Alginate core-shell beads for simplified three-dimensional tumor spheroid culture and drug screening

Abstract

We demonstrate that when using cell-laden core-shell hydrogel beads to support the generation of tumor spheroids, the shell structure reduces the out-of-bead and monolayer cell proliferation that occurs during long-term culture of tumor cells within core-only alginate beads. We fabricate core-shell beads in a two-step process using simple, one-layer microfluidic devices. Tumor cells encapsulated within the bead core will proliferate to form multicellular aggregates which can serve as three-dimensional (3-D) models of tumors in drug screening. Encapsulation in an alginate shell increased the time that cells could be maintained in three-dimensional culture for MCF-7 breast cancer cells prior to out-of-bead proliferation, permitting formation of spheroids over a period of 14 days without the need move the cell-laden beads to clean culture flasks to separate beads from underlying monolayers. Tamoxifen and docetaxel dose response shows decreased toxicity for multicellular aggregates in three-dimensional core-shell bead culture compared to monolayer. Using simple core-only beads gives mixed monolayer and 3-D culture during drug screening, and alters the treatment result compared to the 3-D core-shell or the 2-D monolayer groups, as measured by standard proliferation assay. By preventing the out-of-bead proliferation and subsequent monolayer formation that is observed with core-only beads, the core-shell structure can obviate the requirement to transfer the beads to a new culture flask during drug screening, an important consideration for cell-based drug screening and drugs which have high multicellular resistance index.

Inventions and Innovations in Preclinical Platforms for Cancer Research

“…Droplet uniformity can be enhanced with CS/Gel/CM double-emulsion technique which entraps the cells firmly within the droplet. It is facilitated by encapsulating the cell-containing core droplet within an alginate hydrogel shell [82,86] that acts as an impermeable barrier with respect to the cells.…”

Multiphase microfluidic synthesis of micro- and nanostructures for pharmaceutical applications

“…Various cells have been encapsulated within microgels, such as MCF-7 breast cancer cells (Yu et al, 2015), mesenchymal stem cells (Utech et al, 2015), human umbilical vein endothelial cells , human epithelial carcinoma cells (Miyama et al, 2013), adenoid cystic carcinoma cells (Shi et al, 2013), and Madin Darby canine kidney cells (Eydelnant et al, 2014). 3D cell cultures in microgels offer several advantages, including tunable shear forces imposed on cells, easy visualization (e.g., by conjugating fluorescent agent to the microgel's material (Utech et al, 2015)), easy control over the transport of oxygen, nutrients, growth factors and waste (McGuigan and Sefton, 2007) as well as the mechanical and chemical stability in aqueous media such as buffer or cell culture media.…”

“…Synthetic polymer-based microgels did not gain popularity in cell culture applications due to the harsh conditions involved in the process of microgel preparation, such as strong shear forces, ultraviolet irradiation, and large temperature gradients, which cause severe cell damage or even mortality (Velasco et al, 2012). In contrast, natural polymerbased microgels, such as alginate (Eydelnant et al, 2014;Miyama et al, 2013;Utech et al, 2015;Yu et al, 2015), agarose (Eydelnant et al, 2014;Shi et al, 2013) and chitosan , are widely used for cell encapsulation because of their biocompatibility and mild conditions required to achieve gelation, thus preserving cell viability. Cross-linking methods for gelation affect significantly the network structures of the formed microgels, so the ability to control cross-linking process allows the formation of homogeneous network structure with precise internal structure and tunable stiffness Utech et al, 2015), enabling the stable entrapment of cells in a controlled microenvironment.…”

“…Additionally, the degradability of microgels is essential for enhancing cell viability during long-term cell culture. Moreover, gel matrices can be fabricated to possess various hierarchical structures and any desired shape to 31 support cell culture, such as yarn-ball-shaped (Miyama et al, 2013), core-shell (Yu et al, 2015) or tubular . For example, Miyama et al synthesized yarn-ball-shaped microgels which had an average outer diameter of 200 µm with fibers of 10-30 µm.…”

“…They had a large void volume and a high surface-to-volume ratio, which provided sufficient permeability to promote effective delivery of oxygen, nutrients, and metabolic products to the encapsulated cells (Miyama et al, 2013). Yu et al prepared alginate microgels having a structure of alginate-core/alginate-shell in which the core acted as 3D culturing unit while the shell prevented cells from diffusing out of the microgels and subsequently proliferate and form monolayer in the culture-flask as occurred to the core-only microgels (Yu et al, 2015).…”