Cultured Epithelial Autografts Research Articles

A Case Report of the First CEA Transplant in an HIV-Positive Burn Patient in South Africa Using a Novel Composite Culture Technique

AbstractResource-limited burn centers are usually devoid of cultured epithelial autografts (CEA) owing largely to prohibitive costs. A modified CEA technique at our burn center incorporated cost-effective, routinely-used dressings with favorable outcomes. One unknown concern was the immunosuppressive effect that extensive burns and potentially human immunodeficiency virus (HIV) infection may have on graft take. This case study reports on the graft take outcomes of the first CEA transplanted in an HIV-positive patient with extensive burns in South Africa, using this technique. Graft take was determined after 21 days at 80% and a long-term follow-up of 8 years indicated good pigmentation return and skin pliability. Delayed diagnosis of pyoderma gangrenosum caused partial graft loss; however, successful regrafting occurred after pyoderma gangrenosum treatment and eradication. The case was considered clinically successful. HIV may not directly affect graft take, but may result in immunosuppressive conditions that delay graft take success.

Use of Cultured Epithelial Autograft in Conjunction with Biodegradable Temporizing Matrix in Massive Burns: A Case Series.

Intensive care for massively burn patients has increased survival and highlights the need for a solution to the problem of insufficient donor sites for autologous skin coverage. In this case series we present ten patients with average burn size of 81% TBSA and mean age of 24 years old, who underwent burn excision followed by either immediate or delayed biodegradable temporizing matrix (BTM) placement. After an integration period, the BTM was delaminated either the day before or immediately prior to placement of cultured epithelial autografts (CEA) over a widely meshed (4:1 or 6:1) split thickness skin graft (STSG). One patient had CEA alone, without STSG, placed on integrated BTM and had successful take. Seven patients survived to discharge and had average 95% wound closure at 135±35 days. The patients had on average 10.4 total operations and 8.7 excision and grafting operations. Five patients had complications related to the BTM requiring removal or replacement including three fungal infections, one bacterial infection and one with bleeding and a large clot burden. In conclusion, this surgical strategy is a viable option for patients with massive burns and insufficient donor for autologous skin grafting.

Clinical Indications of Cultured Epithelial Autografts.

Cultured epithelial autografts (CEAs) have been used for decades as a treatment for massive burn injuries. Cultured epithelial autografts allow for wounds to heal by taking a small sample and growing a patient's own epithelium in culture to create large, graftable sheets. This technique is especially useful in large wounds where donor sites are limited compared with conventional skin grafting. However, CEAs have a variety of uses in wound healing and reconstruction and have the potential to aid in the closure of several types of defects. Cultured epithelial autografts have shown applicability in large burns, chronic nonhealing wounds, ulcerating wounds of various etiologies, congenital defects, wounds requiring specialized epithelium to replace like by like, and wounds in critically ill patients. Several factors must be considered when using CEAs, such as time, cost, and outcomes. In this article, we detail the various clinical applications of CEAs and how they can be situationally advantageous outside of their original purpose.

Composition and Performance of Autologous Engineered Skin Substitutes for Repair or Regeneration of Excised, Full-Thickness Burns.

Prompt and permanent wound closure after burn injuries remains a requirement for patient recovery. Historically, split-thickness skin autograft (STAG) has served as the prevailing standard of care for closure of extensive, deep burns. Because STAG availability may be insufficient in life-threatening burns, alternatives have been evaluated for safety and efficacy of wound closure. Since the 1970s, alternatives consisting of cultured epidermal keratinocytes, and/or acellular dermal substitutes were studied and translated into services and devices that facilitated wound closure, survival, and recovery after major burns. Cultured epithelial autografts (CEA) promoted epidermal closure of wounds but were not stable during long-term recovery. An acellular dermal substitute consisting of collagen and glycosaminoglycans (C-GAG) provided more uniform dermal repair, and reduced needs for epidermal harvesting but was subject to loss from microbial contamination. More recently, an autologous engineered skin substitute (ESS) has been reported and includes a C-GAG polymer populated with fibroblasts and keratinocytes which form basement membrane. ESS can be applied clinically over a vascularized dermal substitute and generates stable wound closure that is smooth, soft, and strong. Despite these advances, no current alternatives for permanent wound closure restore the anatomy and physiology of uninjured skin. Current alternatives act by mechanisms of wound healing, not by developmental biology by which skin forms in utero with pigment, hair, sweat and sebaceous glands, microvasculature, and nerve. Until full-thickness burns are restored with all of the normal structures and functions of uninjured skin, regenerative medicine of skin will remain an ambitious aspiration for future researchers and engineers to achieve.

Keratinocyte Culture: Siriraj’s Experience

Objective: Cell-based therapy is gaining increasing prominence in medicine, where it has the potential to replace or repair damaged tissue using new engineered cells. Skin cell engineering, also known as keratinocyte culture or cultured epithelial autograft (CEA), is a promising field in cell-based therapy. CEA is now used in many parts of the world as an alternative treatment for some diseases that require large defects to be covered, such as severe and major burn patients and congenital melanocytic nevus. The use of CEA in conjunction with acellular skin substitution is rapidly expanding.Materials and Methods: This study is an initiative aimed at supporting the production and use of keratinocyte cultures at Siriraj Hospital. This is the first stage of developing sheet keratinocyte culture in vitro.Results: Our study yielded very promising results. As feeder cells, we used irradiated 3T3 murine fibroblasts, as per the standard protocol for keratinocyte culture. The growth duration was four weeks: 2 weeks for the 3T3 murine fibroblasts and 2 weeks for the keratinocytes. The keratinocytes grew rapidly and formed sheets with irradiated 3T3 murine fibroblasts. The retrieval of the cell sheets was straightforward thanks to the temperature-response cell culture dish and halo-ring cell recovery sheet. Flow cytometry revealed that the cells had a very high viability and purity. H&E staining revealed the sheets comprised two to four layers of stratified epithelial tissue.Conclusion: From this study, our method of manufacturing the CEA can offer a promising result. This can be use in the treatment which require large skin coverage. However, we aim to initiate animal and human trial phase next.

794 Use of Cultured Epithelial Autografts After Biodegradable Temporizing Matrix in Massive Burns

IntroductionAs burn care advances, patients are surviving with larger burn injuries, that previously would have been fatal. However, the need for autologous skin coverage continues to be an unmet need for massive burn injuries. Several attempts have been made to address this with various dermal substitutes, temporary coverage, and skin substitutes. For 25 years, Cultured Epithelial Autografts (CEA) have been used to treat large burn injuries, but this was met with variable success and has a mandatory pre-requisite lab time before it is ready for use. In 2018, Biodegradable Temporizing Matrix (BTM) that can be placed immediately on excised burns was first studied in burn patients, which has led to its increased use in subsequent years. This case series seeks to examine our experience using CEA following the application and ingrafting of BTM on large burns.MethodsA retrospective review was conducted from 2017-2020 of adult burn patients admitted to an ABA verified burn center who underwent placement of both BTM and CEA. Demographics, mechanism of injury, burn characteristics, surgeries, and outcome data were collected. Surgical technique was early excision, BTM placement, a BTM integration period, repeat superficial excision, fibrin/thrombin spray, split thickness skin grafting with usually 6:1 mesh autograft, and finally CEA application. CEA was managed per manufacturer protocols. Descriptive statistics and univariate analyses were performed with Microsoft Excel.ResultsEight patients met inclusion criteria. The average age was 29.3±5.3 years, 2nd degree TBSA 22.5±22.6%, 3rd degree TBSA 55.8±21%, and total TBSA was 78.3±4.4%. Four patients died during their hospital course and four survived to discharge. For survivors, the age length of stay was 135±23.6 days and they underwent an average of 8.5±1.5 total excision and/or grafting procedures. All patients had severe complications including severe sepsis/septic shock (n=8), gastrointestinal bleeds (n=2), acute respiratory distress syndrome (n=3), acute kidney injury or renal failure (n=4), pulmonary embolism (n=1) and myocardial infarction (n=1). The average time to 95% wound closure was 5 (79-147) days for survivors.ConclusionsThere continues to be an unmet need for autologous skin coverage in massive burn injuries when there is insufficient donor skin. In this series, we describe eight patients with massive burn injuries who underwent initial BTM placement, followed by 6:1 meshed autograft and CEA application. Although four patients died during their treatment course, the four surviving patients had acceptable wound closure rates and length of stay for their burn size.

663 Application and Management of Cultured Epithelial Autografts (CEAs) on Posterior Burns – A 5-year, Multi-center, Retrospective Review of Outcomes

Abstract Introduction In patients with larger burns, treatment with CEAs have proven to increase survival.1 CEAs, while useful, are not without some disadvantages. For instance, if the injury involves burns to posterior surfaces, the challenge is inherent in that these fragile sheets are easily sheared, and pressure can cause disruption and graft loss. CEA grafting must be managed with specific care and with specialized protocols that help address these challenges. Graft loss due to these and other factors can delay healing, increase hospital length of stay, and increase the cost of care. The purpose of this study is to evaluate the success for graft take in patients with posterior burns treated with CEA and to discuss the techniques, protocols and approaches to managing these patients within our burn network. Methods This retrospective study was granted exemption by IntegReview IRB. Take rate for each application of CEA was not always found for some cases. For purposes of this study, we agreed with methods of other researchers in the literature and adapted the “clinically relevant” assessment that take and final coverage was successful when re-grafting was not required by the time of discharge or death.2 Results Study dates was March 2016 - December 2019 and at this time, is being reported from among 3 of 6 participating centers. Our approach is to provide CEA prep the day before initial placement and then to ensure strict protocols are followed at the bedside post-op and thereafter. Total number of patients considered was 68, 41 were deemed evaluable. See tables for other demographics and results. 31 patients were discharged to rehab (75%), 6 were discharged home (15%) and 4 died (10%). Conclusions Meticulous attention to wound bed preparation and ensuring that post-op care is clearly stated and understood by all clinicians involved in the care of patients with larger burns with posterior trunk involvement is the key to successful coverage with CEA to this challenging anatomical location.

653 Resourceful Surgical Planning for Coverage of Large Burns: Optimizing Cultured Epithelial Autografts (CEAs) Outcomes by Combining with a Modified MEEK Procedure

Abstract Introduction We previously reported a modified MEEK technique providing reliable skin transfer using a specific adhesive called, “The Rule of Sevens”.¹ This innovative approach is now part of our practice and we have experienced good outcomes as a result. With that technique perfected, we have also begun incorporating this method as part of the surgical plan for coverage of large TBSA burns with CEAs. This study is a report of our initial experiences utilizing a combination of our modified MEEK procedure and CEA grafting for larger TBSA burns. Methods This retrospective study was granted exemption by IntegReview IRB. Demographic data was reviewed. In some cases, incomplete documentation related to percentage take was noted. To account for this limitation, we agreed with other investigators in the literature and applied the “clinically relevant” assessment to this study analysis. This approach assumed that take and final coverage were successful when re-grafting was not required by the time of discharge or death.² Results Nineteen (19) patients total were treated with MEEK/CEA from April 2016 – February 2020. One patient was an outlier, acquiring infection, requiring additional surgery to close the wound, and did not meet criteria to evaluate. There were 4 females (25%) and 14 males (74%), age range 9–71, Mean 37, Median 34, Mode N/A. TBSA was 30–92%, Mean 62 Median 60, Mode 55. Length of stay was 20–188 days, Mean 89, Mode 136. This is approximately 1.5 days’ stay, per percent of burn in this group of patients with larger burns. MEEK meshing ratio was documented on 16 patients, range from 6:1 - 9:1 ratio. Five patients had a 6:1 ratio, 11 patients had 9:1 meshing ratio used. There were 6 deaths in the total group of 18 evaluable patients (33%). Of these, all had MEEK performed initially, however; 3 did not live long enough to have CEA placed. One patient died before initial takedown of CEA could be performed. The other two died during treatment, both had documentation supporting 70% and 90% take, respectively. To determine overall take, we determined whether any of the surviving patients treated needed further grafting. None of the 12 remaining patients required further grafting. This met our criteria of successful take and gave us a 100% success rate. There were 9 patients with documentation clearly stating a percent take rate. In this group, the documented percent take range was 60–97%, Mean 84%, Median 85% and Mode 96%,80%. Again, none of these patients required additional surgery. Conclusions A modified MEEK technique in providing coverage of larger burns with CEA has offered our center better options of expansion thus perfecting the technique of transfer. Most importantly, the MEEK/CEA has resulted in excellent outcomes with a documented mean take rate of 84%.

Treatment of giant congenital melanocytic nevi with cultured epithelial autografts: Clinical and histopathological analysis.

IntroductionCurettage and dermabrasion are effective in the treatment of giant congenital melanocytic nevi (GCMN); however, local infection and hypertrophic scar formation are major issues. Thus, we applied cultured epithelial autografts (CEA) on skin defects after curettage or abrasion of GCMN and assessed the postoperative outcomes.MethodsSeven nevi lesions of five patients (aged 3 months to 24 years) were treated with CEA after curettage or abrasion with a dermatome or a surgical bar, respectively. We assessed the postoperative outcomes, including CEA take ratio, erosion and/or ulcer formation in the acute phase, hospitalization days, Vancouver scar scale, and color improvement one year after the operation. In addition, a histological evaluation of a skin biopsy was performed over one year after the operation.ResultsThe CEAs took well on the wound, and the wound surface was mostly epithelized by postoperative day 7 in all cases. While hypertrophic scar formation and slight pigmentation were observed in some lesions, the color was improved in all of the treated lesions. Histopathological examination revealed that the regenerated epidermis had stratified keratinocytes with rete ridges, and the dermal layer without nevus cells regenerated above the remaining dermis layer.ConclusionsIn this study, we found that early epithelialization and regeneration of the dermal layer was achieved after the application of CEA, suggesting that CEA could be an effective option after curettage or abrasion of GCMN.

Using Various Skin Graft Techniques in Major Burn Reconstruction: A Lesson Learned From a Taiwanese Cornstarch Explosion.

Four hundred and ninety-nine patients had burn injuries in an explosion in Taiwan on June 27, 2015, 24 were admitted to the intensive care units of Taipei Veterans General Hospital. This study details our experience with surgical management of these patients, focusing primarily on various skin graft techniques. This single-center retrospective study included patients who underwent at least one of the previously mentioned skin graft techniques because of extensive skin defects. The demography, burn diagram, treatment modalities, postoperative outcome, and costs were all analyzed, and a comparison with traditional mesh skin grafts was performed. The literature was also reviewed. Fourteen patients underwent the Meek skin graft technique. Only 3 received ReCell and 1 cultured epithelial autograft (CEA) at separate time point. Overall, the autologous skin grafts, including Meek/ReCell/CEA were completed within 6 months. The average skin graft success rate was approximately 72.9%, 79.2%, and 38% in Meek, ReCell, and CEA, respectively. The infection rate was approximately 35.7%, 25%, and 100% in Meek, ReCell, and CEA, respectively. The average surgical cost and total medical cost were significantly higher in patients who underwent Meek/ReCell/CEA treatments. In our experience, Meek and ReCell treatments had acceptable success rates, but CEA treatment not. ReCell and CEA treatments are useful in the event of extremely limited donor sites, and they are fragile, easily infected, and technically challenging. These techniques also require longer hospitalization and tend to be more expensive, all factors that should be considered when assessing treatment options.

Place actuelle des cultures d’épidermes autologues dans la prise en charge des brûlures étendues et perspective d’avenir : revue de littérature

Cultured Epithelial Autografts (CEAs), developed at the end of the 1970s from in vitro culture amplification of keratinocytes, have led to a therapeutic revolution in the treatment of major burns. The areas of improvement of the cultures initially involved the manufacturing processes (culture media, support matrices, etc.) and then clinical applications (use of a largely expanded allogeneic or autologous dermal bed). These advances have enabled burn centers (BC) using CEAs to obtain very satisfactory percentages of graft integration and survival of major burns patients. However, since CEAs are not without major drawbacks (fragility, high rate of infection, high cost, unstable scars), these pitfalls have restricted their use worldwide. As of 2014, CEAs produced by Genyzme Tissue Repair are no longer available in Europe, which has considerably reduced an indispensable therapeutic arsenal for severe and extensive burns. To overcome these therapeutic limitations, current research is focusing on techniques combining surgery, tissue engineering and cell therapy. The advent of regenerative medicine, based on the use of stem cells, in particular mesenchymal stem cells (MSC), can contribute to an improvement in the management of these massively burned patients (optimization of the environmental medium, attenuation of the systemic inflammatory response and the immunosuppressive effects of the burn, acceleration of tissue regeneration, etc.). Cell therapy, therefore, offers alternatives to CEAs, which must imperatively retain their place in the therapeutic arsenal, namely an effective emergency coverage technique that can be improved.

Validation of an implantable bioink using mechanical extraction of human skin cells: First steps to a 3D bioprinting treatment of deep second degree burn.

Clinical grade cultured epithelial autograft (CEA) are routinely used to treat burns covering more than 60% of the total body surface area. However, although the epidermis may be efficiently repaired by CEA, the dermal layer, which is not spared in deep burns, requires additional treatment strategies. Our aim is to develop an innovative method of skin regeneration based on in situ 3D bioprinting of freshly isolated autologous skin cells. We describe herein bioink formulation and cell preparation steps together with experimental data validating a straightforward enzyme-free protocol of skin cell extraction. This procedure complies with both the specific needs of 3D bioprinting process and the stringent rules of good manufacturing practices. This mechanical extraction protocol, starting from human skin biopsies, allows harvesting a sufficient amount of both viable and growing keratinocytes and fibroblasts. We demonstrated that a dermis may be reconstituted in vitro starting from a medical grade bioink and mechanically extracted skin cells. In these experiments, proliferation of the extracted cells can be observed over the first 21 days period after 3D bioprinting and the analysis of type I collagen exhibited a de novo production of extracellular matrix proteins. Finally, in vivo experiments in a murine model of severe burn provided evidences that a topical application of our medical grade bioink was feasible and well-tolerated. Overall, these results represent a valuable groundwork for the design of future 3D bioprinting tissue engineering strategies aimed at treating, in a single intraoperative step, patients suffering from extended severe burns.

Cultured Epithelial Autograft Combined with Micropatterned Dermal Template Forms Rete Ridges In Vivo.

For patients with large, full-thickness burn wounds, sufficient donor sites for autografting are not available, and thus, alternate strategies must be used to close these wounds. Cultured epithelial autografts (CEAs) can aid in closing these wounds but are often associated with slow deposition of basement membrane proteins, leading to blistering and graft loss. Rete ridges and dermal papillae present at the dermal-epidermal junction (DEJ) play a key role in epidermal adhesion and skin homeostasis. Promoting the development of an interdigitated DEJ may enhance basement membrane protein deposition and provide enhanced physical interlock of the epidermis and dermis. To develop a dermal template with stable dermal papillae, an electrospun collagen scaffold was seeded with human dermal fibroblasts. Ridged topographies were patterned into the cell-seeded dermal template using laser ablation, creating wide and shallow (ActiveFX) or narrow and deep (DeepFX) wells. Micropatterned or flat (control) dermal templates were combined with CEAs immediately before grafting to full-thickness excisional wounds on immunodeficient mice. CEAs grafted in conjunction with ridged templates showed rete ridge formation at 2 weeks after grafting and led to increased epidermal thickness, proliferation, and stemness compared to templates with a flat DEJ. As this technology is further developed, the dermal papilla-containing dermal templates may be utilized in combination with CEAs to improve adhesion and clinical function. Impact statement Cultured epithelial autografts (CEAs) serve as an adjunct to conventional split-thickness autograft in patients with very large burns, but they are susceptible to blistering that can reduce engraftment. Blistering results, in part, from relatively slow basement membrane deposition after grafting. This study demonstrates that basement membrane deposition and rete ridge formation are enhanced by combination of CEAs with a micropatterned, cell-seeded dermal template. These findings may lead to improved treatment and increased survival in patients with very large burns.

Chemically defined and xenogeneic-free culture method for human epidermal keratinocytes on laminin-based matrices.

The basal keratinocyte progenitor cells in cultured epithelial autografts (CEAs) regenerate human epidermis after transplantation, a curative therapy for severe burns and, recently, diseases with epidermal loss, such as junctional epidermolysis bullosa (EB). Although a culturing technique for skin keratinocytes was developed four decades ago, the xenogeneic nature of that conventional CEA culture system restricts its use to the treatment of critical and life-threatening cases, such as severe burns on >30% of total body surface area and EB. In the present protocol, we describe how to implement a defined, xeno-free culture system that supports long-term ex vivo expansion of functional human epidermal keratinocytes. Skin-specific basement membrane proteins called laminins play important roles in the maintenance of phenotypic integrity and in supporting the survival of keratinocytes that are adhered to them. This fully human keratinocyte culture system is 'regulatory friendly' and increases the potential of epithelial cellular therapy, which can be expanded to treat less severe burns and other skin defects, such as chronic diabetic wounds. It takes between 7 and 14 d to obtain an initial culture. Conservatively, a secondary culture from the primary culture can be expanded up to 20-fold within 4-5 d once cells reach confluency.

Influence of fibrin matrices and their released factors on epidermal substitute phenotype and engraftment.

Cultured epithelial autografts (CEAs) represent a life-saving surgical technique for full-thickness skin burns covering more than 60% total body surface area. However, CEAs present numerous drawbacks leading to heavy cosmetic and functional sequelae. In our previous study, we showed that human plasma-based fibrin matrices (hPBM) could improve the reparative potential of CEAs. Therefore, in the present work, we sought to investigate the role of hPBM compared with fibrin from purified fibrinogen (FPF) or plastic support on epidermal substitute formation and engraftment. The use of hPBM for epidermal substitute culture improved keratinocyte migration, proliferation, and epidermal substitute organization to a better extent than FPF in vitro. Both fibrin matrices favored greater dermal-epidermal junction protein deposition and prevented their degradation. Keratinocyte differentiation was also decreased using both fibrin matrices. Basement membrane protein deposition was mainly influenced by matrix whereas growth factors released from fibrin especially by hPBM were shown to enhance in vitro keratinocyte migration, proliferation, and epidermal substitute organization. Ultimately, epidermal substitutes grown on hPBM displayed better engraftment rates than those cultured on FPF or on plastic support in a NOD-SCID model of acute wound with the formation of a functional dermal-epidermal junction. Together, these results show the positive impact of fibrin matrices and their released growth factor on epidermal substitute phenotype and grafting efficiency. Fibrin matrices, and especially hPBM, may therefore be of interest to favor the treatment of full-thickness burn patients.

350 Combinatorial Use of CEAs with Dermal Substitutes Containing Dermal Papilla-Like Structures

Prompt closure of wounds is critical for recovery from massive burn injuries, but this can be challenging due to lack of available donor skin. Bioengineered therapies have been developed for treating these injuries including cultured epithelial autografts (CEAs). Although CEAs can facilitate wound closure, they are associated with disadvantages including graft fragility and blistering. To combat graft fragility and a weak epidermal-dermal junction, dermal substitutes with papillae-like structures (DS-PLS) were engineered and utilized in conjunction with CEAs. Human CEAs were fabricated by culturing primary epidermal keratinocytes for 19 days followed by release from plastic by brief exposure to dispase. Dermal substitutes were created by seeding electrospun collagen scaffolds with primary human fibroblasts at 500,000/cm2. Dermal substitutes were cultured for 5 days prior to creation of papillae-like structures. The surface of the engineered dermis was CO2 laser ablated to form a series of protrusions on the surface to mimic dermal papillae structure. On the day of surgery, CEAs were placed on the dermal substitutes containing papillae-like structures (DS-PLS) and immediately grafted to full-thickness surgical wounds in athymic mice; flat substitutes served as controls. Graft area, mechanical properties and structure were assessed 2 and 4 weeks post grafting. The DS-PLS promoted the interdigitation of the dermis and epidermis. Though the presence of the papillae-like structures did not significantly alter graft contraction, the epidermis was significantly thicker in this group versus flat controls. In addition, basement membrane deposition was increased in the DS-PLS versus controls and resulted in less delamination of the epidermis and dermis upon tensile testing. Dermal papillae play important roles in skin function; in particular, they enhance epidermal-dermal adhesive strength. The results of this study suggest that engineering dermal papillae-like structures into dermal substitutes can promote interdigitation of CEAs and the dermis and lead to improved epidermal-dermal adhesion. Methods to reduce blistering and enhance outcomes with CEA use may provide additional treatment options for patients with massive burn injuries.

Xeno-free expansion of adult keratinocytes for clinical application: the use of human-derived feeder cells and serum.

Cultured epithelial autograft (CEA) was the birth of skin tissue engineering and encompassed methodologies for the isolation and expansion of autologous basal keratinocytes for burn treatment that are still practiced at some specialised units around the world. One of the limitations of CEA, however, is the reliance on animal-derived material during the manufacturing process and despite all efforts to date, no xeno-free alternative with proven efficacy has been reported. Here, we investigate whether human-derived fibroblast feeder cells and human serum can sufficiently and effectively provide a suitable microenvironment for adult keratinocyte isolation and expansion. Human dermal fibroblasts and epidermal keratinocytes were isolated from discarded skin during abdominoplasty and breast reduction procedures and cultured in xeno-free conditions. We report that these xeno-free adult keratinocytes form similar numbers of colony-forming units as those cultured using the Green's methods; however, xeno-free keratinocytes express lower levels of α6 integrin (CD49f; a progenitor and stem cell marker). We identified IL-8 as a potential growth factor secreted by adult human fibroblasts that may enhance keratinocyte colony formation in human serum. Finally, we propose a step-by-step xeno-free isolation and cultivation methodology for adult keratinocytes that can be tested further in serial cultivation for clinical application.

Does the transplantation of keratinocytes really reduce the risk of death? Survival analysis of patients hospitalized at the Dr Stanisław Sakiel Centre for Burns Treatment in 2008–2015

Keratinocyte transplantation is an adjuvant procedure in the extensive burn therapy method. However, it must be taken into consideration that clinical results of keratinocyte transplantation are ambiguous and progress achieved in this method is still being verified, especially due to the high cost of cultured epithelial autograft (CEA) transplants. The aim of this study was to verify the impact of cultured keratinocyte application on patients' survival. This study included a group of patients with the highest chance for a successful outcome of the therapy and excluded patients with no compelling reason to apply for such an expensive therapy. This study included all the patients with burns diagnosed between January 1, 2008 and January 1, 2016, who were treated with cultured skin cells. Patients' age and gender, percentage of total body surface area (TBSA) affected, percentage of burn depth of the 3rd/4th degree, number of days between admission and surgery, and need for rehabilitation were analyzed. The cultured cell application did not significantly affect the risk of death (p > 0.05). Keratinocytes should be applied as an adjunctive method for the treatment of burns with at least 40% TBSA affected, but with a maximal burn depth of the 2nd degree. In the group of patients below 50 years of age, a higher number of transplants with a cell population above 20 million/mL and a significantly lower mortality rate were observed, which means that in the mentioned age group, this graft was more effective. It has been suggested that patients older than 50 years of age with burns deeper than of the 2nd degree should be treated with more advanced methods like, e.g., the application of stem cells.

Wound healing after cultured epithelial autografting in patients with massive burn injury: A cohort study

Last century, our laboratory produced Cultured Epithelial Autograft (CEA) for clinical use by the affiliated adult burn service and other burn units across the country. Production of CEA for clinical use was discontinued after several years because of a low success rate and subsequent low demand. Recently, at our burns unit, a cell culture program was reintroduced as a direct response to the need for improvement in ongoing deficiencies and clinical requirements in burn wound closure. The aim of this study was to validate the laboratory processes and clinical algorithms established and share our recent clinical experiences involving CEA. This observational cohort study recruited patients with burns exceeding 35% TBSA admitted to the Victorian Adult Burns Service at The Alfred (December 2013-December 2016). Autologous keratinocytes were expanded and delivered through sheets of fibrin carrier. Twelve patients were recruited to participate in the study. Thirty-two sites were treated with CEA. CEA applied in combination with widely meshed SSG led to the highest take rate (90.1%) at 7-10 days. Further, debridement and grafting were necessary in sixteen of thirty-two sites treated, all involving wound beds prepared with Cuono method or sites treated with CEA only. It is important to address the problem of wound bed contamination, either through increased resistance on the part of the construct or wound bed sterilization. Improved understanding of the relative importance of vascularization, control of cell behavior, the extracellular matrix, immune function, and intrinsic antimicrobial capacity for graft take would then inform a more targeted approach to skin tissue engineering for wound closure in severe burns.

From skin allograft coverage to allograft–micrograft sandwich method: A retrospective review of severe burn patients who received conjunctive application of cultured epithelial autografts

A 12-year retrospective review of severe burn patients who received cultured epithelial autografts (CEA) at the Singapore General Hospital Burns Centre from January 2005 to December 2016 was carried out. During this period, two different surgical modalities were employed to manage these burn injuries. In the earlier period, following early excision of the burn wounds, exposed surfaces were covered with a combination of split thickness skin autografts (STSG) and allografts. Surfaces covered with skin allografts were subsequently debrided of the allo-epidermis in about 3 weeks later, exposing the allodermis with granulating tissues for grafting of CEA; a technique known as the Cuono’s method. In the later period, allograft–autologous micrograft sandwich technique was used to graft on the early excised burns with subsequent CEA grafting. The former and latter groups represented by STSG/C (n=10) and M/CEA (n=14) respectively, were compared in terms of clinical profiles, outcomes, allograft/CEA usage and total graft cost. No significant differences were found based on mean age and presence of inhalation burns between the two treatment methods However, percentage total body surface area (TBSA) and Revised Baux Score were significantly higher (p<0.05) in the M/CEA group compared to the STSG/C group. Differences in clinical outcomes of mortality and length of hospital stay between the 2 groups were statistically insignificant. The average area amount of skin allografts used per patient in the M/CEA group was significantly lower compared to the STSG/C method group which contributed to lower total average cost of grafts used per % TBSA in the M/CEA method group. This might be attributed to the presence of micrografts which seemed to improve stabilization of the wound bed resulting in less operating procedures and improving CEA take. To conclude, the M/CEA method introduced was able to treat more severe burn patients at lower graft costs without compromising critical clinical outcomes significantly.