Oligonucleotide delivery

To recreate the PhysioMimix oligonucleotide uptake assay in your laboratory, you need a PhysioMimix OOC Single-organ System, primary human hepatocytes from our catalog of 3D validated cells, and Multi-chip Liver-12 plates. Please contact us for protocol guidance.

One PhysioMimix OOC Controller unit can simultaneously run up to six Multi-chip Liver-12 plates, providing up to 72 samples per run.

In our experiments, we evaluate the characteristics of fluorescently labelled non-conjugated and GalNAc-conjugated oligonucleotides for the target of interest, plus equivalent scrambled oligonucleotides. Collectively, these provide adequate experimental controls to assess differences in the rate and amount of oligonucleotide uptake and the specific effects of gene knockdown.

The PhysioMimix platform is a tissue culture device that recreates and sustains human liver microtissue from primary cells. Soluble biomarker measurements are achieved at various time points by sampling from the culture media. We advise that media samples be collected at every media change, i.e., every 2-3 days starting on day four before oligonucleotide dosing.

Please view the experimental timeline above for a visual representation, but essentially, further samples can be collected for analysis on days six, eight, eleven and fourteen. At each media change, 1.6 mL of media can be collected and frozen for analysis.

To measure oligonucleotide uptake, liver microtissue can be recovered from Multi-chip Liver-12 plates at set time points (to measure uptake over time) from four to fourteen days. Recovered microtissue is fixed before immunofluorescence imaging. Note, that where longitudinal analysis is required over time, one Multi-chip Liver-12 plate is recommended for each time point.

At the end of the experiment, liver microtissues can be removed from the Multi-chip Liver-12 plates and stored for future RNA quantification.

The PhysioMimix platform is a tissue culture device that recreates and sustains human liver microtissue from primary cells. Liver health measurements are achieved at various time points by sampling from the culture media. We advise that media samples be collected at every media change, i.e., every 2-3 days starting on day four before oligonucleotide dosing.

Please view the experimental timeline above for a visual representation, but essentially, further samples can be collected for analysis on days six, eight, eleven and fourteen. At each media change, 1.6 mL of media can be collected and frozen for analysis.

Currently, our PhysioMimix oligonucleotide delivery assay is validated for a primary hepatocyte monoculture. However, a publication by Majer et al., (2024) explored the utility of the assay for characterization of ASO delivery and uptake to primary human hepatocytes using a co-culture model with Kupffer cells. Additionally, a tri-culture liver disease model was utilized by Rao S et al., (2021) to investigate the phenotypic effect of gene knockdown by siRNA (siSPTBN1), see FAQ below for more info.

More information and FAQs relating to our Liver-on-a-chip models can be found here. The model behind the PhysioMimix oligonucleotide assay has been recognized by the US FDA as delivering superior performance for drug metabolism and accumulation studies versus traditional approaches (Rubiano et al., 2020).

The following publication from Majer et al., (2024) evaluates the liver-on-a-chip model for assessing oligonucleotide-based therapeutic uptake using multi-modal imaging techniques.

Please visit our PhysioMimix DILI assay kit: Human 24 and DILI application webpages for more information about recreating this assay in your laboratory. For further questions, please do not hesitate to contact us.

Lipid nanoparticle (LNPs) use has not yet been validated by CN Bio, however, there is no obvious reason why the approach would not be suitable as an alternative to GalNAc-conjugation.

In theory, yes. We have previously explored the efficacy of oligonucleotide therapeutics in our Metabolic dysfunction-associated steatohepatitis (MASH, previously known as Non-alcoholic steatohepatitis) assay for clients.

OOC liver disease models, such as the PhysioMimix Metabolic dysfunction-associated steatosis (MASH) assay and NASH-in-a-box kit, utilize a tri-culture (PHH, Kupffer Cell and Stellate cell) liver-on-a-chip model. The approach offers an alternative for studying oligo efficacy, as explored by Rao S et al., (2021) from George Washington University in a published siRNA study exploring evaluating the effect of treatment to prevent cancer development.

Furthermore, a draft document from the US FDA Clinical Pharmacology Considerations for the Development of Oligonucleotide Therapeutics Guidance for Industry cites that ”when the oligonucleotide therapeutic targets the liver, i.e., the pharmacological target is in the liver or there is active targeting to the liver, the sponsor should consider characterizing the impact of hepatic impairment on tolerability, safety, and pharmacodynamics”

Prior use of in vitro MASH models can provide initial insights to better inform study design.

Within the oligonucleotide-based therapeutic class, we have tested siRNA and ASOs. The approach is expected to be compatible with others in the oligonucleotide drug family e.g., microRNA (miRNA) and aptamers.

More widely, our Liver-on-a-chip models have been utilized for antibody testing and their use has been extensively validated using small molecules.