Elsevier

Tissue and Cell

Volume 67, December 2020, 101412
Tissue and Cell

A novel decellularization method to produce brain scaffolds

https://doi.org/10.1016/j.tice.2020.101412Get rights and content

Highlights

  • A straightforward method for complete decellularization of mice brain is described.

  • Mice brain can be entirely decellularized, while still maintaining ECM components.

  • Successful repopulation of the decellularized brain matrix with Neuro2a cells.

Abstract

Scaffolds composed of extracellular matrix (ECM) can assist tissue remodeling and repair following injury. The ECM is a complex biomaterial composed of proteins, glycoproteins, proteoglycans, and glycosaminoglycans, secreted by cells. The ECM contains fundamental biological cues that modulate cell behavior and serves as a structural scaffold for cell adhesion and growth. For clinical applications, where immune rejection is a constraint, ECM can be processed using decellularization methods intended to remove cells and donor antigens from tissue or organs, while preserving native biological cues essential for cell growth and differentiation. Recent studies show bioengineered organs composed by a combination of a diversity of materials and stem cells as a possibility of new therapeutic strategies to treat diseases that affect different tissues and organs, including the central nervous system (CNS). Nevertheless, the methodologies currently described for brain decellularization involve the use of several chemical reagents with many steps that ultimately limit the process of organ or tissue recellularization. Here, we describe for the first time a fast and straightforward method for complete decellularization of mice brain by the combination of rapid freezing and thawing following the use of only one detergent (Sodium dodecyl sulfate (SDS)). Our data show that using the protocol we describe here, the brain was entirely decellularized, while still maintaining ECM components that are essential for cell survival on the scaffold. Our results also show the cell-loading of the decellularized brain matrix with Neuro2a cells, which were identified by immunohistochemistry in their undifferentiated form. We conclude that this novel and simple method for brain decellularization can be used as a scaffold for cell-loading.

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).

References (60)

  • S. Shojaie

    Acellular lung scaffolds direct differentiation of endoderm to functional airway epithelial cells: requirement of matrix-bound HS proteoglycans

    Stem Cell Reports

    (2015)
  • M. Sondell et al.

    Regeneration of the rat sciatic nerve into allografts made acellular through chemical extraction

    Brain Res.

    (1998)
  • T. Takebe et al.

    Synergistic engineering: organoids meet organs-on-a-chip

    Cell Stem Cell

    (2017)
  • E. Vorotnikova

    Extracellular matrix-derived products modulate endothelial and progenitor cell migration and proliferation in vitro and stimulate regenerative healing in vivo

    Matrix Biol.

    (2010)
  • M.L. Wong et al.

    In vivo xenogeneic scaffold fate is determined by residual antigenicity and extracellular matrix preservation

    Biomaterials

    (2016)
  • E.K.F. Yim et al.

    Synthetic nanostructures inducing differentiation of human mesenchymal stem cells into neuronal lineage

    Exp. Cell Res.

    (2007)
  • Z. Yin

    The regulation of tendon stem cell differentiation by the alignment of nanofibers

    Biomaterials

    (2010)
  • T. Zhu

    An acellular cerebellar biological scaffold: preparation, characterization, biocompatibility and effects on neural stem cells

    Brain Res. Bull.

    (2015)
  • P. Aguiari

    In vitro comparative assessment of decellularized bovine pericardial patches and commercial bioprosthetic heart valves

    Biomed. Mater.

    (2017)
  • J. Bao

    Construction of a portal implantable functional tissue-engineered liver using perfusion-decellularized matrix and hepatocytes in rats

    Cell Transplant.

    (2011)
  • C. Bonnans et al.

    Remodelling the extracellular matrix in development and disease

    Nat. Rev. Mol. Cell Biol.

    (2014)
  • R.G.M. Breuls et al.

    Scaffold stiffness influences cell behavior: opportunities for skeletal tissue engineering

    Open Orthop. J.

    (2008)
  • A. Butter

    Evolution of graft morphology and function after recellularization of decellularized rat livers

    J. Tissue Eng. Regen. Med.

    (2018)
  • B. Chani et al.

    Decellularized scaffold of cryopreserved rat kidney retains its recellularization potential

    PLoS One

    (2017)
  • T. Chow et al.

    Decellularizing and recellularizing adult mouse kidneys

    Kidney Organogenesis

    (2019)
  • P. Crapo

    Biologic scaffolds composed of central nervous system extracellular matrix

    Biomaterials

    (2012)
  • Edgar Ferreira da Cruz et al.

    C.V.R. And n. Bioscaffolds like platform to kidney regeneration using animal models and different time points

    Int. J. Dev. Res.

    (2019)
  • J.A. DeQuach et al.

    Decellularized porcine brain matrix for cell culture and tissue engineering scaffolds

    Tissue Eng. Part A

    (2011)
  • L. Ghasemi-Mobarakeh

    Structural properties of scaffolds: crucial parameters towards stem cells differentiation

    World J. Stem Cells

    (2015)
  • A. Gilpin et al.

    Decellularization strategies for regenerative medicine: from processing techniques to applications

    Biomed Res. Int.

    (2017)
  • Cited by (14)

    • Effect of sterilization methods on the mechanical stability and extracellular matrix constituents of decellularized brain tissues

      2021, Journal of Supercritical Fluids
      Citation Excerpt :

      Besides that, the highest difference was seen in the 1/4 ratio (the highest viability ratio for all groups) of SC-6% group (p < 0.01) with above 115% cell viability, parallel to cell proliferation (Fig. 5D). The reason behind proliferative response of NSC-34 cells in comparison to L929 cells treated with sterilized db-tissues, especially SC-6%, can be justified by preservation of ECM constituents such as neurosupportive proteins, sGAGs and growth factors, that stimulate neural cell growth, proliferation and migration [24,47,51], thereby were adequately providing tissue-specific advantages. Determination of a suitable sterilization technique is critically important in order to effectively sterilize biocompatible db-tissues but at the same time to maintain biochemical, structural and physicomechanical integrities.

    View all citing articles on Scopus
    1

    These authors contributed equally to this work.

    View full text