Page 270 - IJB-9-4

International Journal of Bioprinting 3D acoustically assembled cell spheroids with high-throughput

individual cells as building blocks, the high cell density than 12,000 uniform cell aggregates per operation. One
of spheroids coupled with self-synthesized extracellular problem of this system is that the structure of the loosely
matrix endow fabricated tissues with faster maturation and assembled cell aggregates was easily damaged during
better functionality [11-14] . With these merits, more advances the process of transferring from the channel to the Petri
based on spheroids have been potentially developed in dish, resulting in a low maturation efficiency of spheroids.
several fields of high-throughput screening , precision Besides, the ANs of SAW-based device was located near
[15]
medicine [16,17] , and complex large tissue fabrication , the substrate surface, limiting the capability to create
[18]
however, which puts forward higher criteria on cell more ANs to further improve the yield of aggregates [36,44] .
spheroids with identical morphology and physiology as In contrast, bulk acoustic waves (BAWs)-based devices
well as in sufficient quantity [19,20] . enabled the formation of standing wave fields throughout
the chamber [40,45,46] . Jeger-Madiot presented a multi-trap
To date, many technologies have been developed to
fabricate cell spheroids [21-23] . A hanging drop was frequently acoustic levitation device to create a 1D (1 × 30) plane-
array of ANs, where cells can be levitated into multiple
used, due to easy handling and user-friendliness [24,25] . paralleled layers vertically and self-organized from cell
Based on gravity force, cells in small drops would settle and sheets to cell spheroids . Cai et al. used two standing
[47]
aggregate at the bottom of the drop, and eventually formed BAWs to create a 2D (10 × 10) column-array of ANs so
spheroids. This method generates spheroids in uniform that 100 cell aggregates can be generated horizontally .
[48]
size, but it is labor-intensive, time-consuming, and low- Despite this, the number of ANs produced by existing
throughput. Several commercially available methods, BAW-based devices was still small for mass production of
including spinner flask , rotary cell culture system , and cell spheroids. Nevertheless, these above works inspired
[27]
[26]
low-attachment microplate , enable mass production. us to investigate whether the 2D column-array of ANs in
[28]
However, the shear stress may induce cell damage and BAWs-based devices could be divided into 3D dot-array of
poor size uniformity, and the microplate (e.g., AggreWell) ANs, when applying an acoustic levitation field, to create
is not cost-effective. Other engineering methods, such 10 × 10 × 30 dot-array of ANs so as to enable further
as microfluidic [29-31] , electric , and magnetic-assisted improvement of yield of cell aggregates or spheroids by
[33]
[32]
assembly of spheroids, are capable of size control, whereas more than an order of magnitude.
they have some shortcomings, such as complex device
fabrication, medium modification, and/or cell labeling. For Herein, we developed a 3D acoustic assembly device
example, cells were labeled by magnetic nanoparticles or based on BAWs capable of creating a 3D dot-array of
suspended in a paramagnetic medium for robust assembly levitated acoustic nodes (LANs) for high-throughput
by magnetic forces, which might adversely affect cell fabrication of cell spheroids. Three orthogonal piezoelectric
growth or physiology [34,35] . Therefore, taking into account transducers (PZTs) were employed in this acoustic device
the problems with the above methods, more advanced to generate three orthogonal standing BAWs. We illustrated
spheroid-formation approaches are highly desirable to that these standing BAWs can create 3D dot-array (25 ×
fulfill the requirements, including simplicity, ease of 25 × 22) of LANs, through simulation and experiment
operation, biocompatibility, uniformity in size, and large- validation. In this case, more than 13,000 aggregates were
scale production. generated, and their number can be further increased by
raising the position of the vertical PZT. To improve the
As an alternative solution, acoustic assembly technique maturation efficiency of cell aggregates into spheroids, we
may generate better cell spheroids because it enables adopted GelMA hydrogels that can be rapidly photocured
excellent biocompatibility and provides label-free as supporting scaffolds to maintain and preserve the
manipulation of cells [36-38] . Commonly, this technique structure of cell aggregates after the withdrawal of acoustic
employs acoustic waves to create standing acoustic fields fields or during the process of transferring them into Petri
that have spatial distribution of periodic array of acoustic dish. As a result, most of cell aggregates (>90%) matured
nodes (ANs) [39-41] . In these fields, suspension cells in culture into cell spheroids with uniform size and high viability
medium are moved to the nearby ANs and assembled into (>90%). Finally, we confirmed that these cell spheroids can
cell aggregates under the action of acoustic radiation force, be easily retrieved from the GelMA scaffold for subsequent
thereby rapidly generating cell spheroids with uniform drug testing.
size . So far, several designs of acoustic assembly device
[42]
have been developed for the generation of spheroids. For 2. Materials and methods
instance, standing surface acoustic waves (SAWs) were
combined with a disposable capillary or multi-PDMS 2.1. Acoustic device fabrication
channels to form a 1D (1 × 30) or 2D (200 × 60) dot-array To generate levitated acoustic nodes in three dimensions,
of ANs, respectively [38,43] . The latter can assemble more three orthogonal acoustic standing waves are acquired, thus

Volume 9 Issue 4 (2023) 262 https://doi.org/10.18063/ijb.733