A novel decellularization method to produce brain scaffolds
Section snippets
Impact statement
For the first time, it has been described an easy, fast, effective and low cost method for the complete decellularization of murine brain by the use of only one detergent (SDS) combined with rapid freezing and thawing, that can be used as a 3D scaffold for cell culture of neuronal cells. The results show that the decellularized brains still maintain ECM components essential for cell-loading and survival on the scaffold. Moreover, we found that the decellularized brain matrix can be cell-loaded
Experimental model
Mice (Mus musculus, C57/Bl6 lineage) weighting 25−30 g, were obtained from the University’s Animal Facility (CEDEME/UNIFESP). All protocols were approved by the University’s Committee of Ethics in the Use of Animals (CEUA #2,101,180,516). Every effort was made to minimize animal suffering and reduce the number of animals used. For the surgical proceedings, mice were anesthetized with Ketamine 75 mg/Kg and Xylazine 10 mg/Kg by intraperitoneal injection. After anesthesia, the animals were
Murine brains were fully decellularized by SDS
After fast freezing in liquid nitrogen followed by 24 h in 1 % SDS, the mice brains were analyzed to investigate the remaining of cells, nucleic acids and proteins. Fig. 1A presents a representative image showing that decellularized the brain became smaller, with less blood after the decellularization procedure. Further direct confocal microscopy analysis on a decellularized mouse brain section revealed the absence of nuclei stained with DAPI (Fig. 1D), as compared to the clear presence of
Discussion
In the present work, we investigated the use of ECM scaffolds from murine brains (DBM) as a platform for cell-loading of decellularized tissue. Our results showed a fast and straightforward method based on freezing and thawing murine brains followed by soaking them in a 1 % SDS solution for complete decellularization. This concentration was able to decellularize the brains with minimal residues of DNA reminiscent and preserving structures as the basement membrane, collagen IV, as well as
Conclusion
Decellularized scaffolds derived from an organ can be used as a platform to understand decellularization and cell-loading. Although further studies are still required to validate the present data in translational models, our results are promising due to the fact that we could fully decellularize murine brains with a fast (24 h for complete decellularization) and simple (only one detergent and 3 freezing and thawing processes) method. The decellularization process also retained essential ECM
references
Aguiari et al. (2017), Bonnans et al. (2014), Breuls et al. (2008), Butter et al. (2018), Granato et al. (2018), Hassanein et al. (2017), Haugh et al. (2011), Huang et al. (2017), Lancaster and Knoblich (2014), Lancaster et al. (2013), Lin et al. (2017), Liu et al. (2017), Momtahan et al. (2016), Napierala et al. (2017), Ott (2015), Poornejad et al. (2016), Pu et al. (2018), Qian et al. (2016), Qiao et al. (2018), Roth et al. (2017), Seyler et al. (2017), Takebe et al. (2017), Taylor et al.
CRediT authorship contribution statement
Alessandro E.C. Granato: Conceptualization, Methodology, Software, Data curation, Writing- Original draft preparation, Reviewing and Editing. Edgar Ferreira da Cruz: Conceptualization, Visualization, Investigation, Writing- original draft preparation. Dorival Mendes Rodrigues-Junior: Visualization, Data curation, Validation, Methodology. Amanda Cristina Mosini: Methodology. Henning Ulrich: Supervision. Bruno V.M. Rodrigues: Writing- reviewing and editing. Arquimedes Cheffer: Methodology.
Aknowledgements
This work was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo – FAPESP (2012/00652-5), Conselho Nacional de Desenvolvimento Científico e Tecnológico – CNPq (402319/2013-3; 465656/2014-5), and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – CAPES (Finance Code 001).
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These authors contributed equally to this work.