Validity of DNA analysis to determine cell numbers in tissue engineering scaffolds

Abstract

The potential of a DNA content assay, PicoGreen, for use in 3D bioengineered constructs was examined. The assay was tested on ATDC5 cells in situ during culture in typical tissue engineering 3D constructs. Comparisons of cell standards from cell lines and primary cells to lambdaDNA standards was also conducted. An effective working range of the assay within 3D constructs was shown up to 2.5 x 10(5) cells ml(-1). From significant variation found in DNA content between cell lines and primary cells, it was concluded that the most accurate standard to use for the assay was from the cell type being examined.

Influence of scaffold properties on the inter-relationship between human bone marrow derived stromal cells and endothelial cells in pro-osteogenic conditions

“…To calculate DNA concentrations, a calibration curve was generated using the double stranded lambda DNA standard provided with the kit. Results were divided by the average amount of DNA in a cell [31], to obtain estimates of cell number.…”

“…The color change was measured spectrophotometrically at 405 nm. The amounts of resulting p-nitrophenol released by the cells were quantified by comparison with the standard curve and normalized to DNA content [31].…”

Hollow Fiber Bioreactors for <em>In Vivo</em>-like Mammalian Tissue Culture

“…Subject this to two freeze-thaw cycles in a -80 °C freezer. Measure the DNA content using PicoGreen and determine cell numbers by comparing this value to a standard curve made with the desired cell type 26 .…”

Biological Evaluation of PLDLA Polymer Synthesized as Construct on Bone Tissue Engineering Application

“…PicoGreen reagent (100 µL) was added to the wells containing standard or sample and the plate then read at excitation/emission wavelengths of 485/538 nm in fluorometer (LS-55, PerkinElmer  ). The resulting fluorescence was compared with a standard curve of DNA and the cell number was calculated by assuming 7.7 pg of DNA/cell 22,23 .…”

“…Figure 3 shows the growth curve of cells cultured on PLDLA scaffolds as assessed by DNA quantification using Picogreen reagent. The number of cells was calculated assuming a DNA concentration of 7.7 pg/cell 22,23 . As can be seen, scaffolds not only led cell adhesion on scaffolds surface as well as stimulated a progressive increase in osteoblast proliferation along the first 14 days of culture (p<0.05).…”