Cellevates NanoMatrix as a animal friendly 3D culturing of human cancer- and normal cells
Two-dimensional cell culturing has proven inadequate as a reliable preclinical tumour model due to many inherent limitations. Hence, novel three-dimensional (3D) cell culture models are needed, which in many aspects can mimic a native tumour with 3D extracellular matrix. Here, we present a 3D electrospun polycaprolactone (PCL) mesh mimicking the collagen network of tissue.
In this customer case a University group developing novel anti-cancer compounds was interested in replicating the tumor microenvironment, to increase the relevance of their in-vitro models for breast cancer. The group wanted to mimic the three-dimensional (3D) growth and complex features tumors exhibit in the human body, which is difficult to replicate in a two-dimensional (2D) system and thus approached us.
Specifically, the group was interested in utilizing the model for cytotoxicity assays. Since the customer wanted to work in HTS applications the format was very important to them. Thus, we developed an optimized synthetic polymer scaffold integrated into a multiwell plate, according to SLAS formats.
As cytotoxicity was the desired readout, we provided validation that the scaffold itself was not toxic. An MTT assay was used to investigate the toxicity of extracts from the scaffold components using L929 mouse fibroblasts in compliance with ISO standard 10993–5.
In the image to the left we show biocompatibility results of A) PCL granule and B) PCL fibre mesh. Water soluble extracts were made according to ISO standard 10993–12 and the cells were treated with the serial dilution of extract solutions for 72 h. The results are presented as mean values (n = 3) and the error bars represent ± SD.
Morphological comparison using confocal microscopy of different cells seeded in 3D PCL fibre meshes after 1 week of incubation. Confocal scanning microscopy images of (A) MCF-7 cells, (B) JIMT-1 cells, (C) MCF-10A cells, and (D) human adult dermal fibroblasts on the surface of 3D PCL fibre meshes. The cells were stained with Alexa 488-phalloidin (green) to visualize actin cytoskeleton and DAPI (blue) to visualize nuclei. Scale bars indicate 20 μm.
Confocal microscopy and cryosectioning show that the cells grow deep into the scaffolds after 7 days and exhibit an in-vivo like morphology.
Confocal microscopy z-stack imaging shows infiltration of cells deep into the 3D fibre meshes 7 days after seeding the cells (Fig. 7). Thus, the PCL fibre mesh, which has an average pore size of 50 μm2, provides adequate spacing and porosity permitting cell infiltration (Jakobsson et al., 2017). The breast cancer cells form small tight spheroids at different depths of the mesh while fibroblasts are spread-out in the mesh. The MCF-10A cultures show both clusters of cells and a spread-out morphology of the cells in the mesh.
In this collaboration we successfully developed a new alternative in-vitro model for breast cancer co-culture to act as a potential replacement of animal models and products derived from animals. Overall, we show that normal and cancer cells thrive in the 3D meshes cultured in fetal bovine free medium which eliminates the use of collagen as an extracellular matrix support.
The developed model is currently in use at the academic lab, being utilized in their drug development platform.