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Zhuang, et al.
Integrating acoustics with microfluidics and acoustic perfusion flow that could improve oxygen and nutrient
fluidics have found many applications in cell sorting transportation, sustaining long-term cell culture [107,108] .
and separation . This acoustic-based cell manipulation These continuous flow-based microfluidic platforms
[86]
technique is non-invasive and label-free. By guiding frequently use and require syringe pumps, whereas digital
surface acoustic waves (SAW) to a microfluidic chamber, microfluidic platforms can optimally dispense pico- to
Chen et al. had demonstrated spheroid formation, micro-liter droplets on an electromechanical apparatus;
while simultaneously patterning through a designed 3D therefore, digital microfluidic platform are more portable
acoustic tweezer platform . Guo et al. had developed a and cost-effective [109-111] . Through tight control over fluidic
[47]
high-throughput acoustic fluidic platform for large-scale flow, microfluidic platforms allow the generation of
spheroid formation . By acoustically assembling cancer monodispersed droplets with uniform spheroid formation
[87]
cells, this platform enables high throughput fabrication of coupling a high throughput production output [112,113] .
6000 tumor spheroids per batch within 24 h.
3.7. 3D Bioprinting techniques
3.6. Microfluidic platforms Despite the broad utilization of extrusion-based
By manipulating fluid flow between micro- and nano- bioprinting in building 3D tissue constructs, strong
scales within microchannels, microfluidic platforms interests in inkjet-based bioprinting have been growing
have evolved as powerful tools that could possibly substantially in recent decades. The capability of a
miniaturize significant experimental processes onto “drop-on-demand” style printing to accurately dispense
a microfluidic chip less than the size of a finger [88] . discrete spheroids makes this technique appealing for
Droplet-based microfluidics, which generates high-throughput spheroid formation (Figure 2A-g). By
discrete droplets via immiscible multiphase flows dispensing the cell droplets into an alginate hydrogel
inside microfluidic devices, have gained substantial matrix residing within a 96-well plate, through
interest in past decades. Through adjustments to the microvalve-based printing, Utama et al. had successfully
flow rate of immiscible fluids, this method enables generated spheroids using 3 different cell types, including
generation of highly monodispersed droplets with a neuroblastoma (SK-N-BE(2)), non-small cell lung cancer
production speed spanning from 10-1000 droplets per (H460), and glioblastoma (U87vIII) cells [114] . The size of
second [89] . Typically, there are 3 types of microfluidic these printed spheroids was controlled by adjusting the
configurations for passive droplet generation: cross- initial printing cell density and incubation time, as well as
flowing/T junction [90,91] , flow-focusing [92-95] , and co- the confinement of the printed hydrogel matrix. Evidenced
flowing droplet formation [96] . Flow-focusing with by the expression of Ki67, HIF-1α, and apoptotic marker
single-, double-, and multiple-emulsion designs have cleaved caspase-3, the 3D-printed SK-N-BE(2) spheroids
been extensively utilized (Figure 2A-f) [97-99] . As a result, exhibited similar tumor-like characteristics that resemble
cell-encapsulated capsules with a template of water-in- manually formed spheroids. Similar level of CD133
oil (w/o), oil-in-water-in-oil (o/w/o), water-in-water-in- expression was found in both 3D printed and manually
oil (w/w/o), and water-in-oil-in-water (w/o/w) could be generated neuroblastoma spheroids, indicating a similar
produced [100,101] . By assigning different materials and/or preservation of cancer stemness between both types of
cells to replace each individual phase, microcapsules spheroids. Therapeutic efficacy was also examined by
displaying varied cell/material arrangements could be doxorubicin (DOX) treatment for 2 h. DOX penetration
tailored for diverse applications [102] . For instance, with was found on the periphery of both types of prepared
a double-emulsion, flow-focusing microfluidic device, spheroids, which are also frequently observed in tumors.
Agarwal et al. had developed core-shell microcapsules These results collectively demonstrated the capability
with embryonic stem cell-laden carboxymethyl cellulose of 3D printed spheroids in recapitulating the biological
and alginate in the core and shell, respectively. Other than features of tumors.
alginate [98] , hydrogels such as chitosan, thermosensitive Taking advantage of the thermal property of
gelatin, agarose, Matrigel, collagen, P(NIPAM-AA), gelatin hydrogel, Ling et al. had fabricated concave
photoinitiative gelatin methacrylate (gelMA), PEGDA, wells molded from a polyethylene glycol-dimethacrylate
and hyaluronic acid-MA have all been examined for (PEG-DMA) array, with in situ seeding of human breast
facilitating spheroid formation and growth [94] . In addition cancer cell-laden gelatin for cellular spheroid formation
to droplet-based microfludics, lab-on-a-chip technology on a chip [115] . However, challenges associated with
also can integrate hanging drop networks [103-105] , droplet inconsistency, low cell density, easy nozzle
microwell [50,106] , U-shape microstructure, or micropillar blockage, and physical stresses on cells limit the range
into the platforms for spheroid formation and on-chip of this technique’s applicability. Alternatively, laser-
culture. Microfluidic platforms outperform conventional based bioprinting also enables droplet-based printing for
static culture methods through the introduction of a single cell manipulation or 3D spheroid formation [116,117] .

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