Fabrication of Human Keratinocyte Cell Clusters for Skin Graft Applications by Templating Water-in-Water Pickering Emulsions

Abstract

Most current methods for the preparation of tissue spheroids require complex materials, involve tedious physical steps and are generally not scalable. We report a novel alternative, which is both inexpensive and up-scalable, to produce large quantities of viable human keratinocyte cell clusters (clusteroids). The method is based on a two-phase aqueous system of incompatible polymers forming a stable water-in-water (w/w) emulsion, which enabled us to rapidly fabricate cell clusteroids from HaCaT cells. We used w/w Pickering emulsion from aqueous solutions of the polymers dextran (DEX) and polyethylene oxide (PEO) and a particle stabilizer based on whey protein (WP). The HaCaT cells clearly preferred to distribute into the DEX-rich phase and this property was utilized to encapsulate them in the water-in-water (DEX-in-PEO) emulsion drops then osmotically shrank to compress them into clusters. Prepared formulations of HaCaT keratinocyte clusteroids in alginate hydrogel were grown where the cells percolated to mimic 3D tissue. The HaCaT cell clusteroids grew faster in the alginate film compared to the individual cells formulated in the same matrix. This methodology could potentially be utilised in biomedical applications.

Keywords: DEX; HaCaT; PEO; Pickering emulsions; alginate; hydrogels; keratinocyte; spheroids; tissue engineering; water-in-water emulsions.

Figure 1

Schematics of our high-throughput method for preparation of keratinocyte cell spheroids (A–D). The keratinocyte cells are encapsulated in a dextran-PEO water-in-water emulsion template stabilised by 2 wt% WP particles. The continuous phase is PEO 5.5 wt% and the dispersed phase is composed of cells encapsulated in dextran droplets. Upon emulsification, cells prefer the discontinuous dextran phase, which allow their encapsulation. Adding more concentrated PEO phase causes osmotic shrinking of the cell-rich dextran drops, whose interfacial tension packs the cells into tissue spheroids. The latter are isolated by breaking the emulsion by dilution with culture media.

Figure 2

The average hydrodynamic diameter (A) and zeta-potential (B) for the produced WP particles at pH 5.8. (C) Average DEX droplet diameter for the DEX/PEO Pickering emulsion produced from WP/NaCl 300 mM solution at pH 5.8, 5.5 wt% PEO/5.5 wt% dextran for varying volume fractions of the Dextran phase (average of 200–300 individual drops). The data were obtained by optical microscopy measurements of the DEX droplets for each micrograph with Image J software. (Student’s t-Test, NS: Non-significant, *** p < 0.001).

Figure 3

Optical microscopy images of a DEX/PEO water-in-water Pickering emulsion (PEO 5.5 wt% and DEX 5.5 wt%). (A,B) ф_Dex = 0.2, (C,D) ф_Dex = 0.3, (E,F) ф_DEX = 0.4 stabilized by 2 wt% WP particles at pH 5.8. Scale bars are (A,C,E) 200 µm and (B,D,F) 100 µm.

Figure 4

(A,C) Optical bright field images and (B,D) fluorescence microscope images of single HaCaT cells after being treated with FDA live/dead assay. (E,G) Optical bright field images and (F,H) fluorescence microscope images (F,H) of HaCaT cell clusteroids treated with FDA. FDA treatment was done on after the cell clusteroids fabrication. The fluorescence indicates that both the HaCaT cells preserve their viability during the clusteroids fabrication process, as described in Figure 1. Scale bars are (A,B,E,F,G,H) 200 μm and (C,D) 100 μm.

Figure 5

Optical microscopy images of (A,C,E) HaCaT cell droplets (5.5 wt% PEO/5.5 wt% Dextran) and (B,D,F) HaCaT cell clusteroids (10 wt% PEO/5.5 wt% Dextran) stabilized by 2 wt% WP particles. Here the cell and DEX volume fraction were, ф_HaCaT = 0.15 and ф_DEX = 0.25, respectively. Scale bars are (A,B) 200 µm, (C,D) 100 µm and (E,F) 50 µm.

Figure 6

Average HaCaT spheroid diameter for emulsions 5.5 wt% PEO/5.5 wt% DEX or 10 wt% PEO/5.5 wt% DEX (A) ф_HaCaT = 0.25, ф_DEX = 0.2 and(B) ф_HaCaT = 0.15, ф_DEX = 0.25. The data were obtained by optical microscopy measurements of clusteroids for each micrograph with ImageJ Software (Student’s t-Test, NS: Non-significant, *** p < 0.001).

Figure 7

SEM images of a sample of HaCaT cell clusteroids after being removed from the medium, deposited on a glass substrate and freeze dried before imaging. (A–D) Images correspond to different resolutions. Note that the size of the clusters of cells is slightly lower than the original cell clusteroids due to shrinkage.

Figure 8

Bright field optical microscope images of HaCaT clusteroids isolated by a dilution of the DEX/PEO emulsion by a factor 3 with DMEM medium and incorporated with 0.75 wt% sodium alginate in DMEM media followed by cross-linking with 1M CaCl₂. The HaCaT cells clusteroids were cultured in the alginate film for seven days under DMEM media and images were taken from each well to determine the average clusteroids size. Scale bars are 200 μm (A) and 100 μm (B) images. Clusteroids average size evolution is summarized in Figure 9.

Figure 9

Evolution of the HaCaT clusteroids embedded in a hydrogel composed by different concentrations of sodium alginate and DMEM media for seven days. (A) The average spheroid size vs. time and (B) fractional area of the clusteroids in the alginate film vs. time. Measurements of the clusteroids were made every day with ImageJ software by taking the average of the vertical and horizontal diameter of 500 clusteroids in each well. The area fraction at 0.5 wt% and 0.75 wt% were both significantly different from the area fraction at 1% (p < 0.05).

Figure 10

(A) Digital photograph of the HaCaT clusteroids within an alginate hydrogel film over four days. The film is collected from the bottom of the well plate. (B–F) SEM images of a freeze-dried sample of the film prepared from alginate gel-cultured HaCaT spheroid composite cultured in DMEM media for four days. (C,F) represent a zoom in of the SEM images (B,E), respectively.