3-D microwell culture of human embryonic stem cells.
ABSTRACT Human embryonic stem cells (hESCs) have the ability to proliferate indefinitely and differentiate into each of the embryonic cell lineages. Great care is required to maintain undifferentiated hESC cultures since spontaneous differentiation often occurs in culture, presumably resulting from soluble factors, cell-cell contact, and/or cell-matrix signaling. hESC differentiation is typically stimulated via generation of embryoid bodies (EBs) and lineage commitment of individual cells depends upon numerous cues throughout the EB environment, including EB shape and size. Common EB formation protocols, however, produce a very heterogeneous size distribution, perhaps reducing efficiency of directed differentiation. We have developed a 3-D microwell-based method to maintain undifferentiated hESC cultures for weeks without passaging using physical and extracellular matrix patterning constraints to limit colony growth. Over 90% of hESCs cultured in microwells for 2-3 weeks were viable and expressed the hESC transcription marker Oct-4. Upon passaging to Matrigel-coated tissue culture-treated polystyrene dishes (TCPS), microwell cultured hESCs maintained undifferentiated proliferation. Microwell culture also permits formation of hESC colonies with a defined size, which can then be used to form monodisperse EBs. When cultured in this system, hESCs retained pluripotency and self-renewal, and were able to be passaged to standard unconstrained culture conditions.
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ABSTRACT: Human pluripotent stem cells (hPSCs) provide promising resources for regenerating tissues and organs and modeling development and diseases in vitro. To fulfill their promise, the fate, function, and organization of hPSCs need to be precisely regulated in a three-dimensional (3D) environment to mimic cellular structures and functions of native tissues and organs. In the past decade, innovations in 3D culture systems with functional biomaterials have enabled efficient and versatile control of hPSC fate at the cellular level. However, we are just at the beginning of bringing hPSC-based regeneration and development and disease modeling to the tissue and organ levels. In this review, we summarize existing bioengineered culture platforms for controlling hPSC fate and function by regulating inductive mechanical and biochemical cues coexisting in the synthetic cell microenvironment. We highlight recent excitements in developing 3D hPSC-based in vitro tissue and organ models with in vivo-like cellular structures, interactions, and functions. We further discuss an emerging multifaceted mechanotransductive signaling network – with transcriptional coactivators YAP and TAZ at the center stage – that regulate fates and behaviors of mammalian cells, including hPSCs. Future development of 3D biomaterial systems should incorporate dynamically modulated mechanical and chemical properties targeting specific intracellular signaling events leading to desirable hPSC fate patterning and functional tissue formation in 3D.Biomaterials 06/2015; 52. DOI:10.1016/j.biomaterials.2015.01.078 · 8.31 Impact Factor
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ABSTRACT: We describe a reliable preparation of MgAl-layered double hydroxide (MgAl-LDH) micropatterned arrays on gold substrate by combining SO3--terminated self-assembly monolayer and photolithography. The synthesis route is readily extended to prepare LDH arrays on the SO3--terminated polymer-bonded glass substrate amenable for cell imaging. The anion-exchangeable MgAl-LDH micropattern can act both as bio-adhesive region for selective cell adhesion and as nanocarrier for drug molecules to regulate cell behaviors. Quantitative analysis of cell adhesion shows that selective HepG2 cell adhesion and spreading are promoted by the micropatterned MgAl-LDH, and also suppressed by methotrexate drug released from the LDH interlayer galleries.ACS Applied Materials & Interfaces 02/2015; DOI:10.1021/acsami.5b00145 · 5.90 Impact Factor
Nano Today 01/2015; DOI:10.1016/j.nantod.2014.12.002 · 18.43 Impact Factor