How to Clean a Closed Environment for Cells
Resisting the Misting
In a closed cell environment like a barrier isolator, there are few routes for disinfectants to leave and no way for contaminants to float in.
In an open laboratory, you can spray surfaces liberally with disinfectant and it all evaporates into the room air. Keeping surfaces wet with disinfectant for maximum contact time is the major concern with a closed cell environment.
It is a big change, moving from an open lab, which exists in a constant state of high microbial contamination risks, into the closed Xvivo system, a very low risk environment. A risk-based balance has to be struck between much lower risk of contamination and the higher risk of disinfectant fume build-up.
The Cytocentric Environment is Fundamentally Different from the Open Lab
In an open lab, sterile technique training cultivates an essential awareness of all surfaces as inherently contaminated. In the Xvivo, use of routine cleaning protocols means that everything comes into the unit already surface disinfected. Even the air is changed over to sterile-filtered, tanked gases that are free from particles.
Unlike an open room, in an Xvivo system, there aren’t contaminated particles falling onto work surfaces from workers, non-sterile gloves or sleeves. There aren’t any particles from room air re-contaminating cleaned surfaces after you walk away.
It goes against old thinking to realize that unless you actively contaminate them, disinfected surfaces in an Xvivo System stay clean. In fact, because of the continuously re-circulating HEPA-filtered atmosphere, the inside actually gets cleaner and cleaner the longer it runs, all by itself. It is very much the reverse of years of open-lab training with closed cell environments.
Disinfectants in an Isolator Environment
Vaporized Hydrogen Peroxide and Chlorine Dioxide are gas sterilants that are highly effective at ensuring the internal isolator environment is sterile after equipment set-up. As long as any enclosed equipment can tolerate these, vapor sterilizing is the best way to start sterile. If you cannot vapor sterilize the unit, wiping surfaces with disinfectant can be highly effective. Confirming the sterility of your Xvivo with routine microbial environmental monitoring is a good practice.
Just Say “No” To Alcohols
Since flammable vapor build-up can be explosive in an isolator, we strongly discourage use of any alcohols in isolators. Also, while they are helpful for cleaning, alcohols have limited effectiveness as surface disinfectants as they evaporate too quickly. Leave the alcohol for in the bar after work, and move on to a better disinfectant for the lab.
Hydrogen peroxide and peracetic acid based liquid disinfectants such as SporKlenz are highly effective, even against microbial spores. Phenol-based Sporicidin has also been used in isolators. CleanCide is citric acid based and has also been used.
No Spraying Disinfectant Inside a Closed Isolator. Ever.
Never spray liquid disinfectants inside an isolator. With a high air circulation rate, more disinfectant ends up as re-circulating and accumulating fumes than ends up on the surfaces. After initial set-up, wiping high-risk surfaces with gauze dampened with liquid disinfectant is highly effective at preventing contamination. Also, wiping has the advantage of physically removing contaminants through mechanical action while chemically disinfecting the surface.
Label the disinfectant spray bottle “For External Use Only”, tie the bottle to a string attached to the wall on the other side of the room, do whatever you need to do to keep the spray bottle outside the isolator. It is actually a risk to your cells to spray disinfectant inside of a closed cell environment.
There is a risk to cultures should disinfectant fumes build up too high.
We have had reports from users that experienced problems with cells dying over time, starting from the edge of a plate and moving inward, a gas-mediated effect. If you don’t see high VOC readings, sometimes this is the first indication that there is a problem with your disinfection protocol. Another clue, when you can’t smell the internal air, is oxidation of equipment like the metal on microscope objectives. If you see these effects, ventilate your Xvivo with fresh sterile-filtered tanked gases and re-evaluate your routine cleaning procedures.
Limit Wiping to High-Risk Surfaces
It takes a slightly different mindset to work in an isolator, coming from an open lab. It seems to go against training (more cleaning is better, right?), but because of the trade-off between the risk of contamination and the risk of toxic fume build-up, it really is best to limit wiping to high-risk surfaces.
Certainly if the user knows that there has been a biological spill or splash that could have made contact with the walls, it is important to wipe them. However, could the ceiling of a processing chamber really have become contaminated during routine cell handling when nothing has touched it but sterile-filtered air from tanks? It is much less likely. These walls, doors, and ceilings do not need to be wiped with disinfectant on a routine basis.
It Actually Increases Risks to Cells to Disinfect Surfaces that are at Low Risk for Contamination
Once sterilized, the inner surfaces of a closed system stay sterile unless contaminated by the user. Because there is a risk of too much disinfectant fumes building up in the isolator, it actually increases risks to your cultures to disinfect walls, ceilings, or other low-risk surfaces at every use.
Having too much disinfectant in the unit after the active ingredients break down into water can be risky, too. Any residual water in the processing chamber can become a habitat for potential microbial growth.
The Secrets of the Technique
Unless using manufactured wipes, dampen a stack of gauze outside of the unit (such as in an attached laminar flow hood or LFH), place the gauze in a sealed bag, wipe the outside of the bag with dampened gauze, let it dry in the HEPA-filtered air flow in the LFH, and then send it into the Xvivo for wipe-down of high-risk surfaces only.
When wiping isolator surfaces with disinfectant, gauze should just wet enough to feel cool through gloves, not dripping wet. If the gauze dries out, get another piece of damp gauze out and put the dry one in a waste bag.
Wipe door knobs, gloves (from the fingertips upward), sleeves (from gloves upward), and the work surface floor (side to side, back to front, slightly overlapping strokes) after your work. Keep the damp gauze in a sealed bag or container unless you are actively using it to reduce fumes that might re-circulate inside the unit.
Using this technique, we have cultured cells with no antibiotics for over six years without a single contamination event. One customer with a GMP-compliant process has reported to us as having three isolators up and running for over a year without a single CFU detected inside.
A Different Way of Thinking
So if thinking of everything in the hood as “contaminated by default” is part of “sterile” technique, what do you call thinking of everything as “clean by default?”
Perhaps “Cytocentric” technique.
About the Author
Alicia D Henn, PhD, MBA
Alicia Henn has been the Chief Scientific Officer of BioSpherix, Ltd for two years. Previously, she was a researcher at the Center for Biodefense Immune Modeling in Rochester, NY. Alicia obtained her PhD in molecular pharmacology and cancer therapeutics from Roswell Park Cancer Institute in Buffalo, NY and her MBA from the Simon School at University of Rochester in Rochester, NY.