Introduction
The XA15A1 is a novel preadipocyte cell strain taken from a non-diabetic male aged between 31 and 40. This strain has advantages over 3T3-L1 cells and other lines in that they are derived from primary, normal human preadipocytes. This cell strain is available today for licensing for research, drug discovery, assay development and high throughput screening.
Background
The XA15A1 strain represents a mixed population of conditionally immortalized cells derived from subcutaneous adipose tissue. It is immortalized and expresses temperature sensitive SV 40 Large T- antigen (u19tsA58) using the MMLV-based retroviral vector also possessing selective resistance to Neomycin.
XA15A1 Characteristics
Proliferative capacity - Cells are grown in Clonetics® SKGM®-2 medium at 33ºC. Average proliferation rate is 42 hours/PD.
Transition (optional) - To increase percentage of differentiated cells (especially during later passages) cells are incubated at 37ºC in Poietics® PGM for 24- 72 hours.
Differentiation - Cells are incubated at 37ºC for 12 days in Poietics® PDM medium. No glitazones are necessary for efficient differentiation (they can interfere with PPAR-gamma activation assays).
Fig. 1. Phenotype of differentiated XA15A1 cells. The picture represents the typical phenotype of the differentiated XA15A1 cell strain after 12 days of differentiation.
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Fig.2. Comparison of the life span of functional XA15A1 and primary human adipocytes. The XA15A1 cell strain was maintained continuously at 33ºC and differentiation was initiated at indicated Population Doubling (PD) points. Primary human adipocytes were maintained and differentiated as recommended by Lonza. Differentiated cells were analyzed using the Poietics® Adipo-Red™ assay.
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Fig.3. Comparison of phenotypes of the differentiated XA15A1 cell strain (different Population Doublings) and primary human adipocytes during Adipo-Red assay. For the activities of the corresponding cells refer to Fig. 2.
Fig.3A. 12 Population Doublings
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Fig.3B. 18 Population Doublings
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Fig.3C. 5 Population Doublings
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Fig.4. Leptin and Adiponectin production by the XA15A1 cell strain during differentiation. Cells were differentiated for up to 20 days. Prior to feeding, on every 4th day, media was collected and frozen. Media were analysed for the presence of leptin and adiponectin (R&D Systems). Results are the average of n=3 ± SEM.
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Fig.5. Insulin stimulated glycogen synthesis and the effects of differentiation of XA15A1 cells. Cells were differentiated for up to 14 days in 96-well plates. The rate of insulin-stimulated glycogen synthesis was determined in cells at various stages of differentiation (14 day data shown in graph). The concentration of insulin required for half-maximal stimulation of glycogen synthesis (EC50) is also indicated. Results are the average of n=8 ± SEM.
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Fig.6. Insulin stimulated glucose uptake in XA15A1. Cells were differentiated in 12 well plates for 18 days. After exposure to insulin for 20 minutes, the labeled 2-deoxyglucose uptake was measured over an additional 30 minutes.
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Fig.7A. Expression of differentiation markers in XA15A1 cell strain. Cells were differentiated for up to 12 days in 6-well plates and extracts prepared. Extracts were used to determine a) glycerol-3-phosphate dehydrogenase activity and b) aP2 expression. In a) results are the average of n=3 ± SEM.
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