Table 1 Techniques for 3D OS culture-scaffolds free
From: Relevance of 3d culture systems to study osteosarcoma environment
LIQUID OVERLAY TECNIQUE | METHOD | CELL LINES | REF. |
Agar and Agarose-coated plates | To realize 3D culture: - 5.0x105 cells/well seeded in tissue culture plates (Nunc) covered with 3% agar dissolved in RPMI plus heat-inactivated 10% FBS (37°C, 5% CO2). | MG-63; human fibroblast cell line CRL 7124 | [22] |
To realize 3D culture: - plates were coated with 1.5% (wt/vol) agarose solution (0.15 g agarose and 10 mL PBS). The solution was heated to facilitate agarose dissolution. 70μl of hot agarose solution was pipetted into each well. The solution was kept on a hot plate during the well coating process to prevent premature gelatinization, and then allowed to cool for 20 minutes. - 100 μL of the cell suspension was added to each agarose-coated well. After 72 hours, cells resulted aggregate. | U2OS; MDA-MB-231 | [10] | |
Poly-HEMA-coated plates | Gibbs et al. method was used to form 3D culture: - 60,000 cells/well are seeded in poly-HEMA-coated plates. - Serum-free DMEM/F12 medium with 1% of methylcellulose supplemented with 1% penicillin/streptomycin, 20 nM progesterone, 100μM putrescine, 1% insulin-transferrin-selenium A supplement, 10ng/ml bFGF, and 10ng/ml hrEGF. 3D spheroids were obtained after 7 days in culture. | Human MNNG/HOS | [23] |
Low and Ultralow binding plates | To realize 3D culture: - cells were seeded in NanoCulture ® plates (NCPs; 96-well, low-binding; SCIVAX Corporation) and incubated at 37°C, 5% CO2. | HS-Os-1; NOS-1; SaOS-2; SJSA-1; 143B; HOS; HuO9; KHOS/NP; MG-63; MNNG-HOS; NOS-10. | [18] |
To realize 3D culture: - 2×104 U2OS cells resuspended in 100μl RPMI medium with 10% FBS and penicillin-streptomycin (1% 100 units/mL), were plated on Corning® Spheroid Microplates. - The plates were centrifuged at 1000 RPM x 5 min, and incubated for 72 hrs. | U2OS; A549, C2C12, DU145, F9, GH3, HeLa, HEp2, NIH3T3, PA317, SH-SY5Y, and 293T | [19; 24] | |
|  | Zhang et al. method was used to form 3D cultures: - 10×103 OS cells were grown in Ultra Low Attachment plates, - 2 mL serum-free MEM or McCoy’s medium with 20 ng/mL bFGF, 20 ng/mL EGF, B27, 100 μg/mL gentimycin and antibiotic-antimycotic. - Spheroids are obtained after 7 culture days. | 143B; MNNG/HOS; SaOS-2; MG-63; U2OS; SJSA-1 | [25] |
To realize 3D cultures: - 1.56x104 cells/ml were seeded on ultralow attachment plates (Corning). - Serum-free William’s E medium with GlutaMAX supplemented with putrescine (100 mM), sodium selenite (30 nM), transferrin (25 mg/ml), insulin (20 mg/ml), hr-bFGF (10 ng/ml) and EGF (10 ng/ml) and incubated at 37°C, 5% CO2. - Additional growth factors (100 mg/ml) were added to the media every other day. | Canine osteosarcoma cells; KTOSA5 and CSKOS. | [27] | |
3D OS spheroids were obtained maintaining OS cells in non-adherent round-bottom 96-well plates (Greiner bio-one) for 7, 14 and 19 days. | Canine OS cells (D17) | [20] | |
To realize 3D cultures: - 5000 cells/well cells plated in ultralow attachment plates (Corning). - RPMI-1640 supplemented with B27 Supplement, 10 ng/mL human EGF, and 10 ng/mL human bFGF (fresh aliquots were added every other day), and incubated at 37°C and 5% CO2. - After culture for 2 weeks, spheroids reached > 50 μm in size. | MG63; U2OS; 43B | [29] | |
HANGING DROP TECHNIQUE | METHOD | CELL LINES | REF. |
|  | To realize 3D cultures: - Cells were seeded into Gravity PLUSTM plates (InSphero), at a density of: SaOS-2, 2000 cells/40μl (drop volume); HOS, 2500 cells/40μl; MG-63, 250cells/40μl; OS cells from patients, 2500 cells/40μl (day 0). - DMEM/Ham’s F-12 supplemented with 10% horse serum, 25 mM HEPES buffer and 1× penicillin/streptomycin was used. | SaOS-2; HOS; MG-63; human osteoblastic OS cells. | [21] |
To realize 3D cultures: - 60 cells/μL were seeded to get 400 μm diameter spheroids at the beginning of the treatment with the compound. - After 48 h, the compacted spheroids were transferred to an agar coated plate. - 200 μL of DMEM plus 10 % FBS were added to each well. | MG63 | [32] | |
To realize 3D cultures: - MG63 cells were seeded into Gravity PLUSTM plates (InSphero), at a density of 1x104 cells/40μl (drop volume) to obtain spheroids with a diameter of 400 μm. - DMEM supplemented with 10% FBS, 1mM Na-pyruvate, 2mM glutamine, 250 U ml-1 fungizone, 10 U ml-1 penicillin, 100μg ml-1 streptomycin was used. - 7 μL of culture medium were added to each well every 3-4 days. | MG63, HUVEC | [33] |