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.

Alveolus Lung-Chip Culture Guideline: Co-Culture

Introduction

This guideline covers how to seed and culture an Alveolus Lung-Chip using the Chip-S1® Stretchable Chip.

Chip Cradle Protocol (Supplement to Organ-Chip Culture Protocols)

Introduction

This is a supplemental protocol which outlines the use of the new Chip Cradle. It is not intended to replace current Organ-Chip protocols, but to demonstrate how the new cradle integrates into existing Organ-Chip protocols.

This user guide contains important information to safely and effectively use the Chip Cradle. All users should thoroughly read and understand this guide before use.

Fixed Chip Imaging Adapter User Guide

Introduction

The Fixed Chip Imaging Adapter organizes fixed chips for high-throughput imaging and ensures compatibility with SBS footprint advanced imaging equipment. It can also prevent evaporation for 12+ hours when used with off-the-shelf accessories.

This user guide details what is included with the kit, as well as different ways to configure the adapter to prevent evaporation for different periods.

Compound Distribution Kit Protocol

Introduction

In both in vitro and in vivo experiments, researchers must consider compound distribution within the biological model and experimental setup prior to quantitative drug studies, as distribution determines exposure — the concentration of a compound that cells truly experience.

In in vivo systems, this is addressed by volume of distribution studies, which relate compound dosage to its effective concentration. However, in both in vivo and in vitro studies, the distribution effects of system components, such as infusion tubing, syringes, tissue-culture plates, and pipette tips, are often missed.

With Organ-Chip experiments, we proactively address compound distribution in a number of ways. Several of these are embedded in our protocol designs, where we have selected experimental conditions to optimize compound exposure. Additionally, we have developed the Compound Distribution Kit to directly evaluate distribution and compound exposure.

The Compound Distribution Kit is intended to be used as a specialized control experiment — the distribution control experiment — prior to the intended Organ-Chip study. As such, the contents of the Compound Distribution Kit mirror the contents of the Organ-Chip Bio-Kit, and the protocol used for the distribution control experiment mirrors a simplified version of the intended study (e.g., without cells or ECM coating). The distribution control experiment’s output indicates whether any compound may be distributed into the system and away from cells. Moreover, in some cases, the distribution control can be used to quantitatively correct the experimental results of the intended study and assign it appropriate error bars.

Standard Curve Calculator

Introduction

The measurements made using many analytical instruments, like plate readers, require the conversion from a raw signal to a concentration or other known readout. For example, a ubiquitous Organ-Chip readout is the concentration of a given substance (either biological or pharmaceutical), in the Chip effluent. Typical assays on a plate reader, however, yield a raw signal, like optical density or fluorescence intensity. The conversion from signal intensity to the readout of interest is performed using a standard curve.

The standard curve process requires: first, the preparation of solutions of known concentrations; second, the quantification of the signal intensity of those solutions on an analytical instrument like a plate reader; and third, the establishment of a relationship between the measured signal and known concentrations through some curve fitting method. This relationship can then be used to convert the measured signal intensity from experimental samples to the particular readout, like concentration.

Like other standard curve calculators, the calculator within this excel can be used to establish the relationship between the measured signal from an analytical instrument to known concentrations. After entering the measured signal and the corresponding known concentrations in the “Standard Curve” tab, a curve is fit to the data using a log-log linear regression, which minimizes the percent error between the data and the fitted curve. This ensures a “best-fit” for both relatively low and high concentrations. The equation corresponding to this curve is displayed on a plot of the data/best-fit curve and additionally saved in the calculator’s memory to convert the signal from experimental samples of unknown concentrations.

To analyze the results from an entire plate, simply copy and paste plate reader results/signal into the tab marked “Measurement”; the calculator will take the equation found in the “Standard Curve” tab and apply it to the signal in order to automatically calculate and display the corresponding readout. While this readout will usually be concentration, this calculator applies broadly to any readout and any signal. The results can be copied and pasted for further processing and analysis (e.g. using an output like “effluent concentrations” of a tracer molecule to then calculate apparent permeability).

Glucose Quantification Assay

Introduction

This assay provides a simple and sensitive method to quantify glucose levels from Organ-Chip effluent samples.

Barrier Function Readout Analysis

Introduction

The maintenance or disruption of tissue barriers is an essential part of the pathophysiology of many diseases. The ability to quantitatively characterize tissue barrier is critical in the evaluation of barrier integrity and function.

This protocol is to be used to assess the permeability of an Organ-Chip’s endothelial-epithelial barrier. Apparent permeability (Papp) of tracer molecules is determined by dosing the inlet of one channel, collecting the effluent of both channels, and calculating the amount of compound that crossed through the membrane over time. See full method below and associated Papp Calculator (EC004) for data analysis.

ATP Quantification

Introduction

The CellTiter-Glo® assay kit allows for the quantification of intracellular ATP levels. This endpoint can be used as a marker of cell viability and used in toxicity studies as a measure of cytotoxicity.

Human Cleaved Caspase-3 (Asp 175) Quantification

Introduction

The Cleaved Caspase-3 (Asp175) SimpleStep ELISA® kit is designed for the quantitative measurement of Active Caspase-3 (Asp175) protein in human cells. Caspase-3 is a cytoplasmic cysteine protease involved in the activation cascade of caspases responsible for cell apoptosis.

The SimpleStep ELISA uses an affinity tag labeled capture antibody and a reporter conjugated detector antibody which immunocaptures the sample analyte in solution. This three-part complex is then immobilized via immunoaffinity of an anti-tag antibody coated on the well.

This endpoint can be applied to measure apoptosis in Organ-Chips as part of toxicity testing or other types of studies.