Encapsulation of murine hematopoietic stem and progenitor cells in a thiol-crosslinked maleimide-functionalized gelatin hydrogel.

Biomaterial platforms are an integral part of stem cell biomanufacturing protocols. The collective biophysical, biochemical, and cellular cues of the stem cell niche microenvironment play an important role in regulating stem cell fate decisions. Three-dimensional (3D) culture of stem cells within biomaterials provides a route to present biophysical and biochemical stimuli through cell-matrix interactions and cell-cell interactions via secreted biomolecules. Herein, we describe a maleimide-functionalized gelatin (GelMAL) hydrogel that can be crosslinked via thiol-Michael addition click reaction for the encapsulation of sensitive stem cell populations. The maleimide functional units along the gelatin backbone enables gelation via the addition of a dithiol crosslinker without requiring external stimuli (e.g., UV light, photoinitiator), thereby reducing reactive oxide species generation. Additionally, the versatility of crosslinker selection enables easy insertion of thiol-containing bioactive or bioinert motifs. Hematopoietic stem cells (HSCs) and mesenchymal stem cells (MSCs) were encapsulated in GelMAL, with mechanical properties tuned to mimic the in vivo bone marrow niche. We report the insertion of a cleavable peptide crosslinker that can be degraded by the proteolytic action of Sortase A, a mammalian-inert enzyme. Notably, Sortase A exposure preserves stem cell surface markers, which are an essential metric of hematopoietic activity used in immunophenotyping. This novel GelMAL system enables a route to produce artificial stem cell niches with tunable biophysical properties, intrinsic cell-interaction motifs, and orthogonal addition of bioactive crosslinks. STATEMENT OF SIGNIFICANCE: We describe a maleimide-functionalized gelatin hydrogel that can be crosslinked via a thiol-maleimide mediated click reaction to form a stable hydrogel without the production of reactive oxygen species typical in light-based crosslinking. The mechanical properties can be tuned to match the in vivo bone marrow microenvironment for hematopoietic stem cell culture. Additionally, we report inclusion of a peptide crosslinker that can be cleaved via the proteolytic action of Sortase A and show that Sortase A exposure does not degrade sensitive surface marker expression patterns. Together, this approach reduces stem cell exposure to reactive oxygen species during hydrogel gelation and enables post-culture quantitative assessment of stem cell phenotype.

Efficient Capture and Raman Analysis of Circulating Tumor Cells by Nano-Undulated AgNPs-rGO Composite SERS Substrates.

The analysis of circulating tumor cells (CTCs) in the peripheral blood of cancer patients is critical in clinical research for further investigation of tumor progression and metastasis. In this study, we present a novel surface-enhanced Raman scattering (SERS) substrate for the efficient capture and characterization of cancer cells using silver nanoparticles-reduced graphene oxide (AgNPs-rGO) composites. A pulsed laser reduction of silver nanowire-graphene oxide (AgNW-GO) mixture films induces hot-spot formations among AgNPs and artificial biointerfaces consisting of rGOs. We also use in situ electric field-assisted fabrication methods to enhance the roughness of the SERS substrate. The AgNW-GO mixture films, well suited for the proposed process due to its inherent electrophoretic motion, is adjusted between indium tin oxide (ITO) transparent electrodes and the nano-undulated surface is generated by applying direct-current (DC) electric fields during the laser process. As a result, MCF7 breast cancer cells are efficiently captured on the AgNPs-rGO substrates, about four times higher than the AgNWs-GO films, and the captured living cells are successfully analyzed by SERS spectroscopy. Our newly designed bifunctional substrate can be applied as an effective system for the capture and characterization of CTCs.