Obesity is a prevalent health hazard in many industrialized nations, and its occurrence is closely associated with a number of pathological disorders, such as non-insulin dependent diabetes, hypertension, cancer, and atherosclerosis (1). Because dysregulated adipogenesis and fat cell functions play central roles in these disorders, there has been a surge in efforts to understand the cellular and molecular mechanisms of adipocyte differentiation. Adipocytes are derived from multipotent human mesenchymal stem cells (MSCs), which are found within the bone marrow stroma. MSCs have the potential to differentiate toward many different lineages of mesenchymal tissues including bone, cartilage, muscle, and fat (2).
Human adipose-derived stem cells (ADSCs) are adult stem cells isolated from human lipoaspirates in adipose tissue, and like MSCs, differentiate in vitro toward the osteogenic, adipogenic, myogenic, and chondrogenic lineages when treated with established lineage-specific factors. 3T3-L1 cells are undifferentiated fibroblast-like preadipocytes that are also frequently employed in most adipogenesis studies. These were clonally isolated from Swiss 3T3 cells derived from disaggregated 17- to 19-day-old mouse embryos.
Unique characteristics of adipocyte differentiation include dramatic changes in cell size, shape, membrane potential, metabolic activity, and responsiveness to signals (3). When ADSCs and 3T3-L1 cells commit to the adipocytic lineage, lipid-rich vacuoles accumulate within these cells. We hypothesized that using the Scepter™ cell counter to rapidly assess size distributions of cellular populations would provide a quick, simple method for tracking adipocyte differentiation.
The Scepter™ cell counter (EMD Millipore Cat. No. PHCC20040) captures the ease of automated instrumentation and accuracy of impedance-based counting using the Coulter principle in an affordable, handheld format. The instrumentation has been collapsed into a device the size of a pipette, and uses a combination of analog and digital hardware for sensing, signal processing, data storage, and graphical display in the form of a histogram. The 40 μm- and 60 μm-aperture sensor tips are engineered with a microfabricated, sensing zone that enables discrimination by cell size and cell volume at sub-micron and sub-picoliter resolution, respectively. The histogram output provides a quick snapshot of cell size and density.
This study outlines a method for tracking adipogenic differentiation of ADSCs and 3T3-L1 cells and subsequent sample analysis using the Scepter™ cell counter to measure these changes. We have employed Scepter™ technology for determining cell size and volume, as well as lipid vacuole staining using Oil Red O as a cross-validation histology screen to investigate the relationship between cell differentiation and cell size changes. Although the Scepter™ cell counter was intended primarily as a cell counting device, we demonstrate how this cell counter can also function as a reliable tool to track phenotypic change.Materials and Methods Adipogenesis Differentiation: ADSCs
ADSCs were plated at a density of 50,000 cells per well into gelatin (EMD Millipore Cat. No. ES-006-B)-coated 12-well culture dishes with 1 mL medium per well. Cells were incubated at 37 °C in a 5% CO2 humidified incubator for 4 days. When the cells were 100% confluent, medium was aspirated from each well and replaced with 1 mL Adipogenesis Induction Medium (EMD Millipore Cat. No. SCR020). This medium change was set as differentiation day 1.
Medium was replaced with fresh Adipogenesis Induction Medium every 2-3 days for 14 days. After 0, 7, and 14 days of differentiation, adipocytes were fixed and the lipid droplets stained with Oil Red O Solution. Lipid droplets could be detected by microscopic examination as early as 5 days into the differentiation period.Adipogenesis Differentiation: 3T3-L1 cells
3T3-L1 cells (ATCC Cat. No. CL-173TM) were plated in 10% calf serum/DMEM Medium at a density of 50,000 cells per well into 12-well culture dishes with 2 mL medium per well. Cells were incubated at 37 °C in a 10% CO2 humidified incubator for 4 days.
When the cells were 100% confluent, the medium was aspirated from each well and replaced with 2 mL MDI Induction Medium. This medium change was set as differentiation day 0.
At differentiation day 2, the medium was replaced with Insulin Medium. The medium tended toward greater viscosity as free fatty acids were produced by the cells and secreted into the medium. At differentiation day 4, the medium was replaced with MDI Induction Medium. At differentiation day 6, the medium was replaced with Insulin Medium. At differentiation day 8, the medium was replaced with 10% FBS/DMEM.