Simplification of aggregate culture of human mesenchymal stem cells as a chondrogenic screening assay

Abstract

Aggregate culture provides a three-dimensional (3-D) environment for differentiating or differentiated cells; it is particularly useful to study in vitro chondrogenesis and cartilage biology. We have recently ported this method from a conical tube-based format to a 96-well plate format for the study of mesenchymal stem cell (MSC) chondrogenesis. The microplate format has greatly reduced the workload and materials cost, while maintaining reproducible chondrogenic differentiation. A long-term goal is to fully automate aggregate culture--this requires critically identifying all the indispensable steps of the protocol. Robotic laboratory equipment for manipulating microplate assays are commercially available; however centrifugation steps are difficult to implement automatically. We, therefore, tested whether the centrifugation step can be eliminated, thus significantly streamlining the assay workflow. By comparing aggregates prepared from human bone marrow-derived MSCs (hMSCs) that were formed either through centrifugation or through free sedimentation, we found that both methods produce aggregates with similar formation kinetics, and that there was no perceptible difference in the timing of the appearance of markers of chondrogenesis. Thus, it appears safe to eliminate the centrifugation step from the aggregate culture protocol. This results in significant time and effort savings and paves the way for future full automation of the aggregate assay.

Figure 1. Toluidine blue O stained histological sections of centrifuged (indicated as C) and uncentrifuged (indicated as U) aggregates harvested at various time points

D2 indicates day 2 and so on through day 21. Scale bar, 1 mm.

Figure 2. Immunohistochemically stained sections of centrifuged (indicated as C) and uncentrifuged (indicated as U) aggregates

Immunoreactivity for collagen types I, II, and X was detected using a fluorescein isothiocyanate (FITC)-labeled secondary (green) antibody; 4′,6-diamidino-2-phenyl-indole (DAPI) counter-stain of the nuclei (blue). Scale bar, 1 mm.

Figure 3. Microgram glycosaminoglycan (GAG) per microgram of DNA measured in centrifuged (black bars) and uncentrifuged (gray bars) aggregates harvested at the indicated days

Mean ± sd, average from three preps with six internal replicates each.