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In vitro screening

IMIC WP 6.1 In vitro immunological properties of polyplexes and polymer membranes WP 6.2 In vivo immunological properties of polyplexes and polymer membranes. In vitro screening. Hemolysis Proliferation of mouse spleen cells Proliferation of human peripheral blood mononuclear cells

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In vitro screening

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  1. IMICWP 6.1In vitro immunologicalproperties of polyplexes and polymer membranesWP 6.2In vivoimmunological properties of polyplexes and polymermembranes

  2. In vitro screening • Hemolysis • Proliferation of mouse spleen cells • Proliferation of human peripheral blood mononuclear cells • Cultivation of mouse peritoneal cells • Growth of permanent cell lines • endothelial • fibroblast • monocyte/macrophage

  3. 10 wt% gelatin type B hydrogels - preapared by polymerization of methacrylamide modified gelatin using e-beam - treatment also enables the simultaneous sterilization of the scaffolds 96-well plates and 24-well plates for tissue culture (Nunc, Denmark) Gelatin 1 SAMPLE_IDENTIFICATION_SHEET:_Gelatin_coated_96_well_plates • In vitro screening of biocomatibility and immunocompatibility • Hemolysis • Proliferation of mouse spleen cells • Proliferation of human peripheral blood mononuclear cells • Cultivation of mouse peritoneal cells • Growth of permanent cell lines • endothelial • fibroblast • monocyte/macrophage

  4. OD 545 nm incubation Preincubation of hydrogel-coated plates Optimized protocol: 24 hours in PBS or serum-free medium 2 hours in medium containing 5 – 10% FCS for hemolysis: 3x wash (PBS) Hemolysis spontaneous(PBS) positive control(H2O)

  5. Alamar blue detectin, 24 h Preincubation: serum-free medium, 24 h (37oC, 5 % CO2) medium collected, supplemented with 10 % FCS, used for EAHY cultivation; control: fresh medium

  6. 60,000 gelatin-treated 50,000 untreated 40,000 30,000 fluorescence (U) 20,000 10,000 0 ctrl Con A PWM PHA LPS 8 7 6 5 stimulation index 4 3 2 1 0 ctrl Con A PWM PHA LPS Proliferation of normal mouse spleen cells strains BALB/c, C57BL/6 Mitogens: Con A ........T cells PHA ...........T cells PWM ..........T and B cells LPS ............B cells Detection (viability) XTT ....... colorimetric assay Alamar blue ........fluorescence in vitro stimulation

  7. 0.4 gelatin B 0.3 untreated 0.2 OD 0.1 0 ctrl Con A PWM PHA LPS in vitro stimulation Proliferation of peripheral blood mononuclear cells (PBMC)

  8. stimulation Mouse peritoneal cells (PEC) heterogenous population: granulocytes, macrophages, lymphocytes .... irrigated from peritoneal cavity of normal mice, in vitro cultivated 24 and 48 h detection of nitric oxide (NO) in supernatants unstimulated PEC do not produce NO positive control: lipopolysaccharide (LPS), interferon g IFNg IFNg LPS

  9. Permanent cell lines fibroblasts: mouse 3T3 endothelial: EAHY 926 monocyte/macrophage: mouse RAW 274.6 human THP-1

  10. viability detection: Alamar blue viability detection: Alamar blue Mouse monocytes RAW 274.6 Human THP-1

  11. Human endothelial cells: EAHY 926 Mouse fibroblasts 3T3 viability detection: XTT

  12. viability (XTT) LDH (OD) EAHY cells: 48 h viability (XTT) and cell damage (lactate dehydrogenase = LDH assay)

  13. viability (XTT) LDH (OD) 3T3 cells: 48 h viability (XTT) and cell damage (lactate dehydrogenase = LDH assay)

  14. SUMMARY: Prolonged preincubation of hydrogels is necessary Gelatin B does not induce spontaneous hemolysis of human ery Spontaneous proliferation of mouse spleen cells and human PBMC is not increased within 3 days of cultivation on hydrogel Mitogen-induced proliferation of mouse spleen cells and human PBMC is rather decreased, but SI remain comparable Gelatin B hydrogel does not induce NO production by mouse PEC and mouse macrophage cells RAW, and does not induce TNF production by human THP-1 cells Growth of permanent cell lines is rather limited in monocyte /macrophage cell lines (RAW, THP-1), and almost unchanged in fibroblasts (3T3) and endothelial cells (EAHY) Density of cell seeded on the hydrogel is important Growth and viability of cells (normal, permanent cell lines) is modulated due to the hydrogel surface (clusters)

  15. Cytokine detection in supernatants FLOW CYTOMIX: IFNγ, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12p70, TNFα, TNF β samples: supernatants of mouse spleen cells, unstimulated and mitogen-stimulated, PEC

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