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High-throughput aggregate culture system to assess the chondrogenic potential of mesenchymal stem cells
 
Kitsie J. Penick, Luis A. Solchaga, Jean F. Welter
Case Western Reserve University, Cleveland, OH, USA
BioTechniques, Vol. 39, No. 5, November 2005, pp. 687–691
Full Text (PDF)
Abstract

We have developed an improved method for preparing cell aggregates for in vitro chondrogenesis studies. This method is a modification of a previously developed conical tube-based culture system that replaces the original 15-mL polypropylene tubes with 96-well plates. These modifications allow a high-throughput approach to chondrogenic cultures, which reduces both the cost and time to produce chondrogenic aggregates, with no detrimental effects on the histological and histochemical qualities of the aggregates. We prepared aggregates in both systems with human bone marrow-derived mesenchymal stem cells (hMSC). The aggregates were harvested after 2 and 3 weeks in chondrogenic culture and analyzed for their ability to differentiate along the chondrogenic pathway in a defined in vitro environment. Chondrogenic differentiation was assessed biochemically by DNA and glycosaminoglycan (GAG) quantification assays and by histological and immunohistologic assessment. The chondrogenic cultures produced in the 96-well plates appear to be slightly larger in size and contain more DNA and GAG than the aggregates made in tubes. When analyzed histologically, both systems demonstrate morphological characteristics that are consistent with chondrogenic differentiation and cartilaginous extracellular matrix production.

Introduction

Three-dimensional (3-D) cell aggregate culture has been used to study in vitro chondrogenesis. This system, which dates back to the 1960s when it was used by Holtzer et al. (1) to study organ-cultured chondrocyte aggregates on nutrient agar, has evolved through the years. Terminal differentiation studies of rat (2,3) and rabbit chondrocytes (4) used a modification of this culture system using 15-mL polypropylene centrifuge tubes to establish the aggregates. More recently, the chondrogenic potential of rabbit (5), human bone marrow-derived mesenchymal progenitor cells (6), and rat periosteum-derived progenitor cells (7) have also been studied, using 15-mL polypropylene centrifuge tubes to produce and maintain the aggregates. The unusual use of polypropylene in a cell culture application is a critical component of this technique, because the pelleted cells do not adhere to the tube walls, but rather to one another to form free-floating cell aggregates, in which cell-cell, rather than cell-substrate, interactions take place. One of our research goals is the development of tissue-engineered cartilage implants for the repair of articular cartilage defects. In this context, our group is investigating factors that contribute to the chondrogenic differentiation of human bone marrow-derived mesenchymal stem cells (hMSCs) in an in vitro bioreactor environment (8). We have shown that chondrogenic preconditioning of hMSCs in aggregate culture results in improved chondrogenesis in these tissue engineered constructs (9). This chondrogenic preconditioning paradigm requires the preparation of large numbers of aggregates, but doing so using 15-mL centrifuge tubes is both time-consuming and expensive. We have developed a novel high-throughput method for the preparation and maintenance of cell aggregates that significantly decreases the time required for the preparation of the aggregates and lowers the cost, while maintaining reproducible chondrogenic differentiation. As a result, a more efficient culture protocol to promote chondrogenic differentiation was developed. This method can now be used to efficiently study the effects of growth factors and cytokines on the chondrogenic potential of hMSCs. We have used this system to study the effects of the fibroblast growth factor-2 on the mitogenic and chondrogenic potential of hMSCs (10) and in other experiments that require large quantities of individual aggregates. This paper describes the development of this method in detail and presents a protocol for high-throughput production of chondrogenic cell aggregates.

Materials and Methods

hMSC Isolation

Bone marrow aspirates obtained from the MSC core facility at Case Western Reserve University (CWRU; Cleveland, OH, USA) were used in this study. The cells were derived from bone marrow that was harvested from the posterior iliac crest of healthy volunteer donors. The marrow aspiration procedure was approved by an Institutional Review Board, and informed consent was obtained from all volunteers. The procedure for harvesting hMSCs followed the method described by Haynesworth et al. (11). Briefly, the bone marrow samples were washed with low glucose Dulbecco's modified Eagle medium (DMEM-LG; Invitrogen, Carlsbad, CA, USA) supplemented with 10% of a selected lot of fetal bovine serum (FBS; Invitrogen) (12). To isolate the mononucleated cells, the sample was subjected to a preformed Percoll™ (Sigma, St. Louis, MO, USA) density gradient (1.073 g/mL) centrifugation step. The hMSC primary cultures were seeded at a density of 1.8 × 105 cells/cm2 in serum-supplemented medium in a 10-cm plate. The hMSCs were cultured at 37°C in a humidified atmosphere of 95% air and 5% CO2. Nonadherent cells were removed from the plate after 4 days by changing the medium. The medium was changed every 3 days thereafter. The primary cultures were subcultured after approximately 2 weeks and seeded at 5 × 103 cells/cm2.

Chondrogenic Assay

Culture-expanded hMSCs are typically used at the end of first passage to prepare the cell aggregates. The hMSCs are subcultured at approximately 80% confluence to prevent contact inhibition of growth and spontaneous differentiation (13). The cultures are rinsed with sterile Tyrode's salt solution to remove residual FBS present in the growth medium. The Tyrode's solution is then aspirated, 0.05% trypsin-EDTA (Invitrogen) is added, and the cultures are returned to the incubator for 5–10 min. When the cells have detached, bovine calf serum (Hyclone, Logan, UT, USA) is added to inhibit the trypsin. The cells are transferred to a 50-mL conical tube (BD Biosciences, Bedford, MA, USA) and centrifuged for 5 min at 200× g. The supernatant is discarded, and the cells are resuspended in a defined chondrogenic medium [e.g., that described by Johnstone et al. (5)]. This defined medium is DMEM-HG (DMEM with 4 g/L glucose; Biowhittaker, Walkersville, MD, USA) supplemented with 1% BD™ ITS+ Premix (BD Biosciences; consisting of 6.25 µg/mL insulin, 6.25 µg/mL transferrin, 6.25 ng/mL selenious acid, 1.25 mg/ mL serum albumin, and 5.35 µg/mL linoleic acid), 37.5 µg/mL ascorbate-2-phosphate (WAKO, Richmond, VA, USA), and 10-7 M dexamethasone (Sigma). Additional supplements such as L-glutamine, antibiotic antimycotic (10,000 U/mL penicillin G sodium, 10,000 µg/mL streptomycin sulfate, and 25 µg/mL amphotericin B in 0.85% saline), nonessential amino acids, and sodium pyruvate (all from Invitrogen) are also added to the medium at 1%. The cells are counted using a hemacytometer, and the suspension volume is adjusted to a final cell density of 1.25 × 106 cells/mL. Transforming growth factor β1 (TGF-β1; Peprotech, Rocky Hill, NJ, USA) is added to the cell suspension to a final concentration of 10 ng/mL. The cell suspension is mixed by gentle pipeting to ensure homogeneity. Two hundred-microliter aliquots are then dispensed into the wells of an autoclave-sterilized 96-well, V Bottom, 300-µL polypropylene microplate (Phenix, Hayward, CA, USA) using a repeater pipet (Eppendorf, Westbury, NY, USA) to which a large-orifice tip (Fisher Scientific, Hampton, NH, USA) has been affixed to allow smooth delivery of the aliquots into the wells. A polypropylene lid (Phenix) is placed on the plate, and the plate is centrifuged for 5 min at 500× g and incubated at 37°C in a humidified atmosphere of 95% air and 5% CO2. In the following 12–16 h, the cells coalesce and form a high-density cell aggregate (5). Twenty-four hours after seeding, we ensure that the aggregates can float freely by releasing them from the bottom of the wells. To this end, we aspirate 100 µL of the medium and gently release it back into the wells using an 8-channel pipet (Fisher Scientific). The medium is changed every other day thereafter using DMEM-HG plus supplements plus 10 ng/mL TGF-β1. To avoid possible acidification problems, strict adherence to the medium change regimen is critical with cultures of this size.

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