Supplementary Figure 2: Conditions that affect the successful expansion of human tissues.

(A) Images of human skin samples, both taken by a conventional camera (i, iv) and via fluorescence microscopy (ii-iii, v-vi) when stained with DAPI (gray) and antibodies against vimentin (green) and smooth muscle actin (gene name, ACTA2) (magenta). The samples were digested with 8 units/mL proteinase K solution containing 25 mM Tris (pH 8), 1 mM EDTA, 0.25% Triton X-100, and 0.4 M NaCl at 60°C for 0.5 hour (i-iii) or 2 hours (iv-vi). (i and iv) Conventional camera (i.e., non-fluorescent) photographs of human skin-hydrogel hybrid samples in PBS (~2x expansion), after digestion for 0.5 hour (i) or 2 hours (iv). (ii) Widefield fluorescent image from the sample of (i) pre-digestion. (iii) Post-expansion wide-field fluorescent image of the sample of (ii), with a dashed orange box highlighting regions with autofluorescence in the DAPI channel and distorted vimentin networks post-expansion. (v) As in (ii), for the sample in (iv) pre-digestion. (vi) As in (iii) for the sample of (v) with a dashed orange box highlighting regions with autofluorescence in the DAPI channel. (B) As in (A), except that the samples were digested with 8 units/mL proteinase K solution containing 25 mM Tris (pH 8), 25 mM EDTA, 0.25% Triton X-100, and 0.4 M NaCl, at 60°C for 0.5 hour (i-iii) or 2 hours (iv-vi). Note that in Bi and Biv the samples look quite transparent, due to the heavy digestion. (C) Photographs of human liver samples digested with a 1 mM EDTA-based solution – specifically, 8 units/mL proteinase K solution containing 25 mM Tris (pH 8), 1 mM EDTA, 0.25% Triton X-100, and 0.4 M NaCl at 60°C for 0.5 hour (left) or 2 hours (right). (D) Photographs of human liver samples digested with a 25 mM EDTA-based solution – specifically, 8 units/mL proteinase K solution containing 25 mM Tris (pH 8), 25 mM EDTA, 0.25% Triton X-100, and 0.4 M NaCl at 60°C for 0.5 hours (left) or 2 hours (right). Note that the samples of D are significantly more transparent than those of C. (E) Widefield fluorescent image of a human liver sample stained with DAPI (gray) and an antibody against smooth muscle actin (gene name, ACTA2) (magenta) prior to the ExPath process. (F) Post-expansion wide-field fluorescent image of the sample of E, digested with a 1 mM EDTA-based solution for 1 hour. White dashed line outlines an out-of-focus region caused by distortion. (G) Wide-field fluorescent image of a human liver sample stained with DAPI (gray) and an antibody against smooth muscle actin (gene name, ACTA2) (magenta) prior to the ExPath process. (H) Post-expansion wide-field fluorescent image of the same sample as in G. The sample had been digested with a 25 mM EDTA-based solution for 0.5 hour. (I) Post-expansion confocal image of human lymph node tissue with invaded breast cancer stained with DAPI (blue) and an antibody against vimentin (green), and treated with a 1 mM EDTA-based solution for 3 hours. (J) Post-expansion confocal image of a different sample from the same tissue used in I, treated with a 25 mM EDTA-based solution. (K) Post-expansion confocal image of normal human kidney tissue fixed with acetone, stained with an antibody against collagen IV (magenta), and treated with 0.1 mg/ml Acryloyl-X prior to in situ polymerization. Cracks are indicated by white arrows. (L) Post-expansion confocal image of a different sample from the same tissue used in K, treated with 0.03 mg/ml Acryloyl-X prior to in situ polymerization. Scale bars (yellow scale bars indicate post-expansion images: Aii and iii, 9.2 μm (physical size in iii: 40 μm, expansion factor: 4.33). Av and vi, 9.4 μm (physical size in vi: 40 μm, expansion factor: 4.28). Bii and iii, 9 μm (physical size in iii: 40 μm, expansion factor: 4.41). Bv and vi, 8.9 μm (physical size in vi: 40 μm, expansion factor: 4.51). E and F, 119 μm (physical size in F: 500 μm, expansion factor: 4.22); G and H, 109 μm (physical size in H: 500 μm, expansion factor: 4.58). (I-L) 40 μm, physical size.