A Protocol for Rapid Post-mortem Cell Culture of Diffuse Intrinsic Pontine Glioma (DIPG)

Abstract

Diffuse Intrinsic Pontine Glioma (DIPG) is a childhood brainstem tumor that carries a universally fatal prognosis. Because surgical resection is not a viable treatment strategy and biopsy is not routinely performed, the availability of patient samples for research is limited. Consequently, efforts to study this disease have been challenged by a paucity of faithful disease models. To address this need, we describe here a protocol for the rapid processing of post-mortem autopsy tissue samples in order to generate durable patient-derived cell culture models that can be used in in vitro assays or in vivo orthotopic xenograft experiments. These models can be used to screen for potential drug targets and to study fundamental pathobiological processes within DIPG. This protocol can further be extended to analyze and isolate tumor and microenvironmental cells using Fluorescence-activated Cell Sorting (FACS), which enables subsequent analysis of gene expression, protein expression, or epigenetic modifications of DNA at the bulk cell or single cell level. Finally, this protocol can also be adapted to generate patient-derived cultures for other central nervous system tumors.

Potent antitumor efficacy of anti-GD2 CAR T cells in H3-K27M+ diffuse midline gliomas

“…Patient-derived glioma cell cultures were generated as previously described 40 . Briefly, tumor tissue was dissociated mechanically and enzymatically (Liberase DH, Roche) prior to separation of myelin and debris by sucrose centrifugation.…”

“…Cell surface markers present on DIPG cell cultures were screened using a panel of monoclonal antibodies against human cell surface markers (Lyoplate, BD Biosciences). Low passage (<12) DIPG cultures expanded from tumor tissue collected at autopsy in serum-free, neurosphere forming conditions 40 were allotted to 96 well plates and blocked with 1ug of goat IgG per million cells to reduce nonspecific binding of secondary antibodies subsequently used in the assay. Cells were then incubated sequentially with primary and secondary antibodies with intermediate wash steps according to the manufacturer’s instructions.…”

Neural Precursor-Derived Pleiotrophin Mediates Subventricular Zone Invasion by Glioma

“…DIPG cell cultures were generated as tumor neurospheres from early post-mortem tissue as previously described (Caretti et al, 2014; Lin and Monje, 2017; Monje et al, 2011; Venkatesh et al, 2015). Briefly, tumor tissue was collected under sterile conditions, and transported in Hibernate-A (Thermo Scientific, Waltham, MA) on ice to the laboratory.…”

REST upregulates gremlin to modulate diffuse intrinsic pontine glioma vasculature

“…However, in recent years, availability of biopsy and access to tumor tissue samples resulted in successful development of DIPG cell cultures and high throughput genomic analyses of DIPG tumors. It has now become possible to pursue investigations at the molecular level [ 11 – 18 ].…”