3DCellMaker Poster presentation at Controlled Release Society 2016 Annual Meeting

3DCellMaker (www.3DCellMaker.com) is a thermogelling cell-growth media modifier which allows for culturing cells in 3D structures without requiring specialized plasticware or equipment. At the 2016 CRS meeting, Justin Hadar presented a research poster generated from a collaboration between Akina, Inc. and Purdue University Department of Basic Medical Science’s Professor Sophie Leliévre. For this poster, multiple cell-lines were cultured as well as tests of co-culture between MCF-7 cells with associated fibroblasts and e-cadherin staining to establish formation of cell-to-cell interactions. You can see the full poster and supplemental data here (http://www.3dcellmaker.com/research/). Abstract attached below:

“ABSTRACT: 3DCellMakers: Thermogelling Polymers for 3D Cell Culture, J. Hadar1, J. Garner1 S. Skidmore1, H. Park1, K. Park1,2, B. Han2, F. Atrian2, S.A. Lelièvre2  1Akina, Inc. West Lafayette, IN 47906 USA 2Purdue University West Lafayette, IN 47907 USA jh@akinainc.com Purpose: The goal of this study is to synthesize polymers that provide consistent, reproducible environments for cells to form tumor structures, resulting in more representative drug transport and therapeutic characteristics relevant to clinical applications.  Certain types of inverse thermogelling polymers allow tumor cells as well as non-disease state cells to form three-dimensional (3D) spheroid-like structures with characteristic features that are not observed in the same cells cultured in 2D. The thermogelling property allows mixing cells with polymer solution at room temperature before forming a transparent gel at 37 °C. Methods:  Ethylene oxide sterilized thermogels were dissolved in cell culture medium which consisted of DMEM/F12 + GlutaMAX™ basal medium supplemented with 5% (v/v) fetal bovine serum, penicillin (100 units/ml), and streptomycin (100 µg/ml).  The volume of medium was adjusted for the desired % (w/v) hydrogel, and dissolution was done at 2-8 °C for two days.  Multiple cell lines were used to conduct the experiments. In one approach either breast cancer MCF-7 cells cocultured with human fibroblasts CCD-1068SK or human liver carcinoma HEP G2 cells were mixed with cold hydrogel, and the mixtures were transferred into   a flat bottom polystyrene multiwall plate (uncoated) and incubated at 37 °C. In another approach, the hydrogel solution was prewarmed in polystyrene multiwall plates and the cell suspension was added on top of the solidified gel. At predetermined time points, pictures of cell cultures were taken. During the culture period, the medium was replaced every 48 to 72 hours. Additionally, triple negative breast cancer T4-2 cells typically cultured in a serum-free medium with known additives were seeded onto 100 ul of 3DCellMaker gel per well in a 12-well plate and images were taken after two days of culture.  Results: Three promising polymers for 3D cell culture were identified, and they were named “3DCellMakers”. They include poloxamer-hexamethylene diisocyanate poly(ester-urethane) (PEU), stearate modified methyl cellulose, and poloxamer-methylene diphenyl diisocyanate PEU. In general, seeding the cells onto the prewarmed gels allow 3D structures to form quickly (1~4 days), while mixing the cells directly with cold thermogel solution before heating to gel typically yielded tumors at a later time and of smaller size.  Tumors ranged in size from 40 um to 200 um (Fig. 1).  T4-2 cells that are particularly sensitive to their environment for tumor formation also formed 3D structures in less than 48 hours (Fig. 2). Conclusions: The 3DCellMakers have potential to provide an effective, inexpensive, and easy method for generating 3D multicellular structures.  The new thermogelling polymers provide a new avenue of increased productivity in cell biology research for which multicellular 3D structure formation is critical, e.g., studying the efficacy of various drugs and drug delivery systems for treating tumors.  The ability to co-culture cancer cells with fibroblasts in 3D provides an interesting avenue to study important aspects of the tumor microenvironment, especially if it can be combined with microfluidic devices or high throughput screening systems. Additionally, the new thermogelling polymers allow T4-2 invasive cancer cells, which are known to be sensitive to the microenvironmental conditions and are cultured under serum-free conditions, to thrive. This preliminary result suggests that the 3DCellMaker has potential for broad use. The possibility to culture cells with reproducible and known medium conditions will make easier the study of the effect of specific components on tumor growth.”