Published 3D culture protocols

This section includes peer reviewed references on cell isolation and 3D culture.


This is a seminal paper that talks about culturing MCF10A breast epithelial cells in Matrigel and assessing spheroid formation over time using IF staining techniques.

Debnath J et al.

Morphogenesis and oncogenesis of MCF-10A mammary epithelial acini grown in three-dimensional basement membrane cultures

Isolating small cell populations like stem  cells can be a challenging task especially if you have limited amount of tissue to work with. This latest protocol talks about how to isolate mammary stem cells/progenitor cells for use in culture:

Chen, G et al

Isolation, Culture, and Differentiation of Mammary Epithelial Stem/Progenitor Cells from Fresh or Ex Vivo Cultured Human Breast Tissue

This paper describes culturing breast epithelial cells in laminin rich matrices.

Lee, G. Y. et al.

Three-dimensional culture models of normal and malignant breast epithelial cells



Hepatocyte cell cultures are important for assaying drug metabolism and toxicity. Isolation and culture of primary hepatocytes can be challenging. This paper talks about how to culture and isolate primary hepatocytes.

Kegel, V et al.

Protocol for Isolation of Primary Human Hepatocytes and Corresponding Major Populations of Non-parenchymal Liver Cells


3D pancreatic cell cultures can be useful for answering questions on underlying disease mechanism and measuring drug metabolism. This paper talks about how to isolate pancreaticcells from mice and culturing them in 3D for in well immunostaining analysis

Rezanejad H et al.

Generation of Pancreatic Ductal Organoids and Whole-Mount Immunostaining of Intact Organoids

Esophagus, head and neck

3D cultures of esophogeal cells can mimic features of normal tissues including cell polarization, differentiation and gene expression. This paper talks about how to isolate and characterize mouse and human epithelial cells for 3D organotypic culture.

Kalabis, J et al.

Isolation and characterization of mouse and human esophageal epithelial cells in 3D organotypic culture


Sergiu P. Pasca

The rise of three-dimensional human
brain cultures

Pre-clinical drug development

This paper describes a microplate assay to measure the effects of drugs on tumor spheroid cultures using quantitative imaging techniques in a variety of cell lines. This study compares drug efficacy in 3D vs. 2D cultures. This paper also has a good description of 3D cultures, cell viability assays and immunofluorescence staining techniques.

Vinci, A et al.

Advances in establishment and analysis of three dimensional tumor spheroid-based functional
assays for target validation and drug evaluation