Construction Of 3D Human-Hamster Hybrid Cellular System For Environmental Mutagenic Sensitive Detection

As increasing social economy and exogenous harmful substances,traditional toxicity testing is facing great challenge.Traditional toxicity testing relies on a large number of animal experiments,on the other hand,due to long experimental period,high cost and animal ethics,alternative methods in toxicology has been paid more attention,which hinders the evaluation of exogenous harmful substances.Now researchers have mostly used cell experiments in vitro and other techniques to replace the traditional animal toxicity testing,forming the alternative toxicology gradually.However,alternative toxicology is approaching a pivotal point,and there have been few studies on alternative model for genotoxicity detection efficiently.Since only a small segment of the human chromosome is required for the viability of AL cells,it is sensitive in detecting mutagens.As a result,we assess genotoxic effects by constructing a 3D AL cellular system with low-adhesion agarose culture technology,detecting the morphology,biochemical characterization and the differences in environmental toxicological responses between three-dimensional system and traditional monolayer cells.The results as follow:1.Morphological observation.Through dynamic observation of cell morphology under different conditions,2D cells grew as a monolayer adherent morphology,and 3D cells were approximately spherical suspension in the culture medium.After sieving and analyzing the characteristics of 3D spheroid,large loose aggregates maintained stable 3D morphology.As a result,3D cellular system with highly sensitive mutagenic detection was constructed by the suspension methods in the agarose petri dish.During the cultivation period,the number of 3D spheres increased with increasing incubation time gradually,and the cell cluster size would be more uniform.After culturing for 8 consecutive days,the average diameter of the 3D spheres was found to be approximately 240 μm,and the average cell number of the 3D spheres was found to be approximately 1725.The study support that cell microenvironment can play an important role in the rearrangement of intercellular connections,which would lead to the change of cell morphology and the function.2.Mutagenic background and biochemical characterization.In order to identify this cell aggregates is an excellent three-dimensional culture system.The cell viability and background mutation were analyzed by cell colony formation and CD59 gene loci mutation assay.3D cells with no significant mutation enhancement of background showed better proliferative capacity than 2D cells.Moreover,compare with 2D cells,3D cells have significant differences in the molecular level of cell sternness(Oct4,Nanog,Sox2,Lin28)and cell-associated(Connexin 43,LI-cadherin).The expression of Oct4 and Cx43 gene was about 5.9 and 3.2 times higher than 2D cells,respectively.All of these results showed 3D cells had better proliferative capacity and cell communication.3.Environmental toxicity assessment.With the established 3D cell culture model,we studied the toxic effect of environmental contaminant,including heavy metals,persistent toxic pollutants and nanomaterials.The results showed that the survival rate of 3D cells is higher and the mutation rate is lower,3D cells exhibited better resistance than 2D cells.Quantitative measurement assay demonstrated multilayer structure had reduced uptake of environmental pollutants to inner cells.,We further studied the effects of radiation as its penetrability,and the results showed that 3D spheres had significant radiation resistance due to the high expression of repair proteins and cell cycle arrest.Since the study demonstrated that as multilayer structure was consistent with animal tissue and its biochemical characteristics were similar to that of cells in vivo,the 3D culture system could provide more accurate information in vivo.In summary,3D cellular system of AL cells were obtained by utilizing a low-attachment-cultivation technique.Our results showed that as alternative model for genotoxicity detection efficiently,the 3D cell system reflected more information in vivo.