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Discover Aria Applications

Aria : 1 instrument, a world of applications

Aria performs any protocol with multiple fluid delivery. Replacing and automating manual pipetting reduces errors, increases reproductability and saves time.

Papers

Radtke, A.  <https://arxiv.org/abs/2107.11364> et al. (2021)IBEX: An open and extensible method for high content multiplex imaging of diverse tissues, In Press

Huang, K.  <https://bio-protocol.org/e4128?p=51&way=207> et al. (2021). A Novel Method to Map Small RNAs with High Resolution. Bio-protocol 11(16): e4128. DOI: 10.21769/BioProtoc.4128.

PRESENTATION

Radtke, A. <https://drive.google.com/file/d/1QkJSvtH2oAUFrssg-Sp7jp5XQQysuDP9/view> et al. (2021)IBEX: An open and extensible method for high content multiplex imaging of diverse tissues, Presented at CZI HCA network and HuBMAP consortium

DNA and RNA Hybridization

Over the past decades, in situ hybridization has been used with greater frequency to  capture the localization, structure and expression of specific DNA and RNA sequences within tissues. The high sensitivity and specificity of this technique relies on the hybridization of labelled oligonucleotide probes to targeted DNA or RNA sequences within tissues. Chromosomal microdeletion, amplification, structure, translocation and expression can be easily detected. Compared to PCR analysis, Northern Blotting or a DNA microarray performed on lysed cells, hybridization provides spatial information as specific RNA and DNA are detected within tissues. It has significantly improved gene mapping, cytogenetics and various diagnostic techniques (oncogenic, prenatal, viral infection…).

Recently, a temporal dimension was added to the technology, as it further evolved to be performed on living cells. The spatio-temporal expression, degradation and storage of RNA molecules can now be thoroughly investigated by real time imaging  of the hybridization of labelled oligonucleotides in living cells.

Why use Aria?

DNA and RNA hybridization are often performed under a microscope. Manual injection over a microscope can be risky as :

  • a touch of pipette cone can misplace the dish and recorded position,
  • samples can be flushed away during pipetting,
  • liquid can be spilled over the microscope.

To overcome these issues, Aria automates multiple fluids delivery. The sample is preserved as the flow rate is controlled. Sterility is guaranteed. Reproducibility is increased as inter and intra operator variability are alleviated.

Miniaturizing glass slide protocols into microfluidic chip protocols might require the optimization of several steps mainly due to differential surface properties and the higher volume to surface ratio in microfluidic chips. Higher reagent concentrations might be needed to compensate for non-specific adsorption. On the other hand, the total volume needed might be reduced as well as the duration of the steps as binding kinetics are different under continuous flow-through incubation.

AriaFluigent’s automated perfusion system, has been designed for multi-step protocols and allows for automation of the entire experiment. It integrates all fluidic connections and comes with an intuitive software package.

Following is an example of a DNA hybridization protocol performed on a chip:

  • The ethanol dehydration step is replaced with a continuous washing step using SSC (Saline Sodium Citrate) 2x buffer:
  • Cell adhesion: 50µL cell solution is delivered in the chip at 100µL/min (step 1) and left to incubate for 30 minutes (step 2). Non adhering cells are removed by flowing SSC 2x buffer in the chip at 100µL/min for 2 minutes (step 3).
  • Enzymatic digestion: Pepsin solution is perfused at 100µL/min in the chip for 7 minutes at 37°C (step 4) and then washed with SSC 2x buffer at 100µL/min for 7 minutes (step 5).
  • Fixation: 100µL of Carnoy’s fixative is delivered to the chip at 100µL/min and left to incubate for 20 minutes (step 6). Cells are then washed with buffer under continuous flow at 100µL/min for 20 minutes (step 7).
  • Probe injection and denaturation: 40µL of labeled DNA probes mix solution are delivered to the chip at 100µL/min (step 8). DNA probes are co-denaturated at 75°C for 15 minutes and incubated for 37°C overnight (step 9).
  • Washing: Cells are then washed with SSC x2 at 100µL/min for 20 minutes (step 10).

Using Aria’s software, the protocol would look like this (the number of the steps in Aria coincides with the steps in brackets in the protocol):

Aria Software Dose response

Drug combination and long-term perfusion

In various therapeutic areas (glaucoma, vascular, HIV, oncology), a combination of drugs is typically found to be more effective for disease treatment. Oncology, in particular, has really paved the way for this approach to these new therapies due to the failure of a ‘one size fits all’ approach. The emergence of next-generation sequencing technology profiling has revealed the heterogeneity in many cancers at the origin of the differential response to treatment. As a result, therapeutic strategies are evolving towards multi-targeted drug combinations that effectively inhibit the cancer cells and block the emergence of drug resistance while selectively incurring minimal side effects on healthy cells.

Drug combination therapy is not straight forward as in many cases; the results do not equal the sum of the parts. Cross-reactions are observed and fall into 5 categories: low risk & synergy, low risk & no synergy, caution, unsafe, and dangerous. The contribution and dosage of each active molecule should be closely investigated in terms of dose, frequency and duration to evaluate the efficacy of drug combination.

Why using Aria?

Aria is the perfect system to study drug combinations as up to 10 different active molecules can be perfused at a controlled flow rate over a sample. Frequency of dosage, volume, and incubation time are all parameters that can be easily evaluated using its intuitive software. In addition, the effect of drug combinations can be investigated over long term experiments (up to several weeks) as Aria comes with two refillable reservoirs.

Here is an example of a drug combination protocol:

A combination of 3 drugs administrated successively with 2 hours delay every day for 3 days will be tested. Each drug will be perfused at 100µL/min and left to incubate for 30 minutes. The drugs will be delivered at specific dosages: drug 1: 100µL, drug 2: 50µL and drug 3: 150µL. Tubing is cleared between deliveries and cells are washed under constant perfusion for 10 minutes at 100µL/min. To mimic human uptake of drugs, protocols are planned to start each day at 8 a.m.

To translate the experiment into Aria’s protocol:

First the time of start is set to 8.am:

First step starting 8 AM is the delivery of 100µL of drug 1 at 100µL/min and its incubation for 30 minutes.

Tubing is then cleared. Samples are washed with buffer for 10 minutes at 100µL/min and waiting time is adjusted to 1h20 to reach the 2 hour pause between two successive drug deliveries.

50µL of drug 2 are delivered in the chip at 10 AM. The protocol is repeated for drug 2 and drug 3. The final waiting time is adjusted to 19h18 to repeat the whole protocol at 8 AM on the following day. Meanwhile phenotypic analysis can be run on cells to investigate their response to the repetitive exposure of drug combinations.

Immunostaining

Immunostaining experiments are multistep protocols to detect specific antigens in biological samples. The sample is successively incubated or exposed to fixation agentswashing buffers, and probes.

In conventional protocols on petri dishes or well plates, fluid deliveries are performed manually using pipets. Solutions are directly added to the sample.

Transposing protocols from a petri dish to a microfluidic format usually requires some minor adjustments as cells or tissue samples are exposed to solutions in a different manner. As opposed to petri dishes, microfluidic chips or chambers are closed systems. Therefore solutions cannot be deposited directly on the top of the cells but are perfused over the sample at a controlled flow rate, reaching all cells homogenously. The use of a rotary valve with perfusion systems enables one to perfuse different solutions at given time points in the chip or chamber. In addition, automating the sequential delivery of solutions saves time and avoids creating bubbles inside the chips by disconnecting and reconnecting the traditional pump to the chip.

Immunostaining_Image

Why using Aria?

Historically, performing multi-step protocols has been very time consuming and suffer from reproducibility issues with numerous manual steps. AriaFluigent’s perfusion system has been designed to overcome these issues and allow for automation of the entire protocol. It integrates all fluidic connections and comes with an intuitive software package.

Following is an example protocol using a chip with a volume of 250µL and the following labeling protocol:

  • To fix cells: 300µL of a solution of 4% (v/v) PFA is delivered in the chip (step 1) at 50µL/min and incubated for 30 minutes (step 2)
  • PFA is then washed with PBS under continuous flow for 30 minutes at 100µL/min (step 3)
  • To permeabilize cells, 300µL of Triton solution is delivered in the chip at 50µL/min (step 4) and incubated for 10 minutes (step 5)
  • Triton is then washed for 30 minutes at 100µL/min (step 6)
  • 300µL or primary antibody solution is then delivered in the chip at 50µL/min (step 7) and incubated for 2 hours (step 8).
  • Primary antibodies are then washed for 30 minutes at 100µL/min (step 9)
  • 300µL of secondary antibody solution is then delivered in the chip at 50µL/min (step 10) and incubated for 2 hours (step 11).
  • Secondary antibody solution is then washed over for 30 minutes with PBS (step 12).

Using Aria’s software, the protocol would look like this (the number of the steps in Aria coincides with the steps in brackets in the above protocol). Note that by using Aria, the 8 manual pipetting steps and preparation and waiting time associated are replaced with 3 easy steps at the beginning of the protocols: preparing all solutions, connecting the chips, loading the protocol:

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