Page 350 - IJB-10-5

International Journal of Bioprinting Printing organoids in peptide matrices

In this study, we demonstrate the versatility of USAPs 2. Materials and methods
as a chemical scaffold for the biofunctionalization of
various ligands, while concurrently assessing the tunability 2.1. Peptide synthesis
of mechanical and biochemical properties in the resultant The peptides, IIFK (Ac-Ile-Ile-Phe-Lys-NH ), LAM
2
biofunctional formulations. Our investigation delves into (Ac-Ile-Ile-Phe-Lys-Gly-Gly-Gly-Tyr-Ile-Gly-Ser-
characterizing the gelation properties of these bioinks, Arg-NH ), and FIB (Ac-Ac-Ile-Ile-Phe-Lys-Gly-Gly-
2
and investigating their impact on CRC organoids and Gly-Arg-Gly-Asp-Ser-NH ), were synthesized by
2
assesses the corresponding biological responses across 9-fluorenylmethoxycarbonyl (Fmoc)-based solid phase
various bioink formulations. Our study aims to develop peptide synthesis. The Rink amide-methylbenzhydrylamine
a biofunctional and chemically well-defined alternative (MBHA) resin (GL Biochem, China) was pre-swelled
bioink that solidifies under physiological conditions. with dichloromethane (DCM) (SRL, India) for 30 min,
Hence, we propose the utilization of biofunctional self- followed by deprotection of the Fmoc group with 20% v/v
assembling peptides as a modular and tunable scaffold for piperidine/dimethylformamide (DMF) (Merck, Germany)
bioprinting organoids, with CRC organoids serving as our before the first amino acid was introduced. An N-methyl-
primary model system for this study. 2-pyrrolidone (NMP) solution (SRL, India), containing
2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium
The YIGSR motif is a cell-binding motif found in tetrafluoroborate (TBTU), 1-hydroxy benzotriazole
laminin-derived peptides that can modulate cell adhesion (HOBt), N,N-diisopropylethylamine (DIPEA), and Fmoc-
and spreading. It has been reported to co-localize laminin- protected amino acid, was added to the reaction vessel
binding proteins with two focal adhesion binding sites and agitated for 3 h. The resin was then washed, and a
(actin and vinculin) and promote vinculin expression, capping process was conducted using a mixture of DIPEA
which can direct cell migration. YIGSR-mediated cell and dimethylformamide (Sigma Aldrich, USA). The resin
38
adhesion occurs via the 67-kDa laminin receptor, which was then cleaved using trifluoroacetic acid (TFA) (Thermo
differs from the RGD-mediated cell adhesion mechanism Fisher Scientific, USA), triisopropylsilane (Sigma Aldrich,
through multiple integrins. The YIGSR motif can also USA), and water. The pure peptide was obtained through
39
induce the phosphorylation of cytoplasmic focal adhesion reverse phase-liquid chromatography purification. Purity
kinase, which is vital in regulating cell motility. and peptide mass and structure were confirmed by mass
The RGD motif is also a cell-binding motif found spectra analysis.
in ECM proteins, such as fibronectin, vitronectin,
fibrinogen, osteopontin, and bone sialoprotein. The 2.2. Peptide hydrogel preparation
40
RGD sequence is the attachment site of many adhesive The lyophilized peptide was sterilized under UV light
ECM proteins and cell surface proteins, and nearly half for 30 min and dissolved in MilliQ water to produce a
of the known integrins recognize this sequence in their 2× final concentration. Gelation was initiated by adding
adhesion protein ligands. The integrin-binding activity 2× phosphate-buffered saline (PBS) (Gibco, USA) to the
41
of the RGD motif plays a crucial role in cell adhesion and peptide solution; the hydrogels were incubated at room
migration. Synthetic peptides containing the RGDS motif temperature for 10–30 min. The peptide concentrations
have been used extensively as inhibitors of integrin– used in this manuscript are depicted in Table 1.
ligand interactions in cell adhesion, migration, growth, 2.3. Rheology
and differentiation studies. Several studies have proven Rheology measurements were obtained using an Ares-G2
that the presence of the RGD ligand in artificial matrices rheometer (TA Instruments, USA) and a standard
facilitates gastrointestinal organoid self-organization. 42–44 8-mm parallel plate (TA Instruments, USA). Samples for

This study presents the design of two biofunctional rheology were prepared as described in Section 2.2 and
peptides decorated with the YIGSR and RGD ligands. left at room temperature overnight for gelation. Peptide
The nanofiber-forming properties of these peptides were samples (300 µL) were prepared by casting in a 9 mm glass
®
characterized in various combinations with the parent ring coated with Sigmacote (Sigma Aldrich, USA). Three
peptide and evaluated as matrices for CRC organoid culture. dynamic tests were performed in each sample: a standard
Additionally, we demonstrate the printability of these oscillatory time sweep (time = 300 s, A (amplitude) =
bioactive peptides for the high-throughput fabrication 0.1%, f (frequency) = 1 rad/s), a frequency sweep (t = 400
of CRC organoids for drug screening applications. These s, A = 0.1%, f = 0.1–100 rad/s), and a standard oscillatory
peptides enable the generation of reductive matrices that amplitude sweep (t = 300 s, A = 0.001–100%, f = 1 rad/s).
can stimulate particular cell behaviors in a well-defined Stiffness was calculated based on the average storage
3D environment. modulus from the time sweep. Storage and loss moduli

Volume 10 Issue 5 (2024) 342 doi: 10.36922/ijb.3033