Basolateral Recruitment of Peripheral Blood Mononuclear Cells (PBMCs) on the Colon Intestine-Chip

Introduction

This protocol describes how to induce the migration of peripheral blood mononuclear cells (PBMCs) from the vascular (bottom) channel to the epithelial (top) channel in the Emulate Colon Intestine-Chip and assess downstream effector function.

Quantification of Cytokines and Acute Inflammatory Phase Proteins Secretion

Introduction

This protocol uses the Colon Intestine-Chip as a reference point aiming to the quantitation of the secretion levels of cytokines (IL-1β, IL-6, IL-8, TNFα) and acute inflammatory phase proteins (SAA, CRP, VCAM-1, ICAM-1) under baseline conditions and upon basolateral stimulation with the pro-inflammatory cytokine interferon gamma (IFNγ). These methods and assay conditions could change with a different Organ-Chip.

Assessment of Phosphorylated Signal transducer and activator of transcription 3 (STAT3)

Introduction

This protocol uses the Primary Colon Intestine-Chip as a reference point aiming to the semi-quantitative assessment of the phosphorylated Signal transducer and activator of transcription 3 (STAT3) levels under baseline conditions and upon basolateral stimulation with the cytokine interleukin 22 (IL-22). These methods and assay conditions could change with a different Organ-Chip or stimulant.

Immunofluorescence Staining—Colon Intestine-Chip

Introduction

This is the method we have developed for fixation and immunofluorescence staining for the Colon Intestine-Chip. We realize, however, that users may have their own fixation and staining processes that have been developed for specific cell types, antibodies, or antigens. If users would prefer to use other fixatives, permeabilizing solutions, or blocking buffers, they may do so while following the process outlined below.

Isolation and Purification of RNA Samples

Introduction

This is the method for isolation and purification of the total RNA for the epithelial and endothelial cells of the Colon Intestine-Chip. For isolation and purification of customer originated cell material, this protocol might need to be optimized. Please reach out to Emulate Field Science Support for additional guidance.

Total Protein Quantification

Introduction

This is the method to identify the total amount of protein in epithelial cells lysate and/or effluent samples of the Colon Intestine-Chip. For protein quantification of customer originated cell material, this protocol may need to be optimized. Please reach out to Emulate Field Science Support for additional guidance.

Cytokine-Mediated Barrier Disruption

Introduction

The Colon Intestine-Chip provides a human organoid-based platform to study cell-cell interactions and mechanisms that elicit collapse of the intestinal epithelial homeostasis. We employed the cytokines IFN, a prototype cytokine on intestinal epithelial barrier disruption studies, or IL-22. IL-22 acts as a barrier-insulting cytokine when applied to healthy tissue in our Colon Intestine-Chip, in line with recent findings.

For barrier disruption of customer-originated cell material, this protocol may need to be optimized. Please reach out to Emulate Field Science Support for additional guidance.

Protein Sample Isolation

Introduction

This is the method we have developed for lysing and purification of the protein fraction for the epithelial cells of our Colon Intestine-Chip. For lysing and purification of customer-originated cell material, this protocol may need to be optimized. Please reach out to Emulate Field Science Support for additional guidance. This protocol has been optimized for identification of intracellular proteins using the Meso Scale Discovery technology.

Assessment of Cleaved Caspase-3

Introduction

This protocol uses the Colon Intestine-Chip as a reference point aiming to the semi-quantitative assessment of the cleaved Caspase-3 levels under baseline conditions and upon basolateral stimulation with pro-inflammatory cytokines. These methods and assay conditions could change with a different Organ-Chip. Please reach out to Emulate Field Science Support for additional guidance.

Colon Intestine-Chip Culture Protocol: Co-Culture

Introduction

This protocol explains how to seed and culture a Colon Intestine-Chip using the Chip-S1® Stretchable Chip.