FLUSH BOXES WERE THE ORIGINAL ... AND STILL THE BEST FOR BEGINNERS
Even students can incubate cells in sophisticated
mixtures of O2/CO2...
Good for proof of concept, but not for precision work
A FLUSH BOX IS SIMPLE TO UNDERSTAND
The classic resealable chamber is how cell culture under special atmospheres originated. Whether it was a mason jar or cake tin or tupperware container, over the last 80-100 years flush chambers have been the first place to turn when a special mix of gas for cells is required. Flush boxes are the most economical and straightforward method available. Placing cultures inside sealable containers, flushing air out with the desired gas mixture, then sealing the chamber to hold that atmosphere is basic. Even today flush boxes are used extensively, and for good reason. Virtually any gas mix can be used. Different gas mixes can be used in separate chambers for different protocols. Cultures sealed inside are not disturbed even inside a host incubator that is opened frequently. Cultures can be carried across the room without disturbance to conditions and without exposure to room contaminants. Many labs keep a flush box handy. All sorts of miscellaneous random resealable chambers have been successfully used, but by far the best is probably the only one specifically designed for people doing cell culture. Over 30 years ago Billups-Rothenburger designed a flush chamber built out of rugged autoclavable clear plastic, with internal multi-level racks just right for holding culture vessels, a convenient single stainless steel clamp for easy open and close, with autoclavable seal gaskets, and integral gas flushing ports with hose clamps on both inlet and outlet. No longer did people have to cobb up their own. They started thousands of cell scientists down the path of atmosphere optimization and physiologic simulation.
Sometimes Cells Need Full Time Optimization
One handicap with flush boxes is the reliability of "conditioning", as opposed to real time control. Usually the desired atmosphere is ordered and comes in a pressurized tank of premixed gas. Technician simply flushes the box with the premix, then seals it off. The chamber seal is then supposed to hold the mix. Conditioning establishes desired conditions at the start. Then you depend on the seal to keep those conditions over the full course of culture. Trouble is, if the seal is not perfect, conditions won't hold. They will change over time. Human error, debris blocking the seal, degraded seal gaskets can all prevent a good seal. There is no way to compensate if a leak occurs. Worse, there is no way to tell if something like this happens. There is no way to know what actually did happen. If you get inconsistent results, you don't know if it was due to the lack of control or something else. Now you can prevent these problems by upgrading your flush boxes with feedback control.
CYTOCENTRIC SOLUTION #1 - To make sure your flush box maintains the premix over the full course of every culture, don't condition. Control. Add a ProOx controller. It continuously measures the conditions inside the flush box and stands ready to compensate for any leak, as needed, whenever needed. If conditions start to show drift away from optimum, it will infuse just the right amount of premix to keep conditions optimum.
CYTOCENTRIC SOLUTION #2 - For more versatility than a premix with fixed gas proportions, upgrade with a ProOx C21 controller. Set the O2 and CO2 concentrations to any level you want. Change them anytime you want. Avoid the uncertainty of conditioning. Get more consistent results by directly controlling the atmosphere in real time. No longer depend on the perfect seal. No longer be in the dark about what the cells actually see.
Another handicap is apparent when you access your cells. To examine them under a microscope or to change the media, for example, you have to open the flush box. Optimum conditions are lost. Flush boxes may be good for "hands-off uninterruptibility", but not for "hands-on uninterruptibility". Now meet cell need for uninterruptibility during handling by adding a processing chamber with controlled environment same as flush box. Move the flush box inside. Only open it inside.
CYTOCENTRIC SOLUTION #1 - To keep O2 and/or CO2 constant while accessing your cells incubating in a flush box, move the flush box into the I-Glove or C-Shuttle glove boxes before opening:
CYTOCENTRIC SOLUTION #2 - To keep O2/CO2/temperature/CO/NO etc. constant during handling, or for examination under a microscope, or plate reading, or any other manual or automated processing, move the flush box into a properly configured Xvivo System before opening.(CO2 incubator/Billups with arrow pointing toward small Xvivo System.)
Sometimes Cells Need Dynamics
Cells in culture are not static, yet conditions inside a flush box usually are. Eliminate that handicap by fitting your flush box with a dynamic OxyCycler controller.
CYTOCENTRIC SOLUTION #1 - For programmable dynamic O2 and CO2 control, retrofit a flush box with an OXYCYCLER C42. Up to 2 flush boxes can be fitted and operated simultaneously with different conditions in each: both dynamic, or both static, or one dynamic while the other is static. (CO2 incubator with 2 Billups chambers inside + OxyCycler = new Incubator Subchamber Culture System with Oxycycler on incubator with 2 Billups inside)
CYTOCENTRIC SOLUTION #2 - For programmable dynamic CO and O2 and CO2 control, retrofit flush boxes with an OxyCycler C41OCC. Cells incubating inside now can have dynamic or static conditions. (CO2 incubator with 1 Billups inside + OxyCycler = Sub.Cham.Cult.Syst. of OxyCycler on CO2 incubator with Billups inside)
CYTOCENTRIC SOLUTION #3 - For programmable dynamic NO and O2 and CO2 control, retrofit your flush boxes with an OxyCycler C41OCN. Cells incubating inside now have dynamic or static conditions. (CO2 incubator with 1 Billups inside + OxyCycler = Sub.Cham.Cult.Syst. of OxyCycler on CO2 incubator with Billups inside)
Sometimes Cells Need To Fit
Sometimes cells need to fit the time you have available. Another handicap with a flush box is the work it takes to operate. If the technicians culturing cells are too busy with flush boxes, cell needs can be neglected if they can't fit into your schedule.
To incubate cells in a flush box, you have to first position culture vessels inside open flush box, then close flush box, then clamp seal flush box, then hook up gas hose from gas tank, then open gas tank valve, then set pressure on gas tank pressure regulator, then open gas release valve until proper flush rate is established, then set timer, then wait sometimes 15 minutes until timer goes off to signal flush is complete, then turn off gas flow, then clamp gas outlet port to close, then clamp gas inlet port to close, then disconnect gas hose, then carry loaded flush box to incubator carefully keeping it level so cultures don't spill, then open incubator, then carefully place flush box inside incubator, then finally close incubator door.
To get at cells incubating in a flush box, first open the incubator, then carefully remove the loaded flush box from the incubator, then carry the flush box to the hood carefully keeping it level so cultures don't spill, then carefully place it in the hood, then unclamp the seal, then open by removing the top, finally remove culture vessels from the open flush box. Whew. To incubate after processing, go back to start. Whew again! If you have to process many cultures in many flush boxes, the work can be oppressing. It's no wonder some cells might get neglected. It's no wonder human error can creep in, with all that handling. Spills are more likely. Eliminate this handicap by upgrading to a different type of chamber. Put the flush box on the shelf, or donate it to the beginners in the new lab down the hall.
CYTOCENTRIC SOLUTION #1 - Substitute your flush box with a C-Chamber. These are much more convenient to operate. They sit on a shelf inside your incubator. Instead of all that clamping and unclamping, carrying back and forth, stopping and starting flushing, eliminate it all. With a C-Chamber, simply open the incubator, then open one more small door. There's your cells. You get all the advantages of a closed chamber, with none of the work. Plus C-Chambers are completely compatible with and ready fit for any of the cytocentric controllers.
CYTOCENTRIC SOLUTION #2
Go all the way. You've paid your dues. Eliminate all the work. Eliminate all the limitations. Meet all the needs of your cells. Cytocentric-by-Design. It will take your cells and you to the top. Now meet those cell needs!
SOMETIMES CELLS NEED MORE... than people!