Efficient Generation of Skin Organoids from Pluripotent Cells via Defined Extracellular Matrix Cues and Morphogen Gradients in a Spindle-Shaped Microfluidic Device.

Pluripotent stem cell-derived skin organoids (PSOs) emerge as a developmental skin model that is self-organized into multiple components, such as hair follicles. Despite their impressive complexity, PSOs are currently generated in the absence of 3D extracellular matrix (ECM) signals and have several major limitations, including an inverted anatomy (e.g., epidermis inside/dermis outside). In this work, a method is established to generate PSOs effectively in a chemically-defined 3D ECM environment. After examining various dermal ECM molecules, it is found that PSOs generated in collagen -type I (COLI) supplemented with laminin 511 (LAM511) exhibit increased growth compared to conventional free-floating conditions, but fail to induce complete skin differentiation due in part to necrosis. This problem is addressed by generating the PSOs in a 3D bioprinted spindle-shaped hydrogel device, which constrains organoid growth longitudinally. This culture system significantly reduces organoid necrosis and leads to a twofold increase in keratinocyte differentiation and an eightfold increase in hair follicle formation. Finally, the system is adapted as a microfluidic device to create asymmetrical gradients of differentiation factors and improves the spatial organization of dermal and epidermal cells. This study highlights the pivotal role of ECM and morphogen gradients in promoting and spatially-controlling skin differentiation in the PSO framework.

In vitro induction of hair follicle signatures using human dermal papilla cells encapsulated in fibrin microgels.

Cellular spheroids have been described as an appropriate culture system to restore human follicle dermal papilla cells (hFDPc) intrinsic properties; however, they show a low and variable efficiency to promote complete hair follicle formation in in vivo experiments. In this work, a conscientious analysis revealed a 25% cell viability in the surface of the dermal papilla spheroid (DPS) for all culture conditions, questioning whether it is an appropriate culture system for hFDPc. To overcome this problem, we propose the use of human blood plasma for the generation of fibrin microgels (FM) with encapsulated hFDPc to restore its inductive signature, either in the presence or in the absence of blood platelets. FM showed a morphology and extracellular matrix composition similar to the native dermal papilla, including Versican and Collagen IV and increasing cell viability up to 85%. While both systems induce epidermal invaginations expressing hair-specific keratins K14, K15, K71, and K75 in in vitro skin cultures, the number of generated structures increases from 17% to 49% when DPS and FM were used, respectively. These data show the potential of our experimental setting for in vitro hair follicle neogenesis with wild adult hFDPc using FM, being a crucial step in the pursuit of human hair follicle regeneration therapies.

Single Plane Illumination Microscopy for Microfluidic Device Imaging.

Three-dimensional imaging of live processes at a cellular level is a challenging task. It requires high-speed acquisition capabilities, low phototoxicity, and low mechanical disturbances. Three-dimensional imaging in microfluidic devices poses additional challenges as a deep penetration of the light source is required, along with a stationary setting, so the flows are not perturbed. Different types of fluorescence microscopy techniques have been used to address these limitations; particularly, confocal microscopy and light sheet fluorescence microscopy (LSFM). This manuscript proposes a novel architecture of a type of LSFM, single-plane illumination microscopy (SPIM). This custom-made microscope includes two mirror galvanometers to scan the sample vertically and reduce shadowing artifacts while avoiding unnecessary movement. In addition, two electro-tunable lenses fine-tune the focus position and reduce the scattering caused by the microfluidic devices. The microscope has been fully set up and characterized, achieving a resolution of 1.50 µm in the x-y plane and 7.93 µm in the z-direction. The proposed architecture has risen to the challenges posed when imaging microfluidic devices and live processes, as it can successfully acquire 3D volumetric images together with time-lapse recordings, and it is thus a suitable microscopic technique for live tracking miniaturized tissue and disease models.

Evaluation of different methodologies for primary human dermal fibroblast spheroid formation: automation through 3D bioprinting technology.

Cell spheroids have recently emerged as an effective tool to recapitulate native microenvironments of living organisms in anin vitroscenario, increasing the reliability of the results obtained and broadening their applications in regenerative medicine, cancer research, disease modeling and drug screening. In this study the generation of spheroids containing primary human dermal fibroblasts was approached using the two-widely employed methods: hanging-drop and U-shape low adhesion plate (LA-plate). Moreover, extrusion-based three-dimensional (3D) bioprinting was introduced to achieve a standardized and scalable production of cell spheroids, decreasing considerably the possibilities of human error. This was ensured when U-shape LA-plates were used, showing an 85% formation efficiency, increasing up to a 98% when it was automatized using the 3D bioprinting technologies. However, sedimentation effect within the cartridge led to a reduction of 20% in size of the spheroid during the printing process. Hyaluronic acid (HA) was chosen as viscosity enhancer to supplement the bioink and overcome cell sedimentation within the cartridge due to the high viability values exhibited by the cells-around 80%-at the used conditions. Finally, (ANCOVA) of spheroid size over time for different printing conditions stand out HA 0.4% (w/v) 60 kDa as the viscosity-improved bioink that exhibit the highest cell viability and spheroid formation percentages. Besides, not only did it ensure cell spheroid homogeneity over time, reducing cell sedimentation effects, but also wider spheroid diameters over time with less variability, outperforming significantly manual loading.

Effect of Fibrin Concentration on the In Vitro Production of Dermo-Epidermal Equivalents.

Human plasma-derived bilayered skin substitutes were successfully used by our group to produce human-based in vitro skin models for toxicity, cosmetic, and pharmaceutical testing. However, mechanical weakness, which causes the plasma-derived fibrin matrices to contract significantly, led us to attempt to improve their stability. In this work, we studied whether an increase in fibrin concentration from 1.2 to 2.4 mg/mL (which is the useful fibrinogen concentration range that can be obtained from plasma) improves the matrix and, hence, the performance of the in vitro skin cultures. The results show that this increase in fibrin concentration indeed affected the mechanical properties by doubling the elastic moduli and the maximum load. A structural analysis indicated a decreased porosity for the 2.4 mg/mL hydrogels, which can help explain this mechanical behavior. The contraction was clearly reduced for the 2.4 mg/mL matrices, which also allowed for the growth and proliferation of primary fibroblasts and keratinocytes, although at a somewhat reduced rate compared to the 1.2 mg/mL gels. Finally, both concentrations of fibrin gave rise to organotypic skin cultures with a fully differentiated epidermis, although their lifespans were longer (25-35%) in cultures with more concentrated matrices, which improves their usefulness. These systems will allow the generation of much better in vitro skin models for the testing of drugs, cosmetics and chemicals, or even to "personalized" skin for the diagnosis or determination of the most effective treatment possible.

Lidocaine-Loaded Solid Lipid Microparticles (SLMPs) Produced from Gas-Saturated Solutions for Wound Applications.

The delivery of bioactive agents using active wound dressings for the management of pain and infections offers improved performances in the treatment of wound complications. In this work, solid lipid microparticles (SLMPs) loaded with lidocaine hydrochloride (LID) were processed and the formulation was evaluated regarding its ability to deliver the drug at the wound site and through the skin barrier. The SLMPs of glyceryl monostearate (GMS) were prepared with different LID contents (0, 1, 2, 4, and 10 wt.%) using the solvent-free and one-step PGSS (Particles from Gas-Saturated Solutions) technique. PGSS exploits the use of supercritical CO2 (scCO2) as a plasticizer for lipids and as pressurizing agent for the atomization of particles. The SLMPs were characterized in terms of shape, size, and morphology (SEM), physicochemical properties (ATR-IR, XRD), and drug content and release behavior. An in vitro test for the evaluation of the influence of the wound environment on the LID release rate from SLMPs was studied using different bioengineered human skin substitutes obtained by 3D-bioprinting. Finally, the antimicrobial activity of the SLMPs was evaluated against three relevant bacteria in wound infections (Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa). SLMPs processed with 10 wt.% of LID showed a remarkable performance to provide effective doses for pain relief and preventive infection effects.

Bioprinting for Skin.

We describe an extrusion-based method to print a human bilayered skin using bioinks containing human plasma and primary human fibroblasts and keratinocytes from skin biopsies. We generate 100 cm2 of printed skin in less than 35 min. We analyze its structure using histological and immunohistochemical methods, both in in vitro 3D cultures and upon transplantation to immunodeficient mice. We have demonstrated that the printed skin is similar to normal human skin and indistinguishable from bilayered dermo-epidermal equivalents, previously produced manually in our laboratory and successfully used in the clinic.