Light-mediated Formation and Patterning of Hydrogels for Cell Culture Applications

Abstract

Click chemistries have been investigated for use in numerous biomaterials applications, including drug delivery, tissue engineering, and cell culture. In particular, light-mediated click reactions, such as photoinitiated thiol−ene and thiol−yne reactions, afford spatiotemporal control over material properties and allow the design of systems with a high degree of user-directed property control. Fabrication and modification of hydrogel-based biomaterials using the precision afforded by light and the versatility offered by these thiol−X photoclick chemistries are of growing interest, particularly for the culture of cells within well-defined, biomimetic microenvironments. Here, we describe methods for the photoencapsulation of cells and subsequent photopatterning of biochemical cues within hydrogel matrices using versatile and modular building blocks polymerized by a thiol−ene photoclick reaction. Specifically, an approach is presented for constructing hydrogels from allyloxycarbonyl (Alloc)-functionalized peptide crosslinks and pendant peptide moieties and thiol-functionalized poly(ethylene glycol) (PEG) that rapidly polymerize in the presence of lithium acylphosphinate photoinitiator and cytocompatible doses of long wavelength ultraviolet (UV) light. Facile techniques to visualize photopatterning and quantify the concentration of peptides added are described. Additionally, methods are established for encapsulating cells, specifically human mesenchymal stem cells, and determining their viability and activity. While the formation and initial patterning of thiol-alloc hydrogels are shown here, these techniques broadly may be applied to a number of other light and radical-initiated material systems (e.g., thiol-norbornene, thiol-acrylate) to generate patterned substrates.

Understanding ER+ Breast Cancer Dormancy Using Bioinspired Synthetic Matrices for Long‐Term 3D Culture and Insights into Late Recurrence

“…This approach for matrix formation was selected based on our prior work that shows i) consistent peptide incorporation, ii) independent control of hydrogel mechanical properties and biochemical cues for mimicking different soft tissues like those of interest here, and iii) a uniform encapsulation of single cancer cells within a thin hydrogel layer for quantitative analysis of cell responses in three dimensions using imaging-based techniques. [31,38,39] We previously have established the relevance of this materials system for shortterm studies of the growth and morphology of mesenchymal stem cells or breast cancer cells and now exploit the property control it affords for studying the effects of specific bioinspired matrix compositions on breast cancer dormancy in long-term 3D cultures.…”

Tunable hydrogels for mesenchymal stem cell delivery: Integrin-induced transcriptome alterations and hydrogel optimization for human wound healing

“…Note, the polymerization conditions used here (light wavelength and dose, photoinitiator type, and concentration) were selected to minimize exposure and the potential impact on cell health. Similar conditions have previously been reported to have little impact on human MSCs or other types of stem cells, as determined by assays such as the live/dead cytotoxicity assay, metabolic activity assay, and p53 expression . After gel polymerization, gels were incubated at 37°C and 5% CO 2 , then growth medium was replaced 1 hour after gelation to remove unreacted species and excess LAP.…”

“…Hydrogel synthesis was performed as previously described by Rehmann et al 28 Briefly, hydrogel precursor solutions were prepared by diluting stock solutions to final concentrations of 10 mM norbornene from PEG-8-norbornene (PEG-8-Nb, 40 kDa), 8 mM thiol from the peptide cross-linker (GCRDVPMS#MRGGDRCG that degrades in response to matrix metalloproteinases-MMP-1, MMP-2, MMP-3, MMP-7, and MMP-9), 40 types of stem cells, as determined by assays such as the live/dead cytotoxicity assay, metabolic activity assay, and p53 expression. [41][42][43][44] After gel polymerization, gels were incubated at 37 C and 5% CO 2 , then growth medium was replaced 1 hour after gelation to remove unreacted species and excess LAP. Gels were incubated in complete growth medium at 37 C and 5% CO 2 for subsequent RNA isolation.…”

Hydrogels that listen to cells: a review of cell-responsive strategies in biomaterial design for tissue regeneration

“…Furthermore, spatio-temporal control (via light) of the biomolecule conjugation and gelation has resulted in several novel materials applications in biofabrication. Recent advances include complex patterns within hydrogels [105][106][107] and the development of photo-crosslinkable thiol-ene hyaluronic acid 108 and polyglycidol 109 based bioinks for 3D printing of cellhydrogel mixtures.…”