使用CCCadvanced™FN1无异源耗材培养人多能干细胞（二）
Materials and Methods
Cell culture conditions and surface transition
Cryopreserved hiPSCs (SC102A-1, SBI™, USA) were initially thawed and
pre-cultivated on a Corning Matrigel-coated surface (Corning, USA) in a
xeno-free culture medium specifcally adapted for feeder-free hiPSC
expansion (Gibco® Essential 8™, Thermo Fisher Scientifc®, USA) according
to manufacturer’s recommendations. After 5 passages on the Corning
Matrigel-coated surface, the cells were seeded on the Eppendorf
CCCadvanced FN1 motifs surface using the classic clump passage
procedure. Briefly, cell clumps were harvested using Gentle-Stem™
Enzyme-Free Human ESC/ iPSC Dissociation Solution (SBI, USA) according
to manu facturer’s recommendations. The cell clump suspension was plated
directly into a ready-to-use FN1 motifs 6-well plate and incubated
under standard cell culture conditions (37 °C, 5% CO 2, humidifed
atmosphere).
Starting at 24 h post-seeding, 100% of the culture medium was replaced
with fresh medium daily until the confluency level of interest was
reached. Cells were maintained on the FN1 motifs surface during 25
successive passages. Micro scopic visual assessment was performed daily
to check cell and colony morphologies as well as qualitatively estimate
the spontaneous differentiation percentage per well. In parallel, hiPSCs
were cultivated on the Corning Matrigel coated surface as well as on the
Corning® Synthemax® II-SC Self-Coating Substrate (Corning, USA) in
order to compare hiPSC growth performances on Eppendorf CCCadvanced FN1
motifs with those obtained on a biological feeder-free culture system
and a competitor synthetic surface, respec tively.
Cell growth evaluation
Cell growth and viability were assessed at each passage through cell
counting performed on 3 independent wells. After complete cell
detachment using Accutase® (Merck Millipore™, Germany), the single cell
suspension obtained through gentle pipetting was transferred to a tube.
The well was rinsed with D-PBS to recover any remaining cells. 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
Stability of doubling time development has been compared using Fisher test for equality of variances.
Alkaline Phosphatase (ALP) staining
The Red-Color™ AP Staining Kit (SBI, USA) was used at dif ferent time
points during the long-term expansion process in accordance with the
manufacturer’s recommendations. Pluripotent marker expression assessment using immunostaining
The preservation of a high expression level of several key pluripotent
markers was demonstrated by immunostaining at the beginning and at the
end of the hiPSCs expansion process using the Pluripotent Stem Cell
4-Marker Immuno cytochemistry Kit (Thermo Fisher Scientifc, USA) accord
ing to the manufacturer’s recommendations. This kit allows specifc
fluorescent staining of two specifc nuclear markers (OCT4 and SOX2) and
two specifc cell surface markers (SSEA4 and TRA-1-60) in cells
counterstained with NucBlue® Fixed Cell Stain (DAPI nuclear DNA stain).
Fluorescent stained cells were observed with the Invitrogen® EVOS® FL
Cell Imaging System (Thermo Fisher Scientifc, USA).
Pluripotent marker expression assessment using flow cytometry analysis
The control of the expression level of 3 key pluripotent markers (Nanog,
OCT3/4 and SOX2) was performed through flow cytometry analyses using the
BD™ Pluripotent Stem Cell Transcription Factor Analysis Kit (BD
Biosciences, USA). At the end of the expansion process, viable cell
density was determined from single-cell suspensions by cell count. The
appropriate number of cells (input cells: 10,000) was prepared for FACS
analysis according to the manufacturer’s recommendations. For each cell
type analyzed, a sample of unstained cells and an isotype control were
prepared in or der to measure autofluorescence and non-specifc staining,
respectively. Cells were analyzed with a BD FACSVerse™ flow cytometer (BD
Biosciences, USA), and data analysis was performed using the BD
FACSUITE™ SOFTWARE (BD Biosciences, USA).
Trilineage differentiation potential
To confrm the ability of expanded hiPSCs to differentiate into cells of
each of the three embryonic germ layers (en doderm, mesoderm and
ectoderm), an in vitro non-directed differentiation process was
performed. Briefly, after expan sion, cells were frst used to form
embryoid bodies (EBs) in suspension containing an EB culture medium
without bFGF (basic Fibroblast Growth Factor) before transfer to a
Corning Ultra-Low Attachment vessel (Corning, USA).
EB formation required 4 days of incubation at 37 °C and 5% CO 2 with daily 100% volume medium refreshment.
After 4 days in suspension, these EBs were seeded on the FN1 motifs
surface and allowed to spontaneously differenti ate over 14 days in the
same culture medium, with medium refreshments performed every two days.
Finally, the differenti ated cells obtained were characterized by
immunostaining using the 3-Germ Layer Immunocyto-chemistry Kit (Thermo
Fisher Scientifc, USA), allowing specifc fluorescent staining of
well-established markers characteristic of the three embry onic germ
layers (β-III tubulin (TUJ1)) for ectoderm, smooth muscle actin (SMA)
for meso-derm, and alpha-fetoprotein (AFP) for endoderm) in cells
counterstained with NucBlue Fixed Cell Stain (DAPI nuclear DNA stain).
This kit was used according to the manufacturer’s recommendations, and
fluorescent-stained cells were visualized using the Invitrogen EVOS FL
Cell Imaging System (Thermo Fisher Scientifc, USA).
Karyotype analysis
Karyotype analysis was performed on fxed metaphas blocked cell samples.
In order to arrest the cell cycle in metaphase, cells were incubated for
50 minutes at 37 °C in the presence of KaryoMAX® Colcemid® (Thermo
Fisher Scientifc, USA). After harvesting and hypotonic treatment,
swollen cells were carefully fxed. Fixed cell suspensions were then sent
to an external service (Cell Guidance Systems Ltd, UK) for G-banding
(Giemsa-banding) karyotype analysis. For each sample, 17 G-banded
metaphase spreads were created and analyzed.
Results and Discussion
Robust long-term xeno-free expansion of undifferentiated hiPSCs on the Eppendorf CCCadvanced™ FN1 motifs surface
The hiPSC morphology was monitored daily during the entire expansion
process of 25 successive passages on the FN1 motifs surface (Figure 1).
Their morphology corresponds to the hiPSC morphology expected on a
feeder-free culture system, characterized by compact cells with a high
nuclear to-cytoplasm ratio and a prominent nucleolus, forming flat,
tightly packed, shiny colonies with well-defned boarders [9]. This
morphology remained stable across all passages and it was similar to the
morphology of hiPSCs expanded on the Corning Matrigel-coated surface
used as a reference feeder free culture system.
Figure 1: hiPSCs morphology during long-term expansion
hiPSCs cultured on the Eppendorf CCCadvanced FN1 motifs surface as well
as on the Corning Matrigel-coated surface showed comparable homogenous
flat and shiny colonies with well-defned borders. The images showed
representative areas at passage numbers 9 and 24, respectively. Scale
bar indicates 400 µm.
The FN1 motifs surface supports a linear cumula-tive popula tion
doubling curve during long-term expansion (Figure 2). Between two
successive passages, surface colony coverage levels of 80-90% were
obtained within approximately 4 days. An efcient and stable hiPSC
doubling time was measured at an average value of 24 h. This doubling
time was similar to that of hiPSCs expanded on a Corning Matrigel-coated
sur face, indicating that cells expanded on the FN1 motifs surface
present the same proliferation rate as cells expanded on the reference
biological coating growth surface. Additionally, hiP SCs expanded on the
FN1 motifs surface exhibit a signifcant ly more stable doubling time
compared to cells expanded on a synthetic self-coating competitor
surface, Corning Synthemax II-SC, as confrmed by the F-test for equality
of variances.
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