Mesenchymal Stem Stem Cells (MSCs) could provide
alternative therapeutic solutions for a number of bone and
cartilage tissue diseases, playing an important role in regenerative
medicine. Several sources of stem cells are currently available. In
recent years, researchers attention was focused on the isolation of
Adipose Stem Cells (ASCs) because they are easily obtainable in
large quantity trough a minimally invasive procedure.
Furthermore, Adipose Derived Stem Cells (ADSCs) have multiple
properties, including self-renewal, differentiation potential,
secretion of active factors that might influence surrounding cells
and immune modulatory effects useful in the treatment of several
degenerative diseases. The aim of this study was to present a new
“in vitro” ADSCs isolation and characterization protocol using a
mechanical method in comparison to the enzymatic digestion that
has represented the gold standard procedure to isolate adult
mesenchymal stem cells.
Methods
Lipoaspirate from forty human donors were obtained by
minimally invasive liposuction procedures: by definition,
lipoaspirate contains a relatively high content of ASCs (1-5% of
isolated nucleated cells). A minimum starting volume of 30 mL of
lipoaspirate was obtained in all procedures for a sufficient yield of
mesenchymal stem cells (MSCs). Lipoaspirate was stored at 2-8º
C for a maximum of 24 hours before use. Tissue from each donor
was split in two samples and each sample was treated by two
different in vitro procedures with the goal to isolate ADSCs:
enzymatic digestion and experimental mechanical isolation
procedure. Tissue from both methods were first diluted with an
equal volume of Phosphate-Buffered Saline (PBS), centrifuged at 500 xg at 22° for 5 minutes, digested adding 1 ml of collagenase
digestion solution for every 1 ml volume of tissue, incubated at
37ºC in a pre-warmed orbital shaker for 30 minutes, centrifuged at
22°C for 5 minutes at 500xg and finally suspended for expansion
in alpha-MEM medium adding 10% of bovine fetal serum.
Subsequently, MSCs were characterized following the guidelines
of the Society for Cellular Therapy (SCT).
Results
Characterization of the processed tissue was performed
after digestion and “in vitro” culture expansion of plastic
adherent cells. Our results showed that cells from both study
groups were adherent to the plastic having fibroblast morphology.
The cells were immune-characterized by Flow cytometry. Results
showed a phenotypic mutation during the process of cellular
expansion, from the isolation of the stromal vascular fraction to
the selection of a purely population with mesenchymal stem cells
properties in the subsequent passages. After the isolation of
MSCs, the obtained ADSC showed a subpopulation (immuno
phenotype differentiation) expressing specific markers such us
CD 34, a marker associated with the state of vascular and
endothelial progenitor, and CD 146, a pericytic markers. Finally,
the evaluation of the cells differentiation potential showed that
ADSCs, under controlled condition, were able to differentiate into
adipogenic, chondrogenic and osteogenic cells. The results were
similar for both differential methods.
Conclusion
Our experience suggests that ADSCs enzymatically and
mechanically isolated from Lipoaspirate respond to the three
criteria of the Society for Cellular Therapy (SCT) and, for this
reason, can be considered as MSCs. New methods to treat fat
tissue and obtain ADSCs, as the mechanical method described in this article, can be safely developed to isolate in an easier way
these promising cells. Therefore, improved characterization
protocols are still necessary in order to make a final population of
ADSCs as an homogeneous cellular product ready for clinical
application in the field of osteogenic and chondrogenic tissue
engineering. Finally, more studies are necessary to understand the
immunomodulatory and anti-inflammatory properties of ADSCs
before clinical applications to treat different orthopaedic
pathologies