Flipped Well-Plate Hanging-Drop Technique for Growing Three-Dimensional Tumors

Jeong, Yoon; Tin, Ashley; Irudayaraj, Joseph

doi:10.3389/fbioe.2022.898699

Cited by 16 publications

(12 citation statements)

References 74 publications

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“…PANC-1 3D spheroids were generated by the well-plate flip technique. 47 Briefly, a standard 96-well plate (Thermo Scientific) was used to generate hanging drops that can be formed at the bottom of a flipped well plate. The seeding density of PANC-1 cells varied from 1 × 10 3 to 2 × 10 4 cells per well to assess an optimal concentration of PANC-1 cell densities.…”

Section: Methodsmentioning

confidence: 99%

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Nanocatalase-Based Oxygen-Generating Nanocarriers for Active Oxygen Delivery to Relieve Hypoxia in Pancreatic Cancer

Han

Jeong

Irudayaraj

2022

ACS Appl. Nano Mater.

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Hypoxia of the tumor microenvironment has the potential to give rise to significant radiation and drug resistance that can contribute to tumor recurrence and or low treatment efficacy. If hypoxia can be mitigated, successive treatments can be more efficacious. Herein, we propose a dual oxygen supply strategy afforded by oxygen generation utilizing dextran sulfate-modified catalase as an enzyme in a nano bioreactor that will react with the overexpressed hydrogen peroxide in the tumor microenvironment to generate oxygen. Meanwhile, oxygen loaded onto these nanoparticles could diffuse out of the nanocarriers to deliver a second stage of oxygenation. Our dual oxygen supply platform was found to release oxygen at a sustained rate for over 6 h in PANC-1 pancreatic cancer cells. We further extended our studies to evaluating hypoxia mitigation efficacy in three-dimensional (3D) tumor spheroids fabricated with a flip well plate method. Gene expression studies revealed that the expression of several hypoxia-related genes in both two-dimensional (2D) and 3D culture systems could be restored upon oxygenation to normal levels. The dual oxygen supply platform was synthesized using safe reagents by "green" synthesis and with Food and Drug Administration (FDA)-approved reagents and is expected to have a broad appeal in mitigating hypoxia in cancers as well as in other diseases.

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Section: Methodsmentioning

confidence: 99%

“…PANC-1 3D spheroids were generated by the well-plate flip technique . Briefly, a standard 96-well plate (Thermo Scientific) was used to generate hanging drops that can be formed at the bottom of a flipped well plate.…”

Section: Methodsmentioning

confidence: 99%

Nanocatalase-Based Oxygen-Generating Nanocarriers for Active Oxygen Delivery to Relieve Hypoxia in Pancreatic Cancer

Han

Jeong

Irudayaraj

2022

ACS Appl. Nano Mater.

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“…The hanging-drop technique is widely applied in spheroid and organoid generation due to its simple method of handling, without the requirement of additional equipment, and its applicability of making cocultivation spheroids/organoids with different cell populations [41]. To increase the productivity and standardisation of the method, new devices like hanging-drop plates [42], microfluidic channels [43] and pressure-controlled air chambers [44] for better loading of hanging drops have been recently invented and applied.…”

Section: Self-assembly Culturesmentioning

confidence: 99%

Progress of 3D Organoid Technology for Preclinical Investigations: Towards Human In Vitro Models

Liu

Xu²,

Abraham³

et al. 2022

IJDDP

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Review Progress of 3D Organoid Technology for Preclinical Investigations: Towards Human In Vitro Models Yingjuan Liu *, Honglin Xu, Sabu Abraham, Xin Wang, and Bernard D. Keavney* Division of Cardiovascular Sciences, Faculty of Biology, Medicine and Health, University of Manchester, M13 9PT, UK. * Correspondence: yingjuan.liu@manchester.ac.uk (Yingjuan Liu); bernard.keavney@manchester.ac.uk (Bernard D. Keavney) Received: 1 November 2022 Accepted: 24 November 2022 Published: 21 December 2022 Abstract: Currently, with an increased requirement for new therapeutic strategies, preclinical drug testing or screening platforms have rapidly evolved in recent years. In comparison to traditional 2D cell cultures, 3D organoids or spheroids with or without scaffolds improve the microenvironment of in vitro cultures, advancing the in vitro biological observation and enabling mechanistic studies of drug reactions in the human tissue-like environment. 3D organoids and spheroids are straightforward to produce, and relatively uniform in size and shape. This helps to facilitate high throughput screening requirements. Spheroids and organoids have been applied in anti-cancer drug testing, toxicity evaluations, as well as mechanism studies for variable organ systems, including the intestine, liver, pancreas, brain, and heart. Among 3D cultures of spheroids and organoids, ‘tumour spheroids’ formed by dissociated tumour tissues or cancer cell lines are relatively simple in composition and commonly applied to anticancer drug screening. The ‘healthy organoids’ differentiated from hiPSCs/hESCs are more complex in cell composition, distribution, structure and function with higher similarity to in vivo organs, and have found applications in toxicity tests, personalised medicine, and therapeutic and mechanistic studies. In most cases, the multicellular 3D organoids are more resistant and stable in reaction to stimulations or chemicals in vitro , suggesting more accurate modelling of in vivo responses. Here, we review recent progress in human-origin organoid/spheroid systems and their applications in preclinical studies.

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“…recent work, Jeong et al[21] proposed a flipped well concept that works on the same principal as that of the hanging drop technique. It uses the standard 96-well plate to generate the same contact-free environment as that of the hanging drop technique, but with a larger working volume of more than 1 ml.…”

mentioning

confidence: 99%

“…It uses the standard 96-well plate to generate the same contact-free environment as that of the hanging drop technique, but with a larger working volume of more than 1 ml. The technique generated a large 3D tumor spheroid of over 1.5 mm diameter and stable culture configuration, thus overcoming the technical barrier of low working volume[21].…”

mentioning

confidence: 99%

A particle-based numerical method for modelling of fluid-structure interactions in biomanufacturing processes

Lee¹

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The study of human physiology and target medicine has made a great leap with the discovery of cell culture, an operation under the more general topic of biomanufacturing. One key biomanufacturing technique for the creation of in-vitro threedimensional tissue models and even diseases is the extrusion-based bioprinting, but the associated mechanical stresses can become so high that the cell viability significantly decreases. Therefore, methods that can characterize stresses induced in cell-laden bioinks during the printing process are critical to the advancement of bioprinting, ensuring high efficacy in the biomanufacturing technique. In this dissertation, a particle-based numerical method, commonly known as smoothed particle hydrodynamics (SPH), is developed to emulate the fluid-structure interactions between bioink and cells. To represent the cell deformations and motions, the SPH solver is coupled with an element bending group (EBG) model, which treats the cell as a solid membrane with liquid filling. The method is validated through comparison with experimental data on deformations and transport of a MCF7 cancerous cell through a microchannel. Upon validation, the method was applied on a model extrusion-based bioprinting configuration, considering in addition the use of cytoprotective gel particles to encapsulate the cells in the bioink, thereby maintaining a high cell viability. Using the deformations of the gel particles as boundary conditions, flow-induced stresses on the protective encapsulation can be computed by a separate finite element analysis. Stress analysis suggests the presence of a high stress region near the converging section before the bioprinter nozzle outlet, which is the likely cause of low cell viability in extrusion-based bioprinting. The results also indicate that the protective property from the encapsulating gel particles is due to the substitution of flow-induced stresses with internal stresses on the cells, which can be up to 90% less than the former. The findings from this research demonstrate that the two-dimensional SPH-EBG methodology developed here is a promising tool to predict stresses on cells within bioinks. Such a capability will not only facilitate the design of extrusion-based bioprinters, but also be applicable to other biomanufacturing techniques that involve cell-laden flows.

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Flipped Well-Plate Hanging-Drop Technique for Growing Three-Dimensional Tumors

Cited by 16 publications

References 74 publications

Nanocatalase-Based Oxygen-Generating Nanocarriers for Active Oxygen Delivery to Relieve Hypoxia in Pancreatic Cancer

Nanocatalase-Based Oxygen-Generating Nanocarriers for Active Oxygen Delivery to Relieve Hypoxia in Pancreatic Cancer

Progress of 3D Organoid Technology for Preclinical Investigations: Towards Human In Vitro Models

A particle-based numerical method for modelling of fluid-structure interactions in biomanufacturing processes

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