Nikolay Samusik and Yury Goltsev, co-founders of Biotech startup Parhelia Bio, created (and patented) a plug-in module that turns any robotic liquid handler into an autostainer for microscopy samples. Now they’re using the Opentrons OT-2 liquid handling robot and the Opentrons Python Protocol API to automate staining protocols such as regular immunofluorescence, Akoya’s CODEX, and IHC with antigen retrieval on FFPE samples.
Opentrons: What was the genesis of the Parhelia device to automate slide staining with a liquid handler?
Nikolay Samusik: The idea and need for the Parhelia PAR2 device stemmed from research we did at Stanford in Garry Nolan's lab where we worked on an ultra highly multiplexed discovery tissue staining protocol called CODEX. CODEX and its clinical grade high throughput companion OPAL are currently distributed by another company we founded—Akoya Biosciences.
Yury Goltsev: Working on CODEX, OPAL, and other advanced tissue staining methods taught us that complicated protocols such as RNA FISH and multiplexed IHC are easy for us as developers but hard for adopters, and the learning curve can be quite high. Multi-step treatments take many hours; depending on antibody/probe incubation time, full procedures can easily take more than a day, and avoiding pipetting mistakes is hard for a novice student and experienced technician. We realized we needed a special device to automate the staining, but with a starting price of more than $150,000 there wasn’t a single “full cycle” stainer that our lab could afford. We also could not find an autostainer that could process coverslips—the mainstream planar support media used in CODEX.
Opentrons: Why is staining so difficult?
Yury Goltsev: It's a multi-step process, and learning a new protocol takes dedicated hands-on effort. To run a large, investigational project that involves a lot of tissue sample processing, you’ve got to have a dedicated technician (or even a team of technicians) diligently following all the steps. A post-doc doing sophisticated multi-color staining will spend most of his time at the bench, with no time left to read and analyze the data. That's a lot of unnecessary manual labor leading to problems with reproducibility which, in our opinion, can only be resolved by efficient automation.
Opentrons: How did you integrate the OT-2 into your lab?
Yury Goltsev: Rather than spending $250,000 that we didn’t have on a commercially available autostainer, we bought a $200 3D printer—which essentially does the same job automating XYZ tool positioning. But then we saw the OT-2 and thought it was the sweet spot for us: it's ready-made, looks nice, inexpensive, and we can merge it with our device.
Opentrons: How do you incorporate the OT-2 with the Parhelia PAR2 device?
Nikolay Samusik: We use OT-2 to dispense the staining and washing solutions into our device. We support staining both coverslips and slides, and, to our knowledge, we’re the only company to have automated coverslip staining. The deck of the OT-2 has 11 slots that can fit SBS format 96-well-plates, so when you multiply 12 coverslips—the capacity of the first version of the PAR2 device—by 11, that’s more than 100 total coverslips that can be simultaneously processed.
Opentrons: Were you familiar with other automated liquid handlers before using the OT-2?
Nikolay Samusik: Not really, but we have coding knowledge, so using Python was easy enough. The open nature of the OT-2 platform made it very accessible for us.
Opentrons: What was it like to get your OT-2 up and running?
Yury Goltsev: For us, the OT-2 is very comfortable. The great thing about the OT-2 is that it allows you at least three ways to interact with it: through the Jupyter notebooks, SSH shell, and the Opentrons Python API. The really great thing is that the protocol runs on the robot, not on the computer, so if the computer disconnects from the robot or something fails, the robot continues to run the protocol.
Opentrons: Did you encounter any challenges setting up your OT-2?
Yury Goltsev: We found that depending on the firmware version of the Opentrons App the initial calibration could be a bit challenging. But the customer support was always instant. We think it is a phenomenal device, extremely smooth, and we love it.
Opentrons: Tell us about automating FFPE antigen retrieval with the OT-2.
Nikolay Samusik: One of our biggest achievements so far has been automating FFPE antigen retrieval (an epitope de-masking step requiring precise high temperature treatment) in the context of the IHC protocol. We managed to do that by using the Parhelia PAR2 device with the Opentrons Temperature Module. Very few other stainers in the market have the ability to do both heat treatment and staining, and the big instrument vendors charge a big buck for an ability to chain antigen retrieval with antibody staining in one instrument. The OT-2 allows our device to run temperature-controlled experiments with the on-deck Temperature Module and opens a whole new range of protocols for us, as we can do everything that hi-end autostainers can do.
Opentrons: Tell us about media and processing with the OT-2.
Yury Goltsev: Our device is extremely compact and contained, so we are able to fit everything into the footprint of the 96 well plate (SBS footprint) and make it compatible with any liquid handler. Second, we wanted all the media to reside in the box because it helps maintain the humidity and protects against biohazard spills. Let's say you're working with dangerous samples: they could be contained completely using our device. You can pretty much do everything and safely discard the box if needed. We believe this is especially important in the context of highly transmissible diseases such as COVID-19.
Opentrons: What’s your vision for expanding your geographic market?
Yury Goltsev: Developing country markets are great for us because our product is less expensive than other autostainers—literally 20 times less. It’s a great option for Africa, or Latin America, or any places where research budgeting is a problem.
Opentrons: Is there anything else you’d like to say about using the OT-2?
Nikolay Samusik: The OT-2 and Opentrons have been a big inspiration for us. The way the company has disrupted the liquid handling market is how we want to disrupt the autostaining market. Most labs don't have liquid handlers precisely because they're out of the budget range, and they're complicated and hard to use, but that’s not our experience with the OT-2.
Yury Goltsev: We have big plans. Frankly, our device is much bigger than just slide staining. We plan to combine it with a microscope. Coverslips, for example, are used in various research contexts other than staining tissues. Our device can automate the processing of proteins, DNA, or chemicals arrayed on the glass. You can put in any sample that needs laminar flow liquid exchange, and our device with the OT-2 creates that laminar flow situation to treat it in this system. There is absolutely no doubt about it: Opentrons opens the universe for us.