The Human Neonatal Skin Fibroblast, an Available Cell Source for Tissue Production and Transplantation, Exhibits Low Risk of Immunogenicity In Vitro.

The immunogenicity of allogeneic skin fibroblasts in transplantation has been controversial. Whether this controversy comes from a natural heterogeneity among fibroblast subsets or species-specific differences between human and mouse remains to be addressed. In this study, we sought to investigate whether fibroblasts derived from either adult or neonatal human skin tissues could induce different immune responses toward phagocytosis and T cell activation using in vitro co-culture models. Our results indicate that both phagocytosis and T cell proliferation are reduced in the presence of neonatal skin fibroblasts compared to adult skin fibroblasts. We also show that neonatal skin fibroblasts secrete paracrine factors that are responsible for reduced T cell proliferation. In addition, we show that neonatal skin fibroblasts express less class II human leukocyte antigen (HLA) molecules than adult skin fibroblasts after interferon gamma priming, which might also contribute to reduced T cell proliferation. In conclusion, this study supports the use of allogeneic neonatal skin fibroblasts as a readily available cell source for tissue production and transplantation to treat patients with severe injuries.

Cancer Spheroids Embedded in Tissue-Engineered Skin Substitutes: A New Method to Study Tumorigenicity In Vivo.

Tumorigenic assays are used during a clinical translation to detect the transformation potential of cell-based therapies. One of these in vivo assays is based on the separate injection of each cell type to be used in the clinical trial. However, the injection method requires many animals and several months to obtain useful results. In previous studies, we showed the potential of tissue-engineered skin substitutes (TESs) as a model for normal skin in which cancer cells can be included in vitro. Herein, we showed a new method to study tumorigenicity, using cancer spheroids that were embedded in TESs (cTES) and grafted onto athymic mice, and compared it with the commonly used cell injection assay. Tumors developed in both models, cancer cell injection and cTES grafting, but metastases were not detected at the time of sacrifice. Interestingly, the rate of tumor development was faster in cTESs than with the injection method. In conclusion, grafting TESs is a sensitive method to detect tumor cell growth with and could be developed as an alternative test for tumorigenicity.

Comparison of Two Human Skin Cell Isolation Protocols and Their Influence on Keratinocyte and Fibroblast Culture.

For the development of advanced therapies, the use of primary cells instead of cell lines is preferred. The manufacture of human tissue-engineered skin substitutes requires efficient isolation and culture protocols allowing a massive expansion of the cells in culture from an initial specimen of a minimal size. This study compared two skin cell isolation protocols, routinely applied in two clinical laboratories. Epithelial (keratinocytes) and dermal (fibroblasts) cells were isolated and cultured from three human skin biopsies (N = 3). The two-step digestion protocol (LOEX-Protocol) firstly used thermolysin to enzymatically disrupt the dermal-epidermal junction while, for the one-step digestion protocol (UPCIT-Protocol), mechanical detachment with scissors was applied. Then, the epidermal and dermal layers were digested, respectively, to achieve cell isolation. The cell size, viability, yield and growth were analyzed over five passages (P). The colony-forming efficiency (CFE) and Keratin 19 (K19) expression of epithelial cells were also assessed after P0 and P1. Regarding the dermal cells, no significant differences were observed in the tested parameters of isolation and culture. However, for the epithelial cells, viability was higher (93% vs. 85%) and the number of cells extracted per cm2 of skin was 3.4 times higher using the LOEX-Protocol compared to the UPCIT-Protocol. No significant difference was observed for any parameter once the keratinocytes were cultured from P1 to P4. The CFE and K19 expression decreased from P0 to P1 in both protocols, probably due to the culture process. This study shows that both protocols enable the efficient isolation of skin dermal and epithelial cells and subsequent culture to produce grafts destined for the treatment of patients.

Production of Tissue-Engineered Skin Substitutes for Clinical Applications: Elimination of Serum.

Tissue-engineered skin substitutes (TESs) are used as a treatment for severe burn injuries. Their production requires culturing both keratinocytes and fibroblasts. The methods to grow these cells have evolved over the years, but bovine serum is still commonly used in the culture medium. Because of the drawbacks associated with the use of serum, it would be advantageous to use serum-free media for the production of TESs. In a previous study, we developed a serum-free medium (Surge SFM) for the culture of keratinocytes. Herein, we tested the use of this medium, together with a commercially available serum-free medium for fibroblasts (Prime XV), to produce serum-free TESs. Our results show that serum-free TESs are macroscopically and histologically similar to skin substitutes produced with conventional serum-containing media. TESs produced with either culture media expressed keratin 14, Ki-67, transglutaminase 1, filaggrin, type I and IV collagen, and fibronectin comparably. Mechanical properties, such as contraction and tensile strength, were comparable between TESs cultured with and without serum. Serum-free TESs were also successfully grafted onto athymic mice for a six-month period. In conclusion, Surge SFM and Prime XV serum-free media could be used to produce high quality clinical-grade skin substitutes.

A Newly Developed Chemically Defined Serum-Free Medium Suitable for Human Primary Keratinocyte Culture and Tissue Engineering Applications.

In our experience, keratinocytes cultured in feeder-free conditions and in commercially available defined and serum-free media cannot be as efficiently massively expanded as their counterparts grown in conventional bovine serum-containing medium, nor can they properly form a stratified epidermis in a skin substitute model. We thus tested a new chemically defined serum-free medium, which we developed for massive human primary keratinocyte expansion and skin substitute production. Our medium, named Surge Serum-Free Medium (Surge SFM), was developed to be used alongside a feeder layer. It supports the growth of keratinocytes freshly isolated from a skin biopsy and cryopreserved primary keratinocytes in cultured monolayers over multiple passages. We also show that keratin-19-positive epithelial stem cells are retained through serial passaging in Surge SFM cultures. Transcriptomic analyses suggest that gene expression is similar between keratinocytes cultured with either Surge SFM or the conventional serum-containing medium. Additionally, Surge SFM can be used to produce bilayered self-assembled skin substitutes histologically similar to those produced using serum-containing medium. Furthermore, these substitutes were grafted onto athymic mice and persisted for up to six months. In conclusion, our new chemically defined serum-free keratinocyte culture medium shows great promise for basic research and clinical applications.

Tie-Over Bolster Pressure Dressing Improves Outcomes of Skin Substitutes Xenografts on Athymic Mice.

The efficacy of skin substitutes is established for the treatment of burn injuries, but its use is not limited to this condition. This technology has the potential to improve the treatment of various conditions by offering highly advanced and personalized treatments. In vivo studies are challenging but essential to move to clinical use in humans. Mice are the most widely used species in preclinical studies, but the main drawback of this model is the limited surface area of the graft in long-term transplantation studies caused by the displacement and the contraction of the graft. We improved the conventional surgical procedures by stabilizing the chamber covering the graft with intramuscular sutures and by adding a tie-over bolster dressing. The current study was therefore performed to compare outcomes of skin grafts between the conventional and optimized skin graft model. Human self-assembled skin substitutes (SASSs) were prepared and grafted to athymic mice either by the conventional method or by the new grafting method. Graft healing and complications were assessed using digital photographs on postoperative days 7, 14, and 21. Similar structure and organization were observed by histological staining. The new grafting method reduced medium and large displacement events by 1.26-fold and medium and large contraction events by 1.8-fold, leading to a 1.6-fold increase in graft surface area compared to skin substitutes grafted with the usual method. This innovation ensures better reproducibility and consistency of skin substitute transplants on mice.

Are the Effects of the Cholera Toxin and Isoproterenol on Human Keratinocytes' Proliferative Potential Dependent on Whether They Are Co-Cultured with Human or Murine Fibroblast Feeder Layers?

Human keratinocyte culture has provided the means to treat burns, wounds and skin pathologies. To date, to efficiently culture keratinocytes, cells are cultured on an irradiated feeder layer (iFL), either comprising human (iHFL) or murine (i3T3FL) fibroblasts, and the culture medium is supplemented with a cyclic adenosine monophosphate (cAMP) accumulation inducing agent such as isoproterenol (ISO) or cholera toxin (CT). Previous studies have characterized how the feeder layer type and the cAMP inducer type influence epithelial cells' phenotype independently from one another, but it is still unknown if an optimal combination of feeder layer and cAMP inducer types exists. We used sophisticated statistical models to search for a synergetic effect of feeder layer and cAMP inducer types on human keratinocytes' proliferative potential. Our data suggests that, when culturing human keratinocytes, using iHFL over i3T3FL increases population doublings and colony-forming efficiency through signaling pathways involving Ak mouse strain thymoma (Akt, also known as protein kinase B) isoforms 1 to 3, signal transducer and activator of transcription 5 (STAT5), p53, and adenosine monophosphate activated protein kinase α1 (AMPKα1). Both tested cAMP inducers ISO and CT yielded comparable outcomes. However, no significant synergy between feeder layer and cAMP inducer types was detected. We conclude that, to promote human keratinocyte growth in the early passages of culture, co-culturing them with a human feeder layer is preferable to a murine feeder layer.

Effect of 27-MHz radiofrequency on hair follicles: histological evaluation of skin treated ex vivo.

BACKGROUND: A multitude of methods and treatments exist for cosmetic hair removal. Electroepilation is a commonly performed method of hair removal that is so-called "permanent"; however, there is a paucity of histological studies of the effects of radiofrequency (RF) on hair follicles. OBJECTIVE: This study aimed to observe the destruction of human hair follicles and surrounding tissue after the treatment with 27.12-MHz RF, with more attention paid to the thermal destruction of bulge and bulb/dermal papilla. METHODS: Human scalp specimens obtained during face-lift surgery were treated with 27.12-MHz RF. The probe tip was inserted into hair follicle, RF current was applied, and treated specimens were processed for histological analysis. RESULTS: Significant damages were observed on treated hair follicles. Thermal damage was lance-shaped and extended over several hundred micrometers (100-400 µm). The location of destruction areas varied, likely depending on the point of insertion of the probe. The epidermis remained intact. CONCLUSION: This study shows that the general mechanism of thermolysis is to generate damage to cells and tissues surrounding the insertion point of the filament. The results suggest that if the insertion point is close to the bulge region, there is a risk to destroy hair follicle epithelial stem cells.

Irradiated human dermal fibroblasts are as efficient as mouse fibroblasts as a feeder layer to improve human epidermal cell culture lifespan.

A fibroblast feeder layer is currently the best option for large scale expansion of autologous skin keratinocytes that are to be used for the treatment of severely burned patients. In a clinical context, using a human rather than a mouse feeder layer is desirable to reduce the risk of introducing animal antigens and unknown viruses. This study was designed to evaluate if irradiated human fibroblasts can be used in keratinocyte cultures without affecting their morphological and physiological properties. Keratinocytes were grown either with or without a feeder layer in serum-containing medium. Our results showed that keratinocytes grown either on an irradiated human feeder layer or irradiated 3T3 cells (i3T3) can be cultured for a comparable number of passages. The average epithelial cell size and morphology were also similar. On the other hand, keratinocytes grown without a feeder layer showed heavily bloated cells at early passages and stop proliferating after only a few passages. On the molecular aspect, the expression level of the transcription factor Sp1, a useful marker of keratinocytes lifespan, was maintained and stabilized for a high number of passages in keratinocytes grown with feeder layers whereas Sp1 expression dropped quickly without a feeder layer. Furthermore, gene profiling on microarrays identified potential target genes whose expression is differentially regulated in the absence or presence of an i3T3 feeder layer and which may contribute at preserving the growth characteristics of these cells. Irradiated human dermal fibroblasts therefore provide a good human feeder layer for an effective expansion of keratinocytes in vitro that are to be used for clinical purposes.

Prospective study on the treatment of lower-extremity chronic venous and mixed ulcers using tissue-engineered skin substitute made by the self-assembly approach.

BACKGROUND: Despite present optimal standard treatment of lower-extremity ulceration, a high incidence of recurrence and treatment failure is observed. The objective of this project was to evaluate the effect of a self-assembled skin substitute (SASS) made by tissue engineering as a temporary cutaneous dressing in the treatment of hard-to-heal chronic ulcers. PATIENTS AND METHODS: The prospective uncontrolled case study includes patients suffering from venous or mixed ulcers lasting more than 6 months and unresponsive to compression therapy, with an Ankle Brachial Index greater than 0.5. Compression therapy was combined with the weekly application of SASS, produced from the patient's own skin cells, until healing. A weekly follow-up recorded wound size, skin aspect, pain, drainage, and percentage of wound healing. Photographs were also taken to assess ulcer evolution. RESULTS: Fourteen ulcers present on 5 patients were treated. A mean of 6.7 SASS depositions by ulcer was required for healing. Two ulcers developed a minor wound infection, which was treated with oral antibiotics; another 2 ulcers recurred, and 1 healed with a second course of treatment, whereas 1 ulcer had a small recurrence treated with local wound care. CONCLUSION: The authors' study suggests that the SASS used as a biological dressing is a promising treatment for hard-to-heal chronic venous and mixed ulcers that are unresponsive to compression therapy.

Speeding up the production of clinical-grade skin substitutes using off-the-shelf decellularized self-assembled dermal matrices.

Patients with deep and extensive wounds need urgent skin coverage to re-establish the cutaneous barrier that prevents life-threatening infections and dehydration. However, the current clinically-available skin substitutes intended for permanent coverage are limited in number, and a trade-off between production time and quality must be made. Here, we report the use of decellularized self-assembled dermal matrices to reduce by half the manufacturing process time of clinical-grade skin substitutes. These decellularized matrices can be stored for over 18 months and recellularized with patients' cells in order to generate skin substitutes that show outstanding histological and mechanical properties in vitro. Once grafted in mice, these substitutes persist over weeks with high graft take, few contraction events, and high stem cell content. These next-generation skin substitutes constitute a substantial advancement in the treatment of major burn patients, combining, for the first time, high functionality, rapid manufacturability and easy handling for surgeons and healthcare practitioners. Future clinical trials will be conducted to assess the advantages of these substitutes over existing treatments. STATEMENT OF SIGNIFICANCE: The number of patients in need for organ transplantation is ever-growing and there is a shortage in tissue and organ donors. In this study, we show for the first time that we can preserve decellularized self-assembled tissues and keep them in storage. Then, in only three weeks we can use them to produce bilayered skin substitutes that have properties very close to those of the native human skin. These findings therefore represent a major step forward in the field of tissue engineering and organ transplantation, paving the way toward a universal off-the-shelf biomaterial for tissue reconstruction and surgery that will be beneficial for many clinicians and patients.

In vitro comparison of human plasma-based and self-assembled tissue-engineered skin substitutes: two different manufacturing processes for the treatment of deep and difficult to heal injuries.

Background: The aim of this in vitro study was to compare side-by-side two models of human bilayered tissue-engineered skin substitutes (hbTESSs) designed for the treatment of severely burned patients. These are the scaffold-free self-assembled skin substitute (SASS) and the human plasma-based skin substitute (HPSS). Methods: Fibroblasts and keratinocytes from three humans were extracted from skin biopsies (N = 3) and cells from the same donor were used to produce both hbTESS models. For SASS manufacture, keratinocytes were seeded over three self-assembled dermal sheets comprising fibroblasts and the extracellular matrix they produced (n = 12), while for HPSS production, keratinocytes were cultured over hydrogels composed of fibroblasts embedded in either plasma as unique biomaterial (Fibrin), plasma combined with hyaluronic acid (Fibrin-HA) or plasma combined with collagen (Fibrin-Col) (n/biomaterial = 9). The production time was 46-55 days for SASSs and 32-39 days for HPSSs. Substitutes were characterized by histology, mechanical testing, PrestoBlue™-assay, immunofluorescence (Ki67, Keratin (K) 10, K15, K19, Loricrin, type IV collagen) and Western blot (type I and IV collagens). Results: The SASSs were more resistant to tensile forces (p-value < 0.01) but less elastic (p-value < 0.001) compared to HPSSs. A higher number of proliferative Ki67+ cells were found in SASSs although their metabolic activity was lower. After epidermal differentiation, no significant difference was observed in the expression of K10, K15, K19 and Loricrin. Overall, the production of type I and type IV collagens and the adhesive strength of the dermal-epidermal junction was higher in SASSs. Conclusions: This study demonstrates, for the first time, that both hbTESS models present similar in vitro biological characteristics. However, mechanical properties differ and future in vivo experiments will aim to compare their wound healing potential.

Cultured Autologous Corneal Epithelia for the Treatment of Unilateral Limbal Stem Cell Deficiency: A Case Series of 15 Patients.

Damage to limbal epithelial stem cells can lead to limbal stem cell deficiency (LSCD). Current autologous treatment procedures for unilateral LSCD bear a significant risk of inducing LSCD in the donor eye. This complication can be avoided by grafting a stem cell containing cultured autologous corneal epithelium (CACE). The primary objective of this study was to demonstrate the safety of CACE grafted on eyes with LSCD. The secondary objective was to assess the efficacy of a CACE graft in restoring a self-renewing corneal surface with adequate anatomic structures, as well as improving the best corrected visual acuity (BCVA). Fifteen patients were grafted with a CACE on a fibrin gel produced from a 3 mm2 limbal biopsy harvested from the donor eye. Data were collected at baseline and after grafting. Follow-ups from 1 to 5 years were conducted. No major adverse events related to the CACE graft were observed. For every visit, an anatomic score based on corneal opacity as well as central vascularization and a functional score based on BCVA were determined. Safety was demonstrated by the low occurrence of complications. Anatomical (93%) and functional (47%) results are promising for improving vision in LSCD patients. Combined functional success and partial success rates with inclusion of BCVA were 53% [CI95: 27-79%] one year after CACE grafting. At the last follow-up, 87% [CI95: 60-98%] of the patients had attained corneal clarity. The outcomes demonstrate the safety of our technique and are promising regarding the efficacy of CACE in these patients.

Efficient Gamma-Retroviral Transduction of Primary Human Skin Cells Using the EF-c Peptide as a Transduction Enhancer.

Efficient gene transfer into cultured fibroblasts and keratinocytes during retroviral transduction is a critical step toward the treatment of genodermatoses such as epidermolysis bullosa. However, achieving high transduction rates is still a difficult task, particularly for the insertion of large coding sequences for which high viral titers cannot always be obtained. Multiple polycationic molecules, such as polybrene, which has been used in several clinical trials, have the ability to boost ex vivo retroviral gene transfer. However, the use of polybrene has been associated with a reduction of the proliferation and growth potential of human keratinocytes in culture. We developed a method for the efficient retroviral transduction of primary fibroblasts and keratinocytes using EF-c, a polycationic nanofibril-forming peptide. In comparison with polybrene, we found that the retroviral transduction efficiency with EF-c was increased 2.5- to 3.2-fold for fibroblasts, but not for keratinocytes. Moreover, the use of EF-c did not affect fibroblast proliferation and keratinocyte stem cell content, whereas polybrene induced a decrease in both. This method could have a positive impact on the development of ex vivo gene correction of genodermatoses, allowing for more efficient gene transfer into primary skin cells with little to no effect on proliferation and stem cell content. © 2022 Wiley Periodicals LLC. Basic Protocol: Fibroblast and keratinocyte transduction Support Protocol: Assessment of transduction efficiency through flow cytometry analysis.

Stem cells of the skin and cornea: their clinical applications in regenerative medicine.

PURPOSE OF REVIEW: The use of stem cells is of great interest for the treatment of various pathologies and ultimately for the restoration of organ function. Progress pointing towards future treatments of skin and corneal epithelial stem cell defects are reviewed, including the transplantation of living tissue-engineered substitutes. RECENT FINDINGS: This article focuses on substitutes optimized for permanent replacement of skin and cornea. New skin substitutes for burn care are currently under development. More complex tissue-engineered skin substitutes in which stroma, adipose tissue, capillaries, and neurons are combined with the epithelium are being developed. Some dermal/epidermal substitutes have been applied to the treatment of patients. Cultured corneal epithelial cells have been characterized and more complete corneal substitutes are being designed. Long-term clinical results on the transplantation of cultured corneal stem cells for the treatment of limbal stem cell deficiency have been reported. SUMMARY: Advances in tissue engineering for the development of substitutes that will benefit patients suffering from skin or corneal stem cell deficiencies are reviewed. These products are often a combination of cells, scaffolds and other factors. Key considerations in the development of corneal and skin substitutes for clinical applications are discussed.

Emergency department visits and primary care among adults with chronic conditions.

BACKGROUND: An emergency department (ED) visit may be a marker for limited access to primary medical care, particularly among those with ambulatory care sensitive chronic conditions (ACSCC). OBJECTIVES: In a population with universal health insurance, to examine the relationships between primary care characteristics and location of last general physician (GP) contact (in an ED vs. elsewhere) among those with and without an ACSCC. RESEARCH DESIGN: A cross-sectional survey using data from 2 cycles of the Canadian Community Health Survey carried out in 2003 and 2005. SUBJECTS: The study sample comprised Québec residents aged ≥18 who reported at least one GP contact during the previous 12 months, and were not hospitalized (n = 33,491). MEASURES: The primary outcome was place of last GP contact: in an ED versus elsewhere. Independent variables included the following: lack of a regular physician, perceived unmet healthcare needs, perceived availability of health care, number of contacts with doctors and nurses, and diagnosis of an ACSCC (hypertension, heart disease, chronic respiratory disease, diabetes). RESULTS: Using multiple logistic regression, with adjustment for sociodemographic, health status, and health services variables, lack of a regular GP and perceptions of unmet needs were associated with last GP contact in an ED; there was no interaction with ACSCC or other chronic conditions. CONCLUSIONS: Primary care characteristics associated with GP contact in an ED rather than another site reflect individual characteristics (affiliation with a primary GP and perceived needs) rather than the geographic availability of healthcare, both among those with and without chronic conditions.

Peel Test to Assess the Adhesion Strength of the Dermal-Epidermal Junction in Tissue-Engineered Skin.

Innovative therapies combining gene-corrected stem cells and the production of bioengineered tissues to treat epidermolysis bullosa are emerging. However, quantitative tests to measure the adhesion forces between two highly viscoelastic substrates such as those found in bilayered bioengineered skin are needed and are still lacking. The objective of this study was to develop a mechanical test to measure the dermal-epidermal adhesion strength of our bilayered tissue-engineered skin substitute (TES) produced with the self-assembly method. We developed a peel test, which allows the displacement of both skin layers in a T configuration, based on the ASTM International standard. A MATLAB program was written to process and analyze raw data. The experimental setup was tested by measuring the dermal-epidermal adhesion strength in TESs produced with normal or collagen VII-deficient cells. Our peel testing method allowed us to detect the impact of the absence of collagen VII in the dermal-epidermal adhesion strength of TESs and also to examine the progression of the dermal-epidermal adhesion strength in relation to culture time in normal TES. Impact statement This study describes a method for assessing the adhesion strength at the dermal-epidermal junction of individual tissue-engineered skin substitute (TES). An ASTM standardized protocol of peel testing was designed to measure this important mechanical property. Our innovative approach will serve as a quality control in the production, improvement, and application of TESs for the treatment of pathologies affecting the dermal-epidermal adhesion such as epidermolysis bullosa. Data presented contribute to research on the interfaces between biological substrates and provide a reference factor for the characterization of products derived from tissue engineering.

Vibrissa hair bulge houses two populations of skin epithelial stem cells distinct by their keratin profile.

Defining the properties of postnatal stem cells is of interest given their relevance for tissue homeostasis and therapeutic applications, such as skin tissue engineering for burn patients. In hair follicles, the bulge region of the outer root sheath houses stem cells. We show that explants from the prominent bulge area, but not the bulb, in rodent vibrissa follicles can produce epidermis in a skin model of tissue engineering. Using morphological criteria and keratin expression, we typified epithelial stem cells of vibrissa bulge. Two types of slow-cycling cells (Bb, Bs1) featuring a high colony-forming capacity occur in the bulge. Bb cells are located in the outermost basal layer, express K5, K15, K17, and K19, and feature a loosely organized keratin network. Bs1 cells localize to the suprabasal layers proximal to Bb cells and express K5/K17, correlating with a network of densely bundled filaments. These prominent bundles are missing in K17-null mice, which lack vibrissa. Atypically, both the Bb and Bs1 keratinocytes lack K14 expression. These findings show heterogeneity within the hair follicle stem cell repository, establish that a subset of slow-cycling cells are suprabasal in location, and point to a special role for K5/K17 filaments in a newly defined subset of stem cells. Our results are discussed in the context of long-term survival of engineered tissues after grafting that requires the presence of stem cells.

Regeneration of skin and cornea by tissue engineering.

Progress in tissue engineering has led to the development of technologies allowing the reconstruction of autologous tissues from the patient's own cells. Thus, tissue-engineered epithelial substitutes produced from cultured skin epithelial cells undergo long-term regeneration after grafting, indicating that functional stem cells were preserved during culture and following grafting. However, these cultured epithelial sheets reconstruct only the upper layer of the skin and lack the mechanical properties associated to the connective tissue of the dermis. We have designed a reconstructed skin entirely made from human cutaneous cells comprising both the dermis and the epidermis, as well as a well-organized basement membrane by a method named the self-assembly approach. In this chapter, protocols to generate reconstructed skin and corneal epithelium suitable for grafting are described in details. The methods include extraction and culture of human skin keratinocytes, human skin fibroblasts as well as rabbit and human corneal epithelial cells, and a complete description of the skin reconstructed by the self-assembly approach and of corneal epithelium reconstructed over a fibrin gel.

Qualitatively Monitoring Binding and Expression of the Transcription Factors Sp1 and NFI as a Useful Tool to Evaluate the Quality of Primary Cultured Epithelial Stem Cells in Tissue Reconstruction.

Electrophoretic mobility shift assays and Western blots are simple, efficient, and rapid methods to study DNA-protein interactions and protein expression, respectively. Primary cultures and subcultures of epithelial cells are widely used for the production of tissue-engineered substitutes and are gaining popularity as a model for gene expression studies. The preservation of stem cells through the culture process is essential to produce high quality substitutes. However, the increase in the number of cell passages is associated with a decrease in their ability to proliferate until senescence is reached. This process is likely to be mediated by the altered expression of nuclear-located transcription factors such as Sp1 and NFI, whose expression has been documented to be required for cell adhesion, migration, and differentiation. In some of our recent studies, we observed a correlation between reconstructed tissues exhibiting poor histological and structural characteristics and a low expression of Sp1 in their constituting epithelial cells. Therefore, monitoring both the expression and DNA binding of these transcription factors in human skin and corneal epithelial cells is a useful tool for characterizing the quality of primary cultured epithelial cells.