In the process of xenobiotic toxicity prediction and risk assessment, in vitro cell culture models possess high practical application value. With the rapid development of biological technologies such as three-dimensional (3D) bio-printing, organoid culture and organ-on-a-chip systems, in vitro cell culture models have made great progress. Sharing the similarities in structure, function and the physiological environment with tissues or organs in vivo, hazard identification and dose-response analysis based on 3D cell culture models provide access to more accurate toxicity data as a theoretical basis for risk assessment and risk management of chemicals. This review summarizes the establishment of three typical 3D cell culture models, i.e., human cell line-based co-culture model, 3D-printed scaffold-based cell culture model and organoids, and their application in toxicity tests of xenobiotics.
Cell Culture Techniques ; Cell Culture Techniques, Three Dimensional ; Cell Line ; Humans ; Toxicity Tests ; Xenobiotics/toxicity*

Three-dimensional (3D) cell culture model is a system that co-culture carriers with 3D structural materials and different types of cells in vitro to simulate the microenvironment in vivo. This novel cell culture model has been proved to be close to the natural system in vivo. In the process of cell attachment, migration, mitosis and apoptosis, it could produce biological reactions different from that of monolayer cell culture. Therefore, it can be used as an ideal model to evaluate the dynamic pharmacological effects of active substances and the metastasis process of cancer cells. This paper compared and analyzed the different characteristics of cell growth and development under two-dimensional (2D) and 3D model culture and introduced the establishment method of 3D cell model. The application progress of 3D cell culture technology in tumor model and intestinal absorption model was summarized. Finally, the application prospect of 3D cell model in the evaluation and screening of active substance was revealed. This review is expected to provide reference for the development and application of new 3D cell culture models.
Cell Culture Techniques, Three Dimensional ; Cell Culture Techniques ; Apoptosis ; Cell Proliferation ; Technology

A three-dimensional finite element model of the cell culture membrane was developed in the culture device under tension state made by us. The magnitude of tension and the displacement distribution in the membrane made of silicon rubber under different hydrostatic load were obtained by use of FEM analysis. A comparative study was made between the numerical and the experimental results. These results can serve as guides to the related cellular mechanical research.
Animals ; Biomechanical Phenomena ; Cell Culture Techniques ; Finite Element Analysis ; Imaging, Three-Dimensional ; Membranes, Artificial ; Rats ; Rats, Sprague-Dawley ; Silicone Elastomers

UV photolithography and hydrofluoric acid wet etching were used to produce silicon master molds and polydimethylsiloxane (PMDS)-based soft lithography was adopted to fabricate three-dimensional poly(lactic-co-glycolic acid) (PLGA) and PDMS microwell patterns with high aspect ratio and channel connection. Nine microwell patterns were thus obtained with different structural dimensions. Patterns were treated with oxygen plasma etching and polylysine coating to enhance hydrophilicity and cell compatibility for subsequent culture of C17. 2 neural stem cells. With proliferation during the culture, C17. 2 cells gradually distributed within the microwells, showing an obviously three-dimensional (3-D) growth behavior. The presence of channel structures greatly favored the 3-D growth of C17. 2 neural stem cells on the microwell patterns. Multi-layered scanning with confocal microscopy and 3-D rendering after carboxyfluorescein diacetate succinimidyl ester (CFDA-SE) staining showed that most C17. 2 cells grew within a range of 30 to 90 microm from the microwell bottom. Immunofluorescence staining indicated that C17. 2 cells within 3-D microwell patterns were uniformly nestin-positive on day 2 after cell plating. It could well be concluded that the microwell patterns thus fabricated were suitable for the 3-D culture and subsequent differentiation of C17. 2 neural stem cells. And the cells can be maintained with uniform stemness properties while cultured in these microwell patterns.
Cell Culture Techniques ; methods ; Dimethylpolysiloxanes ; chemistry ; Imaging, Three-Dimensional ; Intermediate Filament Proteins ; metabolism ; Lactic Acid ; chemistry ; Microscopy, Confocal ; Nerve Tissue Proteins ; metabolism ; Nestin ; Neural Stem Cells ; cytology ; Polyglycolic Acid ; chemistry

OBJECTIVETo fabricate an innovative scaffold for lung cancer cell culture and establish a three-dimensional lung cancer model in vitro, and to reveal the differences in biological functions of lung cancer cells under the two-dimensional and three-dimensional culture conditions.

METHODSWe chose agarose and alginate as the scaffold materials, and 3D printing technique was applied to construct cell culture scaffold. 95D cells were co-cultured with this scaffold. The differences of cell morphology, proliferation ability, protein expression, etc. in the cells cultured under 2D and 3D cultural conditions were evaluated by light microscopy using HE staining, MTT assay, scanning electron microscopy, and Western blot analysis.

RESULTSCells cultured in 2D wells displayed a spindle and polygonal morphology, whereas those grown in the 3D culture aggregated into spheroids, which invaded, migrated and disseminated into the surrounding scaffold. MTT assay showed that the proliferation rates of the 3D-cultured cells for 2-6 days were significantly lower than, but those cultured for 8-9 days were significantly higher than that of the 2D-cultured cells, indicating that proliferative activity of the cells grown in 2D cultures for 8-9 days was inhibited. In contrast, cells grown on 3D scaffolds still maintained a higher proliferation. The Western blot assay showed that the expression of Cdc42, p53, mTOR were significantly down-regulated in 3D scaffold-cultured group (0.529±0.103, 0.820±0.038 vs. 1.967±0.066), compared with that of the 2D-cultured group (3.063±0.139, 1.738±0.122 vs. 2.472±0.151) (P<0.05 for all), while the expression of MMP-2 was up-regulated in the 3D-cultured cells (1.110±0.029), significantly higher than that of the 2D-cultured cells (0.017±0.001) (P<0.05).

CONCLUSIONSThe cell morphology, proliferation and associated protein expression of lung cancer cells in 3D-culture systems are distinctively different as compared to those of the 2D-cultural cells. 3D-bioprinted agarose-alginate scaffold can better mimic the growth microenvironment of lung cancer in vivo and may provide a promising model for lung cancer research in vitro.

Alginates ; Carcinoma, Non-Small-Cell Lung ; metabolism ; pathology ; physiopathology ; Cell Culture Techniques ; Cell Movement ; Cell Proliferation ; Cells, Cultured ; Coculture Techniques ; Glucuronic Acid ; Hexuronic Acids ; Humans ; Lung Neoplasms ; metabolism ; pathology ; physiopathology ; Neoplasm Invasiveness ; Neoplasm Proteins ; metabolism ; Printing, Three-Dimensional ; Sepharose ; Spheroids, Cellular ; pathology ; Time Factors ; Tissue Scaffolds ; Tumor Microenvironment