Injection-free, spatially controlled delivery of glioblastoma cells to human brain slice cultures using soft cryogel biomaterials.

Organotypic brain slice culture has emerged as a valuable ex vivo system for studying brain development, function, and disease in a multicellular tissue context. More recently, this has extended to the use of human tissue derived from fetal, post-mortem, or discarded surgical specimens. In glioblastoma research, focal delivery of exogenous cells to brain slices is often technically challenging, particularly when spatial control is required. Here, we present a simple and experimentally accessible method for localized cell delivery to human brain slice cultures using soft donut-shaped cryogel biomaterials. Stereolithography-based 3D printed templates were used to generate well-defined cryogels by sub-zero polymerization of poly(ethylene glycol) diacrylate and 3-sulfopropyl acrylate. The resulting macroporous scaffolds were soft, easy to handle, and could be positioned directly on top of human brain slices prior to loading brain tumor cells into the central lumen. This design enabled focal confinement of the administered cells while permitting invasion into the underlying tissue. The ability to tailor cryogel dimensions, together with the straightforward workflow, makes this approach a versatile platform for spatially controlled cell delivery to brain slice cultures. Under the conditions tested, cryogel placement was not associated with an increase in local GFAP (Glial Fibrillary Acidic Protein) signal, supporting compatibility with the tissue during culture.