The overall goal of this in-vitro model is to observe liver cell function in vitro in a physically relevant environment that has separate sinusoid like and bile canaliculi like compartments. This method can help culture cells in a physiologically relevant environment. For example in order to produce more realistic cell responses to drug dosing in vitro.
The main advantage of this technique is that it can be used by any competent cell biologist and it fits in a standard incubator. The modules used in this study are made from one millimeter thick borosilicate glass and have two sideports. Fibers in this module have an outer service area of 4.95 square centimeters or roughly of a well of a six well plate.
Begin by siliconizing the modules before first use. Coat the inner surface with Sigmacote and allow to dry in a fume hood. Then autoclave the module to increase the life of the treatment.
Using a scalpel, cut 75 millimeter long fibers and insert three fibers into each module leaving approximately seven millimeters excess length at each end. Next add 0.5 milliliters of silicone glue to a weighing boat. Then use a P200 pipette tip to pick up a small amount of silicone and work the glue into the ends of the module around the fibers to form a three to five millimeter plug.
Allow to dry for a minimum of three hours. Once dry, use a scalpel to cut the silicone so that it is flush with the glass module ends. Lastly, wrap about four layers of polytetrafluoroethylene tape around one sideport.
Prior to setup, autoclave all of the autoclavable components. Then while working in a laminar flow hood, add 10 milliliters of 70%ethanol to the reservoir bottle and assemble the reservoir bottle, Q series cap, feed tube, pump, and pump tubing. Now loosely place an endcap over the PTFE taped sideport.
Slide the ends of the LS16 module connectors over the module ends and free sideport and connect a 14 millimeter section of LS13 tubing to the module connector nearest to the capped sideport. Connect the module to the pump tubing orientating to the module so the capped sideport is nearest to the pump. Connect the permeate line and retentate line to the module connectors and to the LS14 of the Y connector on the reservoir bottle.
To sterilize, pump ethanol through the module at 800 microliters per hour for a minimum of 30 minutes. To wash the ethanol out of the system, first turn off the pump. Then detach the pump tubing from the module adapter tubing.
Hold the module aloft to drain the ethanol out of the fibers and retentate line. Remove the sideport endcap from the module to drain the ethanol from the module itself and the permeate line, then reattach the sideport endcap. Reverse the flow of the pump to drain the pump tubig and feed line of ethanol.
Once the lines are empty, turn off the pump and reattach the pump tubing to the module adapter. Next, unscrew the ethanol bottle from the lid and replace with a bottle containing 10 milliliters of cell growth medium without serum. Pump the medium through the system at 800 microliters per hour until the retentate line is full of media.
Clamp the retentate like to force the media to permeate through the fibers to wash the module. Then wash for approximately two hours. Harvest the cell type of choice according to standard protocols.
Re-suspend the cell pellets to four times ten to the six cells per milliliter in growth media supplemented as required for the desired cell type. Turn off the pump and drain the module and tubing as before. Then detach the module from the connectors and detach the sterile module endcaps while leaving one sideport free.
Next, use an 18-gauge needle and a one milliliter syringe to transfer 500 microliters of the cell suspension to the module. Take care to avoid bubble formation and not to damage the fibers. Now cap the sideport with an endcap.
Then incubate the cells at 37 degree celsius and 5%carbon dioxide for two to four hours with manual turning of the module by 180 degrees every five minutes. After the incubation, attach a sterilized injection port with cap to one sideport of the module. Remove the other sideport endcap and slowly drain the cells by injecting air into the attached injection port using a 27-gauge needle and a one milliliter syringe.
Replace the injection port with an endcap. Slowly fill the module with media using the free sideport and an 18-gauge needle with a one milliliter syringe. Then remove the module endcaps and attach the module to the tubing using the module connectors.
Lastly, replace the wash media bottle with one containing 50 milliliters of growth media with supplements. Pump the growth media through the system at 800 milliliters per hour. To excise fibers from the module for analysis at the end of an experiment, first unplug and drain the hollow factor bioreactor.
Then carefully insert a small blade between the glass and silicone without damaging the blade. Turn the module so as to cut away the silicone from the glass. Rotate the module over the blade to cut the silicone glue from the glass.
Using the blade, work out the silicone plug from one end, then grasp with forceps and gently pull. Ensure that the fibers come cleanly with the plug. Cells were grown for 48 hours before fibers were excised, fixed, and nuclei stained with DAPI.
Each of the following images is a composite of 12 focus stacked images to increase the depth of field of the resulting image. Areas of higher and lower cell density are found along the fiber at this time point. Where present fiber boundaries are denoted with a red-dashed line.
The scale bar represents 200 microns. This histogram shows cell densities exceeding and after a seven day proliferation period. 2D cell numbers were determined using a haemocytometer.
Hollow fiber bioreactor cell numbers were determined using the PicoGreen assay and a standard curve produced from C3A cells. Here, population doubling times are shown. Cell viability as determined by Trypan blue exclusion at the end of a seven day proliferation is shown here.
Inlet, central, and outlet represent regions within the hollow fiber bioreactor. This image shows glucose consumption and lactic acid production. Levels were monitored at the reservoir bottle as well as the retentate and permeate outlets and compared to routine culture and tissue culture plastic.
After watching this video, you should have a good understanding of how to setup and run this hollow fiber bioreactor culture system.
