Experiment 3

Microspheres and soluble alginates were incubated with 2 million PBMCs/mLin RPMI supplemented with 30% h.i. serum for 14 days. Supernatants were harvested at selected time points: after 4 hours, 1 day, 5 days, 8 days, 14 days. Figure 3.1.21 shows the experimental layout of experiment 3 and 4.

Figure 3.1.21: Shows the experimental setup of experiment 3 and 4 of the long-termin vitroexperimental model.

The measurement of selected cytokines was based on results from experiment 1 and 2. IL-1ra was added to the cytokine selection. Figures 3.1.22 to 3.1.29 show a comparison of the induced concentration (pg/mL) of selected cytokines in 2D and 3D. The plotted data are given in Table C.1.17 to Table C.1.32 in Appendix C on page xxii. Induced levels of IL-8 were in most cases measured out of range above the standard curve, so the cytokine is not presented in results, but is shown in Figure A.2.10 in Appendix A on page viii.

Figure 3.1.22: Comparison of the secretion of TNF in 2D and 3D culturing induced by the microspheres A (green), AP (red), and SA (blue); the soluble alginates UPLVG (G-rich alginate) (green), LVM (M-rich alginate) (orange), SA (sulfated alginate) (blue), with concentrations of 500 (spotted), 100 (diagonal lines) and 10 (straight lines) µg/mL;

and the controls LPS (black spotted), CC (black squared).

The induction of TNF was observed to be higher in the 3D culture model as compared to the 2D culture for the microspheres. The induction peaked for both models at day 1, were the microspheres induced levels between 6-38pg/mLin 2D, and 10-4500pg/mLin 3D. The induction of TNF induced by the soluble alginates was higher in the 2D model from day 5. The induced level was observed to increase for each time point in 2D, while decreasing for each time point in 3D. SA induced prominent TNF amounts compared to the medium control, UPLVG, and LVM from day 5 in 2D. Following the incubation with CC, the amounts peaked at day 1, following a similar picture observed for the microspheres. The amounts of TNF induced by CC in 2D and 3D were of comparable character, between 1-35pg/mgin 2D and 1-60pg/mLin 3D.

Figure 3.1.23: Comparison of the secretion of IL-1βin 2D and 3D culturing induced by the microspheres A (green), AP (red), and SA (blue); the soluble alginates UPLVG (G-rich alginate) (green), LVM (M-rich alginate) (orange), SA (sulfated alginate) (blue), with concentrations of 500 (spotted), 100 (diagonal lines) and 10 (straight lines) µg/mL; and the controls LPS (black spotted), CC (black squared).

The induction of IL-1βwas higher in 3D vs. 2D for the microsphere AP. On day 1, AP induced 2pg/mLin 2D vs. 120pg/mLin 3D. A and SA induced similar levels in 2D vs. 3D ranging from 0.001-0.3 vs. 0.01-0.2 pg/mL. The amount IL-1β induced by the soluble alginates were higher in 2D on day 1 and day 14. On day 5 and day 8, similar levels of induced IL-1βwere observed. CC and LPS peaked on day 1 and showed a comparable picture in 2D vs. 3D.

Figure 3.1.24: Comparison of the secretion of IL-6 in 2D and 3D culturing induced by the microspheres A (green), AP (red), and SA (blue); the soluble alginates UPLVG (G-rich alginate) (green), LVM (M-rich alginate) (orange), SA (sulfated alginate) (blue), with concentrations of 500 (spotted), 100 (diagonal lines) and 10 (straight lines) µg/mL;

and the controls LPS (black spotted), CC (black squared).

The induction of IL-6 was higher in the 3D culture model after day 1 for the microsphere AP by ranging between 170-5000pg/mLcompared to 6-190pg/mLin 2D. The medium control, A, and SA induced higher amounts of IL-6 in 2D than in 3D, for example on day 1: 25-50 pg/mL vs. 2-7 pg/mL. The amounts of IL-6 induced by the soluble alginates were higher in 2D on day 1 and day 14. Some concentrations of SA induced higher levels on day 8 and day 14 compared to the medium control (and UPLVG and LVM) by ranging between 170-1200pg/mLagainst 1-7pg/mLin 2D. The induction of LPS and CC peaked on day 1 and showed a comparable picture in 2D vs. 3D.

Figure 3.1.25: Comparison of the secretion of IL-10 in 2D and 3D culturing induced by the microspheres A (green), AP (red), and SA (blue); the soluble alginates UPLVG (G-rich alginate) (green), LVM (M-rich alginate) (orange), SA (sulfated alginate) (blue), with concentrations of 500 (spotted), 100 (diagonal lines) and 10 (straight lines) µg/mL;

and the controls LPS (black spotted), CC (black squared).

The induction of IL-10 was observed to be higher in the 3D vs. 2D culture model for the microspheres.

In 2D, the level of IL-10 decreased after day 1. In 3D, the induced level was observed to be stable around 0.40-3 pg/mL from 4 hours towards day 14. AP peaked on day 1 with a concentration around 7 pg/mL before decreasing towards the level of the medium control and the other microspheres. The induction of IL-10 induced by the soluble alginates was observed to be higher in 2D from day 5 towards day 14. This increase followed more or less the induced increase by the medium control. In 3D, IL-10 induced by UPLVG and LVM showed amounts comparable to the medium control: 0.4-1.40 vs. 0.7-1.40pg/mL. SA showed an increase in induced IL-10 compared to the medium control, on day 5 and day 8, 2-2.8 and 1.7-3 pg/mL, where the induction increased with decreasing SA concentration. CC peaked on day 1 around the same amount of IL-10 induced in 3D for each time point. LPS showed partially the same pattern in 2D vs. 3D by increasing towards day 1 before decreasing.

Figure 3.1.26: Comparison of the secretion of MCP-1 in 2D and 3D culturing induced by the microspheres A (green), AP (red), and SA (blue); the soluble alginates UPLVG (G-rich alginate) (green), LVM (M-rich alginate) (orange), SA (sulfated alginate) (blue), with concentrations of 500 (spotted), 100 (diagonal lines) and 10 (straight lines) µg/mL; and the controls LPS (black spotted), CC (black squared).

The induction of MCP-1 was observed to be higher in 2D vs. 3D for the microspheres. In 2D, the induced level increased towards day 1 and 5 before decreasing. A and SA induced levels of MCP-1 comparable to medium, while AP was prominent from day 5 by inducing amounts of 450-1100pg/mLvs. 150-600pg/mL by the medium control. In 3D, the induced level of MCP-1 was more or less stable from day 1, where A and AP were measured around the medium control: 450-900pg/mL; and SA induced amounts above: 1600-2100 pg/mL. The induction of MCP-1 by the soluble alginates was observed to be higher in 2D for the medium control, UPLVG, and LVM from day 5, where UPLVG and LVM showed a similar induction like the medium control. SA was observed to induce lower amounts than the medium control in 2D, and lower amounts than SA in 3D. As LVM and UPLVG induced more or less similar amounts of MCP-1 like the medium control in 3D on day 1, 5, and 8, SA induced high amounts ranging from 1100-2100pg/mLvs. 450-900pg/mLby the medium control. The induction by SA aslo showed a concentration-dependence, where the induction of MCP-1 increased with decreasing SA concentration. CC and LPS induced higher amounts of MCP-1 in 3D.

Figure 3.1.27: Comparison of the secretion of M-CSF in 2D and 3D culturing induced by the microspheres A (green), AP (red), and SA (blue); the soluble alginates UPLVG (G-rich alginate) (green), LVM (M-rich alginate) (orange), SA (sulfated alginate) (blue), with concentrations of 500 (spotted), 100 (diagonal lines) and 10 (straight lines) µg/mL; and the controls LPS (black spotted), CC (black squared).

The induced level of M-CSF by AP was observed to be higher in 3D from day 1. The medium control, A and SA showed a comparable induction by ranging from 6-18pg/mLin 2D and 4-25pg/mLin 3D. The soluble alginates induced higher M-CSF levels in 2D from day 8, where some concentrations of SA were prominent compared to the other conditions. In 3D, it was observed a gradual increase in the induced amounts of M-CSF form day 1 towards day 14, where UPLVG, LVM, and SA induced more or less amounts similar to the medium control. CC and LPS showed an increased induction of M-CSF in 2D from 4 hours towards day 14, together with the amounts induced by the medium control. LPS showed a similar pattern in 3D but induced lower amounts of M-CSF than in 2D. The induction of M-CSF by CC peaked on day 1 in 3D before decreasing.

Figure 3.1.28: Comparison of the secretion of GRO-α in 2D and 3D culturing induced by the microspheres A (green), AP (red), and SA (blue); the soluble alginates UPLVG (G-rich alginate) (green), LVM (M-rich alginate) (orange), SA (sulfated alginate) (blue), with concentrations of 500 (spotted), 100 (diagonal lines) and 10 (straight lines) µg/mL; and the controls LPS (black spotted), CC (black squared).

The induction of GRO-αwas observed to be higher in 3D vs. 2D for the AP microsphere, for example on day 1, the measured amount of GRO-αwas 1500pg/mLin 2D vs. 7000pg/mLin 3D. A and SA induced similar levels in 2D vs. 3D: 50-600 vs. 60-600pg/mL. The secretion of GRO-αinduced by the soluble alginates was higher in 2D vs. 3D. In most cases, the soluble alginates induced levels like the medium control ranging from 350-1600pg/mL, but some outliers of SA on day 8 and 14 induced amounts of 3200-3700pg/mL. In 3D, the soluble alginates induced levels around the medium control, in the range 60-500pg/mL. CC induced higer amounts of GROαin 3D vs. 2D, at top 2700pg/mLcompared to 500pg/mL. LPS induced higher amounts in 3D vs. 2D, ranging from 40-2700 vs. 60-5000pg/mL.

Figure 3.1.29: Comparison of the secretion of IL-1ra in 2D and 3D culturing induced by the microspheres A (green), AP (red), and SA (blue); the soluble alginates UPLVG (G-rich alginate) (green), LVM (M-rich alginate) (orange), SA (sulfated alginate) (blue), with concentrations of 500 (spotted), 100 (diagonal lines) and 10 (straight lines) µg/mL;

and the controls LPS (black spotted), CC (black squared).

The induced level of IL-1ra was observed to be higher in 3D vs. 2D from day 1 for the microspheres. More specific, the amounts of IL-1ra were between 1500-15000pg/mLin 3D vs. 1-1400pg/mLin 2D. The soluble alginates induced higher levels of IL-1ra in 2D from day 5. SA was observed to induce the highest amounts of IL-1ra compared to UPLVG and LVM, on day 5 and 8, by ranging from 3400-12500 vs. 3600-10000pg/mL. SA showed a similar picture in 3D, 4000-6000 vs. 900-5000pg/mL. CC induced higher amounts of IL-1ra in 3D, for example on day 5, 7000 vs. 50pg/mL.

Overall, the measured concentration for most cytokines induced by the microspheres was observed to be higher in 3D. AP showed a prominent induction in 3D compared to the other microspheres for the pro-inflammatory cytokines TNF, IL-1β, and IL-6, as well as, the growth factors M-CSF and GRO-α. For MCP-1, SA was observed to induce the highest levels. In most cases, the soluble alginates induced the highest levels of cytokines in 2D. For some cytokines, the measured level was observed to be in a concentration-dependent manner. For example, the induction of MCP-1 by SA increased with decreasing concentration, both in 2D and 3D. Induction by LPS and CC showed in many cases to be more or less similar in 2D and 3D. However, induction of MCP-1 in 2D and 3D showed some differences, where induction was highest in 3D, and CC was observed to induce levels like SA.