural injuries disrupt the normal functions of the nervous system, whose complexities limit current treatment options. Because of their enhanced therapeutic effects, neurospheres have the potential to advance the field of regenerative medicine and neural tissue engineering. Methodological steps can...
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| Format: | Article |
| Language: | English |
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MDPI AG
2023-08-01
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| Series: | International Journal of Molecular Sciences |
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| Online Access: | https://www.mdpi.com/1422-0067/24/17/13506 |
| _version_ | 1797582414225080320 |
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| author | Meaghan E. Harley-Troxell Madhu Dhar |
| author_facet | Meaghan E. Harley-Troxell Madhu Dhar |
| author_sort | Meaghan E. Harley-Troxell |
| collection | DOAJ |
| description | Neural injuries disrupt the normal functions of the nervous system, whose complexities limit current treatment options. Because of their enhanced therapeutic effects, neurospheres have the potential to advance the field of regenerative medicine and neural tissue engineering. Methodological steps can pose challenges for implementing neurosphere assemblies; for example, conventional static cultures hinder yield and throughput, while the presence of the necrotic core, time-consuming methodology, and high variability can slow their progression to clinical application. Here we demonstrate the optimization of primary neural cell-derived neurospheres, developed using a high-throughput, stress-free, 3D bioreactor. This process provides a necessary baseline for future studies that could develop co-cultured assemblies of stem cells combined with endothelial cells, and/or biomaterials and nanomaterials for clinical therapeutic use. Neurosphere size and neurite spreading were evaluated under various conditions using Image J software. Primary neural cells obtained from the hippocampi of three-day-old rat pups, when incubated for 24 h in a reactor coated with 2% Pluronic and seeded on Poly-D-Lysine-coated plates establish neurospheres suitable for therapeutic use within five days. Most notably, neurospheres maintained high cell viability of ≥84% and expressed the neural marker MAP2, neural marker β-Tubulin III, and glial marker GFAP at all time points when evaluated over seven days. Establishing these factors reduces the variability in developing neurospheres, while increasing the ease and output of the culture process and maintaining viable cellular constructs. |
| first_indexed | 2024-03-10T23:20:53Z |
| format | Article |
| id | doaj.art-c87d5189a3704aac8c27346c546a2a7c |
| institution | Directory Open Access Journal |
| issn | 1661-6596 1422-0067 |
| language | English |
| last_indexed | 2024-03-10T23:20:53Z |
| publishDate | 2023-08-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | International Journal of Molecular Sciences |
| spelling | doaj.art-c87d5189a3704aac8c27346c546a2a7c2023-11-19T08:18:16ZengMDPI AGInternational Journal of Molecular Sciences1661-65961422-00672023-08-0124171350610.3390/ijms241713506Assembling Spheroids of Rat Primary Neurons Using a Stress-Free 3D Culture SystemMeaghan E. Harley-Troxell0Madhu Dhar1Tissue Engineering and Regenerative Medicine, Large Animal Clinical Sciences, College of Veterinary Medicine, University of Tennessee, Knoxville, TN 37996, USATissue Engineering and Regenerative Medicine, Large Animal Clinical Sciences, College of Veterinary Medicine, University of Tennessee, Knoxville, TN 37996, USANeural injuries disrupt the normal functions of the nervous system, whose complexities limit current treatment options. Because of their enhanced therapeutic effects, neurospheres have the potential to advance the field of regenerative medicine and neural tissue engineering. Methodological steps can pose challenges for implementing neurosphere assemblies; for example, conventional static cultures hinder yield and throughput, while the presence of the necrotic core, time-consuming methodology, and high variability can slow their progression to clinical application. Here we demonstrate the optimization of primary neural cell-derived neurospheres, developed using a high-throughput, stress-free, 3D bioreactor. This process provides a necessary baseline for future studies that could develop co-cultured assemblies of stem cells combined with endothelial cells, and/or biomaterials and nanomaterials for clinical therapeutic use. Neurosphere size and neurite spreading were evaluated under various conditions using Image J software. Primary neural cells obtained from the hippocampi of three-day-old rat pups, when incubated for 24 h in a reactor coated with 2% Pluronic and seeded on Poly-D-Lysine-coated plates establish neurospheres suitable for therapeutic use within five days. Most notably, neurospheres maintained high cell viability of ≥84% and expressed the neural marker MAP2, neural marker β-Tubulin III, and glial marker GFAP at all time points when evaluated over seven days. Establishing these factors reduces the variability in developing neurospheres, while increasing the ease and output of the culture process and maintaining viable cellular constructs.https://www.mdpi.com/1422-0067/24/17/135063D cultureneurospheresprimary neural cellsnerve tissue engineering |
| spellingShingle | Meaghan E. Harley-Troxell Madhu Dhar Assembling Spheroids of Rat Primary Neurons Using a Stress-Free 3D Culture System International Journal of Molecular Sciences 3D culture neurospheres primary neural cells nerve tissue engineering |
| title | Assembling Spheroids of Rat Primary Neurons Using a Stress-Free 3D Culture System |
| title_full | Assembling Spheroids of Rat Primary Neurons Using a Stress-Free 3D Culture System |
| title_fullStr | Assembling Spheroids of Rat Primary Neurons Using a Stress-Free 3D Culture System |
| title_full_unstemmed | Assembling Spheroids of Rat Primary Neurons Using a Stress-Free 3D Culture System |
| title_short | Assembling Spheroids of Rat Primary Neurons Using a Stress-Free 3D Culture System |
| title_sort | assembling spheroids of rat primary neurons using a stress free 3d culture system |
| topic | 3D culture neurospheres primary neural cells nerve tissue engineering |
| url | https://www.mdpi.com/1422-0067/24/17/13506 |
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