可靠的CCCadvanced FN1无异源耗材支持人间充质干细...（三）

Results and Discussion
The Eppendorf CCCadvanced™ FN1 motifs surface supports an efcient short-term expansion of hMSC-BM in various xeno-free culture media The ready-to-use FN1 motifs surface supports efcient hMSC-BM growth in combination with different serum-free and xeno-free culture media.
The morphology and proliferation of hMSCs were evaluated 7 days post-seeding on the FN1 motifs surface and on a TC treated surface, in different commercial culture media, respectively (Figure 1 and 2). In traditional serum-contain ing culture systems, hMSC-BM adhered and pro-liferated similarly on the FN1 motifs and the TC treated surface. On both surfaces, cells exhibited their typical fbroblast-like morphology after short-term expansion. In the absence of serum in the culture medium, hMSCs had difculty adher ing and proliferating on the TC treated surface, suggesting the need for additional cell adhesion-promoting coating. By contrast, regardless which of the XF media were tested, the FN1 motifs surface efciently supported hMSC-BM attachment and growth.
As previously described in the literature and by media sup pliers, cells expanded under xeno-free culture conditions exhibited more elongated spindle-shaped cell morphology as compared to cells expanded in the presence of serum and were associated with more compact mono-layers and higher cell densities [12, 13]. Starting from a comparable initial cell density, hMSC-BM yields obtained 7 days post-seeding on the FN1 motifs surface in the three tested XF culture media indicate efcient and similar cell expansion. These results confrm the suitability of the FN1 motifs surface for xeno free expansion of hMSC-BM in combination with various commercial media.

Figure 1: hMSC-BM morphology after short-term expansion on Eppendorf CCCadvanced FN1 motifs surface in different culture media
hMSCs cultured in traditional serum-containing medium display their characteristic fbroblast-like morphology after short-term expansion on both the FN1 motifs surface and a TC treated surface. In contrast to the TC treated surface, the FN1 motifs surface also efciently supports hMSC-BM attachment and growth in various serum-free culture systems, showing a more elongated spindleshaped cell morphology, more compact monolayers and higher cell densities. The images show representative areas at 7 days post-seeding. Scale bar indicates 100 µm.

Figure 2: hMSC-BM proliferation after short-term expansion on Eppendorf CCCadvanced FN1 motifs surface in different culture media

Seeded at comparable cell densities in different commercial serumfree media, the FN1 motifs surface efciently supports xeno-free hMSC expansion with high viable cell numbers 7 days post-seeding. Results are expressed in viable cell number per cm2 and represent a mean of three independent cell counts performed 7 days post-seeding. The dotted line indicates the initial cell seeding density (3,500 cells/ cm2). Average fold inductions are noted above columns.

The Eppendorf CCCadvanced™ FN1 motifs surface supports an efcient long-term expansion of hMSC-BM in a completely defned, animal-component-free culture system
hMSCs were maintained for 10 successive passages on the FN1 motifs surface in order to confrm that this synthetic culture surface supports long-term hMSC-BM expansion in a completely defned, animal-component-free culture system without impacting cell quality. In parallel, hMSC-BM were maintained in an identical ACF culture system on two competitor surfaces, the ready-to-use surface by Competitor A and the self-coated surface by Competitor B. Moreover, cells expanded in a traditional culture system (TC treated surface, serum-containing culture medium and Trypsin/ EDTA as detachment solution) were used as a reference. The morphology of the hMSC-BM was monitored at each passage, under each experimental condition, during the entire expansion process of 10 successive passages, and it was recorded at P4 and P12, respectively, after 2 and 10 successive passages on the different cell culture surfaces (Figure 3).

Figure 3: hMSC-BM morphology after long-term expansion on Eppendorf CCCadvanced FN1 motifs surface and competitor surfaces in animal-component-free conditions
hMSC-BM were expanded for 10 successive passages on the FN1 motifs surface, a ready-to-use surface by Competitor A and a self-coated surface by Competitor B, each showing elongated cells characteristic of hMSC expansion under ACF culture conditions. However, long-term expansion on the surface by Competitor A promoted the formation of large hMSCs such as those observed in a traditional culture system (TC treated surface – serum-containing medium – Trypsin/EDTA), whereas the FN1 motifs surface preserve the characteristic hMSC morphology in restrictive culture conditions during the successive 10 passages. The images show representative areas at the respective passages. Scale bar indicates 400 µm.

At an early passage following seeding (P4), cells displayed the expected hMSC-BM morphology corresponding to a fbroblast-like, spindle-shaped morphology on each tested synthetic surface. As previously described, cells expanded in a serum-containing culture system presented a wider spin dle-shape as compared to the more elongated cells expanded in serum-free conditions. On the FN1 motifs surface, this mor phology remained stable across passages. By contrast, cells expanded in the traditional culture system or on the competi tor A surface exhibited a higher proportion of very large cells at P12, suggesting the emergence of replicative senescence signs [14].
The altered cell morphology was indeed associated with a progressive increase of senescence-associated β-galactosidase positive cells as well as with a decreased proliferation potential during progressive hMSC expansion on a TC treated surface in the serum-containing culture system, especially after passage 8 (Figure 4). By contrast, hMSCs expanded in ACF culture conditions on the FN1 motifs surface maintained a stable doubling time across passages with no signifcant β-galactosidase activity.

Figure 4: hMSC replicative senescence characterization during long-term expansion on Eppendorf CCCadvanced FN1 motifs surface
(A/B) While β-galactosidase staining revealed an increasing number of positive hMSCs in a traditional culture system (TC treated surface – serum-containing medium – Trypsin/EDTA), indicating a permanent cell-cycle arrest from passage 8, hMSCs expanded on the FN1 motifs surface in animal-component-free medium exhibited no senescent phenotype at the same stage. The images show representative areas after β-galactosidase staining of hMSCs, and arrows indicate β-galactosidase positive cells. (C) Expansion on the FN1 motifs surface prevented senescence as characterized by the absence of signifcant β-galactosidase activity and a stable doubling time across passages. Solid lines illustrate doubling times calculated from the proliferation assay. Dotted lines illustrate the percentage of β-galactosidase positive cells. Results represent a mean of three independent cell counts (n=3).

The FN1 motifs surface supported robust and stable hMSC proliferation during the entire culture period of 10 successive passages (Figure 5). Compared with results obtained on other growth surfaces used in combination with animal component-free conditions, the culture of hMSC-BM expand ed on FN1 motifs surface achieved a signifcantly faster pro liferation rate, as shown by a short doubling time and high population doubling number. These results demonstrate that the FN1 motifs surface is a ready-to-use synthetic surface for efcient hMSC expansion in ACF culture conditions.