Microfluidic Cell Culture

From LPDwiki

About this protocol: This assumes the user has created a device following the steps outlined in Microfluidic Device Fabrication. This protocol was written originally for culturing NIH/3T3 fibroblasts.

Necessary Materials

1. NIH/3T3 growth medium (or appropriate cell growth medium)

2. Microfluidic device

3. Syringe pump

  • Currently using Harvard Model 11 Plus syringe pump.

4. 1 mL Covidien Monoject syringe (sterile)

  • Other syringe sizes may be possible depending on required media perfusion rate

5. PVC laboratory tubing (ID x OD: 1/16” x ⅛”)

  • About 9 inches of tubing should suffice.

6. Straight couplets, 1/16” ID, polypropylene (Cole-Parmer)

7. Industrial dispensing tips, 14 Ga. x 1-½”

  • Purchased from CML supply (Item 901-14-150)

8. Cloning cylinders

  • Purchased from Sigma
  • These should be sterilized and dipped in silicon vacuum grease, both sides.

9. PET film, clear, 0.0005” thickness

  • Purchased from McMaster (ID: 8567K102)
  • Cut into approx. 10 x 10 mm squares and sterilized

10. Sterile 50 mL tube

  • Used as a de facto waste container. A sterile petri dish will also work.

Preparing Device

1. If device has not been de-gassed recently, place device in vacuum chamber and evacuate for 30 minutes.

2. Remove device from vacuum chamber, spray a Kimwipe lightly with ethanol solution, and gently wipe down device before placing in BSC. Clean tubing, coupling, and dispensing tip in same way, and place in BSC.

3. Place cloning cylinder over one inlet/outlet port on device. Fill cylinder with ~400 µL culture medium. Let media flow into channel.

Note: it may be necessary to use tubing to pull media into channel, or aspirate the other end.

4. Once media has settled in channel, aspirate liquid in cloning cylinder and place device in incubator for 30 minutes.

5. Turn syringe pump on, and set the diameter and flow rate to appropriate settings (4.65 mm and 0.1 µL/min for initial design). Turn syringe pump off and disconnect power supply.

6. Wipe syringe pump down with ethanol-soaked Kimwipes. Place pump without power supply into BSC.

7. Wipe pump power supply cable down with ethanol-soaked Kimwipes. Thread power supply through back of incubator, removing and replacing plugs on outside and inside of incubator. This must be done quickly and carefully to avoid contamination!

Note: it may be necessary at this point to remove one shelf from the incubator to accommodate the pump.

Cell Seeding

1. Follow the standard procedure for passaging, such as Culture Passaging, and save the suspension of cells. It is worth noting that microchannels often require very high seeding densities, so re-suspending cells in small volumes of media may save time.

2. Dilute cell stock to desired concentration. For initial seeding of fibroblasts, 750,000 cells / mL worked nicely, though this depends on the channel dimensions. Note that if the desired density is higher than the stock density, the cells must be centrifuged again and re-suspended. This may not be possible for all cell lines, however.

3. Pipette ~400 µL cell suspension into cloning cylinder on device. Check under microscope to ensure cells are flowing into channels. Aspirate liquid from cloning cylinder once cells have begun flowing into chamber.

4. Place device back into incubator and let cells settle for 15 minutes.

While cells are settling, syringes should be prepared for media perfusion. Because air has elastic effects, it is important to reduce the volume of air in the line as much as possible.

5. Connect coupling to one end of tubing, and dispensing tip to other end. Fit dispensing tip onto syringe.

6. Place end of tubing into warmed media and pull until media fills tubing up to the tip of the syringe.

7. While keeping a 50 mL tube underneath the syringe as a “waste” container, detach the dispensing tip while keeping the other end of the tubing at an equal height so that media does not spill out.

8. Quickly push all air out of syringe by depressing the plunger. Reattach the dispensing tip to the syringe. Ensure that the tubing is still filled with media up to the end of the coupler. If not, pull a small amount of media in. Keep a Kimwipe in the BSC to prevent media from spilling out of the end.

9. Fasten the syringe to the syringe pump, using the holders to keep in place the end of the syringe body and the end of the plunger. Tighten the top screw to secure the body of the syringe.

10. With syringe(s) in place, carefully move the syringe pump into the incubator. Watch the tubing to make sure no media leaks. Connect to power supply to the back of the incubator.

11. Turn the pump on. The pump should remember the previous setting for diameter and volumetric flow rate.

Important: Note the position of the directionality switch in the back! This is automatically set to the “infuse” direction. To set the pump to “withdraw” mode, the switch must be toggled. This means that the position of the switch in the back is not absolute with regards to the direction of flow.

12. Toggle the switch in the back of the pump to set to withdraw mode. Place the couplers at the end of the tubing into the outlet of the channel.

13. Once the tubing is in place, pipette ~400 µL fresh media into the cloning cylinder at the inlet.

14. After the cells have settled for 15 minutes, press the “run” button on the pump to begin media perfusion. Place ~150 µL fresh media into the cloning cylinder every 24 hours.