Multiplexed, high-throughput analysis of 3D microtissue suspensions.
Three-dimensional (3D) tissue models have significantly improved our understanding of structure/function relationships and promise to lead to new advances in regenerative medicine. However, despite the expanding diversity of 3D tissue fabrication methods, approaches for functional assessment have been relatively limited. Here, we describe the fabrication of microtissue (μ-tissue) suspensions and their quantitative evaluation with techniques capable of analyzing large sample numbers and performing multiplexed parallel analysis. We applied this platform to 3D μ-tissues representing multiple stages of liver development and disease including: embryonic stem cells, bipotential hepatic progenitors, mature hepatocytes, and hepatoma cells photoencapsulated in polyethylene glycol hydrogels. Multiparametric μ-tissue cytometry enabled quantitation of fluorescent reporter expression within populations of intact μ-tissues (n≥ 10²-10³) and sorting-based enrichment of subsets for subsequent studies. Further, 3D μ-tissues could be implanted in vivo, respond to systemic stimuli, retrieved and quantitatively assessed. In order to facilitate multiplexed 'pooled' experimentation, fluorescent labeling strategies were developed and utilized to investigate the impact of μ-tissue composition and exposure to soluble factors. In particular, examination of drug/gene interactions on collections of 3D hepatoma μ-tissues indicated synergistic influence of doxorubicin and siRNA knockdown of the anti-apoptotic gene BCL-XL. Collectively, these studies highlight the broad utility of μ-tissue suspensions as an enabling approach for high n, populational analysis of 3D tissue biology in vitro and in vivo.
Chen
Alice A
AA
Massachusetts Institute of Technology, Cambridge, 02139, USA.
Underhill
Gregory H
GH
Bhatia
Sangeeta N
SN
D000818:Animals; D001672:Biocompatible Materials / Q000737:chemistry; D018929:Cell Culture Techniques / Q000379:methods; D002454:Cell Differentiation / Q000502:physiology; D002470:Cell Survival / Q000187:drug effects / Q000235:genetics; D003131:Combined Modality Therapy; D004317:Doxorubicin / Q000494:pharmacology / Q000627:therapeutic use; D004318:Doxycycline / Q000494:pharmacology; D053595:Embryonic Stem Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism / Q000637:transplantation; D005260:Female; D005347:Fibroblasts / Q000166:cytology; D005434:Flow Cytometry / Q000379:methods; D005456:Fluorescent Dyes / Q000737:chemistry / Q000378:metabolism; D015870:Gene Expression / Q000187:drug effects / Q000235:genetics; D017930:Genes, Reporter / Q000235:genetics; D024002:HMGB Proteins / Q000235:genetics / Q000378:metabolism; D056945:Hep G2 Cells; D022781:Hepatocytes / Q000166:cytology; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D020100:Hydrogels / Q000737:chemistry; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D055616:Microtechnology / Q000379:methods*; D011092:Polyethylene Glycols / Q000737:chemistry; D045663:Quantum Dots; D034741:RNA, Small Interfering / Q000235:genetics / Q000627:therapeutic use; D051381:Rats; D011917:Rats, Inbred Lew; D055760:SOXF Transcription Factors / Q000235:genetics / Q000378:metabolism; D033581:Stem Cell Transplantation; D023822:Tissue Engineering / Q000379:methods*; D051020:bcl-X Protein / Q000235:genetics
C494811:BCL2L1 protein, human; D001672:Biocompatible Materials; D005456:Fluorescent Dyes; D024002:HMGB Proteins; D020100:Hydrogels; D034741:RNA, Small Interfering; D055760:SOXF Transcription Factors; C511606:Sox17 protein, mouse; D051020:bcl-X Protein; C437167:poly(ethylene glycol)diacrylate; D011092:Polyethylene Glycols; D004317:Doxorubicin; D004318:Doxycycline
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