Liver-Chip

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.


← Back to Protocols

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).


← Back to Protocols

Barrier Function 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.


← Back to Protocols

Live Staining of CLF Uptake into Bile Canaliculi

Introduction

Corning® Cholyl-lysyl-fluorescein (CLF) staining is used to visualize the structure and function of bile canaliculi in polarized hepatocytes. CLF is a substrate for the canalicular bile salt export pump (BSEP), thus it can be used to visualize BSEP-mediated canalicular efflux, as well as to label the bile canaliculi structures.


← Back to Protocols

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.


← Back to Protocols

Gsh-Glo™ Glutathione Assay

Introduction

The GSH-Glo™ Assay is a luminescent-based assay for the detection and quantification of glutathione (GSH), an antioxidant that can prevent damage to cellular components caused by reactive oxygen species such as free radicals, peroxides, and lipid peroxides. GSH is involved in the detoxification of both xenobiotic and endogenous compounds. A change in GSH levels can be used as an indicator of toxicity.


← Back to Protocols

Glutathione S-Transferase (αGST) Assay

Introduction

TECO® αGST is an ELISA for the quantitative determination of alpha Glutathione S-Transferase (αGST). GSTs are enzymes that catalyze the conjugation of glutathione to electrophilic centers on a wide variety of substrates in order to make the compounds more water-soluble. The role of GSTs is to detoxify endogenous compounds such as those that enable the breakdown of xenobiotics. High intracellular concentrations of GSTs coupled and released into serum are indicators of hepatocyte injury and toxicity and can be used as biomarkers. They are also used as indicators of renal injury and nephrotoxicity.


← Back to Protocols

Keratin 18 (K18) Assay

Introduction

Keratin 18 (K18) is an intracellular protein expressed at high levels by many types of epithelial cells. During cell death, the cellular content of soluble K18 will be released into the extracellular compartment. The M65 EpiDeath® ELISA measures soluble keratin 18 (K18) (cytokeratin 18 [CK18]) released from dying cells and can be used in the research of overall cell death (due to apoptosis and necrosis) of epithelial cells.


← Back to Protocols