Reproducible generation of human liver organoids (HLOs) on a pillar plate platform <i>via</i> microarray 3D bioprinting

Shrestha, Sunil; Lekkala, Vinod Kumar Reddy; Acharya, Prabha; Kang, Soo-Yeon; Vanga, Manav Goud; Lee, Moo-Yeal

doi:10.1039/d4lc00149d

Cited by 10 publications

(2 citation statements)

References 65 publications

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“…This was achieved by carefully aspirating most of the EB formation medium and then adding the NIM. The NIM is composed of DMEM-F12, 1% (vol/vol) N2 supplement (ThermoFisher, 17502048), 1% (vol/vol) GlutaMAX, 1% (vol/vol) MEM-NEAA, and 1 µg/mL heparin (MilliporeSigma, H3149) (18). NEs were cultured until day 7, after which they were transferred to a 36PillarPlate preloaded with 5 µL of Matrigel.…”

Section: Neuroectoderm (Ne) Formationmentioning

confidence: 99%

“…On day 7, NEs in the ULA 384-well plate were transferred to the 36PillarPlate (Bioprinting Laboratories Inc., 36-01-00) by a sandwiching and inverting method 15 . The 36PillarPlate, manufaced by injection molding of polystyrene, feastures a 6 x 6 array of pillars with a 4.5 mm pillar-to-pillar distance, 11.6 mm pillar height, and 2.5 mm outer and 1.5 mm inner diameter of pillars 13,14,18 . Each pillar is uniquely designed with sidewalls and slits suitable for culturing a single NE or organoid encapsulated in Matrigel.…”

Section: Ne Transfer From the Ula 384-well Plate To The 36pillarplate...mentioning

confidence: 99%

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Cryopreservation of neuroectoderm on a pillar plate andin situdifferentiation into human brain organoids

Zolfaghar,

Acharya,

Joshi

et al. 2024

Preprint

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Cryopreservation in cryovials extends cell storage at low temperatures, and advances in organoid cryopreservation improve reproducibility and reduce generation time. However, cryopreserving human organoids presents challenges due to the limited diffusion of cryoprotective agents (CPAs) into the organoid core and the potential toxicity of these agents. To overcome these obstacles, we developed a cryopreservation technique using a pillar plate platform. To illustrate cryopreservation application to human brain organoids (HBOs), early-stage HBOs were produced by differentiating induced pluripotent stem cells (iPSCs) into neuroectoderm (NEs) in an ultralow atachement (ULA) 384-well plate. These NEs were transferred and encapsulated in Matrigel on the pillar plate. The early-stage HBOs on the pillar plate were exposed to four commercially available CPAs, including PSC cryopreservation kit, CryoStor CS10, 3dGRO, and 10% DMSO, before being frozen overnight at -80C and subsequently stored in a liquid nitrogen dewar. We examined the impact of CPA type, organoid size, and CPA exposure duration on cell viability post-thaw. Additionally, the differentiation of early-stage HBOs on the pillar plate was assessed using RT-qPCR and immunofluorescence staining. The PSC cryopreservation kit proved to be the least toxic for preserving these HBOs on the pillar plate. Notably, smaller HBOs showed higher cell viability post-cryopreservation than larger ones. An incubation period of 80 minutes with the PSC kit was essential to ensure optimal CPA diffusion into HBOs with a diameter of 400 - 600 um. These cryopreserved early-stage HBOs successfully matured over 30 days, exhibiting gene expression patterns akin to non-cryopreserved HBOs. The cryopreserved early-stage HBOs on the pillar plate maintained high viability after thawing and successfully differentiated into mature HBOs. This on-chip cryopreservation method could extend to other small organoids, by integrating cryopreservation, thawing, culturing, staining, rinsing, and imaging processes within a single system, thereby preserving the 3D structure of the organoids.

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Section: Neuroectoderm (Ne) Formationmentioning

confidence: 99%

Section: Ne Transfer From the Ula 384-well Plate To The 36pillarplate...mentioning

confidence: 99%

Cryopreservation of neuroectoderm on a pillar plate andin situdifferentiation into human brain organoids

Zolfaghar,

Acharya,

Joshi

et al. 2024

Preprint

Self Cite

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Integrating 3D Bioprinting and Organoids to Better Recapitulate the Complexity of Cellular Microenvironments for Tissue Engineering

Hu,

Zhu,

Cui

et al. 2024

Adv Healthcare Materials

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Organoids, with their capacity to mimic the structures and functions of human organs, have gained significant attention for simulating human pathophysiology and have been extensively investigated in the recent past. Additionally, 3D bioprinting, as an emerging bio‐additive manufacturing technology, offers the potential for constructing heterogeneous cellular microenvironments, thereby promoting advancements in organoid research. In this review, the latest developments in 3D bioprinting technologies aimed at enhancing organoid engineering are introduced. The commonly used bioprinting methods and materials for organoids, with a particular emphasis on the potential advantages of combining 3D bioprinting with organoids are summarized. These advantages include achieving high cell concentrations to form large cellular aggregates, precise deposition of building blocks to create organoids with complex structures and functions, and automation and high throughput to ensure reproducibility and standardization in organoid culture. Furthermore, this review provides an overview of relevant studies from recent years and discusses the current limitations and prospects for future development.

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Organoids: development and applications in disease models, drug discovery, precision medicine, and regenerative medicine

Yao,

Cheng,

Pan

et al. 2024

MedComm

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Organoids are miniature, highly accurate representations of organs that capture the structure and unique functions of specific organs. Although the field of organoids has experienced exponential growth, driven by advances in artificial intelligence, gene editing, and bioinstrumentation, a comprehensive and accurate overview of organoid applications remains necessary. This review offers a detailed exploration of the historical origins and characteristics of various organoid types, their applications—including disease modeling, drug toxicity and efficacy assessments, precision medicine, and regenerative medicine—as well as the current challenges and future directions of organoid research. Organoids have proven instrumental in elucidating genetic cell fate in hereditary diseases, infectious diseases, metabolic disorders, and malignancies, as well as in the study of processes such as embryonic development, molecular mechanisms, and host–microbe interactions. Furthermore, the integration of organoid technology with artificial intelligence and microfluidics has significantly advanced large‐scale, rapid, and cost‐effective drug toxicity and efficacy assessments, thereby propelling progress in precision medicine. Finally, with the advent of high‐performance materials, three‐dimensional printing technology, and gene editing, organoids are also gaining prominence in the field of regenerative medicine. Our insights and predictions aim to provide valuable guidance to current researchers and to support the continued advancement of this rapidly developing field.

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Reproducible generation of human liver organoids (HLOs) on a pillar plate platform via microarray 3D bioprinting

Abstract: Human liver organoids (HLOs) hold significant potential for recapitulating the architecture and function of liver tissues in vivo. However, conventional culture methos of HLOs, forming Matrigel domes in 6-/24-well plates,...

Cited by 10 publications

References 65 publications

Cryopreservation of neuroectoderm on a pillar plate andin situdifferentiation into human brain organoids

Cryopreservation of neuroectoderm on a pillar plate andin situdifferentiation into human brain organoids

Integrating 3D Bioprinting and Organoids to Better Recapitulate the Complexity of Cellular Microenvironments for Tissue Engineering

Organoids: development and applications in disease models, drug discovery, precision medicine, and regenerative medicine

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