This protocol can be used to investigate numerous culture studies and provide a new tool to investigating direct cell communication. This new device saves significant time in establishing a culture model and is applicable to different cell types requiring a specific coating or not. Indeed, the four compartment of the 3D-printed inserts, allow to culture different cell types in monolayer, or in 3D in the same way with different combinations.
The 3D-printed inserts are more durable, flexible, scalable, and can be used to design experimental models for the studies of physiopathologies, such as immunology, or androgenesis. To begin, mix the two components, food silicon, and catalyst provided in the kit in a ratio of 10:1, using a 70%ethanol sterilized spatula according to the manufacturer's instructions. Apply the inserts on the silicone mix with tweezers so that the mixture is homogenously distributed at the bottom edge of the insert.
Place the inserts into the wells of a six well plate and apply gentle pressure to ensure that the inserts are in close contact with the plate to avoid any leakage of the cell culture medium. Put the plate at 37 degrees Celsius to support the solidification process of the silicon for one hour. After solidification, sterilize the plates with the inserts by immersing them in a 70%ethanol bath for 30 minutes to one hour.
Next, discard the alcohol by pipetting, and let the plate dry overnight in a cell culture hood. To culture the keratinocytes of the outer root sheath and human dermal microvascular endothelial cells, discard the medium from the flasks and add five milliliters of trypsin to detach the cells. Place the flask containing the keratinocytes of the outer root sheath at 37 degrees Celsius with 5%carbon dioxide for five minutes, and incubate the flask containing the human dermal microvascular endothelial cells for five minutes at room temperature.
In a 15 milliliter sterile conical tube, mix the trypsin solution containing the keratinocytes of the outer root sheath with five milliliters of mesenchymal stem cell medium supplemented with 5%FBS and growth factors and the human dermal microvascular endothelial cells with five milliliters of endothelial cell medium supplemented with 5%FBS and growth factors. Centrifuge the keratinocytes of the outer root sheath and human dermal microvascular endothelial cell suspensions at 200 G for five minutes at room temperature. After discarding the supernatant, resuspend the cells in two milliliters of their respective mediums.
Mix 10 microliters of the cell suspension with 10 microliters of trypan blue dye, and add 10 microliters of the mix into the chamber of the counting slide. Insert the counting slide into the cell counting system and detect the cell number and viability. After preparing the cell suspension at a concentration of 50, 000 cells per milliliter in the respective medium, distribute 800 microliters to one milliliter of the cell suspension into the individual large compartments, and 100 to 150 microliters into the individual small compartments with a pipette.
See the keratinocytes in mesenchymal stem cell medium supplemented with 5%FBS and growth factors and human dermal microvascular endothelial cells in endothelial cell medium, supplemented with 5%FBS and growth factors in the small and other big compartments. Place the cell culture plate at 37 degrees Celsius with 5%carbon dioxide, or under the usual conditions, to allow for cell adhesion and or maturation. Empty the culture media of the different compartments of the inserts using a pipette.
Then rinse the cells with one milliliter of 37 degrees Celsius warm PBS in the big compartments, and 200 microliters in the small compartments. Add three milliliters of a common medium selected to be compatible for all cell types. Place the plate containing the co-cultures at 37 degrees Celsius with 5%carbon dioxide for 24 to 48 hours.
Detach the cells using trypsin for cell suspension counting, and check the viability using trypan blue staining. After observing the morphology, count the cell number of the different compartments during the experiment under an optical microscope after 24 to 48 hours of incubation. First, remove the inserts from the six well plate at the end of the experiment by a slight twist, and then remove the silicon from the inserts by pulling it off.
Wash the inserts with conventional cell culture detergents and cleaning materials. Then sterilize the inserts for future use in an autoclave, or by immersing them in a 70%ethanol bath for one hour. The phenotype and the cell viability were compared, where 90%viability was observed in the wells of the six well plates, and at 91%was observed in the 3D-printed inserts for keratinocytes of the outer root sheath.
The viability of human dermal microvascular endothelial cells was 85%in the wells of the six well plate, whereas 86%in the 3D-printed inserts. The indirect cell communications between keratinocytes and endothelial cells in the 3D-printed inserts demonstrated that in the presence of keratinocytes in the inserts the human dermal microvascular endothelial cells'proliferation significantly increased by 1.5-fold after 24 hours, and by 3.1-fold after 48 hours compared to the control. However, the keratinocyte of the outer root sheath condition medium was shown to significantly increase human dermal microvascular endothelial cell proliferation by 1.5-fold after 24 hours, and by 2.1-fold after 48 hours.
The 3D-printed insert co-culture model was tested to determine the effect of keratinocytes on human dermal microvascular endothelial cell migration. In the control condition, endothelial cells migrated and covered approximately 44%of the wound area after 24 hours. In the presence of keratinocytes, a significant increase in human dermal microvascular endothelial cell migration was observed, which shows that 43%67%and 99%of the wound area was covered after three, 12, and 24 hours respectively.
Similarly, the migration of HDMECs increases in presence of keratinocytes of the outer root sheath condition medium. It is crucial to ensure the sealing of the insert to the plate, in order to prevent the leakage of the medium of cells from one compartment to another. Several applications could be made, such as proliferation, migration, sedative formation assays DNA, RNA, protein and other analysis.
