DOE PAGES title logo U.S. Department of Energy
Office of Scientific and Technical Information

Title: Silica bioreplication preserves three-dimensional spheroid structures of human pluripotent stem cells and HepG2 cells

Abstract

Three-dimensional (3D) cell cultures produce more in vivo-like multicellular structures such as spheroids that cannot be obtained in two-dimensional (2D) cell cultures. Thus, they are increasingly employed as models for cancer and drug research, as well as tissue engineering. It has proven challenging to stabilize spheroid architectures for detailed morphological examination. Here we overcome this issue using a silica bioreplication (SBR) process employed on spheroids formed from human pluripotent stem cells (hPSCs) and hepatocellular carcinoma HepG2 cells cultured in the nanofibrillar cellulose (NFC) hydrogel. The cells in the spheroids are more round and tightly interacting with each other than those in 2D cultures, and they develop microvilli-like structures on the cell membranes as seen in 2D cultures. Furthermore, SBR preserves extracellular matrix-like materials and cellular proteins. In conclusion, these findings provide the first evidence of intact hPSC spheroid architectures and similar fine structures to 2D-cultured cells, providing a pathway to enable our understanding of morphogenesis in 3D cultures.

Authors:
 [1];  [1];  [2];  [3];  [1];  [1];  [2];  [1]
  1. Univ. of Helsinki (Finland). Centre for Drug Research
  2. Sandia National Lab. (SNL-NM), Albuquerque, NM (United States). Advanced Materials Lab.; Univ. of New Mexico, Albuquerque, NM (United States). Dept. of Chemical and Biomolecular Engineering
  3. Univ. of New Mexico, Albuquerque, NM (United States). Dept. of Internal Medicine; Univ. of New Mexico, Albuquerque, NM (United States). Center for Micro-Engineered Materials
Publication Date:
Research Org.:
Sandia National Lab. (SNL-NM), Albuquerque, NM (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Basic Energy Sciences (BES)
OSTI Identifier:
1261097
Grant/Contract Number:  
FA9550-14-1-0066; CBET-1344298
Resource Type:
Accepted Manuscript
Journal Name:
Scientific Reports
Additional Journal Information:
Journal Volume: 5; Journal ID: ISSN 2045-2322
Publisher:
Nature Publishing Group
Country of Publication:
United States
Language:
English
Subject:
60 APPLIED LIFE SCIENCES

Citation Formats

Lou, Yan-Ru, Kanninen, Liisa, Kaehr, Bryan, Townson, Jason L., Niklander, Johanna, Harjumäki, Riina, Jeffrey Brinker, C., and Yliperttula, Marjo. Silica bioreplication preserves three-dimensional spheroid structures of human pluripotent stem cells and HepG2 cells. United States: N. p., 2015. Web. doi:10.1038/srep13635.
Lou, Yan-Ru, Kanninen, Liisa, Kaehr, Bryan, Townson, Jason L., Niklander, Johanna, Harjumäki, Riina, Jeffrey Brinker, C., & Yliperttula, Marjo. Silica bioreplication preserves three-dimensional spheroid structures of human pluripotent stem cells and HepG2 cells. United States. https://doi.org/10.1038/srep13635
Lou, Yan-Ru, Kanninen, Liisa, Kaehr, Bryan, Townson, Jason L., Niklander, Johanna, Harjumäki, Riina, Jeffrey Brinker, C., and Yliperttula, Marjo. Tue . "Silica bioreplication preserves three-dimensional spheroid structures of human pluripotent stem cells and HepG2 cells". United States. https://doi.org/10.1038/srep13635. https://www.osti.gov/servlets/purl/1261097.
@article{osti_1261097,
title = {Silica bioreplication preserves three-dimensional spheroid structures of human pluripotent stem cells and HepG2 cells},
author = {Lou, Yan-Ru and Kanninen, Liisa and Kaehr, Bryan and Townson, Jason L. and Niklander, Johanna and Harjumäki, Riina and Jeffrey Brinker, C. and Yliperttula, Marjo},
abstractNote = {Three-dimensional (3D) cell cultures produce more in vivo-like multicellular structures such as spheroids that cannot be obtained in two-dimensional (2D) cell cultures. Thus, they are increasingly employed as models for cancer and drug research, as well as tissue engineering. It has proven challenging to stabilize spheroid architectures for detailed morphological examination. Here we overcome this issue using a silica bioreplication (SBR) process employed on spheroids formed from human pluripotent stem cells (hPSCs) and hepatocellular carcinoma HepG2 cells cultured in the nanofibrillar cellulose (NFC) hydrogel. The cells in the spheroids are more round and tightly interacting with each other than those in 2D cultures, and they develop microvilli-like structures on the cell membranes as seen in 2D cultures. Furthermore, SBR preserves extracellular matrix-like materials and cellular proteins. In conclusion, these findings provide the first evidence of intact hPSC spheroid architectures and similar fine structures to 2D-cultured cells, providing a pathway to enable our understanding of morphogenesis in 3D cultures.},
doi = {10.1038/srep13635},
journal = {Scientific Reports},
number = ,
volume = 5,
place = {United States},
year = {Tue Sep 01 00:00:00 EDT 2015},
month = {Tue Sep 01 00:00:00 EDT 2015}
}

Journal Article:
Free Publicly Available Full Text
Publisher's Version of Record

Citation Metrics:
Cited by: 27 works
Citation information provided by
Web of Science

Save / Share:

Works referenced in this record:

Embryonic Stem Cell Lines Derived from Human Blastocysts
journal, November 1998


Human embryonic stem cells express an immunogenic nonhuman sialic acid
journal, January 2005

  • Martin, Maria J.; Muotri, Alysson; Gage, Fred
  • Nature Medicine, Vol. 11, Issue 2
  • DOI: 10.1038/nm1181

Long-term human pluripotent stem cell self-renewal on synthetic polymer surfaces
journal, December 2010


Engineered polymer-media interfaces for the long-term self-renewal of human embryonic stem cells
journal, October 2011


Synthetic polymer coatings for long-term growth of human embryonic stem cells
journal, May 2010

  • Villa-Diaz, Luis G.; Nandivada, Himabindu; Ding, Jun
  • Nature Biotechnology, Vol. 28, Issue 6
  • DOI: 10.1038/nbt.1631

High-Throughput Discovery of Synthetic Surfaces That Support Proliferation of Pluripotent Cells
journal, February 2010

  • Derda, Ratmir; Musah, Samira; Orner, Brendan P.
  • Journal of the American Chemical Society, Vol. 132, Issue 4
  • DOI: 10.1021/ja906089g

Long-term self-renewal of human pluripotent stem cells on human recombinant laminin-511
journal, May 2010

  • Rodin, Sergey; Domogatskaya, Anna; Ström, Susanne
  • Nature Biotechnology, Vol. 28, Issue 6
  • DOI: 10.1038/nbt.1620

Synthetic peptide-acrylate surfaces for long-term self-renewal and cardiomyocyte differentiation of human embryonic stem cells
journal, May 2010

  • Melkoumian, Zara; Weber, Jennifer L.; Weber, David M.
  • Nature Biotechnology, Vol. 28, Issue 6
  • DOI: 10.1038/nbt.1629

Recombinant Vitronectin Is a Functionally Defined Substrate That Supports Human Embryonic Stem Cell Self-Renewal via αVβ5 Integrin
journal, September 2008


Hyaluronic acid hydrogel for controlled self-renewal and differentiation of human embryonic stem cells
journal, June 2007

  • Gerecht, S.; Burdick, J. A.; Ferreira, L. S.
  • Proceedings of the National Academy of Sciences, Vol. 104, Issue 27
  • DOI: 10.1073/pnas.0703723104

Feeder-free self-renewal of human embryonic stem cells in 3D porous natural polymer scaffolds
journal, January 2010


A 3D microfibrous scaffold for long-term human pluripotent stem cell self-renewal under chemically defined conditions
journal, March 2012


The benefit of human embryonic stem cell encapsulation for prolonged feeder-free maintenance
journal, October 2008


The Use of Nanofibrillar Cellulose Hydrogel As a Flexible Three-Dimensional Model to Culture Human Pluripotent Stem Cells
journal, February 2014

  • Lou, Yan-Ru; Kanninen, Liisa; Kuisma, Tytti
  • Stem Cells and Development, Vol. 23, Issue 4
  • DOI: 10.1089/scd.2013.0314

Xeno-Free Production of Human Embryonic Stem Cells in Stirred Microcarrier Systems Using a Novel Animal/Human-Component-Free Medium
journal, February 2013

  • Marinho, Paulo André Nobrega; Vareschini, Daniel Tait; Gomes, Ismael Carlos
  • Tissue Engineering Part C: Methods, Vol. 19, Issue 2
  • DOI: 10.1089/ten.tec.2012.0141

Effective surface-based cryopreservation of human embryonic stem cells by vitrification
journal, December 2011


Cellular complexity captured in durable silica biocomposites
journal, October 2012

  • Kaehr, B.; Townson, J. L.; Kalinich, R. M.
  • Proceedings of the National Academy of Sciences, Vol. 109, Issue 43, p. 17336-17341
  • DOI: 10.1073/pnas.1205816109

Synthetic fossilization of soft biological tissues and their shape-preserving transformation into silica or electron-conductive replicas
journal, December 2014

  • Townson, Jason L.; Lin, Yu-Shen; Chou, Stanley S.
  • Nature Communications, Vol. 5, Article No. 5665
  • DOI: 10.1038/ncomms6665

Biomimetic synthesis of ordered silica structures mediated by block copolypeptides
journal, January 2000

  • Cha, Jennifer N.; Stucky, Galen D.; Morse, Daniel E.
  • Nature, Vol. 403, Issue 6767
  • DOI: 10.1038/35002038

Surface Patterning of Silica Nanostructures Using Bio-Inspired Templates and Directed Synthesis
journal, September 2004

  • Coffman, Elizabeth A.; Melechko, Anatoli V.; Allison, David P.
  • Langmuir, Vol. 20, Issue 20
  • DOI: 10.1021/la048907o

Hierarchical architectures by synergy between dynamical template self-assembly and biomineralization
journal, May 2007

  • Pouget, Emilie; Dujardin, Erik; Cavalier, Annie
  • Nature Materials, Vol. 6, Issue 6, p. 434-439
  • DOI: 10.1038/nmat1912

Protein-Directed Assembly of Arbitrary Three-Dimensional Nanoporous Silica Architectures
journal, January 2011

  • Khripin, Constantine Y.; Pristinski, Denis; Dunphy, Darren R.
  • ACS Nano, Vol. 5, Issue 2, p. 1401-1409
  • DOI: 10.1021/nn1031774

Cell-Directed Assembly of Lipid-Silica Nanostructures Providing Extended Cell Viability
journal, July 2006

  • Baca, Helen K.; Ashley, Carlee; Carnes, Eric
  • Science, Vol. 313, Issue 5785, p. 337-341
  • DOI: 10.1126/science.1126590

Cell-Directed Assembly of Bio/Nano Interfaces—A New Scheme for Cell Immobilization
journal, September 2007

  • Baca, Helen K.; Carnes, Eric; Singh, Seema
  • Accounts of Chemical Research, Vol. 40, Issue 9, p. 836-845
  • DOI: 10.1021/ar600027u

Cell-Directed Localization and Orientation of a Functional Foreign Transmembrane Protein within a Silica Nanostructure
journal, October 2009

  • Carnes, Eric C.; Harper, Jason C.; Ashley, Carlee E.
  • Journal of the American Chemical Society, Vol. 131, Issue 40
  • DOI: 10.1021/ja906055m

Cell-Directed Integration into Three-Dimensional Lipid−Silica Nanostructured Matrices
journal, September 2010

  • Harper, Jason C.; Khirpin, Constantine Y.; Carnes, Eric C.
  • ACS Nano, Vol. 4, Issue 10, p. 5539-5550
  • DOI: 10.1021/nn101793u

Mechanically Encoded Cellular Shapes for Synthesis of Anisotropic Mesoporous Particles
journal, September 2014

  • Meyer, Kristin C.; Coker, Eric N.; Bolintineanu, Dan S.
  • Journal of the American Chemical Society, Vol. 136, Issue 38, p. 13138-13141
  • DOI: 10.1021/ja506718z

Nanofibrillar cellulose hydrogel promotes three-dimensional liver cell culture
journal, December 2012

  • Bhattacharya, Madhushree; Malinen, Melina M.; Lauren, Patrick
  • Journal of Controlled Release, Vol. 164, Issue 3
  • DOI: 10.1016/j.jconrel.2012.06.039

Engineering liver tissue spheroids with inverted colloidal crystal scaffolds
journal, September 2009


Suspension Culture of Undifferentiated Human Embryonic and Induced Pluripotent Stem Cells
journal, April 2010

  • Amit, Michal; Chebath, Judith; Margulets, Victoria
  • Stem Cell Reviews and Reports, Vol. 6, Issue 2
  • DOI: 10.1007/s12015-010-9149-y

Derivation, propagation and controlled differentiation of human embryonic stem cells in suspension
journal, March 2010

  • Steiner, Debora; Khaner, Hanita; Cohen, Malkiel
  • Nature Biotechnology, Vol. 28, Issue 4
  • DOI: 10.1038/nbt.1616

Method for generation of homogeneous multicellular tumor spheroids applicable to a wide variety of cell types
journal, May 2003

  • Kelm, Jens M.; Timmins, Nicholas E.; Brown, Catherine J.
  • Biotechnology and Bioengineering, Vol. 83, Issue 2
  • DOI: 10.1002/bit.10655

Spheroid Culture for Enhanced Differentiation of Human Embryonic Stem Cells to Hepatocyte-Like Cells
journal, January 2014

  • Subramanian, Kartik; Owens, Derek Jason; Raju, Ravali
  • Stem Cells and Development, Vol. 23, Issue 2
  • DOI: 10.1089/scd.2013.0097

Drying cells for SEM, AFM and TEM by hexamethyldisilazane: a study on hepatic endothelial cells
journal, April 1997


Biomimetic Silicification of Demineralized Hierarchical Collagenous Tissues
journal, April 2013

  • Niu, Li-na; Jiao, Kai; Ryou, Heonjune
  • Biomacromolecules, Vol. 14, Issue 5
  • DOI: 10.1021/bm400316e

Cells made of silica
journal, December 2012


Induced Pluripotent Stem Cell Lines Derived from Human Somatic Cells
journal, December 2007


Cell-Directed Assembly of Bio/Nano Interfaces — A New Scheme for Cell Immobilization
journal, December 2007


Induced Pluripotent Stem Cell Lines Derived From Human Somatic Cells
journal, January 2008


Method for generation of homogeneous multicellular tumor spheroids applicable to a wide variety of cell types
journal, May 2003

  • Kelm, Jens M.; Timmins, Nicholas E.; Brown, Catherine J.
  • Biotechnology and Bioengineering, Vol. 83, Issue 2
  • DOI: 10.1002/bit.10655

Suspension Culture of Undifferentiated Human Embryonic and Induced Pluripotent Stem Cells
journal, April 2010

  • Amit, Michal; Chebath, Judith; Margulets, Victoria
  • Stem Cell Reviews and Reports, Vol. 6, Issue 2
  • DOI: 10.1007/s12015-010-9149-y

The benefit of human embryonic stem cell encapsulation for prolonged feeder-free maintenance
journal, October 2008


Feeder-free self-renewal of human embryonic stem cells in 3D porous natural polymer scaffolds
journal, January 2010


Long-term human pluripotent stem cell self-renewal on synthetic polymer surfaces
journal, December 2010


Engineered polymer-media interfaces for the long-term self-renewal of human embryonic stem cells
journal, October 2011


Effective surface-based cryopreservation of human embryonic stem cells by vitrification
journal, December 2011


Nanofibrillar cellulose hydrogel promotes three-dimensional liver cell culture
journal, December 2012

  • Bhattacharya, Madhushree; Malinen, Melina M.; Lauren, Patrick
  • Journal of Controlled Release, Vol. 164, Issue 3
  • DOI: 10.1016/j.jconrel.2012.06.039

Cell-Directed Assembly of Bio/Nano Interfaces—A New Scheme for Cell Immobilization
journal, September 2007

  • Baca, Helen K.; Carnes, Eric; Singh, Seema
  • Accounts of Chemical Research, Vol. 40, Issue 9, p. 836-845
  • DOI: 10.1021/ar600027u

Mechanically Encoded Cellular Shapes for Synthesis of Anisotropic Mesoporous Particles
journal, September 2014

  • Meyer, Kristin C.; Coker, Eric N.; Bolintineanu, Dan S.
  • Journal of the American Chemical Society, Vol. 136, Issue 38, p. 13138-13141
  • DOI: 10.1021/ja506718z

Cell-Directed Localization and Orientation of a Functional Foreign Transmembrane Protein within a Silica Nanostructure
journal, October 2009

  • Carnes, Eric C.; Harper, Jason C.; Ashley, Carlee E.
  • Journal of the American Chemical Society, Vol. 131, Issue 40
  • DOI: 10.1021/ja906055m

High-Throughput Discovery of Synthetic Surfaces That Support Proliferation of Pluripotent Cells
journal, February 2010

  • Derda, Ratmir; Musah, Samira; Orner, Brendan P.
  • Journal of the American Chemical Society, Vol. 132, Issue 4
  • DOI: 10.1021/ja906089g

Surface Patterning of Silica Nanostructures Using Bio-Inspired Templates and Directed Synthesis
journal, September 2004

  • Coffman, Elizabeth A.; Melechko, Anatoli V.; Allison, David P.
  • Langmuir, Vol. 20, Issue 20
  • DOI: 10.1021/la048907o

Cell-Directed Integration into Three-Dimensional Lipid−Silica Nanostructured Matrices
journal, September 2010

  • Harper, Jason C.; Khirpin, Constantine Y.; Carnes, Eric C.
  • ACS Nano, Vol. 4, Issue 10, p. 5539-5550
  • DOI: 10.1021/nn101793u

Biomimetic synthesis of ordered silica structures mediated by block copolypeptides
journal, January 2000

  • Cha, Jennifer N.; Stucky, Galen D.; Morse, Daniel E.
  • Nature, Vol. 403, Issue 6767
  • DOI: 10.1038/35002038

Synthetic peptide-acrylate surfaces for long-term self-renewal and cardiomyocyte differentiation of human embryonic stem cells
journal, May 2010

  • Melkoumian, Zara; Weber, Jennifer L.; Weber, David M.
  • Nature Biotechnology, Vol. 28, Issue 6
  • DOI: 10.1038/nbt.1629

Synthetic polymer coatings for long-term growth of human embryonic stem cells
journal, May 2010

  • Villa-Diaz, Luis G.; Nandivada, Himabindu; Ding, Jun
  • Nature Biotechnology, Vol. 28, Issue 6
  • DOI: 10.1038/nbt.1631

Synthetic fossilization of soft biological tissues and their shape-preserving transformation into silica or electron-conductive replicas
journal, December 2014

  • Townson, Jason L.; Lin, Yu-Shen; Chou, Stanley S.
  • Nature Communications, Vol. 5, Article No. 5665
  • DOI: 10.1038/ncomms6665

Human embryonic stem cells express an immunogenic nonhuman sialic acid
journal, January 2005

  • Martin, Maria J.; Muotri, Alysson; Gage, Fred
  • Nature Medicine, Vol. 11, Issue 2
  • DOI: 10.1038/nm1181

Hierarchical architectures by synergy between dynamical template self-assembly and biomineralization
journal, May 2007

  • Pouget, Emilie; Dujardin, Erik; Cavalier, Annie
  • Nature Materials, Vol. 6, Issue 6, p. 434-439
  • DOI: 10.1038/nmat1912

Micro-scaffold array chip for upgrading cell-based high-throughput drug testing to 3D using benchtop equipment
journal, January 2014

  • Li, Xiaokang; Zhang, Xinyong; Zhao, Shan
  • Lab Chip, Vol. 14, Issue 3
  • DOI: 10.1039/c3lc51103k

Drying cells for SEM, AFM and TEM by hexamethyldisilazane: a study on hepatic endothelial cells
journal, April 1997


Hyaluronic acid hydrogel for controlled self-renewal and differentiation of human embryonic stem cells
journal, June 2007

  • Gerecht, S.; Burdick, J. A.; Ferreira, L. S.
  • Proceedings of the National Academy of Sciences, Vol. 104, Issue 27
  • DOI: 10.1073/pnas.0703723104

Cellular complexity captured in durable silica biocomposites
journal, October 2012

  • Kaehr, B.; Townson, J. L.; Kalinich, R. M.
  • Proceedings of the National Academy of Sciences, Vol. 109, Issue 43, p. 17336-17341
  • DOI: 10.1073/pnas.1205816109

Spheroid Culture for Enhanced Differentiation of Human Embryonic Stem Cells to Hepatocyte-Like Cells
journal, January 2014

  • Subramanian, Kartik; Owens, Derek Jason; Raju, Ravali
  • Stem Cells and Development, Vol. 23, Issue 2
  • DOI: 10.1089/scd.2013.0097

The Use of Nanofibrillar Cellulose Hydrogel As a Flexible Three-Dimensional Model to Culture Human Pluripotent Stem Cells
journal, February 2014

  • Lou, Yan-Ru; Kanninen, Liisa; Kuisma, Tytti
  • Stem Cells and Development, Vol. 23, Issue 4
  • DOI: 10.1089/scd.2013.0314

Cell-Directed Assembly of Lipid-Silica Nanostructures Providing Extended Cell Viability
journal, July 2006

  • Baca, Helen K.; Ashley, Carlee; Carnes, Eric
  • Science, Vol. 313, Issue 5785, p. 337-341
  • DOI: 10.1126/science.1126590

Works referencing / citing this record:

Quantified forces between HepG2 hepatocarcinoma and WA07 pluripotent stem cells with natural biomaterials correlate with in vitro cell behavior
journal, May 2019


Hepatocyte Growth Factor: A Microenvironmental Resource for Leukemic Cell Growth
journal, January 2019

  • Giannoni, Paolo; Fais, Franco; Cutrona, Giovanna
  • International Journal of Molecular Sciences, Vol. 20, Issue 2
  • DOI: 10.3390/ijms20020292

Composites of Polymer Hydrogels and Nanoparticulate Systems for Biomedical and Pharmaceutical Applications
journal, December 2015


Bioinspired Silicification Reveals Structural Detail in Self-Assembled Peptide Cages
journal, December 2017


Quantified forces between HepG2 hepatocarcinoma and WA07 pluripotent stem cells with natural biomaterials correlate with in vitro cell behavior
journal, May 2019


Composites of Polymer Hydrogels and Nanoparticulate Systems for Biomedical and Pharmaceutical Applications
journal, December 2015