Table 2.
Techniques for 3D OS cultures with scaffolds
| NATURAL SCAFFOLD | METHOD | CELL LINES | REF. |
| Alginate | To realize 3D cultures: - 4 x106 cells/ml were encapsulated in 1.2% low-viscosity sterile alginate in 0.15 M sodium chloride (NaCl). - The cell suspension in alginate solution was slowly pushed through a 21-gauge needle and dropped into a 102 mM CaCl2 solution. Beads were allowed to polymerize in this solution for 10 min before two consecutive washes with 50 ml of 0.15 M NaCl. - Maintained in DMEM supplemented with 10% FBS and gentamicin (50 μg/ml), and incubated at 37°C and 5% CO2. Media were changed daily. |
Dunn; LM8 | [5, 39] |
| Matrigel and Collagen | HOS and MG-63 cells were mixed with matrigel and cultured for 48 h. | HOS; MG-63 | [40] |
| To realize 3D cultures: - 300μl of 3D BME (Cultrex) scaffold were seeded into the plates and transferred in incubator at 37°C for 30 min to promote gel formation. - 2.0x104 cells were seeded on top of the thick gel in each well. - Maintained in DMEM (supplemented with 10% FBS) and incubated at 37°C and 5% CO2, for up to 14 days. Media were replaced every 3 days. |
3AB-OS CSCs | [41] | |
| To realize 3D OS cultures: - 105 OS cells were mixed with 3% Matrigel in complete medium. - Cellular suspensions were loaded into culture plates, allowed to polymerize at 37°C, and then overlaid with complete media. |
RF379L; RF1044; RF43 |
[42] | |
| To realize 3D OS cultures: - 2.4 mg/ml collagen gel solution was prepared for the bottom layer, while a 1.2 or 2.4 mg/ml solution was used for the upper layer. - After gel polymerization in the bottom layer, cells were suspended in collagen gel solution and added to the dish, then transferred to 37°C for 60 min to polymerize, and covered with culture media. |
Dunn; LM8 | [46] | |
| To realize 3D cultures: - 6.0x104 cells were mixed with 50μl neutralized collagen I solution and left for 2h while polymerization took place. - 3D fibrillar collagen I gels were prepared by adding 50 μL neutralized collagen I solution (7:1:1:1 of each 3 mg/mL) collagen I in 0.2% acetic acid, 10x serum-free medium, 1.0mM Hepes, pH 7.3, and 0.33mM NaOH, to each well. |
OHS | [47] | |
| To realize 3D cultures: - 250 μl of Matrigel or Type I collagen was dropped onto glass coverslips and allowed to polymerize for 1 h at 37°C. - 7.5×105cell/ml was transfected and seeded on top of the gels, maintained in DMEM 10% FBS and incubated at 37°C and 5% CO2. |
MG63 | [48] | |
| To realize 3D cultures: - gel isolated from mouse tails was dissolved in 0.013 mol/L HCl (final concentration 5 mg/mL). Type I collagen solution (200 μl) was mixed with 674 μl H2O (on ice). The mixture was added to 26 μl NaOH (0.1 mol/L). The new mixture was added to 100 μl of 10x RPMI 1640 medium, and 5μl of the mixture was added onto each slide and incubated at 37°C for 1h until the gel solidified. - 5x105 LM8 cells/mL seeded on Type I collagen gel, supplemented with a basal medium with or without ZA. |
LM8 | [49] | |
| To realize 3D cultures: - 100,000 cells/ml were added to the unpolymerized collagen solution for proliferation assay and 30,000 cells/ml for migration assay. - Collagen Type 1 stock solution was diluted to 3 or 4 mg/ml, mixing equal volumes of neutralizing buffer (100 mM Hepes in 26 PBS, pH 7.3) with PBS. - To polymerize, gels were placed for 2h in the incubator (37°C, 5% C02), resulting in cell-embedded collagen gels. Medium was replaced daily. |
U20S; MCF7 | [50] | |
| To realize 3D cultures: - OS cells were directly added to the cooled hydrogel solution, composed by Matrigel (Beckton Dickinson) and collagen I. - 14.5μL of gel-cell suspension (2000 cells/well) were transferred in culture plate using a robotic liquid handler (CyBio Selma 96/60). - After polymerization (30 min at 37°C, 5% CO2), growth medium was added. |
MOS; U2OS | [51, 52] | |
| Chytosan | To realize 3D cultures: - composite matrices were made by freezing and lyophilizing a suspension of chitosan (medium-molecular-mass chitosan, 250 kDa; Aldrich) and nanohydroxyapatite (nHA powder; Aldrich) as described by Thein-Han & Misra (2009). The dimensions of pores were variable, with a mean cross-section of ~100 mm. - 106 cells were plated per matrix and their growth was followed. |
U2OS; SaOS-2 | [53] |
| Silk | To realize 3D cultures: - 3% (w/v) Silk fibroin, derived from Bombyx mori silkworms silk solution, was placed in 55mm diameter petri dishes, frozen at 20°C and freeze dried overnight to form porous silk sponges. - The sponges were then soaked in 90% methanol to convert the silk into the b-sheet structure and washed with distilled water. - 0.5x106 cells were suspended in DMEM 10% FBS, 1% penicillin/streptomycin and infused into the silk or nanohydroxyapatite-coated silk scaffolds. - The scaffolds were incubated at 37°C, 5% CO2. |
143.98.2 | [54] |
| To realize 3D cultures: - 0.5x106 U2OS cells or U2OS cocultured with immortalized fibroblasts were seeded in the silk scaffold. - Cultured for 7 days. |
U2OS; HPV-16 E6/E7; HUVEC |
[55] | |
| To realize 3D cultures: - 0.5x106 of OS cells were suspended in 30μl complete culture medium (DMEM 10% FBS, 1% penicillin/streptomycin) and injected into silk scaffolds. |
SaOS2; U2OS,143.98.2 | [56] | |
| Methylcellulose | To realize 3D cultures: - 200 cells/well were mixed with 20% methylcellulose and were seeded into round-bottom Ultra-Low Attachment plates, in 100μl DMEM-F12 containing 20 ng/ml epidermal growth factor (EGF), 20 ng/ml basic fibroblast growth factor (bFGF), 10 ng/ml hepatocyte growth factor (HGF), 10 % B27, 2 % bovine pituitary extract (BPE). - Spheroids were incubated at 37 °C and 5 % CO2, and harvested at various time points for RNA isolation or drug testing as described below. |
RD; HT1080; SW872; HOSS1 | [57] |
| Bacterical cellulose | To realize 3D cultures: - Bacterial cellulose sheets were cut with a biopsy punch to 0.32 cm2 and sterilized in a steam autoclave (at 121°C for 20 min). - 100ul of CSC cells (105cells/mL) suspension was seeded onto the drained scaffolds. - Scaffolds were incubated (37°C, 5% CO2) for 1h to allow cell attachment, and then 1mL of fresh McCoy’s 5A medium, 2.5% FBS and 2 mM L-glutamine were added onto each scaffold. - The scaffolds were placed in their respective hypoxic or control experimental conditions. |
SaOS-2 | [14] |
| SYNTHETIC SCAFFOLD | METHOD | CELL LINES | REF. |
| PEGDA | To realize 3D cultures: - The hydroxyl end-groups of the PEG macromer was made to react with acryloyl chloride to produce PEG diacrylate (PEGDA). - 100 mg PEGDA macromer was dissolved in a photoinitiator solution (10 mg Irgacure-2959 in 1 mL PBS) sterilized by filtration. - Next, tumor cells were uniformly suspended in gel precursor solution by gentle mixing to a final density of 0.3-2.0x106 cells/mL. - The cell-suspended gel precursor solution was crosslinked by UV irradiation, with peak wavelength of 365 nm. - After crosslinking, gels were cut into disks and incubated in stem cell medium to form tumor spheres. |
MDA231; MCF7; HCT116; AGS; U2OS. |
[58] |