Our exogenic-free method for isolation and in vitro expansion of human adipose-derived stem cells is the answer to the biosafety concerns around cell therapy when it's considered for translational application. Our isolation and expansion methods are simple to replicate, quick, and low in technical requirements, making them useful to everyone. We investigate human adipose stem cells-based therapy strategy for peripheral nerve repair.
However, our technique can be widely applied when new adipose stem cells are needed in different clinical research setups. We kept it simple to conserve easier producibility. Sterility should be carefully observed from operating room until cell culture.
For sample manipulation and fragments, micro dissection are at a risk of contamination. After obtaining the adipose tissue sample from abdominal plastic surgery, using sterile scissors, cut it into pieces of approximately one square centimeter. With a scalpel, micro dissect each piece into smaller fragments and disperse five fragments in a 10 centimeter Petri dish.
For fragment attachment, use a scalpel to create incisions on the plastic surface and drag each fragment. Gently, add 10 milliliters of complete culture medium to cover all the fragments without detaching them and incubate the Petri dish at 37 degrees Celsius and 5%carbon dioxide. Monitor the human adipose-derived stem cell, or HASC, expansion daily under an optical microscope until cells become confluent.
Replace the medium with a fresh complete medium every 72 hours to remove cell debris and let them grow until the first passage. Once the HASC culture reaches 70 to 80%confluency, remove and discard all the pieces of tissue using sterile tweezers. Also, discard the exhausted medium avoiding the HASC's detachment from the Petri dish.
Carefully wash the cells with 10 milliliters of PBS. After removing PBS, add one milliliter of animal-free trypsin and incubate at 37 degrees Celsius for five minutes. Check for cell detachment under an optical microscope and incubate for two more minutes, if required.
To support the cell detachment, gently tap the plastic surface. Stop the trypsin action by adding two milliliters of complete medium and collect all the cells in a 15 milliliter tube. Remove the remaining trypsin by centrifugation.
After discarding the supernatant, suspend the cells in five milliliters complete medium and transfer the cell suspension to a new T25 flask. For cryo preservation, count an aliquot of detach detached cells using the cell counter under an optical microscope. Centrifuge the cells at 300 G for five minutes.
After discarding the supernatant, suspend the cells in the freezing mix at a density of one times 10 to the sixth cells per milliliter and transfer one milliliter of the cell suspension into one cryo vial. Put the cryo vials at minus 80 degrees Celsius in a freezing container that decreases the temperature by one degree Celsius per minute and then move the cryo vials to liquid nitrogen for long-term storage. In the initial cluster appearance, the HASCs showed a classical spindle-like shape and were smaller, as well as more elongated compared to the control cells in the presence of FBS.
The cell immunophenotype appeared to be similar between the HASCs cultured with the two supplements. More than 80 to 90%of the HASCs were positive for CD73 and CD105 and less than 5%expressed for CD34 and CD45. Finally, the proliferation assay revealed a significant increase in cell growth when human platelet lysate was added to the medium compared to the FBS control.
The most important step is the micro dissection of adipose fragments and their attachment with scalpel incision on Petri dish. A ready to use human adipose-derived stem cell population is obtained in respect to the GMP biosafety criteria, keeping therapeutic cell properties and enhancing cell proliferation.
