The following protocol provides instructions to generate autologous human-induced pluripotent stem cells through the fast and efficient reprogramming of human dermal fibroblasts isolated from a skin punch biopsy. The procedures can be carried out by researchers with little or no experience in cell reprogramming and with proper adjustment, allows the product of xeno-free induced pluripotent stem cells. Autologous IPS cells might be used for regenerative medicine applications and for modeling diseases like genetic disorders or cancer to investigate on underlying molecular mechanisms and develop more specific therapies.
The workload is high. It is extremely helpful to study the protocol prior to carefully plan the experiment, including the steps during which reagents and materials should be prepared. The visual demonstration of the procedure helps the researches with little or no experience perform the cell isolation and the reprogramming, avoiding common mistakes related to possible microbial and RNA contamination.
Under a sterile hood, wash the freshly obtained sample of human skin from the abdomen in a 100 millimeter dish with HPSS solution. Remove hair and fat using fine forceps and scissors and dissect it with a scalpel to obtain fragments of the size of two millimeters by one millimeters, avoiding scars, dirty or burned areas of the tissue. Place four small fragments in each 35 millimeter dish, cover with a sterile cover glass and add 1.5 millimeters of I-DMEM.
Incubate the plates at 37 degrees Celsius and 5%carbon dioxide for about 15 days or until cells reach 85%confluence. Change the culture medium every three days and check the outgrowth of cells using an inverted phase contrast microscope daily. Now, lift the cover glasses with fine forceps, place them upside down in a 100 millimeter dish and wash with 1X sterile PBS.
Remove the fragments of the samples and discard them. Wash plates with 1X sterile PBS. Remove the 1X PBS and add three millimeters of Trypsin-EDTA to the cover glasses placed in the 100 millimeter dish and add one millimeter of Trypsin-EDTA to the 35 millimeter dishes as well.
Incubate for five minutes at 37 degrees Celsius with 5%carbon dioxide. Then, block the trypsinization by adding five millimeters of TSS to the 100 millimeter dish and two millimeters of TSS to each 35 millimeter dish. Collect the suspension into 15 millimeter sterile tubes, centrifuge at 400 times G at four degrees Celsius for five minutes.
Aspirate the supernatant and resuspend the pellet in 12 millimeters of I-DMEM. Split the cell suspension into three millimeter aliquots and transfer them each into a 60 millimeter plate. Incubate at 37 degrees Celsius with 5%carbon dioxide.
Change the medium daily. Keep cells in culture until they reach a confluence of 75%After that, repeat trypsinization three times to obtain cells that passage four. Then, synchronize the cells by replacing the I-DMEM with S-DMEM and incubate for 48 hours at 37 degrees Celsius with 5%carbon dioxide.
During the cell synchronization, prepare fibroblast expansion medium and total non-modified RNA reprogramming cocktail for fibroblast free programming. To simplify the preparation of the nine tubes of non-modified RNA aliquots that are then split into 36, we used to follow a scheme that we attached to the hood wall. Coat a 24-well plate by transferring 100 microliters of basement membrane matrix thawed on ice to each well to uniformly cover the bottom.
Incubate for at least one hour at 37 degrees Celsius before seeding the cells. Aspirate the medium from plates with cells in culture and quickly wash in 1X PBS. Repeat trypsinization to obtain cells that passage five.
Aspirate the supernatant and resuspend the pellet in an appropriate volume of fibroblast expansion medium. Prepare and clean the hemocytometer with 70%alcohol. Then, prepare a solution by gently mixing 10 microliters of trypan blue and 10 microliters of cell suspension in a 1.5 milliliter tube, and incubate for one to two minutes at room temperature.
Pipette 10 microliters of the solution into the hemocytometer and place it on the stage of an inverted phase contrast microscope to count. Non-viable cells are blue while viable cells are unstained. Count the cells in at least two squares of the hemocytometer chamber.
Perform a dilution to obtain a density of 2.5 times 10 to the fourth cells per 500 microliters of fibroblast expansion medium and resuspend the cell pellet. Pipette 500 microliters of the cell suspension into each well of the 24-well plate. Incubate cells overnight at 37 degrees Celsius and 5%carbon dioxide.
In the morning, remove the old medium from each well and replace it with 500 microliters of pre-zarmed xeno-free factor-free culture medium. Incubate at 37%degrees Celsius under 5%carbon dioxide for at least six hours. Thaw five 15.4 microliter aliquots of total nmRNA reprogramming cocktail at room temperature then place them in ice.
Add 234.6 microliters of reduced serum medium to each aliquot, gently pipette three to five times and label each one as tube A.Label five sterile RNase-free 0.5 milliliter tubes as tube B and mix six microliters of synthetic small interfering RNA transfection reagent with 244 microliters of reduced serum medium. Then, use pipettes to add the contents of each tube B to a tube A drop wise. Mix by tapping the bottom of the tube.
Incubate at room temperature for 15 minutes. After that, to add 125 microliters of nmRNA transfection complex solution from each of the five aliquots to each well, tilt the plate and pipette drop wise into the medium. Mix the contents in the 20 wells by rocking gently.
Use the remaining four wells as a reference. Incubate the plate for 15 hours at 37 degrees Celsius and 5%carbon dioxide. The next day, aspirate the medium.
Under the sterile hood, repeat the transfection procedure, as on day one, every day for three more days. On day five, aspirate the medium and exchange with fresh, pre-warmed XF/FF Culture Medium under a sterile hood. Incubate the plate at 37 degrees Celsius and 5%carbon dioxide.
Monitor the cell culture daily by a phase contrast microscope, until observing the formation of IPSC colonies. The aim of this protocol was to reprogram dermal fibroblasts isolated from abdominal skin using non-integrating reprogramming method. Human fibroblasts outgrew from samples of abdominal skin within one week of culture and reached 85%confluence within two weeks.
Cells were characterized by adhesive growth on plastic culture dishes, and their morphology varied from elongated and spindle-shaped to flattened and star-shaped. Fibroblast morphology and arrangement and culture dramatically changed after seeding on BMM, when they acquired an elongated morphology and arranged to form thin branched structures. Small colonies were already visible in culture at day one from first transfection and their size grew progressively over time, while their number increased til day seven and then remained stable between day seven and day 14, probably as a result of smaller colonies merging to form larger colonies.
Since the reprogramming procedure was performed using antibiotic-free media, in the case where sterile conditions were not guaranteed, microbial contamination became a major issue. Proliferation rate and the plating density impact the reprogramming efficiency, so it is important to plan cell passaging and define the plating density on the basis of cell proliferation rate. Substituting animal-derived reagents for appropriate xeno-free reagents, this protocol allows the reprogramming of adult human fibroblasts into IPS cells in a complete xeno-free culture environment that weren't their clinical use.
Human cells are a potential source of infection with bloodborne pathogens, hence, personal protective equipment should be worn throughout the procedure.
