Cytometry and Antibody Technology

MACSQuant Tyto review part 1 – The Case for Sterility Focused Cell Sorters in Service Laboratories

by | Nov 20, 2019 | Instrument Demo, Learn: Cytometer Setup | 0 comments

Arya Stark from Game of Thrones recites a list of the names of the people she plans to murder at some point or another. I, for one, think that behavior to be odd, somewhat rude and unhealthy. In my mind, I recite a list of the features I’m looking for on cell sorters.

  • Nimble: The sorter should be able to handle any fluorophore we throw at it. It should not mind how many different ones we decide to use and it should be ready to split the sample into as many fractions as we want.
  • Easy to use: I like the idea that my users are able to run a cell sorter when my staff has gone back home. It’s a better use of the instrument, a more efficient way to utilize the hundreds of thousands of dollars we poured in these beasts.
  • Biosafety: The samples we are getting can come from any source, therefore it is necessary—for the safety of the operator—that the instrument operates under BSL2 conditions. You just never know.
  • Sterility: Quite a few of the samples we sort will go right back in petri dishes. So the sorter has to be squeaky clean.

I’d love to see a cell sorter design that answers the call for all four features. But I’m sorry to say that this sorter does not exist right now. We currently run Arias in our Facility and they are doing great for us, but sterility is just not reliable. Arya didn’t get to murder that lady hanging out with that giant weirdo. She got squashed by that collapsing rooftop. The point is that we might not be able to get everything we want.

Here’s how I know: analysis of verbal communication between homo sapiens. For example, a user comes to me and asks: “Can I put my sorted cells back in culture?”

My answer, developed over 14 years of service in the CAT Facility, sounds like: “Well. Look. We use sterile 10X PBS mixed with Mili-Q water in our instruments, the sample lines are bleached between every experiment and we flush PBS with EtOH at the end of every day to prevent anything from growing. The fluidics lines and the sheath tank are bleached at least once a week and the staff runs sterility checks to survey any contamination issue. So sure, you could put the cells back in culture. We definitively encourage you to use antibiotics though.”

William Shakespeare has Polonius instruct us that ‘Brevity is the soul of wit’. You tell me how my users should feel when I present them with this diatribe. Yet it remains an honest answer. What you should understand is:

  1. No, the instrument is not ‘sterile’, we just clean it a lot.
  2. No, I don’t really know if you’ll get contamination, we just try our best to reduce the risk.
  3. If you do get contamination in your cell culture after the sort, I won’t be able to tell you for sure where it might be coming from.

The solutions we have discussed, implemented, and perfected over the years all rely on better cleaning techniques of the cell sorters. Bleach more often. Have an instrument dedicated to sterile cell sorting that we can bleach more often. Best cell culture practices on behalf of the staff: wear gloves sprayed with EtOH, spray EtOH on the working surfaces, limit sample exposure to air. It is not a trivial task. My staff works very hard at keeping a clean working environment. But if the sorters are to be used by all research groups on campus, are exposed to a great range of samples, and are run unassisted by super-users after hours, it becomes an uphill battle.

We have a battery of advice we give our users who inquire about sterile cell sorts:

  • The most important one is to talk to the staff! Make sure the sorter operator knows what you intend to do with the cells after the sort. That person will take every precaution with your samples and do whatever might help to keep the instrument clean.
  • Use antibiotics!! Gentamicyn is your friend. Penicillin and streptomycin seem to have lost the evolutionary battle against bacteria, so they aren’t enough.
  • Sort in the morning. It’s an empirical observation, but we have better success if we run your samples right after the completing the Aseptic Sort Protocol.
  • And if you do get contamination from the sort, let us know!! We’ll figure out what happened and resolve whatever is going on.

The issue should be fairly obvious, but let’s underline it anyway. There is tremendous loss whenever sorted cells turn out to be contaminated. The patient or animal cells are gone and those may have been difficult or time-consuming to obtain. The money used to sort the cells is wasted. All the reagents and chemicals are wasted as well. If the cells had to be injected in immunosuppressed, germ-free, or humanized animals, that’s another huge loss. But overall the biggest loss is that everyone’s time has been wasted.

For years we have dealt with this problem using the temporary gap solutions described above. It’s a sisyphean struggle. To BD’s credit, the aseptic sort protocol on the Aria works very well. But the issue will not go away without a change in the technology. It just so happens that several new instruments have appeared on the market to deal with the situation.

SONY, for example, offers the FX500, a 3 laser / 8 fluorophore chip-based cell sorter where the user can replace key parts of the fluidic lines before every sort. However, it seems that the cost of replacing all that tubing for each sort might be out of reach for most run-of-the-mill flow cytometry service labs. It might be designed for more clinical settings. Nanocellect offers a similar and more affordable 1 laser / 3 detector linked to a neat cell-to-plate deposition system. Again, the entire fluidics can be swapped between each sort. On a different note, the CellRaft technology from Cell Microsystems allows the user to grow and isolate clonal colonies through imaging. We haven’t had a chance to try any of these platforms in the facility.

I have presented a case for the need of cell sorter designed specifically for sterile cell sorting. The question that remains is wether or not such an instrument is viable in the context of a core facility. Presumably, it could not replace the current workhorse that are droplet-based cells sorters. But it would support a niche of cell-sorting users in need of clean cells to put back in culture, inject in animals, etc. The sterile cell sorter would have to be implacable on the sterility front, while keeping some of the characteristics I listed earlier: nimble, safe, and easy to use. I’ll add one more: the management and the instrument usage will have to be reasonably affordable for the core and the researchers.

The Miltenyi MACSQuant Tyto might be a great candidate for that role. We got to demo this instrument twice over the last two years, and I’ll guide you through our experience in part 2 of this review.


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