Skip to main content
Fig. 4 | Biological Procedures Online

Fig. 4

From: Heregulin Activity Assays for Residual Testing of Cell Therapy Products

Fig. 4

Validation rounds of dose-dependent changes elicited by fresh hSC media, conditioned media, and wash media derived thereof. Experiments were conducted and analyzed as described in Fig. 3 using a 30 min time for stimulation. a Dose-dependency curves for culture medium samples highlighting the expected concentration ranges for positive controls (spiking samples) and final wash media (test samples). b-c Original Western blot data (a) and densitometric quantification from 3 rounds of testing (c). The test samples, the negative, and the positive controls were snap-frozen on dry ice and stored at − 80 °C up until stimulation of starved rat SCs. The samples consisted of the following: DMEM/F12 (negative control), serial dilutions (up to 1:100,000) of SC growth medium (standard curve), conditioned medium (i.e., cell culture supernatant exposed to cells) and spiking controls at 1/10 and 1/100 (positive controls). Samples for each experimental condition were obtained and analyzed by Western blot in duplicate. Because duplicate samples rendered nearly identical activation profiles, data from one representative membrane per experimental condition is shown. The spiking controls (depicted in the 2 last lanes from each gel) were included for rounds − 2 and − 3 only. To aid in interpretation, the Western blot profiles were aligned horizontally and the blots from each phospho-antibody (readout signal) were located above the respective total protein, (b). In (c), data from each phospho-kinase were normalized to the respective total levels of protein and expressed as arbitrary units of O.D. Values of significant comparisons between the negative control condition and the culture medium conditions or the test articles conditions were indicated in the graphs according to their degree of significance (* p < 0.05, * * p < 0.01 and * * * p < 0.001). Note that heregulin concentrations in the wash supernatants were not high enough to trigger the activation of ErbB3, ERK or Akt above basal levels. The absence of biological activity in these samples provided a high degree of assurance that the final hSC formulation was free of soluble process residuals

Back to article page