Paper-supported 3D cell culture for tissue-based bioassays

Abstract

Fundamental investigations of human biology, and the development of therapeutics, commonly rely on 2D cell-culture systems that do not accurately recapitulate the structure, function, or physiology of living tissues. Systems for 3D cultures exist but do not replicate the spatial distributions of oxygen, metabolites, and signaling molecules found in tissues. Microfabrication can create architecturally complex scaffolds for 3D cell cultures that circumvent some of these limitations; unfortunately, these approaches require instrumentation not commonly available in biology laboratories. Here we report that stacking and destacking layers of paper impregnated with suspensions of cells in extracellular matrix hydrogel makes it possible to control oxygen and nutrient gradients in 3D and to analyze molecular and genetic responses. Stacking assembles the "tissue", whereas destacking disassembles it, and allows its analysis. Breast cancer cells cultured within stacks of layered paper recapitulate behaviors observed both in 3D tumor spheroids in vitro and in tumors in vivo: Proliferating cells in the stacks localize in an outer layer a few hundreds of microns thick, and growth-arrested, apoptotic, and necrotic cells concentrate in the hypoxic core where hypoxia-sensitive genes are overexpressed. Altering gas permeability at the ends of stacks controlled the gradient in the concentration of the O(2) and was sufficient by itself to determine the distribution of viable cells in 3D. Cell cultures in stacked, paper-supported gels offer a uniquely flexible approach to study cell responses to 3D molecular gradients and to mimic tissue- and organ-level functions.

Fig. 1.

Generation and characterization of 3D cultures of defined physical dimensions. (A) Permeation of Matrigel or other hydrogel precursors into chromatography or filter paper yields paper-supported hydrogels of thickness akin to that of the paper and lateral dimensions determined by the volume spotted. (B–-D) Spotting of a suspension of mCherry-HS5 cells in cold (4 °C) Matrigel labeled with AF488, followed by immersion in 37° C media, led to gelation of Matrigel inside the paper. Using a different volume of spotted solution controlled the spotted area. Using different concentrations of cells controlled the number of cells per spotted area. Imaging with a fluorescent gel scanner visualized cells (B), matrix (C), and their colocalization (D) inside paper. (E) The area that contains HS-5 cells within a paper is similar to the area of spreading of Matrigel. (F) Integrated fluorescent intensity of endogenous mCherry fluorescence correlated linearly with fluorescence of F-actin stain. (G–I) We measured the integrated fluorescent intensity of 3D cultures and interpolated the intensity as a function of area (G) and cell density (I) (n = 4 for each size and concentration; all data are presented). Representative images of each culture are presented in H.

Fig. 2.

Properties of cells after nine days of culture in thick (1,600 μm) 3D cultures made of multiple stacked layers of paper (L1–L8) and comparison with thin 3D cultures comprising a SL of paper. (A) Schematic of the plating, culture and analysis. In B–E, samples were cultured for nine days and fixed. Separated layers were stained with AF633-phalloidin (n = 12) (B), Click-It EdU kit (n = 8) (D), Click-It TUNEL kit (n = 8) (E); in C, separated layers were incubated with calcein solution for 20 min and fixed (n = 6). Intensities of the stains in B–E were normalized to those obtained in SL samples after one day of culture (vertical red line). For n > 7, the average and an error bar equal to one standard deviation are presented. For n < 7, all data are presented (the box highlights the range of data). *, P < 0.05; **, P < 0.01.

Fig. 3.

Isolation of viable cells from defined areas of 3D culture. In A, samples were cultured in stacks for nine days, destacked, and placed in separated wells containing Alamar Blue. (B and C) The rate of substrate turnover determined the number of cells four hours (B) or four days (C) after destacking (n = 4). (D) The ratio of values from B and C. All data are presented (the box highlights the range of data). *, P < 0.05; **, P < 0.01.

Fig. 4.

Expression levels of oxygen-responsive genes in different locations of 3D culture. (A and B) Real-time PCR-quantified levels of expression of VEGF and IGFBP3 transcripts using ΔΔC_t values relative to beta-2 microglobulin and reported as “fold increase” with respect to L1 samples (n = 6). (C–E) In the loosely packed eight-layer stack, no gradient of expression of VEGF and IGFPB3 was observed (n = 3) (D), and the fraction of S-phase cells is the same in all layers LS1–LS8 (n = 8) (E). In D, RNA levels in LS1–LS8 are normalized to those of L1 in B and were significantly different from the corresponding layers L1–L8. All data are presented (the box highlights the range of data). *, P < 0.05; **, P < 0.01.

Fig. 5.

Changing gas permeability of the terminal layers reveals that the distribution of cells in 3D follows that of O₂. (A–D) When a PDMS layer was placed on the bottom of the closely packed eight-layer stack (P1–P8), oxygen could access the bottom (P8) layers; the number of cells (n = 4) and the number of S-phase cells (n = 4) (C) in the bottom layers (P8) was significantly higher than in the middle layers (e.g., P4), although it was lower than in the layer exposed to culture media (P1). (D–F) The number of viable cells in stacks containing a bottom layer made of PDMS (D), cellulose acetate (F), or no layer at all (E) was determined by using Alamar Blue (n = 6 in E and F). All data are presented (the box highlights the range of data). *, P < 0.05; **, P < 0.01.

Fig. 6.

Comparison of distribution of cells in multilayer stacks in vivo and in vitro. (A and B) We cultured LLC-GFP cells in SL and multilayer (L1-L8) constructs for three days in vitro (n = 6) (A) or implanted these constructs s.c. in C57BL mice (n = 5) (B), isolated the layers, and measured GFP fluorescence in every layer. The number of cells in SL and terminal layers (L1 or L8) was significantly higher than that in the middle layers. All data are presented; the overlaid box is centered on the average value, and its width is twice the standard deviation. *, P < 0.05; **, P < 0.01. (C–G) We also analyzed the distribution of cells by sectioning the SL and multilayer (L1–L8) samples from culture in vitro (C–E) and in vivo (F–G). We imaged the sections by using a gel scanner; representative images show inverse intensity of the green fluorescence. (H and I) Hematoxylin and eosin (HE) staining of the 18-μm-thick sections of the samples immediately after plating (H), and after three days of culture in vitro (I). (J–P) HE-staining and immunofluorescent staining of 18-μm-thick sections of multilayer constructs cultured in vitro confirm that GFP-LLC cells (green in all images) reside in terminal layers and also infiltrate the adjacent tissue. Frames K–P demonstrate distribution of CD45(+) cells (M), S-phase cells (N), CD31(+) cells (O), and functional capillaries (red in M–P, white arrows in P and L) stained by i.v. injection of rhodamine-ConA. (Q) High-magnification image of the capillary inside the L1 layer. We acquired images by using confocal (I, M–P) or dissecting (H–K) microscopes with 4×, 10×, 20×, or 67× objective. Blue, DAPI; green, GFP; red, rhodamine-ConA; purple, CD31 (M), EdU (N), CD45 (O).