Article

3-D microwell culture of human embryonic stem cells

Department of Chemical and Biological Engineering, University of Wisconsin–Madison, Madison, Wisconsin, United States
Biomaterials (Impact Factor: 8.56). 01/2007; 27(36):6032-42. DOI: 10.1016/j.biomaterials.2006.07.012
Source: PubMed

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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    • "Such micropatterning technique was also recently applied for controlling EB size for large-scale hPSC production using bioreactors [61]. Instead of using micropatterning, Mohr et al. developed 3D microwell structures of different sizes (with a lateral dimension of 100e500 mm and a height of 120 mm) to control EB size [62]. The highest percentage of contracting EBs undergoing cardiogenesis was observed in microwells of an intermediate size (300 mm). "
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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. Copyright © 2015 Elsevier Ltd. All rights reserved.
    Full-text · Article · Jun 2015 · Biomaterials
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    • "It has been reported that 3D culture is important for the acquisition of mature IPCs (Takeuchi et al., 2014). A 3D culture is advantageous to imitate the in vivo micro environment by enhancing cell-cell and cell-matrix interactions and subsequent cell signaling (Wang et al., 2007; Grayson et al., 2004; Schmeichel and Bissell, 2003; Mohr et al., 2006) To date, a variety of 3D cell culture systems have been developed and adopted for directing stem cell differentiation into various lineages (Levenberg et al., 2003; Liu et al., 2013; Mohr et al., 2006). "
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    ABSTRACT: Currently, culture and growth keratinocytes are important stages in achieving a reliable and reproducible skin tissue. In the present study, two different methods, enzymatic and explant methods, for keratinocytes isolation from human foreskin were compared. Foreskins were cut into 2-3 mm pieces and placed in trypsin at 4°C overnight for separation of the epidermis from the dermis. Subsequently, these samples were divided into two groups: i) Keratinocytes separated from the epidermis by trypsin and ii) by the explant method. These keratinocytes were divided into two groups: i) With no feeder layer and ii) onto a type I collagen scaffold. The cells were evaluated using immunocytochemistry and 4',6-diamidine-2'-phenylindole dihydrochloride (DAPI) staining. In the enzymatic treatment, after 7-10 days no attached cells were found in the cell culture dishes. In the explant method, keratinocytes were separated after ~24 h, attached rapidly and formed big colonies into a collagen scaffold. In the absence of a feeder layer, small colonies were developed with rapid loss of proliferation within 2-3 days. Keratinocytes showed positive immunoreactivity for the pan-cytokeratin marker and keratinocytes' nuclei were clearly observed. This method could be applied and developed as a component of skin substitutes to treat burns and wounds and also in laboratory testing.
    Full-text · Article · Mar 2015
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    • "Such micropatterning technique was also recently applied for controlling EB size for large-scale hPSC production using bioreactors [61]. Instead of using micropatterning, Mohr et al. developed 3D microwell structures of different sizes (with a lateral dimension of 100e500 mm and a height of 120 mm) to control EB size [62]. The highest percentage of contracting EBs undergoing cardiogenesis was observed in microwells of an intermediate size (300 mm). "
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    ABSTRACT: During embryogenesis and tissue maintenance and repair in an adult organism, a myriad of stem cells are regulated by their surrounding extracellular matrix (ECM) enriched with tissue/organ-specific nanoscale topographical cues to adopt different fates and functions. Attributed to their capability of self-renewal and differentiation into most types of somatic cells, stem cells also hold tremendous promise for regenerative medicine and drug screening. However, a major challenge remains as to achieve fate control of stem cells in vitro with high specificity and yield. Recent exciting advances in nanotechnology and materials science have enabled versatile, robust, and large-scale stem cell engineering in vitro through developments of synthetic nanotopographical surfaces mimicking topological features of stem cell niches. In addition to generating new insights for stem cell biology and embryonic development, this effort opens up unlimited opportunities for innovations in stem cell-based applications. This review is therefore to provide a summary of recent progress along this research direction, with perspectives focusing on emerging methods for generating nanotopographical surfaces and their applications in stem cell research. Furthermore, we provide a review of classical as well as emerging cellular mechano-sensing and -transduction mechanisms underlying stem cell nanotopography sensitivity and also give some hypotheses in regard to how a multitude of signaling events in cellular mechanotransduction may converge and be integrated into core pathways controlling stem cell fate in response to extracellular nanotopography.
    Full-text · Article · Jan 2015 · Nano Today
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