automated IF staining of spheroids and organoids
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Automated Biomarker Assays for 3D Cell Models on the Pu·MA System® with Xeno-free VitroGel® Hydrogel

Introduction

For decades cell biology principles, drug activities, cell responses, and tissue morphogenesis have been determined in 2D cell monolayer culture systems. But these 2D cultures lack most of the interactions occurring in 3D in native tissues. 3D in vitro cell culture systems are increasingly popular and have great potential as tools for disease modeling and drug discovery1. Their ability to resemble tissue-like structures, characteristics, interactions, and spatial organization can help accelerate the emergence of novel and effective therapies. For creating physiological in vitro environments, 3D cell culture systems often require either an extracellular matrix (ECM) or hydrogel, to provide structural support and stability for the cells, but also to facilitate cell signaling that influences morphology and function2. The Pu·MA System® is a benchtop instrument that uses unique microfluidic flowchip consumables to automate complex multi-step cell assays and data acquisition from 3D cell models3,4. Here, we have developed an automated workflow using the Pu·MA System while incorporating the xeno-free hydrogel VitroGel® (TheWell Biosciences)5 with the 3D cell models.

In this study, we optimized a workflow for creating minidomes with 3D cell models in VitroGel Hydrogel Matrix within the protected sample chamber of the flowchips. The flowchips were loaded with assay reagents for automated assays using the Pu·MA System, followed by imaging of 3D cell models within the flowchip, providing high-quality imaging data for phenotypic profiling (Figure 1). We have established protocols using either complex, animal-based ECM (e.g., Matrigel®) or the easy-to-use VitroGel hydrogel. Although Matrigel is widely used, it has assay limitations like undefined composition, lot-to-lot variability, murine components, and temperature dependency. Chemically defined, xeno-free VitroGel can be modified for desired outcomes and provides reproducible results. In this application note, we demonstrate applications using ECMs and engineered hydrogels for automated image-based profiling 3D assays (Figure 1). We present the use of the Pu·MA System for two 3D cell culture applications using VitroGel:
1. Formation of 3D cell models
2. Cell biomarker detection of 3D cell models.​

Fig 1. The workflow outlined shows creating minidomes with 3D cell model in VitroGel within the protected sample chamber of the sample well, loading flowchips with assay reagents for automated assays using Pu·MA System, followed by imaging of 3D cell models within the flowchip.

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