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International Journal of Bioprinting Expanding 3D cell proliferation with DLP bioprinting

1. Introduction DLP-bioprinted scaffolds using fish GelMA (F-GelMA),
a photocurable bioink, and to conduct basic exploratory
A new paradigm is emerging in tissue engineering. studies on cell proliferation enhancement by utilizing
Recently, certain high-profile companies such as microchannels and media flow environments.
Steakholder, Aleph Farms, and BlueNalu have utilized
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three-dimensional (3D) bioprinting, a tissue engineering To this end, based on our previous work, we
technology, to produce steak-like cultured meats and fabricated high-resolution 3D DLP-printed scaffolds
1-4
launch industrial-scale prototypes of cultured meats. by evaluating the functionalization of F-GelMA and
As such, 3D bioprinting is progressing rapidly, and optimizing it through rheology analysis. Morphological
numerous researchers have attempted creation of meat analysis of the DLP-printed scaffolds revealed wall-
substitutes utilizing 3D scaffolds with various cells, as like structures of 100 µm, which were limited in mass
documented in several published reports. Digital light transport due to their porous structure. To solve this
5-8
processing (DLP) bioprinting has attracted attention issue, we introduced microchannels into the scaffold and
due to its nozzle-free structure, fast printing speed, observed the difference in cell viability and proliferation
high resolution, and ease of automation for various inside the hydrogel and microchannels over time. We
tissue requirements. 9-12 Gelatin methacrylate (GelMA), also introduced a media flow environment to observe and
a gelatin-derived, biocompatible, biodegradable, analyze the changes in cell attachment and proliferation
and photocurable material, has been utilized in 3D inside and outside the microchannels during long-term
bioprinting technologies such as DLP for applications in cell culture for about 5 weeks. Finally, the analysis of
producing functional tissue analogs. 6,13-15 Nonetheless, DLP-printed hydrogel scaffolds demonstrated significant
enhancing cell proliferation and increasing yield within cell proliferation inside the microchannels, proving the
3D-printed scaffolds remain hurdles to overcome and new function of such structures.
important goals for future applications.
Overall, our results demonstrate the feasibility of
Controlling the scaffold microenvironment is microchannels as a space for cell proliferation in DLP-
crucial for manipulating cell attachment, viability, and printed cell-encapsulated F-GelMA scaffolds, offering a
proliferation, as well as regenerating tissue function. 10,16 new strategy to enhance cell attachment and proliferation.
However, poor transportation and slow diffusion of Our results are expected to serve the basic exploratory
oxygen and nutrients pose notable challenges to the research that utilizes 3D culture techniques for regenerative
viability of encapsulated cells. Furthermore, merely medicine and tissue engineering applications, where
17
controlling scaffold porosity is unable to provide effective cell proliferation relative to the same volume is
sufficient oxygen and nutrients, which leads to reduced required, such as in the cases of artificial organs and disease
diffusion capabilities and waste accumulation, ultimately models fabrication (Figure 1). 6,32-37
causing cell death and apoptosis at the scaffold center. 18,19
However, introducing microchannels and media flow 2. Materials and methods
environments could enhance cell attachment and
proliferation. 20,21 The microchannels effectively enhance 2.1. Materials
cell viability by providing nutrients within the scaffold In this study, we used Dulbecco’s modified Eagle’s
while promoting cell adhesion, growth, and migration, medium (DMEM, high glucose) and Dulbecco’s
as well as providing space for mass transport. 22-25 Media phosphate-buffered saline (DPBS) purchased from
flow environments transport oxygen and nutrients by WelGENE (Daegu, Gyeongbuk, South Korea) for
exposing cells to mechanical stimulation, enhancing cell cell culture. Fetal bovine serum (FBS), penicillin–
viability, ATP production, and mitochondrial activity streptomycin (P/S), L-glutamine, phosphate-buffered
compared to non-shaking culture environments. 20,26,27 saline (PBS, pH 7.4), and 0.05% trypsin-EDTA solution
However, no basic research reported on the implantation were also obtained from WelGENE. Gelatin from cold-
of microchannels within DLP-bioprinted scaffolds via water fish skin, methacrylic anhydride (MA), and lithium
cell encapsulation in GelMA hydrogel and its impact phenyl-2,4,6-trimethylbenzoylphosphinate (LAP; ≥95%)
on cell proliferation differences and enhancements in a were purchased from Sigma Aldrich (St. Louis, MO,
media flow environment. Traditionally, microchannels USA). Tartrazine, the light absorber, was purchased
implanted within a scaffold have been considered empty from GreenTech (Daejeon, South Korea). A Live/Dead
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spaces. 25,28-30 In this paper, we propose a new and expanded cell viability kit containing calcein-AM, ethidium
perspective that these microchannels offer space with the homodimer-1, and fluorescent Alexa Fluor 488 was
®
potential to increase cell proliferation more effectively. purchased from Invitrogen (Carlsbad, CA, USA). All
Therefore, this study aims to fabricate cell encapsulated other chemicals used in this study were of analytical grade.

Volume 10 Issue 3 (2024) 409 doi: 10.36922/ijb.2219