Microfluidic Generation of Gradient Hydrogels to Modulate Hematopoietic Stem Cell Culture Environment

The bone marrow provides spatially and temporally variable signals that impact the behavior of hematopoietic stem cells (HSCs). While multiple biomolecular signals and bone marrow cell populations have been proposed as key regulators of HSC fate, new tools are required to probe their importance and mechanisms of action. Here, a novel method based on a microfluidic mixing platform to create small volume, 3D hydrogel constructs containing overlapping patterns of cell and matrix constituents inspired by the HSC niche is described. This approach is used to generate hydrogels containing opposing gradients of fluorescent microspheres, MC3T3-E1 osteoblasts, primary murine hematopoietic stem and progenitor cells (HSPCs), and combinations thereof in a manner independent of hydrogel density and cell/particle size. Three different analytical methods are described to characterize local properties of these hydrogels at multiple scales: 1) whole construct fluorescent analysis; 2) multi-photon imaging of individual cells within the construct; 3) retrieval of discrete sub-regions from the hydrogel post-culture. The approach reported here allows the creation of stable gradients of cell and material cues within a single, optically translucent 3D biomaterial to enable a range of investigations regarding how microenvironmental signals impact cell fate.

[1]  Abraham D Stroock,et al.  Investigation of the staggered herringbone mixer with a simple analytical model , 2004, Philosophical Transactions of the Royal Society of London. Series A: Mathematical, Physical and Engineering Sciences.

[2]  Mehmet Toner,et al.  Microfluidic system for measuring neutrophil migratory responses to fast switches of chemical gradients. , 2006, Lab on a chip.

[3]  David E Reichert,et al.  Microfluidic labeling of biomolecules with radiometals for use in nuclear medicine. , 2010, Lab on a chip.

[4]  J. Manis,et al.  Focal Adhesion Kinase Regulates the Localization and Retention of Pro-B Cells in Bone Marrow Microenvironments , 2013, The Journal of Immunology.

[5]  C. Beta,et al.  Chemotaxis in microfluidic devices--a study of flow effects. , 2008, Lab on a chip.

[6]  R. Jain,et al.  Convection and diffusion measurements using fluorescence recovery after photobleaching and video image analysis: in vitro calibration and assessment. , 1990, Microvascular research.

[7]  W. Rappel,et al.  Dictyostelium discoideum chemotaxis: threshold for directed motion. , 2006, European journal of cell biology.

[8]  A. Trumpp,et al.  Bone-marrow haematopoietic-stem-cell niches , 2006, Nature Reviews Immunology.

[9]  L. Hennighausen,et al.  SOCS3 protein developmentally regulates the chemokine receptor CXCR4-FAK signaling pathway during B lymphopoiesis. , 2007, Immunity.

[10]  U. Demirci,et al.  Bioprinting for stem cell research. , 2013, Trends in biotechnology.

[11]  B. Porse,et al.  Activation of the canonical Wnt pathway leads to loss of hematopoietic stem cell repopulation and multilineage differentiation block , 2006, Nature Immunology.

[12]  R. Pego,et al.  Analysis of binding reactions by fluorescence recovery after photobleaching. , 2004, Biophysical journal.

[13]  Won Gu Lee,et al.  Generating nonlinear concentration gradients in microfluidic devices for cell studies. , 2011, Analytical chemistry.

[14]  A. Trumpp,et al.  The bone marrow stem cell niche grows up: mesenchymal stem cells and macrophages move in , 2011, The Journal of experimental medicine.

[15]  J. Lippincott-Schwartz,et al.  Studying protein dynamics in living cells , 2001, Nature Reviews Molecular Cell Biology.

[16]  A. Mikos,et al.  Investigating the role of hematopoietic stem and progenitor cells in regulating the osteogenic differentiation of mesenchymal stem cells in vitro , 2011, Journal of orthopaedic research : official publication of the Orthopaedic Research Society.

[17]  I. Weissman,et al.  Hematopoietic stem cells and lymphoid progenitors express different Ikaros isoforms, and Ikaros is localized to heterochromatin in immature lymphocytes. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[18]  Adam J. Engler,et al.  Stiffness Gradients Mimicking In Vivo Tissue Variation Regulate Mesenchymal Stem Cell Fate , 2011, PloS one.

[19]  I. Weissman,et al.  Stem Cells Units of Development, Units of Regeneration, and Units in Evolution , 2000, Cell.

[20]  T. Suda,et al.  Knockdown of N-cadherin suppresses the long-term engraftment of hematopoietic stem cells. , 2010, Blood.

[21]  P. Janmey,et al.  Fibrin gels and their clinical and bioengineering applications , 2009, Journal of The Royal Society Interface.

[22]  B N Chichkov,et al.  Adipogenic differentiation of laser-printed 3D tissue grafts consisting of human adipose-derived stem cells , 2011, Biofabrication.

[23]  Douglas A Lauffenburger,et al.  Microarchitecture of three-dimensional scaffolds influences cell migration behavior via junction interactions. , 2008, Biophysical journal.

[24]  Brian A. Aguado,et al.  Improving viability of stem cells during syringe needle flow through the design of hydrogel cell carriers. , 2012, Tissue engineering. Part A.

[25]  S. E. Jacobsen,et al.  Identification of Lin(-)Sca1(+)kit(+)CD34(+)Flt3- short-term hematopoietic stem cells capable of rapidly reconstituting and rescuing myeloablated transplant recipients. , 2005, Blood.

[26]  A. Aplin,et al.  Rnd3 regulation of the actin cytoskeleton promotes melanoma migration and invasive outgrowth in three dimensions. , 2009, Cancer research.

[27]  Nathan C Boles,et al.  Mouse hematopoietic stem cell identification and analysis , 2009, Cytometry. Part A : the journal of the International Society for Analytical Cytology.

[28]  S. Nishikawa,et al.  In vivo and in vitro stem cell function of c-kit- and Sca-1-positive murine hematopoietic cells. , 1992, Blood.

[29]  G. Whitesides,et al.  Generation of Solution and Surface Gradients Using Microfluidic Systems , 2000 .

[30]  Takashi Nagasawa,et al.  Cellular niches controlling B lymphocyte behavior within bone marrow during development. , 2004, Immunity.

[31]  Ralph G Nuzzo,et al.  Fabrication of patterned multicomponent protein gradients and gradient arrays using microfluidic depletion. , 2003, Analytical chemistry.

[32]  R. Bonnecaze,et al.  Extracellular matrix stiffness and architecture govern intracellular rheology in cancer. , 2009, Biophysical journal.

[33]  Gideon A. Rodan,et al.  Control of osteoblast function and regulation of bone mass , 2003, Nature.

[34]  Roger D Kamm,et al.  In vitro 3D collective sprouting angiogenesis under orchestrated ANG-1 and VEGF gradients. , 2011, Lab on a chip.

[35]  Noo Li Jeon,et al.  Generation of stable complex gradients across two-dimensional surfaces and three-dimensional gels. , 2007, Langmuir : the ACS journal of surfaces and colloids.

[36]  Roger D Kamm,et al.  A high-throughput microfluidic assay to study neurite response to growth factor gradients. , 2011, Lab on a chip.

[37]  Linheng Li,et al.  The stem cell niches in bone. , 2006, The Journal of clinical investigation.

[38]  Jaesoon Choi,et al.  Cellular behavior in micropatterned hydrogels by bioprinting system depended on the cell types and cellular interaction. , 2013, Journal of bioscience and bioengineering.

[39]  David Juncker,et al.  Formation of gradients of proteins on surfaces with microfluidic networks , 2000 .

[40]  Ian A. White,et al.  Endothelial cells are essential for the self-renewal and repopulation of Notch-dependent hematopoietic stem cells. , 2010, Cell stem cell.

[41]  D. Leckband,et al.  Cell migration and polarity on microfabricated gradients of extracellular matrix proteins. , 2006, Langmuir : the ACS journal of surfaces and colloids.

[42]  L. Kaufman,et al.  Rheology and confocal reflectance microscopy as probes of mechanical properties and structure during collagen and collagen/hyaluronan self-assembly. , 2009, Biophysical journal.

[43]  H. Fischer,et al.  Three-dimensional printing of stem cell-laden hydrogels submerged in a hydrophobic high-density fluid , 2012, Biofabrication.

[44]  F. Guillemot,et al.  Laser-assisted bioprinting for creating on-demand patterns of human osteoprogenitor cells and nano-hydroxyapatite , 2011, Biofabrication.

[45]  S. Rafii,et al.  The bone marrow vascular niche: home of HSC differentiation and mobilization. , 2005, Physiology.

[46]  M. Magnusson,et al.  Hematopoietic stem cells in transit--where's the niche? , 2008, Cell stem cell.

[47]  A. Khademhosseini,et al.  Convection-driven generation of long-range material gradients. , 2010, Biomaterials.

[48]  Feng Xu,et al.  Embryonic stem cell bioprinting for uniform and controlled size embryoid body formation. , 2011, Biomicrofluidics.

[49]  Joseph Suhan,et al.  Bioprinting of Growth Factors onto Aligned Sub-micron Fibrous Scaffolds for Simultaneous Control of Cell Differentiation and Alignment , 2022 .

[50]  James G McNally,et al.  FRAP analysis of binding: proper and fitting. , 2005, Trends in cell biology.

[51]  D. Leckband,et al.  Cadherin and integrin regulation of epithelial cell migration. , 2009, Langmuir : the ACS journal of surfaces and colloids.

[52]  Qi Wen,et al.  The hard life of soft cells. , 2009, Cell motility and the cytoskeleton.

[53]  B. Chung,et al.  Generation of stable concentration gradients in 2D and 3D environments using a microfluidic ladder chamber , 2007, Biomedical microdevices.

[54]  Karoly Jakab,et al.  Tissue engineering by self-assembly and bio-printing of living cells , 2010, Biofabrication.

[55]  A. Khademhosseini,et al.  Microfluidic synthesis of composite cross‐gradient materials for investigating cell–biomaterial interactions , 2011, Biotechnology and bioengineering.

[56]  J. Neefjes,et al.  From fixed to FRAP: measuring protein mobility and activity in living cells , 2001, Nature Cell Biology.

[57]  Jonas Jarvius,et al.  Endothelial Cell Migration in Stable Gradients of Vascular Endothelial Growth Factor A and Fibroblast Growth Factor 2 , 2008, Journal of Biological Chemistry.

[58]  Younghun Jung,et al.  Osteoblasts support B-lymphocyte commitment and differentiation from hematopoietic stem cells. , 2007, Blood.

[59]  Deborah E Leckband,et al.  Regiospecific control of protein expression in cells cultured on two-component counter gradients of extracellular matrix proteins. , 2005, Langmuir : the ACS journal of surfaces and colloids.

[60]  D. Kent,et al.  High-throughput analysis of single hematopoietic stem cell proliferation in microfluidic cell culture arrays , 2011, Nature Methods.

[61]  Eric D. Miller,et al.  Microenvironments Engineered by Inkjet Bioprinting Spatially Direct Adult Stem Cells Toward Muscle‐ and Bone‐Like Subpopulations , 2008, Stem cells.