Methods for independent analysis of multiple biological processes in encapsulated 3D cell co-cultures
A biological process and co-cultivation technology, applied in the direction of supporting/immobilizing microorganisms, biochemical equipment and methods, biomaterial analysis, etc., can solve the problems of distinguishing various cysts, not disclosing any means, and not being easy to microscope
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[0080] According to another embodiment of the present invention, said at least one or more cell types are selected from the group comprising cell lines or primary tissue cell subpopulations: hepatocytes, tumor cells, brain cells, epithelial cells, endothelial cells , immune system cells, pluripotent stem cells, embryonic stem cells, lung cells, kidney cells, arterial cells, bone cells, chondrocytes, muscle cells, pancreatic cells, intestinal cells, skin cells, fibroblasts, fungal cells or bacteria.
[0081] Preferably, at least one of said one or more cell types to be analyzed in the in vitro drug testing assay or at least one of the optionally added unlabeled cell types of step c) is a hepatocyte .
[0082] According to one embodiment of the invention, at least one of said one or more types of encapsulated cells is derived from a patient cancer sample.
[0083] According to one embodiment of the invention, at least one of said one or more cell types is a bacterial or fungal ...
Embodiment 1
[0127] describe:
[0128] Monolayers of breast cancer (BC) cells (MELN cells, Tables 1 and 2) stably expressing a luciferase reporter gene under the control of estrogen-responsive elements were co-cultured with or without encapsulated liver cancer (LC) cells and treated with Treatment with increasing concentrations of tamoxifen or 4-hydroxy tamoxifen for 48 hours. The activity of estrogen receptor alpha in BC cells was determined by measuring the activity of luciferase (bioluminescent intensity).
[0129] Materials and Methods:
[0130] Cell seeding of BC cells
[0131] Aspirate the medium on the MELN cells (see Table 1)
[0132] Wash once with PBS, then aspirate the PBS
[0133] Add trypsin on cells, aspirate excess trypsin
[0134] Incubate at 37°C for 5 minutes
[0135] Repeatedly pipette leukocyte medium (DMEM without phenol red 10% charcoal-treated fetal calf serum) onto the cells to dissociate, then collect them into tubes
[0136] Cells were seeded at 10,000 ...
Embodiment 2
[0175] describe:
[0176] Encapsulated BC cells stably expressing the dual reporter gene FUCCI (see Table 2) in far-red-labeled cysts and encapsulated LC cells stably expressing the dual reporter gene FUCCI in unlabeled cysts were incubated at different serum concentrations co-cultivation. The number of actively proliferating and non-proliferating cells (latency) was determined by simultaneously quantifying the ratio of red (G1 phase) and green (G2 / S phase) nuclei in LC cells and BC cells in the same co-culture well.
[0177] Materials and Methods:
[0178] Protocol for establishing a stable reporter cell line:
[0179] HEK293 cells were seeded in 10 cm dishes at 4 million cells / dish.
[0180] Incubate at 37°C for 24 hours.
[0181] Cells were transfected with a mixture of plasmids pMD2G, psPAX2, and a lentiviral reporter plasmid of interest (pBOB-EF1-fastFUCCI-puro) by using the polyethylenimine transfection method.
[0182] The transfection solution was added to the...
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