Operating InstructionsLast Edited by: Collin BurkhartRevision 7, 5/11/15

System AssemblyGeneral preparation

1. Seed slides 24 hours before the experiment begins2. Ensure all parts have been gathered

a. Growth chamber componentsi. Aluminum base x1

ii. Polycarbonate lid x1iii. Aluminum insert x1iv. Polycarbonate protective shield x1v. Slides (seeded) x2

vi. Lid Gasket x1vii. Slide gaskets x2

viii. Washers x10ix. Wing nuts x4x. 1” “long” bolts x4

xi. ¾” “short” bolts x6xii. Threaded luer

adapters x2xiii. Roll of Teflon thread

tape x1xiv. Stopcocks x4xv. Stopcock caps x2

xvi. Barbed adapters x2b. Media reservoir components

i. Glass jar x1ii. Reservoir lid x1

iii. 7’ “long” tube x1iv. 18” “short” tube x1v. 2” “filter” tube x1

vi. 2-inch 0.22 μm filter x1vii. 500 mL of media x1

c. Peristaltic Pump d. Incubatore. Assorted tools

i. Wrenchii. Tweezers

iii. Scissors3. Autoclave all parts except stopcocks to sterilize them4. Soak stopcocks in ethanol to sterilize them

a. Adjust toggle during soak to ensure complete sterilization

5. Move all parts to a biosafety hood for assembly in a sterile environment

Growth chamber assembly1. Insert the two slide gaskets into the slide gasket channels of the growth chamber base

a. Do not stretch the gaskets unnecessarily2. Insert the lid gasket into the lid gasket groove

3. Insert the two seeded slides into their slots on top of the slide gaskets using tweezers

4. Insert the metal insert into the polycarbonate lid, ensure the word “DOWN” is visible

a. The word “DOWN” on the insert will later touch the chamber base

5. Align the lid with the insert groove in the chamber base, with “A” on the lid matched to “A” on the chamber

6. Gently push the lid into placea. It can be difficult to line the slides and insert up with their slots while attaching the lidb. Excessive force on the lid can crack misaligned slidesc. Be sure the lid is fully closed as it is tightened down d. To verify the alignment before adding bolts, the lid can be pushed down by hand to

ensure it becomes flush with the metal chamber body on all sides.7. Seal the lid and base together

a. Push the six short bolts through six washersb. Insert the six pieces into the chamber and tighten in the order shown using the wrenchc. Place the protective polycarbonate shield under the chamber body, aligning the holes

d. Push the four long bolts through the corner holes in the chamber

i. The corner holes are not threadede. Place a washer and wing nut on each of the long bolts and tighten them

i. Place the chamber on the reservoir lid to allow access to the top and bottom of

the chamber simultaneouslyii. Hold the bolt head with the wrench while tightening the wing nut

8. Connect the assorted adapters to the chamber inlet and outleta. Cut an approximately 1½ inch piece of teflon tape and then cut it in half the long way

(hot dog style)

b. Wrap a piece of teflon tape clockwise around each threaded luer as shown

c. Connect a wrapped threaded luer to each end of the chamber

d. Connect a stopcock to each of the threaded luer adaptersi. Use the middle stopcock ports

e. Both stopcocks should be in the closed position

i. “OFF” points toward the sealed pathii. The toggle should point toward the middle connection toward the chamber

f. Connect stopcock caps to the middle ports of two more stopcocks

g. Attach the capped stopcocks to the male end of the stopcocks already on the chamber

i. The toggle on the outer stopcocks should point toward the unattached, uncapped ports

h. Connect a barbed adapter to each of the outer stopcocks

9. The growth chamber is now assembled

Prepare the media reservoir1. Obtain the 500 mL reservoir

2. Fill reservoir with 250 mL of media

3. Insert the filter into one end of the 2” length of tubing

a. Use the end that is not angledb. Words on the filter face upward

4. Insert the other end of the 2” length of tubing into a hole in the reservoir lid

5. Pull the 2” length of tubing approximately halfway through the lid6. Insert one end of the 18” length of tubing into another hole in the lid and pull it a few inches

through so it can touch the bottom of the reservoir

7. Insert one end of the 7’ length of tubing into another hole in the lid and pull it a few inches through so it can touch the bottom of the reservoir

8. Clean the tubing that will be within the reservoir with a chem wipe9. Screw on the lid, adjusting tubing as needed

10. Connect the tubing to the barbed adapters on the chamber

THE SYSTEM SHOULD NOW BE CLOSED

Transfer into the incubator1. Be sure the incubator shelving is adjusted to allow for maximum height clearance2. Carefully move the growth chamber and reservoir into the incubator

3. The growth chamber should be positioned front and centera. This allows for easier viewing during operationb. The 18” tubing connected to the reservoir should exit the right side of the chamberc. The 7’ tubing connected to the reservoir should exit the left side of the chamber

4. The reservoir should be positioned behind the chamber against the left side of the incubatora. The reservoir is still in view for observation over the chamberb. This allows the reservoir to be closer to the pump

5. The incubator door can be closed over the tubing running to and from the pumpa. Be sure both sections of tubing are kept close together toward the bottom of the doorb. This reduces the stress on the door and the compression of the tubing

Pump preparation1. Position the pump outside the incubator 2. Ensure the pump is set to “off”3. Plug in the pump

4. Unlock the pump head by pulling up on the metal lever

5. Run the middle of the 7’ length of tubing through the pump head

a. Make final adjustments to tubing nowb. The incubator door may need to be opened for tubing adjustments

6. Clamp the pump head back down using the metal lever

Operation InstructionsBasic operation1. Be sure the assembly instructions were followed to assemble the system

a. Observe the fluid path of the system to ensure components are connected properlyi. Fluid should be able to flow from the reservoir to the pump, then through the

chamber, and finally back to the reservoirb. Ensure connections are tightly sealedc. Ensure the pump head is clamped onto the tubingd. Ensure the pump is plugged in

2. Prime the inlet tubinga. Have a chem wipe ready in the event of a spillb. Disconnect the chamber 7’ inlet tube from the barbed connectorc. Turn the pump on to a low setting to move fluid through the disconnected tube

d. Turn the pump off right before media leaks from the tubinge. Reconnect the inlet tubingf. The tubing is now primed

3. Open the four stopcocksa. Open the incubatorb. Ensure all four stopcocks are openc. The inner stopcock toggles should point away from the tubingd. The outer stopcock toggles should point toward the capped portse. DO NOT RUN THE SYSTEM WITH THE STOPCOCKS CLOSED!

4. Ensure the pump directional toggle will circulate fluid through the system in the correct direction5. Adjust the pump flow rate to a low setting for initialization6. Turn on the pump7. Observe the flow chamber to verify that there is no leaking and fluid is flowing as expected8. Increase the pump flow rate to the desired setting for the experiment9. Allow the system to run for the duration of the experiment10. Be sure to check on the system periodically to ensure it is operating correctly11. Be sure to check the media in the reservoir periodically to check if it must be cycled

Cycling media1. In the event of a lengthy experiment, the media in the reservoir may need to be cycled2. If the media in the reservoir appears to be exhausted of nutrients, begin the cycling process

a. This is also a good time to view the cells under a microscope3. Turn the pump off4. Disengage the pump head from the tubing5. Remove the tubing from the pump6. Open the incubator7. Close all four stopcocks

a. The chamber stopcock toggles should point toward the chamberb. The tubing stopcock toggles should point toward the tubing

8. Unscrew the connection between each pair of stopcocksa. A very small amount of media may leak outb. Be sure to have a chem wipe ready beneath the connections during separation in the event

of a minor leak9. Remove the media reservoir and attached tubing from the incubator10. Spray the reservoir and tubing down with ethanol11. Move the reservoir and tubing into a biosafety hood12. Unscrew and remove the lid13. Remove the exhausted media from the reservoir14. Add 250 mL of fresh media into the reservoir15. Screw the lid back on16. Move the reservoir and tubing back into the incubator17. Gently spray the tubing stopcocks with ethanol just outside the incubator18. Reattach the stopcocks19. Open the four stopcocks

a. DO NOT RUN THE SYSTEM WITH THE STOPCOCKS CLOSED!20. Adjust the pump flow rate to a low setting for initialization21. Turn on the pump22. Observe the flow chamber to verify that there is no leaking and fluid is flowing as expected23. Increase the pump flow rate to the desired setting for the experiment24. Resume the experiment

Viewing cells1. During the course of an experiment, the cells may need to be observed2. The system must be stopped so the chamber can be separated3. Turn the pump off4. Disengage the pump head from the tubing5. Remove the tubing from the pump6. Open the incubator7. Close all four stopcocks

a. The chamber stopcock toggles should point toward the chamberb. The tubing stopcock toggles should point toward the tubing

8. Unscrew the connection between each pair of stopcocksa. A very small amount of media may leak outb. Be sure to have a chem wipe ready beneath the connections during separation in the event

of a minor leak9. Remove the growth chamber from the incubator10. Carefully bring the chamber to an approved laboratory microscope for observation

a. The system is designed to work with the standard inverted, EVOS, and fluorescent microscopes in the BME department

b. The system can be slid from side to side on the microscope stage to view the majority of the cell growth area

c. Do not leave the chamber over a microscope light for more than five minutesi. This may damage the cells

11. When observation is complete, move the chamber back outside the incubator12. Spray the chamber down with ethanol

a. Be sure to spray the exposed stopcocks13. Move the chamber back into its place within the incubator14. Reconnect and open the stopcocks

a. DO NOT RUN THE SYSTEM WITH THE STOPCOCKS CLOSED!15. Adjust the pump flow rate to a low setting for initialization16. Turn on the pump17. Observe the flow chamber to verify that there is no leaking and fluid is flowing as expected18. Increase the pump flow rate to the desired setting for the experiment19. Resume the experiment

Disassembly Instructions1. Initial breakdown

a. At the completion of an experiment, the system must be disassembledb. Turn the pump offc. Disengage the pump head from the tubingd. Remove the tubing from the pumpe. Open the incubatorf. Close all four stopcocks

i. The chamber stopcock toggles should point toward the chamberii. The tubing stopcock toggles should point toward the tubing

g. Unscrew the connection between each pair of stopcocksi. A very small amount of media may leak out

ii. Be sure to have a chem wipe ready beneath the connections during separation in the event of a minor leak

2. Growth chambera. Remove the growth chamber from the incubator

i. Now is the time to observe the cells for a final time within the chamberb. Bring the chamber to a sinkc. Carefully open the chamber stopcocks to drain the chamber fluids into a beakerd. Unscrew all ten bolts sealing the chambere. Remove the protective shieldf. Remove the lidg. Separate the metal insert from the lidh. Remove the outer gasketi. Gently remove the slides

i. They can be pushed up from below if necessaryii. These may be used for further experimentation

j. Remove the slide gasketsk. Unscrew the chamber stopcocksl. Unscrew the threaded luer adaptersm. Remove any teflon film left in the chamber

i. Use tweezers if necessaryn. Rinse all componentso. Scrub the chamber, lid, shield, and insert with soapy waterp. Soak the remaining small components in soapy waterq. Rinse and dry all components

3. Media reservoir and tubinga. Remove the media reservoir and attached tubing from the incubatorb. Move the components to a sinkc. Carefully remove the 7’ and 18” tubing from the lid

i. Tubing may leak as handledii. Tubing still contains media

d. Remove barbed adapters and stopcocks from the longer tubing lengthse. Separate barbed adapters from the stopcocksf. Remove and dispose of the syringe filterg. Drain all tubing into a beaker

h. Run water through the tubing to remove leftover mediai. Tubing can be connected to an air nozzle in a biosafety hood to expel remaining

fluidi. Drain the reservoir into a beakerj. Rinse and scrub the reservoir, lid, and tubing with soapy waterk. Soak the remaining small components in soapy waterl. Ring and dry all components

4. Final cleanupa. Store all system components away as necessaryb. Spray down all biosafety hood and tables usedc. If necessary, sterilize the incubator