On-chip assay of the effect of topographical microenvironment on cell growth and cell-cell interactions during wound healing

Abstract

Wound healing is an essential physiological process for tissue homeostasis, involving multiple types of cells, extracellular matrices, and growth factor/chemokine interactions. Many in vitro studies have investigated the interactions between cues mentioned above; however, most of them only focused on a single factor. In the present study, we design a wound healing device to recapitulate in vivo complex microenvironments and heterogeneous cell situations to investigate how three types of physiologically related cells interact with their microenvironments around and with each other during a wound healing process. Briefly, a microfluidic device with a micropillar substrate, where diameter and interspacing can be tuned to mimic the topographical features of the 3D extracellular matrix, was designed to perform positional cell loading on the micropillar substrate, co-culture of three types of physiologically related cells, keratinocytes, dermal fibroblasts, and human umbilical vein endothelial cells, as well as an investigation of their interactions during wound healing. The result showed that cell attachment, morphology, cytoskeleton distribution, and nucleus shape were strongly affected by the micropillars, and these cells showed collaborative response to heal the wound. Taken together, these findings highlight the dynamic relationship between cells and their microenvironments. Also, this reproducible device may facilitate the in vitro investigation of numerous physiological and pathological processes such as cancer metastasis, angiogenesis, and tissue engineering.

FIG. 1.

Schematic illustration of the experiment procedures and characterization of the microfluidic device. (a) The microfluidic device used in the current study, which was composed of four layers, a PDMS stencil, a PDMS micropillar substrate, a thin PDMS membrane (not shown), and a polystyrene culture dish. The PDMS stencil was sealed to the micropillar substrate, and each type of cells was seeded into the appropriate region (from left to right, HaCaT, ESF-1, and HUVEC cells, respectively). (b) The PDMS stencil was peeled off after the cells were attached and cultured on the micropillar substrate for 24 h. (c) A typical fluorescence image of well-distributed cells after the stencil was just peeled off. To visualize clearly the coexistence of cells, HaCaT (left), ESF-1 (middle), and HUVEC (right) cells were specifically stained using CellTracker Orange, CellTracker Green, and CellTracker Orange, respectively. (d) A typical fluorescence image of cells after wounding, corresponding to 36 h. (e) SEM image of the micropillar substrate. (f) Enlarged image of the square in the dotted lines in (e).

FIG. 2.

Quantitative assessment of the cell attachment on the different micropillar substrates. Histograms present cell numbers of (a) HaCaT, (b) ESF-1, and (c) HUVEC cells. Cell numbers were obtained by manual counting of AO stained cells. For clarity, the different diameters (15, 18, and 21 μm) and spacing (15, 18, and 21 μm) of the micropillars were denoted as d15, d18, and 21 μm and s5, s18 and s21 μm, respectively.

FIG. 3.

Comparison of cell morphology and cytoskeleton after 24-h culture on the flat and micropillar substrates. (a) Fluorescence image of HaCaT cells cultured on the flat substrate. (a') Fluorescence image of HaCaT cells cultured on the d18s18-μm micropillar substrate. (b) Fluorescence image of ESF-1 cells cultured on the flat substrate. (b') Fluorescence image of ESF-1 cells cultured on the d18s18-μm micropillar substrate. (c) Fluorescence image of HUVEC cells cultured on the flat substrate. (c') Fluorescence image of HUVEC cells cultured on the d18s18-μm micropillar substrate. Cells were stained for actin filament (red) and nuclei (blue).

FIG. 4.

Quantitative analysis of cell morphology on different substrates, including the flat substrate and micropillar substrates with different diameters (15, 18, and 21 μm) and spacing (15, 18, and 21 μm). (a) Spreading area and (a') circularity of HaCaT cells on different substrates. (b) Spreading area and (b') aspect ratio of ESF-1 cells on different substrates. (c) Spreading area and (c') circularity of HUVEC cells on different substrates. Orange bars within box plots indicate the mean values.

FIG. 5.

SEM images of the three types of cells on d18s15-μm (a)–(c) and d18s21-μm (a')–(c') micropillar substrates. (a) and (a') HaCaT cells. (b) and (b') ESF-1 cells. (c) and (c') HUVEC cells.

FIG. 6.

Dynamic cell migration under different conditions after the wound. (a) Dynamic migration of HaCaT and ESF-1 cells in their mono-culture and co-culture. (b) Real-time migration of ESF-1 and HUVEC cells in their mono-culture and co-culture. (e) Dynamic migration of HaCaT and HUVEC cells in their mono-culture and co-culture. (c), (d), and (f) Quantitative analysis of cell migration distances during (a), (b), and (e). The initial leading edges (shown as white dashed lines) represent baselines for the migration assay.

FIG. 7.

Comparison of the migration direction of ESF-1 cells in the co-culture and mono-culture during the wound healing. (a) Fluorescence image of ESF-1 cells during HaCaT, ESF-1, and HUVEC cell co-culture, showing ESF-1 cells migrated towards HaCaT cells. (b) Fluorescence image of ESF-1 cells during HaCaT, ESF-1, and HUVEC cell co-culture, showing that ESF-1 cells migrated towards HUVEC cells. (c) Fluorescence image of ESF-1 cells during their mono-culture, showing that ESF-1 cells migrated towards the wound. (a')–(c') The orientation of ESF-1 cells in the co-culture and mono-culture during the wound healing, corresponding to (a), (b), and (c), respectively. The direction of each bar in the rose plots indicates the angular ESF-1 cell orientation, whereas the magnitude of each bar shows the fraction of cells with the indicated ESF-1 cell orientation.