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Three-dimensional spheroid culture targeting versatile tissue bioassays using a PDMS-based hanging drop array.

ABSTRACT: Biomaterial-based tissue culture platforms have emerged as useful tools to mimic in vivo physiological microenvironments in experimental cell biology and clinical studies. We describe herein a three-dimensional (3D) tissue culture platform using a polydimethylsiloxane (PDMS)-based hanging drop array (PDMS-HDA) methodology. Multicellular spheroids can be achieved within 24?h and further boosted by incorporating collagen fibrils in PDMS-HDA. In addition, the spheroids generated from different human tumor cells exhibited distinct sensitivities toward drug chemotherapeutic agents and radiation as compared with two-dimensional (2D) cultures that often lack in vivo-like biological insights. We also demonstrated that multicellular spheroids may enable key hallmarks of tissue-based bioassays, including drug screening, tumor dissemination, cell co-culture, and tumor invasion. Taken together, these results offer new opportunities not only to achieve the active control of 3D multicellular spheroids on demand, but also to establish a rapid and cost-effective platform to study anti-cancer therapeutics and tumor microenvironments.

SUBMITTER: Kuo CT

PROVIDER: S-EPMC5491519 | biostudies-literature | 2017 Jun

REPOSITORIES: biostudies-literature

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Three-dimensional spheroid culture targeting versatile tissue bioassays using a PDMS-based hanging drop array.

Kuo Ching-Te CT Wang Jong-Yueh JY Lin Yu-Fen YF Wo Andrew M AM Chen Benjamin P C BPC Lee Hsinyu H

Scientific reports 20170629 1

Biomaterial-based tissue culture platforms have emerged as useful tools to mimic in vivo physiological microenvironments in experimental cell biology and clinical studies. We describe herein a three-dimensional (3D) tissue culture platform using a polydimethylsiloxane (PDMS)-based hanging drop array (PDMS-HDA) methodology. Multicellular spheroids can be achieved within 24 h and further boosted by incorporating collagen fibrils in PDMS-HDA. In addition, the spheroids generated from different huma ...[more]

PMID: 28663555

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Project description:Targeted intracellular delivery of biomolecules and therapeutic cargo enables the controlled manipulation of cellular processes. Laser-based optoporation has emerged as a versatile, non-invasive technique that employs light-based transient physical disruption of the cell membrane and achieves high transfection efficiency with low cell damage. Testing of the delivery efficiency of optoporation-based techniques has been conducted on single cells in monolayers, but its applicability in three-dimensional (3D) cell clusters/spheroids has not been explored. Cancer cells grown as 3D tumor spheroids are widely used in anti-cancer drug screening and can be potentially employed for testing delivery efficiency. Towards this goal, we demonstrated the optoporation-based high-throughput intracellular delivery of a model fluorescent cargo (propidium iodide, PI) within 3D SiHa human cervical cancer spheroids. To enable this technique, nano-spiked core-shell gold-coated polystyrene nanoparticles (ns-AuNPs) with a high surface-to-volume ratio were fabricated. ns-AuNPs exhibited high electric field enhancement and highly localized heating at an excitation wavelength of 680 nm. ns-AuNPs were co-incubated with cancer cells within hanging droplets to enable the rapid aggregation and assembly of spheroids. Nanosecond pulsed-laser excitation at the optimized values of laser fluence (45 mJ cm-2), pulse frequency (10 Hz), laser exposure time (30 s), and ns-AuNP concentration (5 × 1010 particles per ml) resulted in the successful delivery of PI dye into cancer cells. This technique ensured high delivery efficiency (89.6 ± 2.8%) while maintaining high cellular viability (97.4 ± 0.4%), thereby validating the applicability of this technique for intracellular delivery. The optoporation-based strategy can enable high-throughput single cell manipulation, is scalable towards larger 3D tissue constructs, and may provide translational benefits for the delivery of anti-cancer therapeutics to tumors.

Project description:Background:Mass production of exosomes is a prerequisite for their commercial utilization. This study investigated whether three-dimensional (3D) spheroid culture of mesenchymal stem cells (MSCs) could improve the production efficiency of exosomes and if so, what was the mechanism involved. Methods:We adopted two models of 3D spheroid culture using the hanging-drop (3D-HD) and poly(2-hydroxyethyl methacrylate) (poly-HEMA) coating methods (3D-PH). The efficiency of exosome production from MSCs in the 3D spheroids was compared with that of monolayer culture in various conditions. We then investigated the mechanism of the 3D spheroid culture-induced increase in exosome production. Results:The 3D-HD formed a single larger spheroid, while the 3D-PH formed multiple smaller ones. However, MSCs cultured on both types of spheroids produced significantly more exosomes than those cultured in conventional monolayer culture (2D). We then investigated the cause of the increased exosome production in terms of hypoxia within the 3D spheroids, high cell density, and non-adherent cell morphology. With increasing spheroid size, the efficiency of exosome production was the largest with the least amount of cells in both 3D-HD and 3D-PH. An increase in cell density in 2D culture (2D-H) was less efficient in exosome production than the conventional, lower cell density, 2D culture. Finally, when cells were plated at normal density on the poly-HEMA coated spheroids (3D-N-PH); they formed small aggregates of less than 10 cells and still produced more exosomes than those in the 2D culture when plated at the same density. We also found that the expression of F-actin was markedly reduced in the 3D-N-PH culture. Conclusion:These results suggested that 3D spheroid culture produces more exosomes than 2D culture and the non-adherent round cell morphology itself might be a causative factor. The result of the present study could provide useful information to develop an optimal process for the mass production of exosomes.

Dataset Information

Three-dimensional spheroid culture targeting versatile tissue bioassays using a PDMS-based hanging drop array.

Publications

Three-dimensional spheroid culture targeting versatile tissue bioassays using a PDMS-based hanging drop array.

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