Nanofiber Fabrication and Seeding of Dental Pulp Stem Cells on Nanofiber
Materials Reagents
Polycaprolactone (PCL) Glacial Acetic Acid
Porcine Gelatin Acetonitrile
4” Blade Scalpel 70% Ethanol
Tweezers Dulbecco’s Modified Eagle Medium
24 Well Plate Trypsin
Aluminum Foil Phosphate Buffer Wash (PBS)
Flasks Dental Pulp Stem Cell Differentiation Medium
Protocol-Intro
-Cells were grown in large tissue culture flasks with a filter cap in DMEM. Medium was changed
regularly every two weeks.
- 500 thousand cells were extracted from the flasks and 30,000 cells were seeded into 12 wells.
-Wells were separated into DMEM for dental pulp stem cells (regular growth media) and
Differentiated DMEM for dental pulp stem cells.
Protocol-Solution for Electrospinning
-Measure out 0.4 grams of polycaprolactone (PCL) and dissolve in 2.5 ml acetonitrile and 2.5 ml
acetic acid solution in a 10 ml beaker.
-Measure out 0.5 grams of porcine gelatin and dissolve in 2.5 ml acetonitrile and 2.5 ml acetic
acid solution in a separate 10 ml beaker.
-Add a stir bar to each beaker and spin at 700-800 rpm at room temperature for 24 hours
-Draw up each solution into a separate 5ml syringe
-Place nozzles into the tip of the syringes
-Turn on the Nanofiber Electrospinning Machine and set up parameters as follows:
Nanofiber Electrospinning Machine Parameters---Co-Axial Method
-Collect fibers on the Collect Plating covered in Aluminum foil.
-Set distance from needle tip from collecting plate at 12 cm.
-Set speed to 0.034 ml/min for the PCL mixture.
-Set speed to 0.017 ml/min for the gelatin mixture.
-Set moving platform distance 45-115 cm.
-Set Positive Voltage for 16KV and Negative Voltage 0 KV
-Spin for 60 mins
-Approximately 1.78 ml solution used
Protocol-Preparing Well Plate
-Cut Nanofiber sheet into 10 circles for well plate using a scalpel
-Use tweezers to pick up nanofiber shapes and place into a 24 well plate
-Place rubber rings on top of samples and leave 1mm room
-Spray 70% Ethanol on to well plate and close well plate
-Move well plate into hood and allow the well plate to dry
-Add 500 ul of sterilized PBS and remove it. Be sure to change pipette tips between each well.
-Allow well plate to dry
Protocol-Seeding cells
-Passage the cells per protocol-wash with PBS, trypsinize, suspend in media, and count cells
-Add 600 ul of regular growth media to 6 wells
-Add 600 ul of differential growth media to 6 wells.
-Seed 30,000 cells per well
-Change media every 2-3 days for 14 days.
Protocol-Cell Staining-Immunocytochemistry
-Remove medium and rinse cells with PBS.
-Fix the cells with 4% paraformaldehyde in PBS for 20 minutes (200 ul/well).
-Remove paraformaldehyde
-Wash with PBS twice
-Permeabilize cells with 1% Triton X-100 in PBS (200 ul/well) for 10 minutes
-Remove Triton X-100
-Wash with PBS twice
-Block cells with 10% FBS in PBS solution at room temperature for 30 minutes.
-Incubate the cells for 15 mins
-Remove FBS and wash with PBS
-Add primary antibody and incubate for 30 mins
-Remove primary antibody
-Wash cells with PBS
-Apply secondary antibody and incubate for 30 mins
-Remove secondary antibody
-Wash with PBS
-Apply DAPI staining (10 ul) and incubate for 10 mins and cover with aluminum foil
-Rinse with PBS three times
-Add 200 ul PBS and store at 4℃
Protocol-Cell Staining-Actin Staining
-Remove media and rinse cells with PBS
-Fix cells with 4% paraformaldehyde in PBS for 20 mins (200 ul/well)
-Remove paraformaldehyde
-Gently wash cells with PBS twice and remove PBS
-Permeabilize cells with 1% Triton X-100 in PBS (200 ul/well) for 10 minutes
-Remove Triton X-100
-Wash with PBS twice
-Incubate in rhodamine in PBS for 1 hour and cover well plate with aluminum foil
-Remove rhodamine
-Wash with PBS
-Add DAPI stain and incubate
-Cover with aluminum foil
-Wash with PBS three times
-Add PBS
-Cover with aluminum foil and store at 4℃.
Protocol-SEM Preparation
-Remove PBS from culture wells
-Dehydrate with alcohol at room temperature
-50% ethanol (0.2 ml/well) for 5 mins twice
-70% ethanol (0.2 ml/well) for 5 mins twice
-90% ethanol (0.2 ml/well) for 5 mins twice
-100% ethanol (0.2 ml/well) for 10 mins twice
-Drying with Hexamethyldisilazane
-1:1 HMDS (0.1ml/well) : ethanol (100%) (0.1ml/well) for 10 minutes in fume hood
-100% HMDS (0.2 ml/well) for 10 minutes in fume hood
-Remove HMDS and dry samples in fume hood.
