Page 353 - IJB-10-5

International Journal of Bioprinting Printing organoids in peptide matrices

1× PBS, an RNA purification kit (Thermo Fisher Scientific, microscope (EVOS; Thermo Fisher Scientific, USA). The
USA) was used to lyse the cells and extract RNAs, according proliferation of cells after 3D printing was performed
to the manufacturer’s instructions (n = 5). using an adenosine triphosphate (ATP) cell proliferation
assay (CellTiter-Glo 3D; Promega, USA), following the
2.13.2. Quantitative reverse transcription polymerase manufacturer’s recommendations. Briefly, 100 µL of
chain reaction proliferation reagent was added to each printed structure in
Complementary DNA was transcribed from extracted the 96-well plate, mixed vigorously, and left for incubation
RNA with a High Capacity cDNA Reverse Transcription in the dark for 30 min. Luminescence was measured using
Kit (Thermo Fisher Scientific, USA) using a Mastercycler the PHERAstar FS Microplate Reader (BMG LABTECH,
proS (Eppendorf, Germany). Thereafter, polymerase chain Germany). GraphPad prism (Dotmatics, USA) was used
reaction (PCR) was conducted using a QuantStudio 7 Flex for data analysis, and results are represented as mean ±
Real-Time PCR System with TaqMan Gene Expression standard deviation. Statistical analysis was performed
Assay (GAPDH, CHGA, EPCAM, LGR5, and MUC2) and using two-way analysis of variance (ANOVA), and values
TaqMan Fast Advanced Master Mix from Thermo Fisher with p < 0.05 were considered statistically significant.
Scientific (USA). GAPDH was used as a housekeeping
gene for normalization. Matrigel is used as the control to 3. Results
calculate Cycle threshold (CT). A t-test was conducted to
determine statistically significant differences at p = 0.01, 3.1. Physicochemical characterization of peptide
0.001, and 0.0001 (n = 15). combinations
Previous studies have analyzed the fiber-forming capacity
2.14. Evaluation of printability and of the parent tetrapeptide IIFK. This peptide comprises a
36
bioprinted organoids hydrophobic tail of nonpolar amino acids (Ile [I] and Phe
Printing and bioprinting processes were conducted with [F]) and a positively charged amino acid (Lys [K]) at the
an in-house-developed robotic 3D bioprinter composed of C-terminal, making it an amphiphilic sequence. This parent
a five-degrees-of-freedom robotic arm, a custom-designed peptide self-assembles into well-ordered supramolecular
dual-coaxial nozzle, microfluidic pumps, and a stirring hot nanofibers. 34,36 Peptides FIB and LAM were obtained after
plate. The robotic arm was interfaced with Repetier-Host. the parent peptide was decorated with sequences RGDS
®
The printing files were designed in SolidWorks (Dassault and YIGSR, respectively. Based on previous studies, we
37
Systemes, France) and sliced into G-codes using Slic3r determined that mixtures of decorated peptides and the
and Repetier software (Hot-World GmbH, Germany). The parent peptide enabled the formation of fibrous structures.
dual-coaxial nozzle was fabricated according to literature. Therefore, we generated specific combinations utilizing the
25
The commercial microfluidic pumps were controlled parent peptide and biofunctional peptides (Table 1) to be
simultaneously using the automated pulse mode. The evaluated. We defined mixtures based on low (0.5 mg/mL)
selected flow rates were 40–45 μL/min (peptide), 20–25 μL/ and high (1.0 mg/mL) concentrations of biofunctional
min (5× or 7× PBS, depending on the peptide sequence), peptides FIB and LAM. These were mixed with a fixed
and 15 μL/min (1× PBS with the extracted cells) for each of concentration (1.0 mg/mL) of parent peptide.
the pumps, respectively.
The physicochemical characterization of peptides
Gelation tests of different peptide concentrations after and peptide combinations matrices was performed to
printing were conducted using 2, 4, and 6 mg/mL FIB (low) elucidate the properties of the hydrogels formed. SEM
peptide mixture and 1.5, 3, and 6 mg/mL LAM (high) and AFM were used to characterize the morphology
peptide mixture (Table 1). Bioprinting was conducted of the hydrogels; Raman spectroscopy was used to
using 4.5 mg/mL LAM and 6 mg/mL FIB. The cells were characterize the secondary structure of the hydrogel
mixed with 600 μL of 1× PBS and loaded into the printer. nanofibers; rheology assessment was performed to get
The bioprinted constructs were cultured for 7 days and an insight into the mechanical properties of the different
subsequently analyzed with phase contrast (EVOS M7000; peptide combinations. Images obtained by SEM and AFM
Invitrogen, Invitrogen, USA) and laser scanning confocal demonstrated that all conditions can form supramolecular
microscopes (ZEISS LSM 880 with Airyscan) (ZEISS, assemblies in the form of long nanofibers (Figure 1a and
Germany), as described above. b). SEM imaging further suggests that fibers intertwine
The viability of 3D-bioprinted cells was assessed using in multiple dimensions. AFM topography derived the
the LIVE/DEAD Viability/Cytotoxicity Kit (Thermo Fisher characteristic nanofiber lengths from combined solutions
Scientific, USA) as described above. Stained printed cell- (Table 2). For instance, FIB-containing nanofibers have
laden constructs were imaged using an immunofluorescent a larger diameter than LAM-containing nanofibers.
Interestingly, the highest variability in helical periodicity

Volume 10 Issue 5 (2024) 345 doi: 10.36922/ijb.3033