Ovarian Tissue Culture to Preserve Fertility in Transgender Male Patients after Hormonal Treatment / Cultura de tecido ovariano para preservação da fertilidade em pacientes transexuais masculinos após tratamento hormonal

Abstract Objective To evaluate the reproductive and histological characteristics of fresh cultured ovarian tissue from transgender male patients. Methods An in vitro pilot study in which samples were collected during sex reassignment surgery for transgender male patients. The ovarian cortex was cut into fragments of 2 mm, 3mm, and 4 mm, and placed in a 96-well plate suitable for cultivation at days 0, 2, 4, 6, and 8, when the histology was analyzed. Results Stromal hyperplasia was observed in all samples, and it was not associated with the obtainment of primordial or primary follicles. Peripheral reduction in cell count was also a recurrent finding. Primordial and primary follicles were identified with a heterogeneous pattern in fragments from the same patient and from different patients, and follicles in more advanced stages of development (secondary and antral) were not found. There was an association between the diameter of the ovarian fragments and the identification of primary follicles (p=0.036). The number of days in culture was associated with histological signs of tissue damaging in the fragments (p=0.002). The total number of follicles identified in the samples with 2mm in diameter was significantly lower than in those that measured 4mm in diameter (p=0.031). Conclusion A diameter of 4mm is suitable for ovarian tissue culture with the benefit of ease of handling. Even after prolonged exposure to testosterone, the ovarian fragments presented primordial and primary follicles, maintaining viability throughout the days they were exposed to the culture. Freezing the ovarian cortex of transgender patients who will undergo surgery for gender reassignment would be an interesting option, in the future, for the preservation of fertility.

Resumo Objetivo Avaliar as características reprodutivas e histológicas de tecido ovariano cultivado a fresco de pacientes transexuais masculinos. Métodos Estudo experimental in vitro e piloto, no qual amostras foram coletadas durante a cirurgia de redesignação de sexo para pacientes transexuais masculinos. O córtex ovariano foi cortado em fragmentos de 2mm, 3mm, e 4mm, e colocado em placa de 96 poços própria para cultivo nos dias 0, 2, 4, 6 e 8, quando a histologia foi analisada. Resultados Hiperplasia estromal foi observada em todas as amostras, e não esteve associada à obtenção de folículos primordiais ou primários. A redução periférica no número de células também foi um achado recorrente. Folículos primordiais e primários foramidentificados com padrão heterogêneo emfragmentos domesmo paciente e em fragmentos de pacientes diferentes, não sendo encontrados folículos em estágios mais avançados de desenvolvimento (secundários e antrais). Houve associação entre o diâmetro dos fragmentos ovarianos e a identificação dos folículos primários (p=0,036). O número de dias de cultura esteve associado a sinais histológicos de lesão tecidual nos fragmentos (p=0,002). O número total de folículos identificados nas amostras de 2mm de diâmetro foi significativamente menor do que nas de 4mm de diâmetro (p=0,031). Conclusão O diâmetro de 4mm parece ser mais adequado para a cultura de tecido ovariano com a vantagem de fácil manejo. Mesmo após exposição prolongada à testosterona, os fragmentos ovarianos apresentavam folículos primordiais e primários, e manteve a viabilidade ao longo dos dias de exposição à cultura. No futuro, o congelamento da cortical do ovário de pacientes transgêneros que se submeterão à cirurgia de redesignação sexual poderia ser uma opção interessante para a preservação da fertilidade.

Aloinjertos análogos de mucosa bucal en ratas no consanguíneas / Oral mucosa analog allografts in non-consanguineous rats

Resumen Introducción: A pesar de que existen opciones terapéuticas para el tratamiento de defectos de la mucosa bucal, persiste la necesidad de encontrar sustitutos funcionales, anatómicos y estéticamente similares al tejido que se va a reemplazar, así como soluciones que reduzcan la morbilidad de los injertos autólogos. Objetivo: Determinar la compatibilidad clínica e histológica de aloinjertos equivalentes de mucosa bucal elaborados mediante ingeniería tisular en ratas no consanguíneas. Materiales y métodos: Se utilizó una muestra de mucosa bucal de ratas Sprague Dawley para la obtención de un cultivo de fibroblastos y otro de queratinocitos y fibroblastos. En ambos casos, se usó una membrana de colágeno comercial como soporte. Después de diez semanas de cultivo, las membranas resultantes se injertaron en cuatro ratas Wistar. La primera fase del estudio consistió en la elaboración de los tejidos análogos de mucosa bucal mediante ingeniería tisular, los cuales se implantaron en ratas Wistar inmunocompetentes; posteriormente, se evaluaron las características clínicas e histológicas del aloinjerto. Resultados: La evaluación in vivo de los tejidos análogos demostró que se habían integrado correctamente en los huéspedes inmunocompetentes, y se había logrado el aumento del biotipo periodontal y la creación de una zona con mayor queratinización. Desde el punto de vista histológico, el tejido adquirió características similares a las de la muestra de mucosa bucal de control, sin ningún tipo de reacción inflamatoria ni signos clínicos o histológicos de rechazo. Conclusión: Hubo compatibilidad clínica e histológica de los aloinjertos equivalentes de mucosa bucal obtenidos mediante ingeniería tisular.

Abstract Introduction: Although there are therapeutic options for the treatment of oral mucosa defects, the need for functional, anatomical and aesthetically similar substitutes persists, as well as for solutions to reduce autologous grafts morbidity. Objective: To determine clinical and histological compatibility of equivalent oral mucosa allografts generated through tissue engineering in non-consanguineous rats. Materials and methods: We used a sample of oral mucosa from Sprague Dawley rats to obtain a fibroblast culture and a keratinocytes and fibroblasts co-culture. In both cases, we used a commercial collagen membrane as "scaffold". After ten weeks of culture, we grafted the resulting membranes into four Wistar rats. The first phase of the study was the development of the oral mucosa equivalents generated by tissue engineering. Then, we implanted them in immunocompetent Wistar rats, and finally we evaluated the clinical and histological features of the allografts. Results: In vivo evaluation of mucosal substitutes showed a correct integration of artificial oral mucosa in immunocompetent hosts, with an increase in periodontal biotype and the creation of a zone with increased keratinization. Histologically, the tissue was similar to the control oral mucosa sample with no inflammatory reaction nor clinical or histological rejection signs. Conclusion: The equivalent oral mucosa allografts generated by tissue engineering showed clinical and histological compatibility.

Estudio preliminar del tratamiento de úlceras crónicas con equivalentes cutáneos obtenidos por ingeniería de tejidos / Preliminary Study of the Treatment of Chronic Skin Ulcers with Tissue-engineered Skin Equivalents

Las úlceras de las extremidades inferiores son afecciones de incidencia y prevalencia elevada, cuya cronicidad y persistencia en hacer recidivas son dos de las características evolutivas más relevantes. A pesar de un tratamiento etiológico correcto, a lo cual se suma una gran variedad de apósitos disponibles, el porcentaje de curación y la velocidad de cicatrización siguen siendo bajos. Por tanto se realizó un estudio con el objetivo de obtener un sustituto de piel para su aplicación en la clínica. Se aislaron y cultivaron queratinocitos, que fueron sembrados a una alta densidad, en un sustrato optimizado de fibrina con fibroblastos. De esta forma se desarrolló un nuevo equivalente cutáneo más práctico y de bajo costo, cuyo transplante es directo y de fácil aplicación. Antes de comenzar los estudios clínicos para evaluar sus ventajas y su alcance, se hizo una prueba preliminar con el fin de ver si los cambios efectuados en la técnica estándar no impedían su viabilidad y su acción. A tal fin fueron seleccionaron seis pacientes con ulceras crónicas, sin curación mediante todo tratamiento, a quienes se les aplicaron los nuevos equivalentes. Los pacientes tratados mostraron que los equivalentes eran viables, efectivos, de fácil manipulación y de rápida acción sobre los tejidos afectados. Todos los pacientes que completaron el procedimiento se recuperaron, fue excelente ejemplo un paciente con una lesión crónica de seis meses de evolución que había sido refractaria a los múltiples tratamientos convencionales empleados.

Ulcers of the lower extremities have high prevalence and incidence. Their chronicity and frequent recurrences are two of their most important characteristics. Despite proper etiologic treatment and the great variety of dressings available, the healing rate and recovery speed remain low. Therefore, we conducted a study to obtain skin substitutes for clinical application by isolating and cultivating keratinocytes, which were seeded on a fibroblast-containing fibrin gel at high density. Hence, a new practical and inexpensive skin equivalent of straightforward and easy-to-perform transplantation was developed. Before starting the clinical trials to evaluate its advantages and applicability, we conducted a preliminary test to determine if the changes to the standard technique affected its feasibility and action. For this purpose, we implanted the new equivalents into six patients with chronic ulcers unresponsive to treatment. Outcomes in these patients showed that skin equivalents were feasible, effective, easy to handle and had rapid action on target tissues. All patients who completed the procedure recovered. An excellent example was a patient with a chronic lesion refractory to multiple conventional treatments that had been present for six months.

Prolonged viability of human organotypic skin explant in culture method (hOSEC)

Abstract BACKGROUND: Currently, the cosmetic industry is overwhelmed in keeping up with the safety assessment of the increasing number of new products entering the market. To meet such demand, research centers have explored alternative methods to animal testing and also the large number of volunteers necessary for preclinical and clinical tests. OBJECTIVES: This work describes the human skin ex-vivo model (hOSEC: Human Organotypic Skin Explant Culture) as an alternative to test the effectiveness of cosmetics and demonstrate its viability through cutaneous keratinocytes' proliferative capacity up to 75 days in culture. METHODS: The skin explants obtained from surgeries were cultured in CO2-humid incubator. After 1, 7, 30 and 75 days in culture, skin fragments were harvested for analysis with histomorphological exam (HE staining) on all days of follow-up and immunohistochemistry for Ck5/6, Ck10 and Ki-67 only on the 75th day. RESULTS: On the 7th day, the epidermis was perfect in the dermoepidermal junction, showing the viability of the model. On the 30th day, the epidermis was thicker, with fewer layers on the stratum corneum, although the cutaneous structure was unaltered. On the 75th day, the skin became thinner but the dermoepidermal junctions were preserved and epidermal proliferation was maintained. After the 75th day on culture, the skin was similar to normal skin, expressing keratinocytes with Ck5/6 on supra-basal layers; Ck10 on differentiated layers; and viability could be assessed by the positivity of basal cells by Ki-67. CONCLUSION: The hOSEC model seems a good alternative to animal testing; it can be used as a preclinical test analogous to clinical human skin test with similar effectiveness and viability proven by immunohistological analyses. .

Usefulness of Skin Explants for Histologic Analysis after Fractional Photothermolysis

BACKGROUND: Fractional laser resurfacing treatment has been extensively investigated and is widely used. However, the mechanism underlying its effects is poorly understood because of the ethical and cosmetic problems of obtaining skin biopsies required to study the changes after laser treatment. OBJECTIVE: To evaluate the usefulness of human skin explants for the investigation of fractional photothermolysis. METHODS: Full-thickness discarded skin was treated in 4 ways: no treatment (control), fractional carbon dioxide laser, fractional Er:YAG laser, and fractional 1,550-nm erbium-doped fiber laser. Both treated and non-treated skin samples were cultured ex vivo at the air-medium interface for 7 days. Frozen tissue was sectioned and stained with hematoxylin & eosin for histologic examination and nitro blue tetrazolium chloride for viability testing. RESULTS: Skin explants cultured for up to 3 days exhibited histologic changes similar to those observed in in vivo studies, including microscopic treatment zones surrounded by a thermal coagulation zone, re-epithelialization, and formation of microscopic epidermal necrotic debris. However, the explant structure lost its original form within 7 days of culture. The viability of skin explants was maintained for 3 days of culture but was also lost within 7 days. CONCLUSION: The skin explant model may be a useful tool for investigating the immediate or early changes following fractional photothermolysis, but further improvements are required to evaluate the long-term and dermal changes.

In vitro regeneration and ploidy level analysis of Eulophia ochreata Lindl.

Various parameters including explant-type, medium compositions, use of phytohormones and additives were optimized for direct and indirect regeneration of E. ochreata, a medicinal orchid under threat. Protocorm-like-bodies (PLBs) proved to be the best explants for shoot initiation, proliferation and callus induction. Murashige and Skoog’s (MS) medium containing 2.5 mg L-1 6-benzylaminopurine (BAP), 1.0 mg L-1 kinetin (Kin) and additives (adenine sulfate, arginine, citric acid, 30 mg L-1 each and 50 mg L-1 ascorbic acid) was optimal for shoot multiplication (12.1 shoots and 7.1 PLBs per explant with synchronized growth), which also produced callus. Shoot number was further increased with three successive subcultures on same media and ~40 shoots per explant were achieved after 3 cycles of 30 days each. Additives and casein hydrolysate (CH) showed advantageous effects on indirect shoot regeneration via protocorm-derived callus. Optimum indirect regeneration was achieved on MS containing additives, 500 mg L-1 CH, 2.5 mg L-1 BAP and 1.0 mg L-1 Kin with 30 PLBs and 6 shoots per callus mass (~5 mm size). The shoots were rooted (70% frequency) on one by fourth-MS medium containing 2.0 mg L-1 indole-3-butyric acid, 200 mg L-1 activated charcoal and additives. The rooted plantlets were hardened and transferred to greenhouse with 63% survival rate. Flow-cytometry based DNA content analysis revealed that the ploidy levels were maintained in in vitro regenerated plants. This is the first report for in vitro plant regeneration in E. ochreata.

Fatty Acid Composition of Tissue Cultured Breast Carcinoma and the Effect of Stearoyl-CoA Desaturase 1 Inhibition / 한국유방암학회지

PURPOSE: Stearoyl-CoA desaturase 1 (SCD1) is a novel therapeutic target in various malignancies, including breast cancer. The present study was designed to investigate the effect of the pharmacologic inhibition of SCD1 on fatty acid composition in tissue explant cultures of human breast cancer and to compare these effects with those in adjacent nonneoplastic breast tissue. METHODS: Paired samples of tumor and adjacent noncancerous tissue were isolated from 12 patients with infiltrating ductal breast cancer. Samples were explant cultured in vitro, exposed to the highly selective SCD1 inhibitor CAY10566, and examined for fatty acid composition by gas liquid chromatography. The cytotoxic and antigrowth effects were evaluated by quantification of lactate dehydrogenase release and by sulforhodamine B (SRB) measurement, respectively. RESULTS: Breast cancer tissue samples were found to have higher levels of monounsaturated fatty acids (MUFA) (p<0.001) and arachidonic acid (20:4n-6, p<0.001) and a lower level of linoleic acid (18:2n-6, p=0.02) than the normal-appearing breast tissues. While exhibiting no evident cytotoxicity, treatment with the SCD1 inhibitor, CAY10566 (0.1-1 microM), for 48 hours significantly increased 18:2n-6 levels in both the tumor and adjacent normal-appearing tissue (approximately 1.2 fold, p<0.05). However, the breast cancer tissue samples showed significant increases in the levels of MUFA and 20:4n-6 compared to the normal-appearing breast tissues (p<0.05). The SRB growth assay revealed a higher rate of inhibition with the SCD1 inhibitor in breast cancer tissues than in normal-appearing tissues (p<0.01, 41% vs. 29%). The SCD1 inhibitor also elevated saturated fatty acid (1.46-fold, p=0.001) levels only in the tumor tissue explant. CONCLUSION: The fatty acid composition and response to SCD1 inhibition differed between the explant cultures from breast cancer and the adjacent normal-appearing tissue. Altered fatty acid composition induced by SCD1 inhibition may also, in addition to Delta9 desaturation, modulate other reactions in de novo fatty acid synthesis and lipogenesis, and subsequently affect the overall survival and progression of breast cancer.

An outcome analysis and long-term viability of cryopreserved cultured epidermal allografts: assessment of the conservation of transplantable human skin allografts

PURPOSE: To assess the viability of cultured epithelium and preserved by freezing for periods varying from one month to one year. METHODS: Samples of cultured epithelium were incubated in cryoprotectant medium (Group A), packed in aluminum envelopes and packed in polystyrene boxes. The boxes were subjected to a temperature of-70ºC. After freezing for a period of time ranging from one to 12 months, cultured epithelial samples were assessed for their viability by vital staining (Trypan blue) and metabolic analysis based on glucose consumption and lactate production. Samples of not frozen cultured epithelium (Group B) were also tested for viability and the results obtained were used as comparison parameter for the variation of viability. RESULTS: Statistical analysis between the group A and B indicate that the mean age of the donors (p=0.51) and the culture time (p=1.18) showed no statistical difference. In 30 days we obtained 37% of the original viability of cultured epithelium, 25% at six months and one year, less than 15%. This trend was confirmed statistically with a reduction of approximately 1.8% of the original viability epithelium cultured every 30 days of storage. In the analysis by lactate production, similar results were observed. In the analysis by the glucose consumption results were not significant. The viability indices show statistically significant difference between the group A and B (p<0.0001). CONCLUSIONS: Although cryopreserved cultured epithelium showed significant reduction of viability, all samples remained viable. It was also found that the viability of cryopreserved cultured epithelial decreased as a function of storage time.

Ultradiluição homeopática de triiodotironina altera a apoptose celular da cauda de girinos de Rana catesbeiana: in vitro / Ultra high dilution of triiodothyronine modifies the cellular apoptosis of Rana catesbeiana tadpole tail: in vitro

Ultradiluição (UHD) é o efeito de uma solução, diluída acima do número de Avogrado, que na dependência da sua dinamização (diluição com sucussão) induz um efeito celular supressivo ou estimulante, com conseqüente obtenção de uma curva dose-efeito oscilatória. Por outro lado, a 3,3,5 Triiodo-L-Tironina (T3) é o hormônio mais importante na indução e manutenção das mudanças metamórficas dos girinos, nelas incluídas a absorção da cauda. O presente estudo, cego e randomizado, tem como objetivo comprovar que o T3 5.10-24M (10ª cH) altera a apoptose induzida pelo T3 100 nM na cauda de girinos de Rana catesbeiana, in vitro. Foram distribuídos 60 explantes em três grupos: Grupo A: sem o estímulo do T3 em dose farmacológica e em UHD; Grupo B (teste): sob a ação de T3 100 nM e T3 10ª cH (5.10-24 M); Grupo C (controle): sob a ação do T3 100 nM e etanol 70% sem sucussão. A análise estatística da área dos explantes, no primeiro e ultimo dia do experimento, e do índice apoptótico foi realizado através do teste t Student e foi considerado estatisticamente significante quando p<0,05. Embora sem diferenças significativas na área dos explantes do grupo teste e no grupo controle, um maior e significante índice apoptótico foi identificado nos explantes do grupo teste. Este resultado confirma que o T3 na 10ª cH altera a ação do T3 em dose farmacológica. Futuros experimentos serão realizados, com diferentes dinamizações, com o objetivo da parametrização da curva dose-efeito.

Ultra High Dilution (UHD) is the effect of a solution, beyond the Avogadro limits, that in the dependence of the applied dinamization (dilution with succussion) elicits a suppressive or a stimulant effect on a living cell, with a consequent generation of an oscillatory dose-effect curve. The entire process of anuran amphibian metamorphosis is under thyroid hormones control, included the complete resorption of the tadpole tail. A random and blind study was performed, with the intent to prove that T3 5.10-24 M (10ª cH) modifies the apoptosis induction of T3 100 nM in Rana catesbeiana tadpoles tail tips, in vitro. 60 Explants were distributed in three ways: Group A: without T3 action, at pharmacological and UHD dose; Group B (test): under the action of T3 100 nM and treated with T3 10ª cH (UHD); Group C (control): under the action of T3 100 nM and treated with ethanol 70% unsuccussed. In order to identify significant differences in the area of the remainder explants, at the first and final day of the experiment, and in the apoptotic index we used a student t-test. Although we didnt find statistical difference in macroscopic tadpoles tail tips area from test and control groups, a high and significant (p<0,05) index of apoptosis in histology was found in explants of test group. This data confirms that T3 10 cH modifies the effect of T3 at pharmacological dose. More studies will be necessary, using different dinamizations, to the parameterization of the dose-effect curve proceeding from these experiments.

Cultivo primario de queratinocitos humanos sembrados en submucosa intestinal porcina / Primary culture of human keratinocytes planted on pig's intestine Submucosa

En el campo de la regeneración de piel, la ingeniería de tejidos busca superar las limitaciones asociadas con el uso de autoinjertos inmediatos, dado que la elección de una región donante en el paciente, constituye un riesgo para el mismo, además de ser insuficiente cuando la lesión es extensa. Se ha comprobado que el empleo de la submucosa del intestino delgado de cerdo (SIS) (por la sigla en inglés small intestinal submucosa), por su especial composición, como biomaterial de relleno para tratar lesiones, disminuye el dolor y la inflamación desde su primera aplicación y favorece la movilidad temprana de la región lesionada. Con el fin de determinar la utilidad de SIS, como sustituto epidérmico, en el presente estudio se desarrolló un protocolo para el cultivo primario de queratinocitos humanos, provenientes de prepucios infantiles, sobre una matriz de SIS como soporte. Se evaluó el potencial de adherencia y la capacidad de proliferación de queratinocitos sobre este sustrato.

Tissue engineering, in the fields of skin regeneration, seeks to overcome the limitations associated with the use of immediate auto-grafts, since choosing the donor region of the patient constitutes a risk for the patient himself and is insufficient if the injury that has to be repaired is very large. The use of the pig’s small intestine submucosa (SIS) has being proved as a filling biomaterial to treat injuries, because of its special composition, it lowers pain and inflammation since its first application and it favours early mobility to the wounded area. The present study developed a protocol for the primary culture of human keratinocytes from infant foreskins, and used small intestinal submucosa (SIS) like culture substrate. Cellular adherence potential and proliferation capability of the keratinocytes over this substrate was evaluated.

Aplicação de substituto de pele em oncologia cutânea: estudo experimental com derme acelular e ceratinócitos cultivados / Application of skin substitutes in skin oncology: experimental study using acellular dermis and cultured keratinocytes

FUNDAMENTOS: As neoplasias malignas da pele de grandes dimensões apresentam dificuldades de reconstrução após a excisão. OBJETIVO: O objetivo deste estudo foi avaliar a exeqüibilidade de uma nova proposta de cobertura para feridas cirúrgicas criadas após a ressecção de grandes tumores cutâneos, a combinação da derme acelular humana com epitélio autólogo cultivado. MÉTODOS: A aplicação dos substitutos de pele foi feita em quatro pacientes com área de implante variando de 33 a 120 cm2. Além da observação dos resultados clínicos, realizou-se estudo morfológico para avaliação da integração dos implantes. RESULTADOS: Ceratinócitos autólogos cultivados foram enxertados em dois pacientes e não demonstraram integração. A derme acelular foi aplicada em quatro pacientes, sendo que em um deles foram feitas duas aplicações. Dos cinco implantes de derme acelular realizados, dois não apresentaram integração, em dois a integração foi de 70 por cento, e de 50 por cento no último. CONCLUSÃO: A cobertura imediata e definitiva de defeitos cirúrgicos através da aplicação de derme acelular humana combinada com epitélio autólogo cultivado é exeqüível. Em oncologia cutânea apenas em situações especiais o uso de substitutos de pele pode ser conveniente no sentido de evitar reconstruções mais complexas.

BACKGROUND: Reconstruction difficulties may arise after excision of large malignant skin neoplasms.a OBJECTIVE: The objective of this study was to assess the feasibility of a new coverage for surgical wounds following resection of large skin tumors: a combination of human acellular dermis with cultured autologous epithelium. METHODS: The skin substitute was implanted in four patients, one of them received two implants and the area ranged from 33 to 120 cm2. Clinical results and morphologic studies were assessed as to implant integration. RESULTS: Cultured autologous epithelium was grafted in two patients and no integration was observed. The acellular dermis was applied to four patients. Out of five acellular dermis implants, two did not present integration, two presented 70 percent integration and the remaining, 50 percent integration. CONCLUSION: The immediate and definite coverage of surgical defects by means of application of human acellular dermis combined with cultured autologous epithelium is feasible. In skin oncology, the use of skin substitutes might be convenient only in special situations to avoid more complex reconstructions.

Three-dimensional culture of the primary lung adenocarcinoma tissues / 肿瘤

Objective: To explore the three-dimensional (3-D) culture technique of the lung adenocarcinoma tissues in vitro and further evaluate its application foreground. Methods: In order to mimick the tumor growth in vivo, primary lung cancer tissues were cultured in well-prepared 3-D collagen-based gel in vitro. Optical microscopy, electron microscopy, immunohistochemistry and tumor transplantation techniques were uesd to observe the growth of the tumor tissues. Results: Primary lung adenocarcinoma tissues were grown in collagen mixed with fibroblasts in 3-D space. After the culture period was prolonged, the volume of tumor tissues turned small. Connection was established among adjacent tumor tissues by migration of tumor cells. Some tumor cells permeated the gel, migrated to the bottom of the culture plate, and grew on it. Immunohistochemistry analysis indicated the well growth of tumor tissues. Conclusion: Primary lung adenocarcinoma tissues grow well in 3-D culture enviroment and keep the invasion features. The 3-D culture technique supplies a research platform for mimicking the real enviroment of tumor in vivo.

A Study of Reconstructed Human Epidermis Model in vitro / 中华皮肤科杂志

Objective To establish a reconstructed human epidermis model in vitro, and to study the process of re-epithelialization. Methods A dermal substrate devoid of epidermis was prepared; a 2 mm skin biopsy explant was transplanted onto the dermal substrates. Visualization of epidermal cell migration was carried out by fluorescence imaging. The proliferation and differentiation of the new epithelial cells were observed using histopathological and immunohistochemical staining (Ki67). EGF was added to the culture medium of the experimental samples but not to that of the controls. Results After 3 days of culture, re-epithelialization was observed on the surface of the dermal substrate. A complete structure resembling regular epidermis was noted in 10 days. As compared to control samples, EGF-treated samples had larger area of re-epithelialization (t= 3.02, P