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% CO_2, 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 % CO_2, and harvested at various time points for RNA isolation or drug testing as described below.

RD; HT1080; SW872; HOSS1

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

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]