A Novel Three-Dimensional Glioma Blood-Brain Barrier Model for High-Throughput Testing of Tumoricidal Capability.

The blood-brain barrier (BBB) limits passage of substances between general circulation and the brain extracellular fluid, maintaining homeostasis in neural tissues and providing a defense against potential toxins. However, the protection provided by the BBB often prevents conventional chemotherapeutics from reaching brain tumors which makes brain cancers one of the most difficult cancers to treat (1). Traditionally, high-throughput testing of compound permeability through the BBB in vitro has been limited to assay of radio- or fluorophore-labeled compounds as they pass a cell monolayer growing on a permeable support system. Unfortunately, the labels themselves may negatively impact the assay, and the ability to determine resulting tumor cytotoxicity must be studied independently. The present study demonstrates proof-of-concept of a three-dimensional (3D) model to study label-free BBB transport as well as the resulting brain tumor cytotoxicity by combining two commercially available products: Corning® HTS Transwell®-96 tissue culture system and Corning 96-well spheroid microplates. Transwells are permeable support systems commonly used for drug transport and migration/invasion studies (2, 3). Corning spheroid microplates are cell culture microplates with round well-bottom geometry coated with Corning Ultra-low Attachment surface, enabling the formation of a single multicellular tumor spheroid centered in each well in a highly reproducible manner. By replacing the standard flat-bottom Transwell receiver plate with a Corning spheroid microplate, the resulting system-which can be tailored to any number of cell types and screening applications-enables a more comprehensive assay to study drug transport across the BBB and the resulting 3D glioma spheroid toxicity in an easy-to-use 3D high-throughput assay.

A Novel Three-Dimensional Immune Oncology Model for High-Throughput Testing of Tumoricidal Activity.

The latest advancements in oncology research are focused on autologous immune cell therapy. However, the effectiveness of this type of immunotherapy for cancer remediation is not equivalent for all patients or cancer types. This suggests the need for better preclinical screening models that more closely recapitulate in vivo tumor biology. The established method for investigating tumoricidal activity of immunotherapies has been study of two-dimensional (2D) monolayer cultures of immortalized cancer cell lines or primary tumor cells in standard tissue culture vessels. Indeed, a proven means to examine immune cell migration and invasion are 2D chemotaxis assays in permeabilized supports or Boyden chambers. Nevertheless, the more in vivo-like three-dimensional (3D) multicellular tumor spheroids are quickly becoming the favored model to examine immune cell invasion and tumor cell cytotoxicity. Accordingly, we have developed a 3D immune oncology model by combining 96-well permeable support systems and 96-well low-attachment microplates. The use of the permeable support system enables assessment of immune cell migration, which was tested in this study as chemotactic response of natural killer NK-92MI cells to human stromal-cell derived factor-1 (SDF-1α). Immune invasion was assessed by measuring NK-92MI infiltration into lung carcinoma A549 cell spheroids that were formed in low-attachment microplates. The novel pairing of the permeable support system with low-attachment microplates permitted simultaneous investigation of immune cell homing, immune invasion of tumor spheroids, and spheroid cytotoxicity. In effect, the system represents a more comprehensive and in vivo-like immune oncology model that can be utilized for high-throughput study of tumoricidal activity.

Automation of cell-based drug absorption assays in 96-well format using permeable support systems.

Cell-based drug absorption assays, such as Caco-2 and MDCK-MDR1, are an essential component of lead compound ADME/Tox testing. The permeability and transport data they provide can determine whether a compound continues in the drug discovery process. Current methods typically incorporate 24-well microplates and are performed manually. Yet the need to generate absorption data earlier in the drug discovery process, on an increasing number of compounds, is driving the use of higher density plates. A simple, more efficient process that incorporates 96-well permeable supports and proper instrumentation in an automated process provides more reproducible data compared to manual methods. Here we demonstrate the ability to perform drug permeability and transport assays using Caco-2 or MDCKII-MDR1 cells. The assay procedure was automated in a 96-well format, including cell seeding, media and buffer exchanges, compound dispense, and sample removal using simple robotic instrumentation. Cell monolayer integrity was confirmed via transepithelial electrical resistance and Lucifer yellow measurements. Proper cell function was validated by analyzing apical-to-basolateral and basolateral-to-apical movement of rhodamine 123, a known P-glycoprotein substrate. Apparent permeability and efflux data demonstrate how the automated procedure provides a less variable method than manual processing, and delivers a more accurate assessment of a compound's absorption characteristics.