Encapsulation of murine hematopoietic stem and progenitor cells in a thiol-crosslinke d maleimide-functionalize d gelatin hydrogel

Biomaterial platforms are an integral part of stem cell biomanufacturing protocols. The collective bio-physical, 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 biomateri-als 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, pho-toinitiator), 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 typ-ical in light-based crosslinking. The mechanical properties can be tuned to match the in vivo bone mar-row 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 expo -sure 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. (c) 2021 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Automated summary

Biomaterial platforms are crucial in stem cell biomanufacturing, with GelMAL hydrogel providing a versatile route for encapsulating stem cell populations and mimicking the bone marrow niche. The maleimide-functionalized gelatin allows for stable crosslinking without reactive oxide species generation, demonstrating potential for artificial stem cell niche construction.