Table 1.
Summary of the main methods for establishing 3D models for drug screening.
| Type of 3D Technique | Name of the Technique | Endpoint Assay and Data Acquisition | Ref. |
|---|---|---|---|
| Scaffold-free | Hanging drop | Viability/Cytotoxicity: CellTiter-Glo® 3D, LIVE/DEAD (Calcein AM/ethidium homodimer); Trypan blue; Perfecta3D®; Other Analysis: WB, IHC, IF and LS-FM. |
[31,32] |
| Forced floating (e.g., Ultra-low attachment plates) | Viability/Cytotoxicity: CellTiter-Glo™ 3D, LIVE/DEAD (Calcein AM/ethidium homodimer); ViaLight™ Plus. Other Analysis: WB, qPCR, IF, IHC, HCI (Software: Cytation 3, CellInsight NXT, MetaXpress 6). |
[31,55,56,57,58] | |
| Micromolding | Viability/Cytotoxicity: LIVE/DEAD (Calcein AM/propidium iodide), CCK-8, MTT. Other Analysis: WB, qPCR, Flow Cytometry, Hematoxylin and eosin staining. |
[59,60] | |
| Agitation-based techniques | Viability/Cytotoxicity: CellTiter-Glo® 3D; LIVE/DEAD (Calcein AM/ethidium homodimer); Trypan blue; Perfecta3D®. Other Analysis: IF and LS-FM. |
[32] | |
| Magnetic levitation or bioprinting | Viability/Cytotoxicity: CellTiter-Glo® 3D; LIVE/DEAD (Calcein AM/ethidium homodimer); Trypan blue; Perfecta3D®. Other Analysis: Reporter transgene, IF, LS-FM, ELISA. | [32,61,62,63] | |
| Microfluidics | Viability/Cytotoxicity: LIVE/DEAD (Calcein AM/ethidium homodimer); Calcein AM (LIVE) and 7-Amino-ActinomycinD (DEAD) staining Other Analysis: Flow Cytometry, SEM, PCM, Reporter transgene, IF, qPCR, Actin Cytoskeleton and Focal Adhesion Staining Kit |
[64,65,66] | |
| Pellet Culture | Viability/Cytotoxicity: CellTiter-Glo® 3D; LIVE/DEAD (Calcein AM/ethidium homodimer), Trypan blue; Perfecta3D®; Other Analysis: IF and LS-FM. | [32] | |
| Scaffold-based | 3D-bioprinting | Viability/Cytotoxicity: LIVE/DEAD (Calcein AM/propidium iodide); Alamar Blue, CCK-8, LDH. Other Analysis: MMP Zymography Assay Kit (for matrix metalloproteinase characterization), SEM, Histology, IHC, IF, qPCR. |
[34,67,68,69] |
| Microfluidics | Viability/Cytotoxicity: LIVE/DEAD (Calcein AM/ethidium homodimer), CCK-8. Other Analysis: IF, MMP Zymography Assay Kit, FACS, Caspase 3/7 activity assay, CellTrace™ CFSE Cell Proliferation Kit |
[69,70] | |
| Hydrogel | Viability/Cytotoxicity: CellTiter-Glo® 3D, LIVE/DEAD (Calcein AM/ethidium homodimer); Other Analysis: qPCR, IF |
[58,71] |
CCK-8: Cell Counting kit-8 cell proliferation assay; ELISA: Enzyme-Linked Immunosorbent Assay; FACS: Fluorescence-Activated Cell Sorting; HCI: High-Content Imaging; IF: Immunofluorescence; IHC: Immunohistochemistry; LDH: Lactate Dehydrogenase; LS-FM: Light-Sheet Fluorescence Microscopy; qPCR: Quantitative Real-time Polymerase Chain Reaction; PCM: Phase-Contrast Microscopy; SEM: Scanning Electron Microscopy; WB: Western Blotting.