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Cited by 117Tissue Engineering and Regenerative Medicine3D Printing in Biomedical ResearchHydrogels: synthesis, properties, applications

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Brain organoid formation on decellularized porcine brain ECM hydrogels

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Simsa, Robin, Rothenbücher, Theresa, Gürbüz, Hakan, Ghosheh, Nidal, Emneus, Jenny, Jenndahl, Lachmi, Kaplan, David L., Bergh, Niklas, Serrano, Alberto Martinez, Fogelstrand, Per·2021·PLOS ONE
DOI 10.1371/journal.pone.0245685PMC PMC7842896
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Abstract

Human brain tissue models such as cerebral organoids are essential tools for developmental and biomedical research. Current methods to generate cerebral organoids often utilize Matrigel as an external scaffold to provide structure and biologically relevant signals. Matrigel however is a nonspecific hydrogel of mouse tumor origin and does not represent the complexity of the brain protein environment. In this study, we investigated the application of a decellularized adult porcine brain extracellular matrix (B-ECM) which could be processed into a hydrogel (B-ECM hydrogel) to be used as a scaffold for human embryonic stem cell (hESC)-derived brain organoids. We decellularized pig brains with a novel detergent- and enzyme-based method and analyzed the biomaterial properties, including protein composition and content, DNA content, mechanical characteristics, surface structure, and antigen presence. Then, we compared the growth of human brain organoid models with the B-ECM hydrogel or Matrigel controls in vitro. We found that the native brain source material was successfully decellularized with little remaining DNA content, while Mass Spectrometry (MS) showed the loss of several brain-specific proteins, while mainly different collagen types remained in the B-ECM. Rheological results revealed stable hydrogel formation, starting from B-ECM hydrogel concentrations of 5 mg/mL. hESCs cultured in B-ECM hydrogels showed gene expression and differentiation outcomes similar to those grown in Matrigel. These results indicate that B-ECM hydrogels can be used as an alternative scaffold for human cerebral organoid formation, and may be further optimized for improved organoid growth by further improving protein retention other than collagen after decellularization.

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Fig 1 DNA and ECM content of decellularization pig brain. (A) Native pig brains were decellularized with detergents, lyophilized, and solubilized in a pepsin-HCl mix. After neutralizing the solution (15 mg/mL) to physiological salt and pH concentration, hydrogel formation at 37°C was observed. (B) DAPI staining of whole tissue samples en face (upper panel) and tissue sections (lower panel). (C) DNA content in native brain and B-ECM samples (n = 9) was measured after extraction of DNA from wet tissue with a commercial kit. (D) GAGs from wet tissue (n = 8) and (E) collagen from dry tissue (n = 15) were quantified by extraction of proteins with commercial kits.
Fig 1 DNA and ECM content of decellularization pig brain. (A) Native pig brains were decellularized with detergents, lyophilized, and solubilized in a pepsin-HCl mix. After neutralizing the solution (15 mg/mL) to physiological salt and pH concentration, hydrogel formation at 37°C was observed. (B) DAPI staining of whole tissue samples en face (upper panel) and tissue sections (lower panel). (C) DNA content in native brain and B-ECM samples (n = 9) was measured after extraction of DNA from wet tissue with a commercial kit. (D) GAGs from wet tissue (n = 8) and (E) collagen from dry tissue (n = 15) were quantified by extraction of proteins with commercial kits.CC BY · PubMed Central
Fig 2 Staining of native, B-ECM and B-ECM hydrogel samples. Histological and immunostaining was performed on paraffin-embedded samples after rehydration and antigen retrieval (n = 3). For general morphology, the samples were stained with hematoxylin-eosin (H&E). Fibronectin, vitronectin, laminin, myelin basic protein (MBP) and human leucocyte antigen (HLA-DR) were detected with antibodies (immunofluorescence). Scale bar represents 100 μm.
Fig 2 Staining of native, B-ECM and B-ECM hydrogel samples. Histological and immunostaining was performed on paraffin-embedded samples after rehydration and antigen retrieval (n = 3). For general morphology, the samples were stained with hematoxylin-eosin (H&E). Fibronectin, vitronectin, laminin, myelin basic protein (MBP) and human leucocyte antigen (HLA-DR) were detected with antibodies (immunofluorescence). Scale bar represents 100 μm.CC BY · PubMed Central
Fig 3 Protein analysis performed by mass spectrometry on samples from native brain, B-ECM, B-ECM hydrogel, and on Matrigel. (A) Stacked bar chart of the abundance of the matrisome-proteins that were detected in the analyzed samples, categorized in different matrisome categories (Collagens, ECM-affiliated, Glycoproteins, Proteoglycans, ECM Regulators or Secreted Factors) (B) The abundance of ECM proteins found in the B-ECM hydrogel sample compared to Native, B-ECM, and Matrigel samples, sorted by ECM categories (collagens, glycoproteins, and proteoglycans) with their gene identifier. Besides, the abundances of the brain-specific ECM proteins brevican (BCAN), aggrecan (ACAN), and versican (VCAN) in the different samples are shown as well. Y-axis is in the logarithmic scale.
Fig 3 Protein analysis performed by mass spectrometry on samples from native brain, B-ECM, B-ECM hydrogel, and on Matrigel. (A) Stacked bar chart of the abundance of the matrisome-proteins that were detected in the analyzed samples, categorized in different matrisome categories (Collagens, ECM-affiliated, Glycoproteins, Proteoglycans, ECM Regulators or Secreted Factors) (B) The abundance of ECM proteins found in the B-ECM hydrogel sample compared to Native, B-ECM, and Matrigel samples, sorted by ECM categories (collagens, glycoproteins, and proteoglycans) with their gene identifier. Besides, the abundances of the brain-specific ECM proteins brevican (BCAN), aggrecan (ACAN), and versican (VCAN) in the different samples are shown as well. Y-axis is in the logarithmic scale.CC BY · PubMed Central
Fig 4 SEM of native, B-ECM and B-ECM hydrogel samples. Images taken with 4000x (upper panel) and 1000x (lower panel) magnification.
Fig 4 SEM of native, B-ECM and B-ECM hydrogel samples. Images taken with 4000x (upper panel) and 1000x (lower panel) magnification.CC BY · PubMed Central
Fig 5 Growth and gene expression of brain organoids embedded in B-ECM hydrogel or Matrigel. (A) Timeline of experimental steps of human brain organoid maturation embedded in Matrigel or B-ECM hydrogel. (B) Brain organoids (n = 4) on day 10 in Matrigel (top pictures) or B-ECM hydrogel (bottom pictures). On day 40, no significant difference is apparent. White arrows at day 10 point to ventricular-like zones located in the formed neuronal buds White arrowhead at day 40 indicates residual B-ECM hydrogel. Scale bar: 500 μm. (C) Expression of the genes NEST, TUBB3, DCX, MAP2 and GFAP was measured by extraction of RNA from organoids grown in the presence of B-ECM hydrogel (n = 13) or Matrigel (n = 12) in technical triplicates with RT-qPCR. Statistical analysis performed with unpaired t-test. (D) Tissue sections of brain organoids cultured in Matrigel (left panels) or B-ECM hydrogel (right panels) for 40 days were immunostained for neuronal progenitor markers (SOX2, Nestin, PAX6) and mature neuronal markers (DCX, β-Tubulin III, MAP2). Images were taken with a 10x objective (10x, scale bar: 300 μm) and 40x objective (40x, scale bar: 100 μm). DAPI was used as nuclear counterstaining. White arrowheads indicate ventricular-like zones that were chosen for magnification.
Fig 5 Growth and gene expression of brain organoids embedded in B-ECM hydrogel or Matrigel. (A) Timeline of experimental steps of human brain organoid maturation embedded in Matrigel or B-ECM hydrogel. (B) Brain organoids (n = 4) on day 10 in Matrigel (top pictures) or B-ECM hydrogel (bottom pictures). On day 40, no significant difference is apparent. White arrows at day 10 point to ventricular-like zones located in the formed neuronal buds White arrowhead at day 40 indicates residual B-ECM hydrogel. Scale bar: 500 μm. (C) Expression of the genes NEST, TUBB3, DCX, MAP2 and GFAP was measured by extraction of RNA from organoids grown in the presence of B-ECM hydrogel (n = 13) or Matrigel (n = 12) in technical triplicates with RT-qPCR. Statistical analysis performed with unpaired t-test. (D) Tissue sections of brain organoids cultured in Matrigel (left panels) or B-ECM hydrogel (right panels) for 40 days were immunostained for neuronal progenitor markers (SOX2, Nestin, PAX6) and mature neuronal markers (DCX, β-Tubulin III, MAP2). Images were taken with a 10x objective (10x, scale bar: 300 μm) and 40x objective (40x, scale bar: 100 μm). DAPI was used as nuclear counterstaining. White arrowheads indicate ventricular-like zones that were chosen for magnification.CC BY · PubMed Central

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Citations

Cites

Show all 72 references

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Metadata

Authors
Simsa, R., Rothenbücher, T., Gürbüz, H., Ghosheh, N., Emneus, J., Jenndahl, L., Kaplan, D. L., Bergh, N., Serrano, A. M., & Fogelstrand, P.
Affiliations
  • Tufts University (US)
  • Centro de Biología Molecular Severo Ochoa (ES)
  • Technical University of Denmark (DK)
  • University of Skövde (SE)
  • Sahlgrenska University Hospital (SE)
Corresponding author
  • Simsa, Robin
  • Rothenbücher, Theresa
  • Gürbüz, Hakan
  • Ghosheh, Nidal
  • Jenndahl, Lachmi
  • Kaplan, David L.
  • Bergh, Niklas
  • Fogelstrand, Per
ORCID
Venue
PLOS ONE
Publisher
Public Library of Science (PLoS)
Published
2021-01-28
Volume
16
Issue
1
Pages
e0245685
ISSN
1932-6203 (ISSN-L)
DOI
10.1371/journal.pone.0245685
PMC
PMC7842896
OpenAlex
W3124422938
Cited by
114
References
72
Citations by year
2026: 10 · 2025: 26 · 2024: 24 · 2023: 27 · 2022: 21 · 2021: 6
FWCI
18.32
Type
journal article
Language
en
Open access
Licence
Locations
  • PLoS ONE (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • Diva portal (Dalarna University Library) (submittedVersion, other-oa)
  • DIGITAL.CSIC (Spanish National Research Council (CSIC)) (submittedVersion, cc-by)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
  • Technical University of Denmark, DTU Orbit (Technical University of Denmark, DTU) (submittedVersion, other-oa)
Funders
  • Horizon 2020 (722779)
  • MINECO SAF (2017-83241-R)
  • ISC-III RETICS TerCel (RD16/0011/0032)
  • ASCTN-Training (813851)
  • National Institutes of Health (P41EB027062, R01NS092847)
Topics
  • Health Sciences › Medicine › Surgery › Tissue Engineering and Regenerative Medicine
  • Physical Sciences › Engineering › Biomedical Engineering › 3D Printing in Biomedical Research
  • Physical Sciences › Materials Science › Biomaterials › Hydrogels: synthesis, properties, applications
Keywords
brain organoids, porcine brain, hydrogels, scaffold materials, human embryonic stem cells, Matrigel alternative, decellularization, collagen, protein retention, hydrogel rheology, organoid differentiation
MeSH terms
Human Embryonic Stem Cells, Animals, Brain, Brain Chemistry, Cell Line, Extracellular Matrix, Humans, Organoids, Swine, Hydrogels

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