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Title: Optimizing cell encapsulation condition in ECM-Collagen I hydrogels to support 3D neuronal cultures

Abstract

Background The emergence of three-dimensional (3D) cell culture in neural tissue engineering has significantly elevated the complexity and relevance of in vitro systems. This is due in large part to the incorporation of biomaterials to impart structural dimensionality on the neuronal cultures. However, a comprehensive understanding of how key seeding parameters affect changes in cell distribution and viability remain unreported. New method In this study, we systematically evaluated permutations in seeding conditions (i.e., cell concentration and atmospheric CO 2 levels) to understand how these affect key parameters in 3D culture characterization (i.e., cell health and distribution). Primary rat cortical neurons (i.e., 2×10 6, 4×10 6, and 1×10 7 cells/mL) were entrapped in collagen blended with ECM proteins (ECM-Collagen) and exposed to atmospheric CO 2 (i.e., 0 vs 5% CO 2) during fibrillogenesis. Results At 14 days in vitro (DIV), cell distribution within the hydrogel was dependent on cell concentration and atmospheric CO 2 during fibrillogenesis. A uniform distribution of cells was observed in cultures with 2×10 6 and 4×10 6 cells/mL in the presence of 5% CO 2, while a heterogeneous distribution was observed in cultures with 1×10 7 cells/mL or in the absence of CO 2. Furthermore, increased cellmore » concentration was proportional to the rise in cell death at 14 DIV, although cells remain viable >30 DIV. Comparison with existing methods -Collagen gels have been shown to increase cell viability of neurons long-term. Conclusion In using ECM-collagen gels, we highlight the importance of optimizing seeding parameters and thorough 3D culture characterization to understand the neurophysiological responses of these 3D systems.« less

Authors:
ORCiD logo [1];  [1];  [1];  [1];  [1];  [1];  [1];  [1];  [1]; ORCiD logo [1]
  1. Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States)
Publication Date:
Research Org.:
Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States)
Sponsoring Org.:
USDOE National Nuclear Security Administration (NNSA)
OSTI Identifier:
1581896
Report Number(s):
LLNL-JRNL-775640
Journal ID: ISSN 0165-0270; 968570
Grant/Contract Number:  
AC52-07NA27344
Resource Type:
Accepted Manuscript
Journal Name:
Journal of Neuroscience Methods
Additional Journal Information:
Journal Volume: 329; Journal Issue: C; Journal ID: ISSN 0165-0270
Publisher:
Elsevier
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; Entrapment; Cortical neurons; 3D Neuronal culture; Hydrogel; ECM-collagen hydrogel

Citation Formats

Lam, Doris, Enright, Heather A., Peters, Sandra K. G., Moya, Monica L., Soscia, David A., Cadena, Jose, Alvarado, Javier A., Kulp, Kristen S., Wheeler, Elizabeth K., and Fischer, Nicholas O. Optimizing cell encapsulation condition in ECM-Collagen I hydrogels to support 3D neuronal cultures. United States: N. p., 2019. Web. doi:10.1016/j.jneumeth.2019.108460.
Lam, Doris, Enright, Heather A., Peters, Sandra K. G., Moya, Monica L., Soscia, David A., Cadena, Jose, Alvarado, Javier A., Kulp, Kristen S., Wheeler, Elizabeth K., & Fischer, Nicholas O. Optimizing cell encapsulation condition in ECM-Collagen I hydrogels to support 3D neuronal cultures. United States. doi:10.1016/j.jneumeth.2019.108460.
Lam, Doris, Enright, Heather A., Peters, Sandra K. G., Moya, Monica L., Soscia, David A., Cadena, Jose, Alvarado, Javier A., Kulp, Kristen S., Wheeler, Elizabeth K., and Fischer, Nicholas O. Tue . "Optimizing cell encapsulation condition in ECM-Collagen I hydrogels to support 3D neuronal cultures". United States. doi:10.1016/j.jneumeth.2019.108460. https://www.osti.gov/servlets/purl/1581896.
@article{osti_1581896,
title = {Optimizing cell encapsulation condition in ECM-Collagen I hydrogels to support 3D neuronal cultures},
author = {Lam, Doris and Enright, Heather A. and Peters, Sandra K. G. and Moya, Monica L. and Soscia, David A. and Cadena, Jose and Alvarado, Javier A. and Kulp, Kristen S. and Wheeler, Elizabeth K. and Fischer, Nicholas O.},
abstractNote = {Background The emergence of three-dimensional (3D) cell culture in neural tissue engineering has significantly elevated the complexity and relevance of in vitro systems. This is due in large part to the incorporation of biomaterials to impart structural dimensionality on the neuronal cultures. However, a comprehensive understanding of how key seeding parameters affect changes in cell distribution and viability remain unreported. New method In this study, we systematically evaluated permutations in seeding conditions (i.e., cell concentration and atmospheric CO2 levels) to understand how these affect key parameters in 3D culture characterization (i.e., cell health and distribution). Primary rat cortical neurons (i.e., 2×106, 4×106, and 1×107 cells/mL) were entrapped in collagen blended with ECM proteins (ECM-Collagen) and exposed to atmospheric CO2 (i.e., 0 vs 5% CO2) during fibrillogenesis. Results At 14 days in vitro (DIV), cell distribution within the hydrogel was dependent on cell concentration and atmospheric CO2 during fibrillogenesis. A uniform distribution of cells was observed in cultures with 2×106 and 4×106 cells/mL in the presence of 5% CO2, while a heterogeneous distribution was observed in cultures with 1×107 cells/mL or in the absence of CO2. Furthermore, increased cell concentration was proportional to the rise in cell death at 14 DIV, although cells remain viable >30 DIV. Comparison with existing methods -Collagen gels have been shown to increase cell viability of neurons long-term. Conclusion In using ECM-collagen gels, we highlight the importance of optimizing seeding parameters and thorough 3D culture characterization to understand the neurophysiological responses of these 3D systems.},
doi = {10.1016/j.jneumeth.2019.108460},
journal = {Journal of Neuroscience Methods},
number = C,
volume = 329,
place = {United States},
year = {2019},
month = {10}
}

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