Self-sustaining media-reconstituted GelMA bioink supports printable hematopoietic cultures

Tunable, bioprintable hydrogels are essential for mechanistic studies of hematopoietic stem and progenitor cells (HSPCs) fate because stiffness, porosity, and dimension of culture are crucial for HSPC expansion and differentiation. Collagen-based 3D hydrogels are the current gold standard for in vitro hematopoietic stem and progenitor cell culture, closely mimicking the bone marrow microenvironment (BMME), but lack printability and tunability in stiffness and porosity. Gelatin methacryloyl (GelMA) retains the benefits of collagen-based systems while allowing fine control over the mechanical properties, enabling biofabrication strategies that allow spatial distribution of multiple cell types. Here, we characterize a GelMA-based bioink, prepared in complete media, rather than DPBS, that supports the growth and function of human erythroblast leukemia cells for at least a week without the need for being submerged in a culture medium. By optimizing GelMA's mechanical and physical properties to resemble the native BMME, and incorporating a thermal gelation step, we demonstrate it remains printable at low concentrations that better reflect physiological stiffness. This improved approach demonstrates the potential of GelMA-based constructs to better approximate key physical and mechanical features of the BMME and to support week-long, self-sustaining hematopoietic cultures for drug screening assays that can reduce the reliance on animal models for early-stage research.