Materials and Methods
Short-term cell growth evaluation
Lonza? Poietics? human mesenchymal stem cells (hMSC-BM, PT-2501, Lonza)
derived from normal adult bone marrow were thawed at passage 2 and
pre-cultivated as recommended by the supplier. In order to evaluate the
ability of the Eppendorf CCCadvanced FN1 motifs surface to efciently
support hMSC-BM short-term expansion under di?erent xeno-free (XF)
culture conditions, hMSC-BM (P3) were plated at the initial cell density
of 3,500 cells/cm2, either on a TC treated culture surface or on the
ready-to-use FN1 motifs surface in the presence of 3 di?erent XF culture
media: StemPro? MSC SFM XenoFree medium (A1067501, ThermoFisher
Scientifc?), MesenCult? ACF Medium (05449, STEMCELL? Technologies) and
Corning? stemgro? hMSC Medium (40-410-KIT, Corning). In parallel, cells
were similarly plated on both surfaces in a serum-containing medium
(MSCGM?, PT-3001, Lonza). After seeding in the appropriate culture
medium, hMSCs were incubated under standard cell culture conditions (37
°C, 5% CO2, humidifed atmosphere) and fed every 3 days by refreshment of
the entire volume of culture medium. At day 4 and day 7 post seeding,
after microscope analysis, cells were harvested with 0.25% Trypsin/EDTA
according to manufacturer’s recommendations. After centrifugation, three
independent cell counts were performed on each cell suspension using
the Vi-CELL? automated cell counting device (Beckman Coulter?, USA). The
average viable cell density was then used to evaluate the viable cell
number/cm2 in each of the growth conditions.
Long-term cell growth evaluation
A unique pool of human bone marrow-derived mesenchymal stem cells
(hMSC-BM, PT-2501, Lonza) was thawed at pas sage 2 and seeded directly
at the initial cell density of 3,500 cells/cm2, either on the FN1 motifs
surface or on two other synthetic culture surfaces, the ready-to-use
surface by Competitor A and the surface by Competitor B which o?ers a
self-coating solution. In order to ensure a com pletely defned
animal-component-free (ACF) culture system during the entire expansion
process, cells were expanded in StemPro MSC SFM XenoFree medium and
harvested for subculture using TrypLE? Select 10x (A12177-01, Thermo
Fisher Scientifc). As a reference, hMSC-BM were expanded in parallel in a
traditional culture system consisting of a TC treated surface, 10%
FBS-containing culture medium and 0.25% Trypsin/EDTA as detachment
solution. After seeding in the appropriate culture medium, hMSC-BM were
incubat ed under standard cell culture conditions (37 °C, 5% CO2,
humidifed atmosphere) and fed every 3 days by refresh ment of the entire
culture medium volume until a con?uence level of 70-80% was reached.
For each experimental condition, cells were cultured for 10 successive
passages (from P3 to P12). At each passage, cell morphology was examined
with the EVOS? FL Cell Im aging System (Thermo Fisher Scientifc, USA).
Cell growth and viability were assessed on 3 independent T75 ?asks per
experimental condition. After complete cell detach ment, a cell count
was performed on each homogenized cell suspension using the Vi-CELL
automated cell counting device (Beckman Coulter, USA). Population
doubling (PD) and doubling time (DT) were calculated using the
respective formula:
PD = (log10(NH)-log10(Ni))/log10(2)
DT = time in culture (hours) x (LN(2)/ LN(NH/Ni))
NH = total number of harvested viable cells
Ni = initial number of seeded cells
For the purpose of evaluating statistical significance, a Student t-test
was performed on normalized doubling time data ob tained during 10
successive passages.
β-galactosidase staining
At every two passages within the expansion process, the
senescence-associated β-galactosidase (SA-β-gal) activity was evaluated
on expanded hMSC-BM using the Senes cence Cells Histochemical Staining
Kit (CS0030, Sigma) in accordance with the manufacturer’s instructions.
hMSC-BM surface marker expression analysis by ?ow cytometry
The preservation of the hMSC-specifc immunophenotype was assessed on the
initial cell population (input cells) and at the end of the long-term
expansion process for each experimental condition. The positive and
negative expression levels of several key surface markers (positive
markers: CD44, CD73, CD90 and CD105; negative markers: CD11b, CD19,
CD34, CD45, and HLA-DR) were evaluated through ?ow cytometry analyses
using the BD Stem?ow? Human MSC Analysis Kit (562245, BD Biosciences).
Brie?y, viable cell density was determined from a single-cell suspension
via cell count, and the appropriate number of cells (input cells:
10,000) was prepared for FACS analyses according to the procedure
recommended by the antibody kit manufacturer. For each cell type
analyzed, a sample of unstained cells, as well as an isotype control,
were prepared in order to measure auto-?uorescence and non-specifc
staining, respectively. Cells were analyzed with a BD FACSVerse ?ow
cytometer (BD Biosciences, USA), and data analysis was performed using
the BD FACSUITE? SOFTWARE (BD Biosciences, USA).
hMSC-BM multi-lineage di?erentiation potential
Preservation of the multipotent di?erentiation potential was evaluated
on hMSC-BM expanded for 5 passages on the FN1 motifs surface in an ACF
culture system. Osteo genic, adipogenic and chondrogenic di?erentiation
was induced by using the MesenCult? Osteogenic Stimulatory Kit (Human)
(05434, STEMCELL Technologies), the hMSC Adipogenic Di?erentiation
Bulletkit? (PT-3004, Lonza) or the hMSC Chondrogenic Di?erentiation
Bulletkit (PT-3003, Lonza), respectively, according to the manu
facturer’s instructions. As negative controls, uninduced cells were
maintained in parallel in their initial culture medium. Respective
di?erentiation efciencies were assessed by specifc stainings. The
osteogenic di?erentiation and mineralized matrix accumulation were
highlighted by Alizarin Red staining performed 21 days post-induction.
The intracellular lipid droplet accumulation associated to the
adipogenic di?erentiation was confrmed by Oil Red O staining per formed
21 days post-induction and the glycosaminoglycans secreted by
chondrocytes were observed through Alcian blue specifc staining
performed 14 days post-induction.
