Aspectos biotecnológicos: Aplicaciones preclínicas y clínicas de piel generada a partir de células madre epidérmicas

A la vista de la rapidez con que se avanza en el esclarecimiento de las propiedades de las células madre, tanto embrionarias como adultas, y en su manejo, es de prever que, en un plazo no excesivamente largo, se llegue a generar a partir de ellas terapias innovadoras para enferme- dades que en el momento presente no tienen tratamiento. Junto al sis- tema hematopoyético, la epidermis es uno de los tejidos cuyas células madre se han caracterizado mejor en los últimos años, tanto in vitro como in vivo. Este conocimiento ha dado lugar a aplicaciones clínicas tanto de Terapia Celular como de Terapia Génica. En este capítulo se pretende examinar la situación en este campo basándoscia que el autor y sus colaboradores tienen en él

Real-Time Impedance Monitoring of Epithelial Cultures with Inkjet-Printed Interdigitated-Electrode Sensors

From electronic devices to large-area electronics, from individual cells to skin substitutes, printing techniques are providing compelling applications in wide-ranging fields. Research has thus fueled the vision of a hybrid, printing platform to fabricate sensors/electronics and living engineered tissues simultaneously. Following this interest, we have fabricated interdigitated-electrode sensors (IDEs) by inkjet printing to monitor epithelial cell cultures. We have fabricated IDEs using flexible substrates with silver nanoparticles as a conductive element and SU-8 as the passivation layer. Our sensors are cytocompatible, have a topography that simulates microgrooves of 300 µm width and ~4 µm depth, and can be reused for cellular studies without detrimental in the electrical performance. To test the inkjet-printed sensors and demonstrate their potential use for monitoring laboratory-growth skin tissues, we have developed a real-time system and monitored label-free proliferation, migration, and detachment of keratinocytes by impedance spectroscopy. We have found that variations in the impedance correlate linearly to cell densities initially seeded and that the main component influencing the total impedance is the isolated effect of the cell membranes. Results obtained show that impedance can track cellular migration over the surface of the sensors, exhibiting a linear relationship with the standard method of image processing. Our results provide a useful approach for non-destructive in-situ monitoring of processes related to both in vitro epidermal models and wound healing with low-cost ink-jetted sensors. This type of flexible sensor as well as the impedance method are promising for the envisioned hybrid technology of 3D-bioprinted smart skin substitutes with built-in electronics.The work by D.M.-M. has been performed in the frame of an FPU Program, FPU015/06208, and a Mobility Fellows Program, both granted by the Spanish Ministry of Education, Culture and Sports. This work has been funded by the Comunidad de Madrid under the grant BIOPIELTEC-CM (P2018/BAA-4480) and the Ministerio de Ciencia e Innovación under the grant PARAQUA (TEC2017-86271-R)

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

This article belongs to the Special Issue Advanced Biomaterials for Wound Healing 2021.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.This research was funded by Programa de Actividades de I+D entre Grupos de Investigación de la Comunidad de Madrid, S2018/BAA-4480, Biopieltec-CM; by Programa Estatal de I+D+i Orientada a los Retos de la Sociedad, RTI2018-101627-B-I00; by Programa de Apoyo a la Realización de Proyectos Interdisciplinares de I+D para Jóvenes Investigadores de la Universidad Carlos III de Madrid (project: BIOMASKIN); and by Cátedra Fundación Ramón Areces

A new microfluidic method enabling the generation of multi-layered tissues-on-chips using skin cells as a proof of concept

Microfluidic-based tissues-on-chips (TOCs) have thus far been restricted to modelling simple epithelia as a single cell layer, but likely due to technical difficulties, no TOCs have been reported to include both an epithelial and a stromal component despite the biological importance of the stroma for the structure and function of human tissues. We present, for the first time, a novel approach to generate 3D multilayer tissue models in microfluidic platforms. As a proof of concept, we modelled skin, including a dermal and an epidermal compartment. To accomplish this, we developed a parallel flow method enabling the deposition of bilayer tissue in the upper chamber, which was subsequently maintained under dynamic nutrient flow conditions through the lower chamber, mimicking the function of a blood vessel. We also designed and built an inexpensive, easy-to-implement, versatile, and robust vinyl-based device that overcomes some of the drawbacks present in PDMS-based chips. Preliminary tests indicate that this biochip will allow the development and maintenance of multilayer tissues, which opens the possibility of better modelling of the complex cell–cell and cell–matrix interactions that exist in and between the epithelium and mesenchyme, allowing for better-grounded tissue modelling and drug screening.This work was supported by the "Programa de Actividades de I+D entre Grupos de Investigación de la Comunidad de Madrid" project S2018/BAA-4480, Biopieltec-CM and the Cátedra Fundación Ramón Areces

Technological advances in fibrin for tissue engineering

Fibrin is a promising natural polymer that is widely used for diverse applications, such as hemostatic glue, carrier for drug and cell delivery, and matrix for tissue engineering. Despite the significant advances in the use of fibrin for bioengineering and biomedical applications, some of its characteristics must be improved for suitability for general use. For example, fibrin hydrogels tend to shrink and degrade quickly after polymerization, particularly when they contain embedded cells. In addition, their poor mechanical properties and batch-to-batch variability affect their handling, long-term stability, standardization, and reliability. One of the most widely used approaches to improve their properties has been modification of the structure and composition of fibrin hydrogels. In this review, recent advances in composite fibrin scaffolds, chemically modified fibrin hydrogels, interpenetrated polymer network (IPN) hydrogels composed of fibrin and other synthetic or natural polymers are critically reviewed, focusing on their use for tissue engineering.The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This research was supported by Programa de Actividades de I + D entre Grupos de Investigación de la Comunidad de Madrid, S2018/BAA-4480, Biopieltec-CM, Programa Estatal de I + D + i Orientada a los Retos de la Sociedad, RTI2018-101627-B-I00, Proyectos de Generación de Conocimiento 2021, PID2021-126523OB-I00, Proyectos en colaboración público-privada 2021, CPP2021-008396, LOLICOMB Project, PID2020-116439GB-I00 and Cátedra Fundación Ramón Areces. Grant PID2021-126523OB-I00 funded by MCIN/AEI/10.13039/501100011033 and, as appropriate, by “ERDF A way of making Europe.” Grant CPP2021-008396 funded by MCIN/AEI/ 10.13039/501100011033 and by the European Union “NextGenerationEU/PRTR.”Publicad

3D bioprinting of functional human skin: production and in vivo analysis

Significant progress has been made over the past 25 years in the development of in vitro-engineered substitutes that mimic human skin, either to be used as grafts for the replacement of lost skin, or for the establishment of in vitro human skin models. In this sense, laboratory-grown skin substitutes containing dermal and epidermal components offer a promising approach to skin engineering. In particular, a human plasma-based bilayered skin generated by our group, has been applied successfully to treat burns as well as traumatic and surgical wounds in a large number of patients in Spain. There are some aspects requiring improvements in the production process of this skin; for example, the relatively long time (three weeks) needed to produce the surface required to cover an extensive burn or a large wound, and the necessity to automatize and standardize a process currently performed manually.This work was partially supported by grant DPI2014-61887-EXP from the Spanish Ministerio de Economía y Competitividad

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 an in vitro scenario, 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.We kindly thank Daniel García for their guidance with the rheological experiments. This work was supported by Programa de Actividades de I + D entre Grupos de Investigación de la Comunidad de Madrid, S2018/ BAA-4480, Biopieltec-CM, Programa Estatal de I + D + i Orientada a los Retos de la Sociedad, RTI2018-101627-B-I00 and Cátedra Fundación Ramón Areces. The experimental techniques used during this study were performed in the CleanRooms of Bioengineering, Universidad Carlos III de Madrid, Madrid, Spain

Skin gene therapy for acquired and inherited disorders

The rapid advances associated with the Human Genome Project combined with the development of proteomics technology set the bases to face the challenge of human gene therapy. Different strategies must be evaluated based on the genetic defect to be corrected. Therefore, the re-expression of the normal counterpart should be sufficient to reverse phenotype in single-gene inherited disorders. A growing number of candidate diseases are being evaluated since the ADA deficiency was selected for the first approved human gene therapy trial (Blaese et al., 1995). To cite some of them: sickle cell anemia, hemophilia, inherited immune deficiencies, hyper-cholesterolemia and cystic fibrosis. The approach does not seem to be so straightforward when a polygenic disorder is going to be treated. Many human traits like diabetes, hypertension, inflammatory diseases and cancer, appear to be due to the combined action of several genes and environment. For instance, several wizard gene therapy strategies have recently been proposed for cancer treatment, including the stimulation of the immune system of the patient (Xue et al., 2005), the targeting of particular signalling pathways to selectively kill cancer cells (Westphal and Melchner, 2002) and the modulation of the interactions with the stroma and the vasculature (Liotta, 2001; Liotta and Kohn, 2001).Our work is supported by grants SAF-2004-07717 from Ministerio de Ciencia y Tecnología (Spain) and LSHG-512073 from UE to M. Del Rio, LSHG-503447 from UE to J.L. Jorcano and LSHG-512102 from UE to F. Larcher. We express our gratitude to Dr. Y. Gache, Dr. F. Spirito and Dr. G. Meneguzzi for providing EM pictures to illustrate this work

Hyaluronic acid-fibrin hydrogels show improved mechanical stability in dermo-epidermal skin substitutes

Human plasma-derived bilayered skin substitutes have been successfully used by our group in different skin tissue engineering applications. However, several issues associated with their poor mechanical properties were observed, and they often resulted in rapid contraction and degradation. In this sense, hydrogels composed of plasma-derived fibrin and thiolated-hyaluronic acid (HA-SH, 0.05–0.2% w/v) crosslinked with poly(ethylene glycol) diacrylate (PEGDA, 2:1, 6:1, 10:1 and 14:1 mol of thiol to moles of acrylate) were developed to reduce the shrinking rates and enhance the mechanical properties of the plasma-derived matrices. Plasma/HA-SH-PEGDA hydrogels showed a decrease in the contraction behaviour ranging from 5% to 25% and an increase in Young's modulus. Furthermore, the results showed that a minimal amount of the added HA-SH was able to escape the plasma/HA-SH-PEGDA hydrogels after incubation in PBS. The results showed that the increase in rigidity of the matrices as well as the absence of adhesion cellular moieties in the second network of HA-SH/PEGDA, resulted in a decrease in contraction in the presence of the encapsulated primary human fibroblasts (hFBs), which may have been related to an overall decrease in proliferation of hFBs found for all hydrogels after 7 days with respect to the plasma control. The metabolic activity of hFB returned to the control levels at 14 days except for the 2:1 PEGDA crosslinking ratio. The metabolic activity of primary human keratinocytes (hKCs) seeded on the hydrogels showed a decrease when high amounts of HA-SH and PEGDA crosslinker were incorporated. Organotypic skins formed in vitro after 21 days with plasma/HA-SH-PEGDA hydrogels with an HA content of 0.05% w/v and a 2:1 crosslinking ratio were up to three times thicker than the plasma controls, evidencing a reduction in contraction, while they also showed better and more homogeneous keratin 10 (K10) expression in the supra-basal layer of the epidermis. Furthermore, filaggrin expression showed the formation of an enhanced stratum corneum for the constructs containing HA. These promising results indicate the potential of using these biomimetic hydrogels as in vitro skin models for pharmaceutical products and cosmetics and future work will elucidate their potential functionality for clinical treatment.We kindly thank Rebeca Hernández for their guidance with the rheological experiments and Cristina Moral for her technical assistance with the SEM. This work was supported by Programa de Actividades de I+D entre Grupos de Investigación de la Comunidad de Madrid, S2018/BAA-4480, Biopieltec-CM, Programa Estatal de I+D+i Orientada a los Retos de la Sociedad, RTI2018-101627-B-I00, Madrid Government (Comunidad de Madrid) under the Multiannual Agreement with UC3M in the line of "Fostering Young Doctors Research" (BIOMASKIN-CM-UC3M) and in the context of the V PRICIT (Regional Programme of Research and Technological Innovation) and Cátedra Fundación Ramón Areces.Publicad

Skin-on-a-chip models: General overview and future perspectives-ERRATUM

Original article: Skin-on-a-chip models: General overview and future perspectives. In APL Bioengineering (Vol. 5, Issue 3, p. 030901). AIP Publishing. https://doi.org/10.1063/5.0046376This work was supported by the Programa de Actividades de I+D entre Grupos de Investigación de la Comunidad de Madrid, S2018/BAA-4480, Biopieltec-CM, and Cátedra Fundación Ramón Areces