Low Cell Binding Property of LIPIDURE -COAT

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1 Technical Note_1 ver.1 Low Cell Binding Property of LIPIDURE -COAT 1. LIPIDURE -COAT MULTI DISH A-6MD (Cat. No ) 2. Cell; NIH 3T3 (Fibroblast, mouse) %CS-DMEM; DMEM (Dulbecco's Modified Eagle's Medium) containing 10 %CS (calf serum), 50 IU/mL penicillin, 50 µg/ml streptomycin. 1. NIH-3T3 mouse fibroblasts were maintained in the 10 %CS-DMEM and were passaged every 3 days. Low-cell-binding test 1. The cells were trypsinized and plated at a seeding density of 1 x 10 4 cells/well (2 ml/well) on LIPIDURE -COAT MULTI DISH-6MD. 2. The plate was incubated at 37 in 5 %CO 2 incubator. 3. The typical spheroids-shape cells were observed after 4 days incubation. 4. The plate was washed with PBS and low cell binding property was estimated. Result LIPIDURE -COAT Non-treated plate Wash with PBS Wash with PBS No cell adhesion was observed. The cells adhered to the plate.

2 Spheroid Formation and in Anti-Cancer Drug test using LIPIDURE -COAT 2. Cell; Hep G2 (Hepatocellular carcinoma, human ) %FCS-DMEM; DMEM (Dulbecco's Modified Eagle's Medium) containing 10 %FCS (fetal calf serum), 50 IU/mL penicillin, 50 µg/ml streptomycin. 1. Hep G2 cells were maintained in the 10 %FCS-DMEM and were passaged every 4~7 days. 1. The cells were trypsinized and plated at a seeding density of 2,000 cells/well (0.1 ml/well) on LIPIDURE -COAT Plate. 2. The plate was Incubated at 37 in 5 %CO 2 incubator for 1~5 days. * The medium was gently changed every other day by pipetting ( Recommend the exchange of only half volume of medium). for cancer research 1. Mitomycin-C was added and the plate was incubated for 2 days. 2. Cell viabilities were measured by WST assay. Anticancer activity of Mitomycin-C to Hep G2 cells LIPIDURE -COAT (3D) Monolayer culture (2D) IC50(ug/mL) Technical Note_2 ver.1 Hep G2 (40X) Hep G2 (100X) 1 0 LIPIDURE- COAT(3D) Monolayer culture(2d) Other Cell Lines You can form the corresponding spheroids from the following cell lines by using LIPIDURE - COAT. You would be able to form spheroids from other cell lines which are not shown here. human ES, mouse ES, human ips mouse ips HepG2 NIH3T3 preadipocyte and cancer cell.

3 Technical Note_3 ver.2 2. Cell; PC12 (Pheochromocytoma, Rat ) 3. Poly L-Lysine coated plate or dish;for PC12 culture (Not for spheroid formation) %FBS-10 %HS-DMEM; DMEM (Dulbecco's Modified Eagle's Medium) containing 10 %FBS (Fetal Bovine Serum), 10%HS(Horse Serum), 50 IU/mL penicillin and 50 µg/ml streptomycin. 1. PC12 cells were maintained in the 10 %FBS-10 %HS-DMEM on poly L-Lysine coated dish and were passaged every 4~7 days. 1. The cells were trypsinized and plated at a seeding density of 200 cells/well (0.2 ml/well) on LIPIDURE -COAT Plate. 2. The plate was incubated at 37 in 5 %CO 2 incubator for 12 days. * The medium was gently changed every other day by pipetting. (We recommend the change of half volume of medium.). PC12 spheroids formed on LIPIDURE -COAT Day 3 Day 6 Day 12 Seeding density:500 cells/well

4 Technical Note_4 ver.1 2. Cell; MCF7 (breast adenocarcinoma, Human ) %FBS-MEM; DMEM (Dulbecco s Modified Eagle s Medium) containing 10 %FBS (Fetal Bovine Serum), 1mM NEAA (Non Essential Amino Acids), 1mM Sodium Pyruvate, 50 IU/mL penicillin and 50 µg/ml streptomycin. 1. MCF7 cells were maintained in the 10 %FBS-MEM on Tissue Culture Treated dish and were passaged every 7 days. 1. The cells were trypsinized and plated at a seeding density of 500~10,000 cells/well (0.1 ml/well) on LIPIDURE -COAT Plate. 2. The plate was incubated at 37 in 5 %CO 2 incubator for 1~5 days. * The medium was gently changed every other day by pipetting. (We recommend the change of half volume of medium.). MCF7 spheroids formed on LIPIDURE -COAT Seeding density:500 cells/well Average diameter:176.5±8.9 µm Seeding density:5000 cells/well Average diameter:390.5±13.5 µm Seeding density:10000 cells/well Average diameter:481.8±16.5 µm Day 1 Day 1 Day 1

5 Technical Note _5 ver.1 2. Cell; Rat Hippocampus Neuronal Cell (DS pharma Biomedical Cat No. MB-X0402D ) 3. Human recombinant EGF 4. Human recombinant bfgf 1. Trial Set for Neuronal Cell Culture (DS pharma Biomedical Cat No. MB-X0601D) 1. Neuronal cells were cultured according to Trial Set for Neuronal Cell Culture. 1. The cells were enzyme-treated and plated at a seeding density of 200, 1000, 5000 cells/well (0.1 ml/well) on LIPIDURE -COAT Plate. 2. The plate was incubated at 37 in 5 %CO 2 incubator for 1~7 days. 3. From day 1, the single spheroid were formed in each well. * For spheroid formation, the media consisting with 50ng/mL human recombinant EGF and 25ng/mL human recombinant bfgf was used. * * The medium was gently changed every other day by pipetting. (We recommend the change of 70μL/well.). Neurospheres formed on LIPIDURE -COAT on day 5 Seeding density:200 cells/well Seeding density:1000 cells/well Seeding density:5000 cells/well Scale bar= 200 µm All photos were supplied by Dr. Ijima at Kyusyu University

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