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. Author manuscript; available in PMC: 2019 Jul 23.
Published in final edited form as: J Vis Exp. 2019 May 3;(147):10.3791/59293. doi: 10.3791/59293

Figure 2. Organoid Plating in 3D Protein Matrices and on Tissue Culture Treated Plastic.

Figure 2.

Organoids seeded in collagen (A) and basement membrane (B), imaged after 84 hours of growth. Outgrowth of fibroblasts occurred in matrix plated organoids (C, D). Phase contrast images of organoids sorted through filtration were obtained 192 hours after seeding. No major differences between the filtrate (E) and retentate (F) were observed, with both resulting in confluent fibroblast growth. Cells in E and F were seeded on tissue culture treated plastic. After trypsinizing for 5 min at RT, fibroblasts were removed via aspiration; however, remaining epithelial cells formed a monolayer culture instead of three-dimensional organoids (G). Scale bars for A-G represent 100 μm. (H) Different collagenase types (I (CI) and VIII (CVIII)) and cell processing methods (filtration and centrifugal differentiation (Cent Diff)) were tested, and organoid yield per mammary gland was quantified (n = 2 glands for CI, Filter; 2 glands for CVIII, Filter; 4 glands for CI, Cent Diff, and 12 glands for CVIII, Cent Diff). Statistical significance was determined using a two-tailed, unpaired t-test, ***p<0.0001. Error bars represent standard error. Please click here to view a larger version of this figure.