Bioprinting Perfusion-Enabled Liver Equivalents for Advanced Organ-on-a-Chip Applications.

Many tissue models have been developed to mimic liver-specific functions for metabolic and toxin conversion in in vitro assays. Most models represent a 2D environment rather than a complex 3D structure similar to native tissue. To overcome this issue, spheroid cultures have become the gold standard in tissue engineering. Unfortunately, spheroids are limited in size due to diffusion barriers in their dense structures, limiting nutrient and oxygen supply. Recent developments in bioprinting techniques have enabled us to engineer complex 3D structures with perfusion-enabled channel systems to ensure nutritional supply within larger, densely-populated tissue models. In this study, we present a proof-of-concept for the feasibility of bioprinting a liver organoid by combining HepaRG and human stellate cells in a stereolithographic printing approach, and show basic characterization under static cultivation conditions. Using standard tissue engineering analytics, such as immunohistology and qPCR, we found higher albumin and cytochrome P450 3A4 (CYP3A4) expression in bioprinted liver tissues compared to monolayer controls over a two-week cultivation period. In addition, the expression of tight junctions, liver-specific bile transporter multidrug resistance-associated protein 2 (MRP2), and overall metabolism (glucose, lactate, lactate dehydrogenase (LDH)) were found to be stable. Furthermore, we provide evidence for the perfusability of the organoids' intrinsic channel system. These results motivate new approaches and further development in liver tissue engineering for advanced organ-on-a-chip applications and pharmaceutical developments.

Photochemical formation of quinone methides from peptides containing modified tyrosine.

We have demonstrated that quinone methide (QM) precursors can be introduced in the peptide structure and used as photoswitchable units for peptide modifications. QM precursor 1 was prepared from protected tyrosine in the Mannich reaction, and further used as a building block in peptide synthesis. Moreover, peptides containing tyrosine can be transformed into a photoactivable QM precursor by the Mannich reaction which can afford monosubstituted derivatives 2 or bis-substituted derivatives 3. Photochemical reactivity of modified tyrosine 1 and dipeptides 2 and 3 was studied by preparative irradiation in CH3OH where photodeamination and photomethanolysis occur. QM precursors incorporated in peptides undergo photomethanolysis with quantum efficiency ΦR = 0.1-0.2, wherein the peptide backbone does not affect their photochemical reactivity. QMs formed from dipeptides were detected by laser flash photolysis (λmax ≈ 400 nm, τ = 100 µs-20 ms) and their reactivity with nucleophiles was studied. Consequently, QM precursors derived from tyrosine can be a part of the peptide backbone which can be transformed into QMs upon electronic excitation, leading to the reactions of peptides with different reagents. This proof of principle showing the ability to photochemically trigger peptide modifications and interactions with other molecules can have numerous applications in organic synthesis, materials science, biology and medicine.

Toward intrinsically colored peptides: Synthesis and investigation of the spectral properties of methylated azatryptophans in tryptophan-cage mutants.

Tryptophan has been taken as the basic scaffold for a chromophore whose indole residue can be further functionalized by the introduction of endocyclic nitrogen atoms or by N-methylation. When compared with exocyclic modifications, modifying tryptophan in an endocyclic fashion (through atomic substitution) should not perturb the steric profile of the amino acid side chain to such a large extent as that of an exocyclic modification, while simultaneously modulating the polarity, hydrogen-bonding ability, and spectral properties of the amino acid. Of particular interest is that the spectral properties can be tailored such that the chromophore can be monitored at wavelengths that exceed natural protein fluorescence. Ideally, the optimum excitation wavelength should be between 300 and 350 nm, and the emission wavelength should be ≥500 nm such that no cross-excitation/fluorescence occurs. Here, we report the synthesis of amino acid labels that exhibit large red shifts in their fluorescence profiles and their use in peptides.