Tissue engineering (TE) for regenerative medicine represents a challenge for the biomedical field with a wide-range of applications, including repair, replacement and regeneration of damaged tissues and organs [1
]. A lack of available organs for transplants or tissue reconstruction slows down recovery of multiple patients. Moreover, major risks of these procedures include infection or rejection [2
]. TE usually needs three components: Cells, scaffolds and growth factors [3
]. Three-dimensional (3D) cell culture allows better results as the microenvironment is represented more accurately [4
]. Cells usually interact with each other in the extracellular matrix (ECM), a non-cellular scaffold, which plays an important role in multiple processes such as cell proliferation, differentiation or migration [5
] To mimic these conditions, different methods of 3D cell culture were developed: (i) Scaffold based: Polymeric hard scaffolds, biological scaffolds, micro patterned surface microplates and (ii) non-scaffold based: Microfluidic 3D cell culture, hanging-drop microplates, spheroids microplates containing ultra-low attachment (ULA) coating [7
] The two last techniques mentioned are used for the production of cell spheroids, along with suspension culture in bioreactor (spinner flask, microgravity bioreactor) and micro-patterned surfaces [8
] Cell spheroids are based on the self-assembling properties of cells, which leads to 3D cell aggregates [10
]. The low cost of these techniques, their ability to generate complex cellular structures and the accuracy of the cell microenvironment represent a promising method for 3D cell culture [8
] despite challenges in controlling their size and avoiding core necrosis [10
]. Fibroblast cell spheroids can be formed using cell clustering processes where they adhere to each other rather than to a substrate. This involves methods such as spinner culture, NASA rotary culture and non-adhesive surfaces [9
]. Size control of the individual spheroids was reported by using the hanging-drop culture technique [12
], 3D culturing in microwells [17
], micro-rotational flow [18
] and the magneto-Archimedes effect [19
]. Recently reported methods for the production of cellosomes [20
] can also serve as a possible route for the larger scale production of desired spheroids. Encapsulation of cells in such structures has also been done using natural biopolymers [22
]. Most of these methods are very limited by the amount of cell spheroids that can be produced. Nguyen et al. [23
] and Ganley et al. [24
] showed that water-in-water Pickering emulsions could be stabilized by solid particles. A more comprehensive review on stabilization of w/w Pickering emulsion with colloid particles was published by Dickinson [25
]. Singh et al. [26
] also reported encapsulation of viable microbial cells probiotic bacteria in carboxymethyl cellulose-gelatin water-in-water emulsions.
Aqueous two-phase systems (ATPS) have been previously used to create cancer spheroids [27
] primarily by dispensing dextran rich drops (DEX) with tumour cells into polyethylene glycol rich media (PEG). The dispersed tumour cells have been grown in these DEX drops to form individual spheroids, which were further studied for response to drug treatments [27
] As may be expected a higher resistance to chemotherapy agents was observed when spheroids created with this technique were compared to 2D culture [30
]. The techniques used previously produced effective tumour spheroids to study drug effects, however, the aim of those studies was to create large spheroids, which by their nature should have a necrotic core and proliferating cells on the spheroid surface. However, for tissue engineering (TE) applications, the need to dispense individual DEX drops with cells into the PEG phase practically limits the scalability of these methods in their scope to produce large quantities of cell spheroids [27
]. Recently, Das et al. [31
] reported a rapid and scalable production technique for tissue spheroids of human embryonic kidney cells (HEK 293) by trapping them in w/w emulsion droplets stabilized by protein particles. Their technique is based on w/w emulsion formation where the cells effectively distribute in the emulsion droplets, which are stabilised by protein particles. This method is based on using water-in-water droplets to encapsulate the cells, and then shrinking the droplets by changing the ATPS equilibrium, which rapidly drives the cells to form large clusters that are harvested by breaking down of the w/w emulsion.
In the current study, we aimed to form not true spheroids but dense clusters of cells (termed here as clusteroids) that could have downstream applications where preserving cell viability is required such as skin grafts. Human skin represents an important physical barrier protecting the internal environment from external attacks and regulating body homeostasis. Regenerative skin surgery requites a replacement of the epidermis, which consists mostly of keratinocytes, but also melanocytes and Langerhans cells [32
]. TE uses cells distributed in naturally derived biocompatible hydrogels as scaffolds for 3D culturing due to their ability to retain water, provide structured environment suitable for cell growth and proliferation [34
]. Various parameters are evaluated to provide the best conditions for cell culture, such as choice of polymer, concentration, molecular weight and chemical composition. Alginate is a polysaccharide composed of (1–4) β-d
-mannuronic acid (M) units and α-l
-glucuronic acid (G) units derived from brown algae and is used in the biomedical field for its biocompatibility, low toxicity, non-immunogenicity and low cost [35
]. Crosslinking of sodium alginate hydrogels allows a mild gelation with cations such as Ca2+
, which serve as ion junctions forming a 3D network by binding two COO−
groups. Calcium chloride is typically used as cross-linking agent, releasing Ca2+
ions in the hydrogels by a diffusion mechanism [36
Here, we developed further the method of Das et al. [31
] for the preparation of human keratinocyte cell clusters (clusteroids) using w/w Pickering emulsions as templates and explored their formulation in suitable hydrogel matrices for 3D tissue engineering of skin grafts. 3D cell cultures of human adult keratinocytes (HaCaT) cells were used for the production of spheroids [38
]. HaCaT cells are known as non-tumorigenic and have been successfully transplanted on nude mice resulting in an epidermal tissue layer close to normal keratinocytes [39
]. HaCaT cells can still express differentiation genes like keratin 1 and keratin 10, unlike keratinocyte cell lines virally transformed [39
]. This makes HaCaT cell clusteroids an excellent model system for exploring their growth in alginate hydrogels as a 3D cell culture model.
The schematics of our keratinocyte clusteroids fabrication method are briefly sketched in Figure 1
. A water-in-water Pickering emulsion was formed to encapsulate the cells by mixing two aqueous solutions containing incompatible polymers: Polyethylene oxide (PEO) solution as a continuous phase and dextran solution (DEX) as a dispersed phase. The DEX-in-PEO emulsions were stabilized using whey protein (WP) particles. We found that the keratinocyte cells prefer to accumulate in the DEX phase, which allowed their effective encapsulation in the DEX emulsion drops. In this environment, they were further “compressed” against each other osmotically with the addition of more concentrated PEO phase to the emulsion and adhered to their neighbouring cells in the emulsion drops to form cell clusteroids. The formulation and growth in alginate hydrogel films as model skin grafts is schematically shown in the Supporting Information, Figure S1
The paper is organized as follows. In the following section we discuss the materials and techniques used to control the size of the emulsion drop templates and the average size of the resulting keratinocyte clusteroids. The results section shows typical keratinocyte clusteroids produced by this technique and demonstrates that they consist of viable cells.
We also developed keratinocyte clusteroids composite formulation alginate gels and compare their growth with individual keratinocytes in the same scaffolding media. We found that HaCaT clusteroids can grow faster than the individual cells in alginate films. This methodology could be used as blueprint to enable growth of patients’ own skin cells and to fabricate autologous cell clusteroids for potential clinical applications.
2. Materials and Methods
Deionised water purified by reverse osmosis and ion exchange from a Milli-Q water system (Millipore) was used in all our studies. Its surface tension was 71.9 mN m−1 at 25 °C, with measured resistivity less than 18 mΩ cm−1. Dextran (MW 500 kDa) and PEO (MW 200 kDa) were both purchased from Sigma-Aldrich. Whey protein was sourced from (No1. Supplements, Suffolk, UK). Fluorescein diacetate (FDA, 98%) and sodium alginate and Corning® Transwell® polyester membrane cell culture inserts (12 mm, 12 well plates) were purchased from Sigma-Aldrich, UK. Sodium chloride (99.8%) and calcium chloride were purchased from Fisher Scientific. Dulbecco’s Modified Eagle Medium (DMEM) and Trypsin-EDTA were sourced from Gibco®, Fisher Scientific. NUNC Cell culture 24-well plates were purchased from Thermo Fisher Scientific.
2.2. 2D HaCaT Cell Culture
HaCaT cell line culture was kindly provided by the Skin Research Group at St James University Hospital at Leeds and was cultured from samples frozen in liquid nitrogen. The cells were cultured in high-glucose DMEM media supplemented with 10% foetal bovine serum (FBS, Labtech, Heathfield, UK) and 1% antibiotics (Penicillin Streptomycin, Lonza) and placed in an incubator (37 °C, 5% CO2). After reaching 80% confluence, HaCaT cells were carefully washed with phosphate buffer saline (PBS) for 10 s then incubated with 0.25% Trypsin-EDTA (1X, Lonza), which allowed us to detach the cells from their support after 5 min. Its action was neutralized by adding complete DMEM medium before a centrifugation at 400× g for 4 min. After resuspension in fresh medium, the HaCaT cells were reseeded in tissue culture flasks (Sarstedt). All cell waste was left for at least 30 min in 1% Virkon viricidal disinfectant (Fisher Scientific) before its disposal.
2.3. Preparation of the Whey Protein (WP) Particles
Whey protein powder was dissolved in water at a concentration of 2 wt% for 2 h under agitation. The solution was placed at 4 °C for 12 h to hydrate the whey protein. Then, the solution was centrifuged at 10,800× g for 1 h and the supernatant was collected. A solution of 300 mM NaCl was prepared and mixed with an equal volume of WP solution. The pH was adjusted to 5.8 by drop-wise addition of filtered 0.5 M HCl aqueous solution. After heating the WP/NaCl solution in an oil bath at 85 °C for 15 min, it was left to cool at 4 °C. This precipitation process produced WP particles, which were used in our protocol as stabilizers for the water-in-water emulsions; these emulsions were used as a template for the encapsulation of the keratinocyte cells into clusteroids.
2.4. Production of w/w Pickering Emulsions, Cell Encapsulation and Clusteroid Isolation
PEO aqueous solution (5.5 wt%) was prepared by dissolving PEO into the heat-treated solution of WP, which constituted the continuous phase of the water-in-water emulsion. A centrifugation of the PEO solution was done beforehand at 5000× g for 7 min to remove the silica nanoparticles from the PEO solution, which were added by the manufacturer. A solution of 5.5 wt% dextran (in total) in high-glucose DMEM complete medium under sterile conditions was used as a disperse phase (DEX phase) together with the keratinocytes. The DEX phase with the keratinocytes formed typically a total volume fraction ф = 0.0909 with respect to the DEX/PEO w/w emulsion. To form the latter, the DEX phase (plus the cells) were transferred to the WP/NaCl/PEO solution and gently homogenized using two pumps with a BD Microlance™ 3 needle (21G ½, internal diameter 0.512 mm) and a BD Plastipak™ syringe of 5 mL by two pumps (Becton Dickinson, Wokingham, UK). The emulsion was prepared using PEO phase of volume fraction ф = 0.9181 and DEX phase (with the cells) of volume fraction (ф = 0.0909). This resulted in the cells encapsulation in the emulsion drops due to a higher affinity of the HaCaT cells to the DEX phase compared to the PEO phase. A more concentrated PEO solution was added to the emulsion (to adjust the final PEO concentration to a total of 8 wt%), which allowed the DEX droplets to shrink osmotically and obtain densely packed encapsulated HaCaT cells. The cell–cell adhesion in these structures resulted in the formation of cell clusteroids in approximately 15 min. To break the water-in-water emulsion and release the produced clusteroids, the solution was further diluted with high-glucose DMEM medium by a factor of 10. The clusteroids were left to sediment down by gravity for 15 min. After discarding the supernatant, clusteroids were carefully re-suspended in a fresh DMEM medium.
2.5. Cell Viability Assay
Cell clusteroids were treated with a 5 g L−1
solution of fluorescein diacetate in acetone (10 μL per 1 mL of the dispersion of re-suspended clusteroids; Sigma-Aldrich) to evaluate cell viability. After 10 min of incubation at room temperature in the dark, the sample was observed under an Olympus BX-51 fluorescence microscope (Olympus) with a DP70 digital camera and FITC fluorescence filter set. Fluorescein diacetate (FDA) is known to diffuse through cell membranes, only viable cells are able to hydrolyse non-fluorescent FDA internally to fluorescein by intracellular esterase activity. Since fluorescein dissociates in water, it’s crossing of the cell membranes is hindered by its charge, which results in the accumulation of green fluorescent fluorescein inside intact cells [25
]. This indicates that the cell membranes are intact, and the cells are viable.
2.6. 3D Keratinocyte Clusteroids Culture
A solution of 1.5 wt% sodium alginate was prepared by dissolution in water and then sterilized in an autoclave. DMEM medium was mixed with the sodium alginate 1.5 wt% solutions at different volumes to vary the gelling agent concentration. HaCaT cell clusteroids were then carefully resuspended in this solution and seeded on 24-well tissue culture plates (Sarstedt). Addition of 2 wt% CaCl2 (aq) allowed crosslinking of the alginate chains in the media and formation of the hydrogel. After incubation, the CaCl2 solution was carefully pipetted out without compromising the integrity of the hydrogel-clusteroids composite and the wells were topped up with DMEM medium.
2.7. Fabrication of HaCaT Cell Clusteroids
A sample of 0.3602 g wet weight (approx. 5.5 × 106 cells) of the HaCaT cells was produced by centrifugation at 400× g for 4 min from the culture medium and re-suspended the cells in 540 μL of a solution of DMEM + Dextran 5.5 wt% (DEX dispersed phase). The cell volume fraction in the DEX phase was 0.25, i.e., approximately a quarter of the DEX emulsion drops volume was occupied by cells. Then, we added 1.295 mL of a WP solution to 2%/NaCl 300 mM + PEO 5.5 wt% (PEO continuous phase). The PEO concentration in the emulsion continuous phase was further increased (by adding 1.765 mL 10 wt% PEO in DMEM media) to shrink the DEX droplets with the cells and obtain HaCaT cell clusteroids. After 30 min, the emulsion was broken by 10-fold dilution with DMEM media to release the obtained clusteroids from the DEX drops. This procedure was used to isolate the HaCaT clusteroids prior to their imaging and viability characterisation in DMEM media.
2.8. Preparation of Model HaCaT Clusteroids in Alginate Hydrogels Formulations
A stock solution of 1.5 wt% sodium alginate was prepared by dissolution in water and then sterilized in an autoclave. A 0.5 wt% sodium alginate solution was prepared by mixing DMEM medium with the stock solution of 1.5 wt% sodium alginate solution. A 24-well plate was used to seed HaCaT clusteroids in sodium alginate hydrogels. HaCaT cells were resuspended in a solution 5.5 wt% Dextran in DMEM media (dispersed phase). 5.5 wt% PEO was added as a continuous phase and the emulsion was homogenized with a BD Microlance 3 needle (21G ½) and a BD Plastipak 5 mL syringe (needle internal diameter 0.512 mm) by two pumps. A more concentrated solution of PEO was used to shrink the cells in order to obtain good quality clusteroids. The sample was left to sediment over two hours after the emulsion was broken down by dilution with DMEM media. In the final formulation, the concentrations of sodium alginate and HaCaT cell clusteroids were 0.5 wt% and 0.12 g/mL, respectively. The composite alginate-HaCaT clusteroids formulations were seeded in the wells with a total volume of 100 µL. To crosslink the hydrogel matrix around the clusteroids, 100 µL of 2 wt% CaCl2 was added with a pipette on top of the alginate layer and was incubated for two hours in order to fortify the alginate hydrogel, after which the CaCl2 solution was carefully discarded without damaging the hydrogel. 1.00 mL of 90 vol% high-glucose DMEM medium supplemented with 10 vol% FBS and antibiotics was added in each well plate and the culturing was done at 37 °C over the course of seven days. The growth media was changed every two days. The growth of the clusteroids in the alginate film was monitored every day by taking multiple images by optical microscope in each well and the average size calculated after sizing a minimum 500 clusteroids from each well both in x-and y-direction.
2.9. SEM Imaging of HaCaT Clusteroids
Scanning electron microscope (SEM) images of the clusteroids were taken in order to reveal their morphology. Samples were prepared for SEM images after fixing the cell clusteroids to avoid them to break apart during the evaporation of the aqueous solution. Of the HaCaT cell clusteroids in media, 1 mL was deposited on dry Aclar™ sheets (Agar Scientific Ltd., Essex, UK) or poly-lysine coated glass coverslips and treated with 2 wt% glutaraldehyde for 2 h. This was followed by washing with cacodylate buffer, rinsing with serial ethanol-water solutions of increasing ethanol concentration, starting from 50% ethanol and moving up to washing with an absolute ethanol and then drying by using a critical point dryer. In the case of clusteroids within an alginate film the 5 mm × 5 mm sample was washed with deionised water and deposited on the SEM stub and freeze dried at critical point temperature. The samples were imaged using a scanning electron microscope SEM (ZEISS EVO 60 EP-SEM).
2.10. Statistical Analysis
Comparisons were made using unpaired t-tests or analysis of variance as appropriate with significance set at p ≤ 0.05.
We developed a new technique for the high throughput preparation of keratinocyte clusteroids with the aim of producing 3D tissues. HaCaT cells, a spontaneous immortalized human keratinocyte cell line, was used with the intention of growing skin, as keratinocyte are a major component of the epidermis [32
]. This method is based on cell encapsulation within w/w Pickering emulsion droplets of an aqueous solution of Dextran in aqueous PEO solution stabilized by WP particles [31
Our results showed that gentle homogenization with a syringe by two pumps was enough to avoid cell clumps and limit spheroid disintegration by shear stress. Increasing the HaCaT volume fraction to 0.2 and PEO final concentration to 10 wt% resulted in formation of more compact clusteroids, as shrinking of DEX droplets by osmotic pressure was more efficient. These droplets were about 2.5 times larger than the control with фDEX = 0.1819. This difference was statistically significant, as it was for the results with фDEX = 0.4. However, the average DEX droplet size was lower, 85 ± 20.8 µm. A DEX phase volume fraction of фDEX = 0.3 was selected for the fabrication of large HaCaT clusteroids by templating DEX/PEO Pickering emulsions.
After production of alginate films from keratinocyte clusteroids, various experiments could be elaborated to further control their proliferation and potential use as autologous skin grafts. A major issue with large spheroids is hypoxia, due to a decreased diffusion of oxygen and nutrients resulting in a necrotic core [4
]. Hypoxia inducible factor (HIF)-1a, a survival factor among others, is likely to be activated in response to this lack of oxygen. Activation of these factors could be investigated in further experiments [44
]. Studies have previously demonstrated that 3D skin models derived from HaCaT cells were lacking a stratum corneum and some abnormalities in protein expressions were detected like filaggrin, loricrin and involucrin [38
]. Our method for fabrication of keratinocyte clusteroids could be adapted to primary cells directly derived from patients to produce autologous skin grafts.
The air–liquid interface (ALI) is a technique used to develop artificial skin. It consists of exposing keratinocytes to an ALI in order to induce their differentiation [45
]. The quality of the artificial epidermis could be evaluated by detecting the expression of various specific keratins such as K5 and K14 for basal keratinocytes or K1, K10 and involucrin for late differentiation markers [46
]. In case of deep wounds, a dermal substitute is used as a scaffold before applying and epidermal cover [32
]. Bio-inks based on viable keratinocyte clusteroids could also potentially allow them to be bio-printed into customized 3D skin grafts where they can proliferate and grow more efficiently than single cells [47
The in vitro culture of keratinocytes and fibroblasts for use in autologous grafts can take up to 21 days, during which time the patient must be cared for [48
] although there was no relationship between admission and time to resection of tissue/application of grafts with hospital length of stay. The majority of graft procedures were carried out over 14 days after admission thus allowing enough time for in vitro cultured cells to potentially be available. It has also been noted that patients treated with neonatal fibroblasts for wound infection had a higher patient satisfaction and other parameters such as infection and graft take rates were comparable with allograft [49