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Fig. 4 | Journal of Translational Medicine

Fig. 4

From: Preclinical good laboratory practice-compliant safety study to evaluate biodistribution and tumorigenicity of a cartilage advanced therapy medicinal product (ATMP)

Fig. 4

Biodistribution study: Detection of the huChon spheroids within the mouse skin. Detection of implanted huChon spheroids within mouse subcutis; the figures show a representative example of a normally shaped huChon spheroid (a–c) and a fragmented spheroid (d–f). The huChon spheroid was macroscopically visible as faint yellowish stain (a, arrow). Light microscopic view of the spheroid (b). HLA-ABC staining of this specimen (c, red-brown staining). Haematoxylin was used as a counterstain. The fragmented huChon spheroid is only very weakly visible macroscopically (d, arrow). A Light microscopic view of a fragmented spheroid is shown in (e). BV: Blood vessel. HLA-ABC staining of a fragmented spheroid is shown in (f). The arrows indicate the two fragments. As positive control, HLA-ABC staining of a single huChon spheroid without mouse tissue was used (g). IgG2a isotype control of this specimen is shown in (H). Scale bars indicate 100 μm (g, h), 200 μm (b, c, e, and f) or 500 μm (a, d). A 3D reconstruction of a representative huChon spheroid from nine serial sections was performed (i). The digitized images were registered onto another to realign adjacent slices. Afterwards, each image was binarized to a separate background. An anisotropic Gaussian filter was used to smooth the data for visualization and afterwards the surface of the volume was reconstructed using the ListContourPlot3D algorithm implemented in Mathematica 9.0.1. Depending on the section level (grey arrows), this huChon spheroid appeared fragmented (j) or not (k). The white arrows in (i) and (j) indicate “bulges” of the spheroid. Scale bars indicate 100 μm. The determination of the lower detection limit for DNA extraction and the Plexor® HY PCR (l). The sensitivity and reproducibility of the DNA extraction and qPCR were tested in parallel by adding increasing numbers of human chondrocytes to 50 mg mouse skin tissue. Five cells represented the lowest cell number at which almost all of the reactions were reproducibly positive

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