| Increase developmental maturity of pluripotent stem cell–derived organoids |
Long-term culture (Nicholas et al., 2013; Takasato et al., 2016; Tzatzalos et al., 2016) |
Stem cells are highly sensitive to culture conditions, impeding robust protocols |
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In vivo maturation (Huch et al., 2013; Watson et al., 2014; Takebe et al., 2015) |
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Mechanical or electrical conditioning of muscle and cartilage (Ruan et al., 2016) |
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Improved culture conditions and differentiation protocols |
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Acceleration by small molecules (Chambers et al., 2012) |
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| Source culturable, age-varied human cells |
Surgical discards from elective surgery, transplant trimmings, and tissue peripheral to tumors |
Hard to source certain tissues, especially healthy, culturable adult cells |
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Cells adapt to culture and are not infinitely renewable |
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Nontumor peripheral tissue may differ from healthy tissue |
| In vitro aging to model the effects of specific age-related lesions and provide a source of artificially aged cells |
Induced senescence by DNA damage or environmental stress (Busuttil et al., 2003; Parrinello et al., 2005; Nasto et al., 2013) |
Controversial which treatments best phenocopy aging |
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Long-term culture (Dos Santos et al., 2015) |
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Progeria mutations (Liu et al., 2011; Zhang et al., 2011; Miller et al., 2013) |
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Direct reprogramming of aged cells (Mertens et al., 2015) |
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| Mimic the effects of the aged ECM in vitro |
ECM from aged donors (Gullapalli et al., 2005; Williams et al., 2014; Stearns-Reider et al., 2017) |
Controversial which treatments best phenocopy aging |
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Glycation crosslinking (Rodriguez-Teja et al., 2015) |
ECM extraction from tissues can alter its mechanical properties, microstructure, and composition |
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Enzymatic crosslinking (Levental et al., 2009) |
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| Develop aging-relevant experimental readouts |
Epigenetic clock (Hannum et al., 2013; Horvath, 2013) |
Unclear which signs of aging are most important |
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Mutational analysis (Blokzijl et al., 2016) |
Require signs of aging that change appreciably across the span of an experiment |
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Protein/DNA oxidation |
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Protein aggregation |
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Tissue-specific functional assays |
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| Long-term maintenance of organoids in a stable, growth-arrested state |
Improved culture media |
Relatively few published maintenance conditions |
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Improved ECM and bioreactors |
Vetting culture conditions is lengthy |
| Complete modeling of whole organs and physiological systems |
Co-culture with immune cells, stromal cells, and microbiome bacteria (Parrinello et al., 2005; Engevik et al., 2015; Plaks et al., 2015) |
Few good techniques for sophisticated organoid construction |
| Increase throughput and reproducibility |
Vascularization (Auger et al., 2013) |
Powerful techniques are often cumbersome and low-throughput |
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Innervation (Workman et al., 2017) |
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In vivo implantation (Watson et al., 2014; Takebe et al., 2015) |
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Microfluidic access to apical/basal fluid reservoirs and fluid transport between organ systems (Vernetti et al., 2017) |
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Organ-specific ECM (Voytik-Harbin et al., 2007; Zhang et al., 2009; O’Brien et al., 2010) |
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Defined artificial ECMs may decrease lot-to-lot variability (Gjorevski et al., 2016) |
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Growth factor distribution within ECM gels for spatial control over growth and differentiation (Wylie et al., 2011) |
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Cell patterning for control over initial organoid shape and composition (Nelson et al., 2008; Todhunter et al., 2015) |
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3D-printed gels amenable to perfusion with control over large-scale tissue structure (Kolesky et al., 2016) |
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Morphological screening and sorting to enrich for correctly formed organoids (Arora et al., 2017) |
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