Biotechnology Journal
Biotechnol. J. 2015, 10, 1513–1514
DOI 10.1002/biot.201500308
www.biotechnology-journal.com
COMMENTARY
Interior decoration: Adapting multiwell plates for high throughput mechanobiology Hamish T. J. Gilbert and Joe Swift Wellcome Trust Centre for Cell-Matrix Research, Faculty of Life Sciences, University of Manchester, Manchester, UK See accompanying article by Harkness et al. DOI 10.1002/biot.201400756
In this issue of Biotechnology Journal, Harkness et al. [1] demonstrate a technology that combines patterned substrates, for the purpose of controlling cell geometry, with multi-well plate layouts to allow a high-throughput analysis of the inputs that affect cell behavior.
Much of our current understanding of cell biology is based on studies of cells cultured in vitro on flat and featureless glass or plastic; however, in recent years, there has been a greater appreciation for the ways in which cells perceive and respond to the mechanical properties [2], geometry [3] and dimensionality [4] of their surroundings. It is becoming apparent that a cell does not choose to respond to a mechanical or a chemical input, but rather interprets a combination of both [5]. Thus the cellular response to
a drug or soluble factor may be amplified, or indeed suppressed, when combined with a physical constraint. Furthermore, cell populations can be highly diverse, with significant variation in concentrations of protein and transcript even within genetically identical cells [6]. To understand the pathways that allow cells to produce a concerted response to input, for example, to achieve ordering or synchronization within a tissue, therefore requires study of single cells as well as population averages.
Figure 1. The combination of micropatterned substrates with multi-well plates will allow mechanical and chemical perturbations of cell behaviour to be systematically characterized on a cell-by-cell basis, for example by high-resolution microscopy. Harkness et al. [1] report on the effects of substrate pattern shape on cytoskeletal ordering, nuclear shape and the mobile fraction of histone H2B.
© 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Micropatterning is a technique that enables micron-scale, cell-adhesive features to be placed on a substrate to allow control of cell shape and area. While powerful, these methods were not previously amenable to highthroughput and automated analysis. The requirement to screen combinations of physical and chemical cellular inputs along with the need to understand how robust responses can be achieved in heterogeneous cell populations motivates methodological development such as described by Harkness et al. [1]. In this work, micropattern printing technology was adapted to place patterned substrates with features of varied shape within the wells of standard multi-well plates. A next experimental step could take advantage of the multi-well format to systematically multiplex chemical and mechanical inputs (Fig. 1). The behavior of cells is affected when their shape is constrained by the geometry of the underlying substrate. This mechanobiological input has been demonstrated to propagate into the cell, affecting cytoskeletal organization, nuclear morphology, chromatin packing [7] and potentially influencing decisions of cell fate [8]. Harkness et al. [1] modified human induced pluripotent stem cells (iPSCs) using CRISPR/Cas 9 and lentiviral transduction to express fluorescent histone H2B and actin reporter constructs. A stable fibroblast line was derived from the iPSC clones, cultured on patterned substrates and the cells analyzed by high-resolution microscopy. High-content imaging, which parameterizes features such as cell and nuclear shape, has been shown to
1513
Biotechnology Journal
Biotechnol. J. 2015, 10, 1513–1514 www.biotecvisions.com
COMMENTARY
www.biotechnology-journal.com
be highly diagnostic, and in some cases predictive, of cell behavior [9]. The authors also examined chromatin dynamics (using fluorescence recovery after photobleaching, FRAP), finding that the mobile fraction of histone H2B increased in cells on circular versus elongated patterned substrates [10]. Consistent with expectation, H2B mobility decreased as order in the actomyosin network increased, but intriguingly, inhibition of myosin-II activity led to reduced mobility in circular-patterned cells. While much work is still needed to understand the mechanisms that underpin mechanotransduction signalling pathways, this work demonstrates a powerful, scalable platform on which to base such studies. HTJG and JS are supported by the BBSRC.
References [1] Harkness, T., McNulty, J. D., Prestil, R., Seymour, S. K. et al., Micropatterned multiwell plates for high-content imaging and mechanobiology of human cells. Biotechnol. J. 2015, DOI 10.1002/biot.201400756. [2] Engler, A. J., Sen, S., Sweeney, H. L., Discher, D. E., Matrix elasticity directs stem cell lineage specification. Cell 2006, 126, 677–689. [3] Chen, C. S., Mrksich, M., Huang, S., Whitesides, G. M., Ingber, D. E., Geometric control of cell life and death. Science 1997, 276, 1425–1428. [4] Baker, B. M., Chen, C. S., Deconstructing the third dimension – how 3D culture microenvironments alter cellular cues. J. Cell Sci. 2012, 125, 3015–3024. [5] Dingal, P. C. D. P., Discher, D. E., Combining insoluble and soluble factors to steer stem cell fate. Nat. Mater. 2014, 13, 532–537. [6] Ahrends, R., Ota, A., Kovary, K. M., Kudo, T. et al., Controlling low rates of cell differ-
entiation through noise and ultrahigh feedback. Science 2014, 344, 1384–1389. [7] Talwar, S., Jain, N., Shivashankar, G. V., The regulation of gene expression during onset of differentiation by nuclear mechanical heterogeneity. Biomaterials 2014, 35, 2411–2419. [8] Dalby, M. J., Gadegaard, N., Oreffo, R. O. C., Harnessing nanotopography and integrin-matrix interactions to influence stem cell fate. Nat. Mater. 2014, 13, 558–569. [9] Treiser, M. D., Yang, E. H., Gordonov, S., Cohen, D. M. et al., Cytoskeleton-based forecasting of stem cell lineage fates. Proc. Natl. Acad. Sci. U.S.A. 2010, 107, 610–615. [10] Khatau, S. B., Hale, C., Stewart-Hutchinson, P., Patel, M. S. et al., A perinuclear actin cap regulates nuclear shape. Proc. Natl. Acad. Sci. U.S.A. 2009, 106, 19017–19022.
Correspondence: Dr. Joe Swift E-mail: [email protected]
The authors declare no financial or commercial conflict of interest.
1514
© 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Cover illustration Special Issue: Stem Cell Engineering. This special issue, edited by Joaquim Cabral and Sean Palecek, contains articles on culturing stem cells, their differentiation as well as on imaging techniques. The cover shows primary hematopoietic stem cells and lineage-committed cells from the bone marrow stained with Hoechst 33342 (blue), CMFDA (green) and CMTPX (red). Stem cells (stained blue) are in contact with neighboring niche cells (stained red and green) that regulate stem cell fate decisions. Image provided by Harley et al.
Biotechnology Journal – list of articles published in the October 2015 issue.
Editorial Stem Cell Engineering
http://dx.doi.org/10.1002/biot.201500531
Research Article High-content imaging with micropatterned multiwell plates reveals influence of cell geometry and cytoskeleton on chromatin dynamics
Commentary Engineering at the microscale: A step towards single-cell analysis of human pluripotent stem cells
http://dx.doi.org/10.1002/biot.201400756
Joaquim M. S. Cabral and Sean P. Palecek
Tiago G. Fernandes
http://dx.doi.org/10.1002/biot.201500307 Commentary Interior decoration: Adapting multiwell plates for high throughput mechanobiology
Ty Harkness, Jason D. McNulty, Ryan Prestil, Stephanie K. Seymour, Tyler Klann, Michael Murrell, Randolph S. Ashton and Krishanu Saha
Research Article Inkjet-bioprinted acrylated peptides and PEG hydrogel with human mesenchymal stem cells promote robust bone and cartilage formation with minimal printhead clogging
Hamish T. J. Gilbert and Joe Swift
Guifang Gao, Tomo Yonezawa, Karen Hubbell, Guohao Dai, and Xiaofeng Cui
http://dx.doi.org/10.1002/biot.201500308
http://dx.doi.org/10.1002/biot.201400635
Review Advanced imaging approaches for regenerative medicine: Emerging technologies for monitoring stem cell fate in vitro and in vivo
Research Article Neural commitment of human pluripotent stem cells under defined conditions recapitulates neural development and generates patient-specific neural cells
Molly E. Kupfer and Brenda M. Ogle
Tiago G. Fernandes, Sofia T. Duarte, Mehrnaz Ghazvini, Cláudia Gaspar, Diana C. Santos, Ana R. Porteira, Gonçalo M. C. Rodrigues, Simone Haupt, Diogo M. Rombo, Judith Armstrong, Ana M. Sebastião, Joost Gribnau, Àngels GarciaCazorla, Oliver Brüstle, Domingos Henrique, Joaquim M. S. Cabral and Maria Margarida Diogo
http://dx.doi.org/10.1002/biot.201400760 Review Engineering the hematopoietic stem cell niche: Frontiers in biomaterial science Ji Sun Choi, Bhushan P. Mahadik and Brendan A. C. Harley
http://dx.doi.org/10.1002/biot.201400758 Research Article Clonal analysis of individual human embryonic stem cell differentiation patterns in microfluidic cultures Darek J. Sikorski, Nicolas J. Caron, Michael VanInsberghe, Hans Zahn, Connie J. Eaves, James M. Piret and Carl L. Hansen
http://dx.doi.org/10.1002/biot.201400751 Research Article A reproducible and versatile system for the dynamic expansion of human pluripotent stem cells in suspension Andreas Elanzew, Annika Sommer, Annette Pusch-Klein, Oliver Brüstle and Simone Haupt
http://dx.doi.org/10.1002/biot.201400757
http://dx.doi.org/10.1002/biot.201500035
© 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.biotechnology-journal.com
Research Article Enzymatic passaging of human embryonic stem cells alters central carbon metabolism and glycan abundance
Research Article Compartment-specific metabolomics for CHO reveals that ATP pools in mitochondria are much lower than in cytosol
Mehmet G. Badur, Hui Zhang and Christian M. Metallo
Jens-Christoph Matuszczyk, Attila Teleki, Jennifer Pfizenmaier and Ralf Takors
http://dx.doi.org/10.1002/biot.201400749 Research Article Spatial and temporal control of cell aggregation efficiently directs human pluripotent stem cells towards neural commitment Cláudia C. Miranda, Tiago G. Fernandes, Jorge F. Pascoal, Simone Haupt, Oliver Brüstle, Joaquim M.S. Cabral and Maria Margarida Diogo
http://dx.doi.org/10.1002/biot.201400846 Research Article Microarray profiling of preselected CHO host cell subclones identifies gene expression patterns associated with increased production capacity
http://dx.doi.org/10.1002/biot.201500060 Research Articel The stage-specific in vitro efficacy of a malaria antigen cocktail provides valuable insights into the development of effective multi-stage vaccines Holger Spiegel, Alexander Boes, Robin Kastilan, Stephanie Kapelski, Güven Edgue, Veronique Beiss, Ivana Chubodova, Matthias Scheuermayer, Gabriele Pradel, Stefan Schillberg, Andreas Reimann and Rainer Fischer
http://dx.doi.org/10.1002/biot.201500055
Eva Harreither, Matthias Hackl, Johannes Pichler, Smriti Shridhar, Norbert Auer, Paweł P. Łabaj, Marcel Scheideler, Michael Karbiener, Johannes Grillari, David P. Kreil and Nicole Borth
http://dx.doi.org/10.1002/biot.201400857
© 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.biotechnology-journal.com
Biotechnol. J. 2015, 10, 1513–1514
DOI 10.1002/biot.201500308
www.biotechnology-journal.com
COMMENTARY
Interior decoration: Adapting multiwell plates for high throughput mechanobiology Hamish T. J. Gilbert and Joe Swift Wellcome Trust Centre for Cell-Matrix Research, Faculty of Life Sciences, University of Manchester, Manchester, UK See accompanying article by Harkness et al. DOI 10.1002/biot.201400756
In this issue of Biotechnology Journal, Harkness et al. [1] demonstrate a technology that combines patterned substrates, for the purpose of controlling cell geometry, with multi-well plate layouts to allow a high-throughput analysis of the inputs that affect cell behavior.
Much of our current understanding of cell biology is based on studies of cells cultured in vitro on flat and featureless glass or plastic; however, in recent years, there has been a greater appreciation for the ways in which cells perceive and respond to the mechanical properties [2], geometry [3] and dimensionality [4] of their surroundings. It is becoming apparent that a cell does not choose to respond to a mechanical or a chemical input, but rather interprets a combination of both [5]. Thus the cellular response to
a drug or soluble factor may be amplified, or indeed suppressed, when combined with a physical constraint. Furthermore, cell populations can be highly diverse, with significant variation in concentrations of protein and transcript even within genetically identical cells [6]. To understand the pathways that allow cells to produce a concerted response to input, for example, to achieve ordering or synchronization within a tissue, therefore requires study of single cells as well as population averages.
Figure 1. The combination of micropatterned substrates with multi-well plates will allow mechanical and chemical perturbations of cell behaviour to be systematically characterized on a cell-by-cell basis, for example by high-resolution microscopy. Harkness et al. [1] report on the effects of substrate pattern shape on cytoskeletal ordering, nuclear shape and the mobile fraction of histone H2B.
© 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Micropatterning is a technique that enables micron-scale, cell-adhesive features to be placed on a substrate to allow control of cell shape and area. While powerful, these methods were not previously amenable to highthroughput and automated analysis. The requirement to screen combinations of physical and chemical cellular inputs along with the need to understand how robust responses can be achieved in heterogeneous cell populations motivates methodological development such as described by Harkness et al. [1]. In this work, micropattern printing technology was adapted to place patterned substrates with features of varied shape within the wells of standard multi-well plates. A next experimental step could take advantage of the multi-well format to systematically multiplex chemical and mechanical inputs (Fig. 1). The behavior of cells is affected when their shape is constrained by the geometry of the underlying substrate. This mechanobiological input has been demonstrated to propagate into the cell, affecting cytoskeletal organization, nuclear morphology, chromatin packing [7] and potentially influencing decisions of cell fate [8]. Harkness et al. [1] modified human induced pluripotent stem cells (iPSCs) using CRISPR/Cas 9 and lentiviral transduction to express fluorescent histone H2B and actin reporter constructs. A stable fibroblast line was derived from the iPSC clones, cultured on patterned substrates and the cells analyzed by high-resolution microscopy. High-content imaging, which parameterizes features such as cell and nuclear shape, has been shown to
1513
Biotechnology Journal
Biotechnol. J. 2015, 10, 1513–1514 www.biotecvisions.com
COMMENTARY
www.biotechnology-journal.com
be highly diagnostic, and in some cases predictive, of cell behavior [9]. The authors also examined chromatin dynamics (using fluorescence recovery after photobleaching, FRAP), finding that the mobile fraction of histone H2B increased in cells on circular versus elongated patterned substrates [10]. Consistent with expectation, H2B mobility decreased as order in the actomyosin network increased, but intriguingly, inhibition of myosin-II activity led to reduced mobility in circular-patterned cells. While much work is still needed to understand the mechanisms that underpin mechanotransduction signalling pathways, this work demonstrates a powerful, scalable platform on which to base such studies. HTJG and JS are supported by the BBSRC.
References [1] Harkness, T., McNulty, J. D., Prestil, R., Seymour, S. K. et al., Micropatterned multiwell plates for high-content imaging and mechanobiology of human cells. Biotechnol. J. 2015, DOI 10.1002/biot.201400756. [2] Engler, A. J., Sen, S., Sweeney, H. L., Discher, D. E., Matrix elasticity directs stem cell lineage specification. Cell 2006, 126, 677–689. [3] Chen, C. S., Mrksich, M., Huang, S., Whitesides, G. M., Ingber, D. E., Geometric control of cell life and death. Science 1997, 276, 1425–1428. [4] Baker, B. M., Chen, C. S., Deconstructing the third dimension – how 3D culture microenvironments alter cellular cues. J. Cell Sci. 2012, 125, 3015–3024. [5] Dingal, P. C. D. P., Discher, D. E., Combining insoluble and soluble factors to steer stem cell fate. Nat. Mater. 2014, 13, 532–537. [6] Ahrends, R., Ota, A., Kovary, K. M., Kudo, T. et al., Controlling low rates of cell differ-
entiation through noise and ultrahigh feedback. Science 2014, 344, 1384–1389. [7] Talwar, S., Jain, N., Shivashankar, G. V., The regulation of gene expression during onset of differentiation by nuclear mechanical heterogeneity. Biomaterials 2014, 35, 2411–2419. [8] Dalby, M. J., Gadegaard, N., Oreffo, R. O. C., Harnessing nanotopography and integrin-matrix interactions to influence stem cell fate. Nat. Mater. 2014, 13, 558–569. [9] Treiser, M. D., Yang, E. H., Gordonov, S., Cohen, D. M. et al., Cytoskeleton-based forecasting of stem cell lineage fates. Proc. Natl. Acad. Sci. U.S.A. 2010, 107, 610–615. [10] Khatau, S. B., Hale, C., Stewart-Hutchinson, P., Patel, M. S. et al., A perinuclear actin cap regulates nuclear shape. Proc. Natl. Acad. Sci. U.S.A. 2009, 106, 19017–19022.
Correspondence: Dr. Joe Swift E-mail: [email protected]
The authors declare no financial or commercial conflict of interest.
1514
© 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Cover illustration Special Issue: Stem Cell Engineering. This special issue, edited by Joaquim Cabral and Sean Palecek, contains articles on culturing stem cells, their differentiation as well as on imaging techniques. The cover shows primary hematopoietic stem cells and lineage-committed cells from the bone marrow stained with Hoechst 33342 (blue), CMFDA (green) and CMTPX (red). Stem cells (stained blue) are in contact with neighboring niche cells (stained red and green) that regulate stem cell fate decisions. Image provided by Harley et al.
Biotechnology Journal – list of articles published in the October 2015 issue.
Editorial Stem Cell Engineering
http://dx.doi.org/10.1002/biot.201500531
Research Article High-content imaging with micropatterned multiwell plates reveals influence of cell geometry and cytoskeleton on chromatin dynamics
Commentary Engineering at the microscale: A step towards single-cell analysis of human pluripotent stem cells
http://dx.doi.org/10.1002/biot.201400756
Joaquim M. S. Cabral and Sean P. Palecek
Tiago G. Fernandes
http://dx.doi.org/10.1002/biot.201500307 Commentary Interior decoration: Adapting multiwell plates for high throughput mechanobiology
Ty Harkness, Jason D. McNulty, Ryan Prestil, Stephanie K. Seymour, Tyler Klann, Michael Murrell, Randolph S. Ashton and Krishanu Saha
Research Article Inkjet-bioprinted acrylated peptides and PEG hydrogel with human mesenchymal stem cells promote robust bone and cartilage formation with minimal printhead clogging
Hamish T. J. Gilbert and Joe Swift
Guifang Gao, Tomo Yonezawa, Karen Hubbell, Guohao Dai, and Xiaofeng Cui
http://dx.doi.org/10.1002/biot.201500308
http://dx.doi.org/10.1002/biot.201400635
Review Advanced imaging approaches for regenerative medicine: Emerging technologies for monitoring stem cell fate in vitro and in vivo
Research Article Neural commitment of human pluripotent stem cells under defined conditions recapitulates neural development and generates patient-specific neural cells
Molly E. Kupfer and Brenda M. Ogle
Tiago G. Fernandes, Sofia T. Duarte, Mehrnaz Ghazvini, Cláudia Gaspar, Diana C. Santos, Ana R. Porteira, Gonçalo M. C. Rodrigues, Simone Haupt, Diogo M. Rombo, Judith Armstrong, Ana M. Sebastião, Joost Gribnau, Àngels GarciaCazorla, Oliver Brüstle, Domingos Henrique, Joaquim M. S. Cabral and Maria Margarida Diogo
http://dx.doi.org/10.1002/biot.201400760 Review Engineering the hematopoietic stem cell niche: Frontiers in biomaterial science Ji Sun Choi, Bhushan P. Mahadik and Brendan A. C. Harley
http://dx.doi.org/10.1002/biot.201400758 Research Article Clonal analysis of individual human embryonic stem cell differentiation patterns in microfluidic cultures Darek J. Sikorski, Nicolas J. Caron, Michael VanInsberghe, Hans Zahn, Connie J. Eaves, James M. Piret and Carl L. Hansen
http://dx.doi.org/10.1002/biot.201400751 Research Article A reproducible and versatile system for the dynamic expansion of human pluripotent stem cells in suspension Andreas Elanzew, Annika Sommer, Annette Pusch-Klein, Oliver Brüstle and Simone Haupt
http://dx.doi.org/10.1002/biot.201400757
http://dx.doi.org/10.1002/biot.201500035
© 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.biotechnology-journal.com
Research Article Enzymatic passaging of human embryonic stem cells alters central carbon metabolism and glycan abundance
Research Article Compartment-specific metabolomics for CHO reveals that ATP pools in mitochondria are much lower than in cytosol
Mehmet G. Badur, Hui Zhang and Christian M. Metallo
Jens-Christoph Matuszczyk, Attila Teleki, Jennifer Pfizenmaier and Ralf Takors
http://dx.doi.org/10.1002/biot.201400749 Research Article Spatial and temporal control of cell aggregation efficiently directs human pluripotent stem cells towards neural commitment Cláudia C. Miranda, Tiago G. Fernandes, Jorge F. Pascoal, Simone Haupt, Oliver Brüstle, Joaquim M.S. Cabral and Maria Margarida Diogo
http://dx.doi.org/10.1002/biot.201400846 Research Article Microarray profiling of preselected CHO host cell subclones identifies gene expression patterns associated with increased production capacity
http://dx.doi.org/10.1002/biot.201500060 Research Articel The stage-specific in vitro efficacy of a malaria antigen cocktail provides valuable insights into the development of effective multi-stage vaccines Holger Spiegel, Alexander Boes, Robin Kastilan, Stephanie Kapelski, Güven Edgue, Veronique Beiss, Ivana Chubodova, Matthias Scheuermayer, Gabriele Pradel, Stefan Schillberg, Andreas Reimann and Rainer Fischer
http://dx.doi.org/10.1002/biot.201500055
Eva Harreither, Matthias Hackl, Johannes Pichler, Smriti Shridhar, Norbert Auer, Paweł P. Łabaj, Marcel Scheideler, Michael Karbiener, Johannes Grillari, David P. Kreil and Nicole Borth
http://dx.doi.org/10.1002/biot.201400857
© 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.biotechnology-journal.com
