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Next generation seuquencing of 3D breast cancer cell culture in synthetic hydrogels presenting either a collagen mimic or laminin mimic with low and high mechanical properties

ABSTRACT: MDA-MB-231 (ER-) and T47D (ER+) breast cancer cells were encapsualted in synthetic hydrogels, formed with a multi-arm bioinert polymer poly(ethylene glycol), cell-degradable peptide crosslinks, and receptor-binding pendant peptides, and cultured for 3 days and up to 40 days to assess initial cell responses to different matrix compositions. The synthetic matrices had a Young's modulus (E) ~ 0.6 kPa (low wt%) and # ~ 5 kPa (high wt%), in the range of bone marrow or healthy mammary tissue and lung tissue, respectively, and Biochemcial cues to mimic binding sites found in collagen (GFOGER peptide) or laminin (IKVAV peptide). After 3 days and 40 days in culture, mRNA was collected and RNA-seq performed to compare cell responses between the collagen and laminin mimetic cultures overtime in both low wt% and high wt% matrices.

ORGANISM(S): Homo sapiens

PROVIDER: GSE145696 | GEO | 2022/03/01

REPOSITORIES: GEO

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Project description:I. Exp Design 1. Type of experiment: Comparison of native versus cultured RPE cells 2. Experimental factors: Native RPE versus ARPE-19 cells grown on different matrices 3. How many hybridizations in exp: 21 4. If a common reference used for all the hybs: no 5. Quality control steps: three independent arrays for each condition 6. Description: The expression profile of ARPE-19 cells grown on different matrices were compared to morphologically normal native macular RPE cells that were laser capture microdissected from 3 donors. II. Samples used, extract prep, and labeling 1. Biosource: Human donor globes from NDRI (63, 71, 74 years old) and ARPE-19 cells. 2. Manipulations: Human donor globes were cryopreserved, and morphologically normal RPE cells from the macula were laser capture micodissected. ARPE-19 cells were grown on different matrices (plastic, Matrigel, collagen I, collagen IV, laminin, and fibronectin). 3. Extract preparation: Total RNA from cells were extracted with the RNeasy kit (Qiagen) using the manufacturerâs instructions. 4. Labeling protocol: Total RNA from cells was reverse transcribed with 33P-dCTP and 33P-dATP, and second strand cDNA was labeled with 33P-dCTP and 33P-dATP. 5. No external controls were added. III. Hybridization procedures and parameters 1. Sample, array type, batch and serial # used 2. Hybridization protocol: Hybridization was carried out using the manufacturerâs recommendations. Arrays were prehybridized with Microhyb solution containing denatured Cot-1 DNA and poly dA at 42oC for two hours. Hybridization was carried out at 42oC overnight using a hybridization oven set at 8-10 rpm. Arrays were washed twice at 50oC for 20 minutes using 2x SSC, 1%SDS and once at room temperature for 15 minutes using 0.5x SSC, 1%SDS. IV. Measurement data and specifications of data processing 1,2. Arrays were exposed to a phosphorimaging screen for 3 days and scanned at 50 mm resolution with a BioRad FX Pro-Plus phosphorimager. TIFF images from the phosphorimager were exported into ResGen Pathways 3 software for analysis. 3. Data processing: A gene was expressed if its background subtracted intensity was greater than 1.4 fold background. The data were normalized using a simple global scaling procedure, and Cluster/Treeview and Statistical Analysis of Microarrays (SAM version 1.12) programs were used for analysis.

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Next generation seuquencing of 3D breast cancer cell culture in synthetic hydrogels presenting either a collagen mimic or laminin mimic with low and high mechanical properties

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