Background
Search strategies
Results and discussion
3D CELL culture system - scaffold free
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] |
Liquid-overlay
Agar and agarose
Poly-HEMA
Low and ultralow binding plates
Hanging drop
3D CELL culture system - with scaffold
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] |