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Using Spheroids to build 3D Bioprinted Tumor Microenvironment
ECM, as well as tumor-immune interactions in a more Given its potentiality in recapitulating the key features of
physiologically relevant microenvironment. the avascular tumor in vivo, spheroids have been identified
Recent progress in bioprinting techniques, as a promising tool for understanding tumor biology
biomaterial science and cell biology have generated and anticancer therapeutic development. However,
3D tumor models with greatly enhanced robustness the random structural organization yields inconsistent
and physiological relevance. Such models with higher therapeutic outcomes, which greatly precludes the model
fidelity hold great potential not only in unraveling from translational clinical applications. Featuring with
the underlying mechanism, but also facilitating the excellent control system, bioprinting has been greatly
anticancer drug screening before entering clinical trials. beneficial to the tissue engineering field. The existing
For instance, to validate the reliability of the printed 3D printed models are serving as promising platforms,
co-culture platform in the above-mentioned study [183] , providing deeper insights into some critical aspects
immunotoxins EGF4KDEL and CD22KDEL which of tumor progression, including but not limited to
target EGFR-overexpressing A549s and an off-target tumor heterogeneity resemblance, tumor angiogenesis,
parallel control, respectively, were introduced through metastasis, and anticancer therapeutic development.
the vascular conduit. Specifically, EGF4KDEL greatly However, the major issue in the current 3D printed models
suppressed the tumor growth, invasion, and migration, is balancing the mechanical properties of exogeneous
while negligible effect was observed with the treatment bio-inks with the biological functions of sportingly
of CD22KDEL. By interlacing cancer cells, stromal involved cellular components. To this end, there is a
cells, and vascular networks, this bioprinted model was growing interest in adopting spheroids as building blocks
reminiscent of the native TME, providing a valuable within 3D bioprinting, possibly achieving large-scale
reference for anticancer drug screening. Similarly, tissue construction. Harnessing the power from spheroids
several models displaying varied complexities have been and 3D bioprinting would likely circumvent associated
constructed for anticancer drug testing [184-187] ; yet, the shortcomings from using foreign bio-inks (mismatched
majority were utilizing animal cells or immortal human mechanical stiffness and degradation rate, etc.), while
cell lines, which could be less effective in therapeutic maintaining structural guidance for spheroid growth.
prediction. Notably, in a recently published study, Therefore, tumor models with increased authenticity,
Xie et al. generated a patient-derived hepatocellular including well-organized structure, maximized cell-to-
carcinoma (HCC) model using cell-laden gelatin/alginate cell interaction, cell-secreted ECM, and multicellular
as bio-ink [188] . Immunofluorescence staining revealed the environment, could be fabricated in a high-throughput
stabilized expression of α-fetoprotein in HCC model over manner. Although, promising progresses have been
a 2-week culture period. In addition, evidenced by whole- achieved in 3D printing spheroids so far, current
exome sequencing and RNA-sequencing; a high level of existing technologies could not accurately position the
concordance for single nucleotide variants was observed spheroids. In addition, spheroid processing (including
between 3D printed HCC model and the corresponding spheroidization, assembly process, tissue fusion, and
original HCC tissue, indicating the retained genetic maturation) is time consuming. Using spheroids as tissue
alterations and expression profiles. Overall, the results building blocks in 3D bioprinting is still in its infancy.
demonstrated that the 3D-printed HCC model could Conventional spheroid generation strategies have
preserve the features of the original tumor during long- offered wealthy information on parameters that affect
term culture. The printed models were subsequently spheroid formation but are not scalable. For instance, 96-
subjected to the treatments with 4 commonly used, dose- well U bottom well plate are very expensive, and worsens
dependent targeting drugs. The patient-specific response when a large amount of spheroids are required -a large
suggested the potentiality of the 3D-printed model as a amount of spheroids require more 96 well plates, which
drug prediction model for personalized medicine. is very expensive [189] . Recent advent of microfluidic

6. Outlooks and challenges platforms and drop-on-demand bioprinting are
promising on improving the high throughput fabrication
TME is now recognized as a highly dynamic and of spheroids, yet the spheroid uniformity is far from
heterogeneous environment with reciprocal interactions satisfactory. New methods, such as machine learning,
between cellular and acellular components. Although bring emerging solutions by integrating imaging and
massive efforts have been dedicated to emulating the screening of functional modules into the current systems
key features of TME, the majority of these studies are for spheroid selection through morphological feature
focusing on a single aspect within TME, i.e. a true-to- analysis. A fully automated system can significantly
life tumor model. This golden model could recapitulate improve the fabrication performance and lessen the time.
all the essential characteristics of TME, but currently, its Lee et al. integrated a machine learning model using
construction is restricted by technological limitations. least general generalization algorithm combined with

16 International Journal of Bioprinting (2021)–Volume 7, Issue 4