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.
Aggregate culture is a cell culture technique that has been used widely to provide a three-dimensional (3-D) environment for differentiating cells, particularly to study in vitro chondrogenesis. This system was used in the 1960s to study organ-cultured chondrocyte aggregates on nutrient agar (1). A version of this culture system, in which the aggregates are established in 15-mL polypropylene centrifuge tubes, has been used to study terminal differentiation of rat (2,3) and rabbit chondrocytes (4). More recently, the chondrogenic potential of rabbit (5) and human bone marrow-derived mesenchymal progenitor cells (6) and rat periosteum-derived progenitor cells (7) have also been studied using this culture technique. In this approach, an aliquot of cell suspension is centrifuged in a 15-mL tube, and the resulting cell pellet is left undisturbed at the bottom of the tube. The cells form free-floating cell aggregates in which cell-cell, rather than cell-substrate, interactions take place. Polypropylene tubes are a critical component of this assay, because the cells do not adhere to the tube walls, but rather to one another. Our group is investigating factors that contribute to the chondrogenic differentiation of human mesenchymal stem cells (hMSCs) in vitro (8). These experiments require us to prepare aggregates in large numbers. Generating large numbers of aggregates using 15-mL conical tubes is both time-consuming and expensive. We have recently simplified the aggregate culture method by substituting polypropylene 96-well plates for the conical tubes (9). The microplate-based methodology has enabled us to greatly reduce work load and materials costs, while maintaining reproducible chondrogenic differentiation. It can now be used to efficiently study combinatorial effects of growth factors and cytokines on the chondrogenic potential of hMSCs (10) using large sample sizes.
Fully automating this culture system seems a logical next step. Robotic laboratory equipment for handling, stacking, shaking, sampling, and washing wells in the microplate format are commercially available. Although centrifuges as part of a robotic workstation do exist, they are complex and expensive. In this study, we tested whether the centrifugation step could be eliminated outright, thus significantly streamlining the workflow in this assay, saving time and effort, and making automation simpler.Materials and Methods Materials
Cell culture medium [Dulbecco's modified Eagle's medium (DMEM) with either 4.5 g/L DMEM-HG or 1.5 g/L glucose DMEM-LG], trypsin, L-glutamine, antibiotic/antimycotic (105 U/mL penicillin G sodium, 10 mg/mL streptomycin sulfate, and 25 µg/mL amphotericin B in 0.85% saline), nonessential amino acids, and sodium pyruvate were obtained from Invitrogen (Carlsbad, CA, USA) or Mediatech (Herndon, VA, USA). Transforming growth factor β-1 (TFG-β1) was obtained from PeproTech (Rocky Hill, NJ, USA), and recombinant human fibroblast growth factor-2 (FGF-2) was generously provided by the Biological Resources Branch of the National Cancer Institute (www.ncifcrf.gov/research/brb/site/home.asp). ITS+ Premix was obtained from Collaborative Biomedical Products (Bedford, MA, USA), and ascorbate-2-phosphate from Wako Chemicals (Richmond, VA, USA). Fetal bovine serum (FBS) was obtained from Invitrogen, after screening as described by Lennon et al. (11). Bovine calf serum (BCS) was from Hyclone (Logan, UT, USA). Collagenase was from Worthington Chemical Corporation (Lakewood, NJ, USA). Percoll®, Hoechst 33258 dye, dexamethasone, papain, and Tyrode's salt solution were obtained from Sigma-Aldrich (St. Louis, MO, USA). DNA standards were from Amersham Biosciences (Piscataway, NJ, USA), while chondroitin sulfate C standards were from Seikagaku America (East Falmouth, MA, USA). All cell culture plasticware was from BD Biosciences (San Jose, CA, USA), except for the polypropylene 96-well plates, which were sourced from Phenix Research Products (Hayward, CA, USA). Antibodies to collagen type I were from Sigma-Aldrich, anti-collagen type II from the Developmental Studies Hybridoma Bank at the University of Iowa (dshb.biology.uiowa.edu), and anti-collagen type X was a gift from Dr. Gary J. Gibson. Mouse pre-immune immunoglobulin G (IgG) was purchased from Vector Laboratories (Burlingame, CA, USA). Software packages used for data acquisition and analysis include ImageJ (rsb.info.nih.gov/ij), Microsoft® Excel®, and Sigmaplot/Sigmastat (Systat Software, San Jose, CA, USA).