The utility of biomedical scaffolds laden with spheroids in various tissue engineering applications

Abstract

A spheroid is a complex, spherical cellular aggregate supporting cell-cell and cell-matrix interactions in an environment that mimics the real-world situation. In terms of tissue engineering, spheroids are important building blocks that replace two-dimensional cell cultures. Spheroids replicate tissue physiological activities. The use of spheroids with/without scaffolds yields structures that engage in desired activities and replicate the complicated geometry of three-dimensional tissues. In this mini-review, we describe conventional and novel methods by which scaffold-free and scaffolded spheroids may be fabricated and discuss their applications in tissue regeneration and future perspectives.

Keywords: scaffold-free; scaffolded; spheroid; spheroid application; tissue engineering.

Figure 1

Schematic of various spheroid fabrication methods. A. Hanging drop. B. Nonadherent surface. C. Spinning flask. D. Rotating vessel. E. Micro-patterned mold.

Figure 2

Spheroid fabrication methods. A. Schematic images of microfluidic device (top) and optical images of the fabricated spheroids (bottom). Adapted with permission from , copyright 2018 MDPI. B. Schematic illustration of the spheroid fabrication process using micromolded non-adhesive hydrogel. Adapted with permission from , copyright 2016 Public Library of Science. C. Schematic of cell aggregation caused by acoustic stimulation. Adapted with permission from , copyright 219 Springer Nature. D. Schematic illustration of water-in-water emulsion method using phase separation between PEO and dextran. Adapted with permission from , copyright 2020 Royal Society of Chemistry.

Figure 3

Scaffold-free spheroid applications for bone regeneration. A. The experimental concept (left) and micro-CT images of a rat skull at 8 weeks after spheroid implantation (right). Adapted with permission from , copyright 2015 Springer Nature. B. Schematic overview of the aggregation of gingival stem cells, HA, and an adhesive hydrogel . C. Schematic illustration and osteogenic differentiation of a spheroid building block including biominerals and PDGF-coated PLLA fibers . D. Schematic illustration of spheroid encapsulation within a collagen/fibrin matrix .

Figure 4

Scaffold-free spheroid applications for cartilage regeneration. A. Schematic illustrations of a cylindrical construct composed of a fused spheroid and implantation . B. Schematic of MSC spheroid injection with collagen in a cartilage defect model . C. Illustration (left) and an optical image of cartilaginous tubes fabricated using the Kenzan method (middle), and an H&E-stained image of the graft at 35 days after transplantation (right). Adapted with permission from , copyright 2019 John Wiley and Sons.

Figure 5

Scaffold-free spheroid applications for liver, nerve, and skin regeneration. A. Spheroids stained for CPS1 [a periportal marker (brown)] and with hematoxylin (blue) (left). An MRP-2 (green)/Hoechst (blue) immunofluorescence image of 750 C3A cells in 2D culture (middle) and a spheroid observed after 18 days of culture (right). Adapted with permission from , copyright 2016 Oxford University Press. B. Optical image of 3D-bioprinted scaffold-free nerve constructs obtained from GMSC spheroids using a needle array (top left), and SMI-31/32/fluoromyelin- (top right), DAPI/β-tubulin III- (bottom left), and DAPI/S-100β-immunostained images (bottom right) (scale bars: 200 mm). Adapted with permission from , copyright 2018 Springer Nature. C. Macroscopic image of spheroids robotically inserted into the needle array (top left) and the constructs obtained after spheroid fusion, with the histology of regenerated nerves visualized using toluidine blue (top middle and right) and the number of axons observed in each group (bottom) (scale bar: 1 mm). Adapted with permission from , copyright 2019 John Wiley and Sons. D. DAPI-, caspase-3-, HNA -immunostained images obtained on day 14 (right) (scale bar: 100 mm), and H&E- (left) and Masson's trichrome-stained (middle) images of the wound bed on day 14 (scale bars: 500 mm). Adapted with permission from , copyright 2015 Public Library of Science.

Figure 6

Scaffold-free spheroid applications for vessel regeneration. A. DAPI/PCNA and DAPI/PCNA/HNA immunofluorescence-stained images at 3 days after transplantation of monolayer MSCs or MSC spheroids cultured for 7 days (scale bar: 50 mm). Adapted with permission from , copyright 2016 Korean Society of Lipidology and Atherosclerosis. B. Schematic overview of the bioreactor system (left) and an optical image of the fabricated vascular graft (middle). Von Willebrand factor-stained images taken pre- and post-implantation (right). Adapted with permission from , copyright 2015 Public Library of Science. C. Schematic illustrations of the tube fabrication composed of multicellular spheroids and agarose rods. Adapted with permission from , copyright 2013 John Wiley and Sons.

Figure 7

Scaffolded spheroid applications for bone tissue regeneration. A. Schematic illustration of macroporous scaffold preparation and macroscopic image of a porous scaffold . B. Schematic overview of the preparation of free-standing, patterned, electrospun, PLGA/ collagen/nHAp fiber mats .

Figure 8

Scaffolded spheroid applications for cartilage tissue regeneration. A. Schematic overview of the fabrication of solid freeform scaffolds via frozen liquid deposition (left), with safranin O-stained (red), and type II collagen-stained (green) images, and prelabeled MSC fluorescence images of structures implanted in NOD/SCID mice for 4 weeks (right). Adapted with permission from , copyright 2013 AO Research Institute. B. Schematic illustration of the fabrication of a bilayer scaffold, and a confocal laser scanning microscopy image of cell distribution in the scaffold at 28 days (right). Adapted with permission from , copyright 2020 American Chemical Society.

Figure 9

Scaffolded spheroid applications for liver tissue regeneration. A. Schematic illustration of in situ formation of spheroids (left) and a graph showing albumin secretion by conventional (red color bar) and biodot-printed phenyl-methylene hydantoin spheroids (blue color bar) (right). Adapted with permission from , copyright 2020 John Wiley and Sons. B. Macroscopic image of the bioreactor culture platform (top) and images of spheroids printing process (bottom). Adapted with permission from , copyright 2020 John Wiley and Sons.

Figure 10

Scaffolded spheroid applications for the regeneration of nerves, skin, vessels, and other tissues. A. Schematic illustration of hybrid alginate scaffold fabrication to support ASC spheroids (top), with an SEM image of the fabricated scaffold (bottom left). Optical images of vessel tube formation in ASC spheroid scaffold-driven conditioned medium (right). Adapted with permission from , copyright 2020 IOP Publishing. B. Schematic diagram of the experiment (top), and wound closure/angiogenesis analysis of a rat wound model (bottom). Adapted with permission from , copyright 2014 John Wiley and Sons. C. Schematic diagram of NF/hMSC, composite, multicellular spheroid preparation (top left) and 3D biohybrid assembly of NF/hMSC composite spheroids and the scaffold microstructure (bottom left), with a macroscopic image and perilipin/PECAM-1- and bFGF-immunostained confocal microscope images of the implanted biohybrid construct (right). Adapted with permission from , copyright 2010 John Wiley and Sons. D. Schematic illustration of the cellular structure of a 3D CS/PVA NF sponge and transplantation of DP microtissue (left), with a macroscopic image (top right) (scale bar: 500 mm), H&E-stained image (middle right) (scale bar: 200 mm), and Hoechst (blue)/K14 (green) immunofluorescence image (bottom right) (scale bar: 200 mm) of 3D spheroid-treated samples. Adapted with permission from , copyright 2020 American Chemical Society.