Hematopoietic Stem and Progenitor Cell Expansion in Contact with Mesenchymal Stromal Cells in a Hanging Drop Model Uncovers Disadvantages of 3D Culture

Figure 1

MSCs cultured in hanging drop plates form stable spheroids and do not proliferate. 5 × 10³ mesenchymal stromal cells seeded in a hanging drop plate aggregate into a spheroid. (a) Hematoxylin/eosin staining of a spheroid cryosection shows a typical morphology with vast intercellular spaces in the central region and a tight peripheral MSC ring after 3 days of culture. (b) Human bone marrow MSCs express the cell adhesion molecules N-cadherin and cadherin-11 as shown by immunoblotting of lysates from confluent cell layers of two different donors. Using immunofluorescence staining, both cadherins were clearly detectable in MSC spheroids. (c) Aggregation of MSCs into spheroids was investigated in the presence of antibodies against N-cadherin and cadherin-11. On day 1, additional smaller aggregates were detectable as compared to the untreated cells, while all spheroids showed a similar morphology on the second culture day. (d) Determination of the diameter of the formed MSC spheroids with the AxioVision software revealed a continual decrease over two weeks. Spheroid sizes of four different donors were analyzed. Data are means ± SD. (e) Cell numbers of MSCs cultured in GMP-SFEM-CC100 medium as hanging drops (3D) or in conventional 2D plates were quantified in triplicate and are shown as means ± SD. The data are representative of three donors with comparable results. (f) Apoptotic cells in 3- and 6-day-old spheroids were detected by staining with specific anti-caspase-3 and anti-PARP antibodies. Cell nuclei were counterstained with DAPI. The immunofluorescence and light microscopy pictures are representative for MSC spheroids of at least three different donors. Scale bars: 100 μm (a, b, and f) and 250 μm (c).

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