Comparison of visible and UVA phototoxicity in neural culture systems micropatterned with digital projection photolithography

Abstract

Photopolymerization provides a favorable method for hydrogel formation due to its simplicity, convenience, and versatility. However, the light exposure required to initiate photopolymerization is known to have a cytotoxic effect on encapsulated cells. Here, a 3D in vitro model of the nervous system microenvironment, micropatterned through the use of digital projection photolithography using a single hydrogel formulation that cross-links similarly under ultraviolet A (UVA, 315-400 nm) and visible light (400-700 nm) exposure, is presented. This setup allowed for the investigation of neuronal responses to different light wavelengths and exposure times during photoencapsulation, while ruling out effects due to the hydrogel formulation or photoinitiators used. Cellular studies-including neurite viability, DNA fragmentation, and neurite outgrowth for both UVA and visible light irradiation, the most common spectra used in biological photomicropatterning applications-were performed to assess the effect of light source on neuronal cultures. These studies indicated that while cell death occurs after exposure to either spectrum, visible light was less phototoxic than UVA, when using comparable levels of irradiation, and interestingly, glial cells were more susceptible to phototoxicity than neuronal cells. Thus, while utilizing visible light for micropatterning and cell encapsulation for nervous system applications is beneficial, it is helpful to keep the light exposure low to ensure optimal neuronal survival and growth.

Morphine-sensitive synaptic transmission emerges in embryonic rat microphysiological model of lower afferent nociceptive signaling

“…S1D), indicating that any physiological differences among conditions are not a result of declining cell health. Consistent with previous reports ( 41 – 46 ), immunofluorescent staining of total nerve growth with the neuron-specific cytoskeletal protein, β-III-tubulin, confirmed that stiff, gelatin methacrylate–based ECM promoted DRG neurite outgrowth and inhibited SCDH neurite outgrowth in monoculture (fig. S1E).…”

“…We first sought to identify an optimal ECM composition that promotes the emergence of the appropriate structure-function relationship between DRG and SCDH nerve tissue. Our laboratory has previously had success growing three-dimensional nerve cultures in both gelatin methacrylate and Matrigel-based hydrogels and hypothesized on the basis of previous reports ( 41 – 46 ) that modulation of gel stiffness could be used to direct neurite growth. Therefore, we designed gelatin methacrylate and Matrigel-based gel formulations that would result in ECM of various stiffness and compared morphology and physiology of cultures across gel formulations to identify the optimal ECM.…”

A comprehensive library of human transcription factors for cell fate engineering

“…To facilitate the preparation of cross-sections of myelinated axons, these cells were co-cultured within a microchannel mold that promotes the alignment of axons along the channel in one direction. The microchannel mold was constructed by adding a 10% (w/v) PEG-diacrylate (Mn 1000; Polysciences Inc., Warrington, PA) and 0.5% (w/v) Irgacure 2959 in PBS solution to a collagen-coated transwell 43 , 84 , 85 . A negative mask was used to create the microchannel, followed by irradiation of the light-sensitive media with 181 mW/cm2 UV light for 30 seconds.…”

Methods for fabrication and evaluation of a 3D microengineered model of myelinated peripheral nerve

“…This resulted in glycidyl methacrylate-dextran (MeDex), the composition of which was confirmed using 1 H NMR [(D 2 O) 𝛿 6.1–5.7 (m, 2H, CH 2 ), 𝛿 5.2 (m, 1H, CH), δ 4.9 (m, 1H, CH), δ 1.9 (s, 3H, CH 3 )] with substitution degree of 42% 32,34,35 . Finally, a gel composition of MeDex 50% (w/v), arginine (0.1% (w/w) of MeDex), riboflavin (0.001% (w/w) of MeDex), TEMED (0.2% (v/v) of the final solution) was prepared for photocrosslinking 27 .…”

“…However, this objective was challenged by a number of conflicting design constraints, including hydrogel stability, cell viability, transparency, and ease of histology. Self-assembling hydrogels were susceptible to detaching from PEG hydrogel molds 26 while photocrosslinking was found to be disproportionately harmful to Schwann cells 27 . Preliminary work with alternative hydrogels showed that methacrylated dextran cured with visible light and a riboflavin-arginine photoinitiator was less phototoxic to Schwann cells, but its yellowish translucent color made it difficult to visualize the tissue for placement of extracellular field electrodes.…”