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1.
Science ; 375(6576): eaaw9021, 2022 Jan 07.
Artigo em Inglês | MEDLINE | ID: mdl-34990240

RESUMO

Epithelial organoids are stem cell­derived tissues that approximate aspects of real organs, and thus they have potential as powerful tools in basic and translational research. By definition, they self-organize, but the structures formed are often heterogeneous and irreproducible, which limits their use in the lab and clinic. We describe methodologies for spatially and temporally controlling organoid formation, thereby rendering a stochastic process more deterministic. Bioengineered stem cell microenvironments are used to specify the initial geometry of intestinal organoids, which in turn controls their patterning and crypt formation. We leveraged the reproducibility and predictability of the culture to identify the underlying mechanisms of epithelial patterning, which may contribute to reinforcing intestinal regionalization in vivo. By controlling organoid culture, we demonstrate how these structures can be used to answer questions not readily addressable with the standard, more variable, organoid models.


Assuntos
Mucosa Intestinal/crescimento & desenvolvimento , Organogênese , Organoides/crescimento & desenvolvimento , Engenharia Tecidual , Animais , Diferenciação Celular , Forma Celular , Células Epiteliais/citologia , Hidrogéis , Mucosa Intestinal/anatomia & histologia , Mucosa Intestinal/citologia , Mucosa Intestinal/metabolismo , Camundongos , Organoides/anatomia & histologia , Organoides/citologia , Organoides/metabolismo , Celulas de Paneth/citologia , Receptores Notch/metabolismo , Transdução de Sinais , Células-Tronco/citologia , Células-Tronco/fisiologia , Técnicas de Cultura de Tecidos , /metabolismo
3.
Biol Reprod ; 104(2): 282-293, 2021 02 11.
Artigo em Inglês | MEDLINE | ID: mdl-33009568

RESUMO

The endometrium is the inner lining of the uterus that undergoes complex regeneration and differentiation during the human menstrual cycle. The process of endometrial shedding, regeneration, and differentiation is driven by ovarian steroid hormones and prepares the endometrium and intrauterine environment for embryo implantation and pregnancy establishment. Endometrial glands and their secretions are essential for pregnancy establishment, and cross talk between the glandular epithelium and stromal cells appears vital for decidualization and placental development. Despite being crucial, the biology of the human endometrium during pregnancy establishment and most of pregnancy is incomplete, given the ethical and practical limitations of obtaining and studying endometrium from pregnant women. As such, in vitro models of the human endometrium are required to fill significant gaps in understanding endometrial biology. This review is focused on the evolution and development of in vitro three-dimensional models of the human endometrium and provides insight into the challenges and promises of those models to improve women's reproductive health.


Assuntos
Endométrio/anatomia & histologia , Endométrio/fisiologia , Organoides/anatomia & histologia , Organoides/fisiologia , Feminino , Humanos , Gravidez
4.
Nat Biomed Eng ; 5(1): 11-25, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33318650

RESUMO

Engineered human mini-brains, made possible by knowledge from the convergence of precision microengineering and cell biology, permit systematic studies of complex neurological processes and of pathogenesis beyond what can be done with animal models. By culturing human brain cells with physiological microenvironmental cues, human mini-brain models reconstitute the arrangement of structural tissues and some of the complex biological functions of the human brain. In this Review, we highlight the most significant developments that have led to microphysiological human mini-brain models. We introduce the history of mini-brain development, review methods for creating mini-brain models in static conditions, and discuss relevant state-of-the-art dynamic cell-culture systems. We also review human mini-brain models that reconstruct aspects of major neurological disorders under static or dynamic conditions. Engineered human mini-brains will contribute to advancing the study of the physiology and aetiology of neurological disorders, and to the development of personalized medicines for them.


Assuntos
Encéfalo , Modelos Biológicos , Organoides , Técnicas de Cultura de Tecidos , Doença de Alzheimer , Encéfalo/anatomia & histologia , Encéfalo/citologia , Encéfalo/efeitos dos fármacos , Encéfalo/fisiologia , Lesões Encefálicas Traumáticas , Neoplasias Encefálicas , Técnicas de Cultura de Células , Células Cultivadas , Humanos , Miniaturização , Organoides/anatomia & histologia , Organoides/citologia , Organoides/efeitos dos fármacos , Organoides/fisiologia
5.
Curr Protoc Pharmacol ; 85(1): e54, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30920154

RESUMO

Human intestinal organoids have enabled performance of functional epithelial studies and modeling of human diseases of the intestine. This unit describes 1) a method to isolate and culture crypts from human intestinal tissue, 2) use of combinatorial methods to expand stem cell-enriched spheroids and differentiate them into organoids composed of various intestinal epithelial cell types, and 3) methods to stimulate these organoids with and measure their responsiveness to external stimuli. To validate the differentiation, organoids can be stained to qualitatively evaluate the presence of colonic crypt morphology and specialized epithelial cell markers. These organoids are responsive to challenge with tumor necrosis factor α (TNFα), resulting in cytokine-induced apoptosis. TNFα-driven apoptosis can be blocked by a small-molecule inhibitor of Ire1α (4µ8C), an endoplasmic-reticulum stress sensor. This is one example of how the human intestinal organoid model can be a powerful tool to elucidate important biological pathways involved in human disease in intestinal epithelial cells. © 2019 by John Wiley & Sons, Inc.


Assuntos
Colo , Organoides , Apoptose/efeitos dos fármacos , Colo/anatomia & histologia , Colo/efeitos dos fármacos , Expressão Gênica , Humanos , Himecromona/análogos & derivados , Himecromona/farmacologia , Técnicas de Cultura de Órgãos , Organoides/anatomia & histologia , Organoides/efeitos dos fármacos , RNA/análise , Fator de Necrose Tumoral alfa/farmacologia
6.
Nat Commun ; 9(1): 4559, 2018 11 19.
Artigo em Inglês | MEDLINE | ID: mdl-30455465

RESUMO

Epigenetic regulators are attractive anticancer targets, but the promise of therapeutic strategies inhibiting some of these factors has not been proven in vivo or taken into account tumor cell heterogeneity. Here we show that the histone methyltransferase G9a, reported to be a therapeutic target in many cancers, is a suppressor of aggressive lung tumor-propagating cells (TPCs). Inhibition of G9a drives lung adenocarcinoma cells towards the TPC phenotype by de-repressing genes which regulate the extracellular matrix. Depletion of G9a during tumorigenesis enriches tumors in TPCs and accelerates disease progression metastasis. Depleting histone demethylases represses G9a-regulated genes and TPC phenotypes. Demethylase inhibition impairs lung adenocarcinoma progression in vivo. Therefore, inhibition of G9a is dangerous in certain cancer contexts, and targeting the histone demethylases is a more suitable approach for lung cancer treatment. Understanding cellular context and specific tumor populations is critical when targeting epigenetic regulators in cancer for future therapeutic development.


Assuntos
Progressão da Doença , Histona Desmetilases/metabolismo , Histona Metiltransferases/metabolismo , Neoplasias Pulmonares/metabolismo , Adenocarcinoma de Pulmão/metabolismo , Animais , Carcinogênese , Linhagem Celular Tumoral/efeitos dos fármacos , Sobrevivência Celular , Modelos Animais de Doenças , Matriz Extracelular/genética , Histona Desmetilases/efeitos dos fármacos , Histona-Lisina N-Metiltransferase/efeitos dos fármacos , Histona-Lisina N-Metiltransferase/metabolismo , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Organoides/anatomia & histologia , Fenótipo , Proteínas Proto-Oncogênicas p21(ras)/genética
7.
Cell Mol Gastroenterol Hepatol ; 6(3): 301-319, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30123820

RESUMO

Background & Aims: The human gut microbiota is becoming increasingly recognized as a key factor in homeostasis and disease. The lack of physiologically relevant in vitro models to investigate host-microbe interactions is considered a substantial bottleneck for microbiota research. Organoids represent an attractive model system because they are derived from primary tissues and embody key properties of the native gut lumen; however, access to the organoid lumen for experimental perturbation is challenging. Here, we report the development and validation of a high-throughput organoid microinjection system for cargo delivery to the organoid lumen and high-content sampling. Methods: A microinjection platform was engineered using off-the-shelf and 3-dimensional printed components. Microinjection needles were modified for vertical trajectories and reproducible injection volumes. Computer vision (CVis) and microfabricated CellRaft Arrays (Cell Microsystems, Research Triangle Park, NC) were used to increase throughput and enable high-content sampling of mock bacterial communities. Modeling preformed using the COMSOL Multiphysics platform predicted a hypoxic luminal environment that was functionally validated by transplantation of fecal-derived microbial communities and monocultures of a nonsporulating anaerobe. Results: CVis identified and logged locations of organoids suitable for injection. Reproducible loads of 0.2 nL could be microinjected into the organoid lumen at approximately 90 organoids/h. CVis analyzed and confirmed retention of injected cargos in approximately 500 organoids over 18 hours and showed the requirement to normalize for organoid growth for accurate assessment of barrier function. CVis analyzed growth dynamics of a mock community of green fluorescent protein- or Discosoma sp. red fluorescent protein-expressing bacteria, which grew within the organoid lumen even in the presence of antibiotics to control media contamination. Complex microbiota communities from fecal samples survived and grew in the colonoid lumen without appreciable changes in complexity. Conclusions: High-throughput microinjection into organoids represents a next-generation in vitro approach to investigate gastrointestinal luminal physiology and the gastrointestinal microbiota.


Assuntos
Colo/citologia , Microbioma Gastrointestinal/fisiologia , Trato Gastrointestinal/fisiologia , Microinjeções/métodos , Organoides/citologia , Animais , Bifidobacterium adolescentis/genética , Bifidobacterium adolescentis/crescimento & desenvolvimento , Bifidobacterium adolescentis/isolamento & purificação , Colo/anatomia & histologia , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Fezes/microbiologia , Humanos , Masculino , Camundongos , Organoides/anatomia & histologia , Análise de Célula Única , Gravação em Vídeo , Yersinia pseudotuberculosis/genética , Yersinia pseudotuberculosis/crescimento & desenvolvimento
8.
Poult Sci ; 97(5): 1536-1543, 2018 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-29509914

RESUMO

The integrity and normal function of the small intestinal epithelium depends critically on the rapid renewal of epithelial cells from basal stem cells. The intensive proliferation that fuels this self-renewal process is confined to the intestinal crypts. Establishment of suitable protocols for crypt isolation and culture is pivotal for the studies of intestinal self-renewal mechanisms. In this study, chicken small intestinal crypts were isolated, purified, and further cultured in a Matrigel 3-D culture system. The growth factor concentration assay on the fourth d of culture showed that Group C (50 ng/mL epidermal growth factor (EGF), 100 ng/mL Noggin, and 500 ng/mL R-spondin 1) supplement in culture medium could significantly enlarge the diameter of organoids when compared with Group A (5 ng/mL EGF, 10 ng/mL Noggin, 50 ng/mL, and R-spondin 1) and Group B (10 ng/mL EGF, 20 ng/mL Noggin, and 100 ng/mL R-spondin 1) by 188.4% (P = 0.026) and 176.9% (P = 0.034), respectively. Transmission electron microscopy, neutral red staining, and 5-ethynyl-2΄-deoxyuridine incorporation demonstrated the integrated structure, high viability, and proliferative activity in cultured chicken intestinal organoids. In addition, intestinal stem cell marker genes (Olfm4, Znrf3, Hopx, and Lgr5) also could be detected in cultured intestinal organoids. Furthermore, CHIR99021 (a glycogen synthase kinase 3ß inhibitor) could enhance the expression of Olfm4, Znrf3, Hopx, and Lgr5 by 750% (P = 0.001), 467% (P < 0.001), 450% (P < 0.001), and 333% (P = 0.008), respectively, indicating the responsiveness of the cultured chicken intestinal organoids to exogenous stimulus. This study modified a murine culture model and optimized it to provide a chicken intestinal organoid model for use as a physiological or pathological research platform in vitro.


Assuntos
Técnicas de Cultura de Células/métodos , Galinhas , Intestino Delgado/fisiologia , Organoides/fisiologia , Animais , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Intestino Delgado/anatomia & histologia , Intestino Delgado/ultraestrutura , Organoides/anatomia & histologia , Organoides/ultraestrutura
9.
Curr Protoc Toxicol ; 75: 22.6.1-22.6.7, 2018 02 21.
Artigo em Inglês | MEDLINE | ID: mdl-29512123

RESUMO

In vitro analysis of intestinal epithelium has been hindered by a lack of suitable culture systems useful for gastrointestinal research. To overcome the problem, an air liquid interface (ALI) method using a collagen gel was established to culture three-dimensional primary cells containing both primary epithelial and mesenchymal components from mouse gastrointestinal tissues. ALI organoids accurately recapitulate organ structures, multilineage differentiation, and physiology. Since ALI organoids from human tissues have not been produced, we modified the previous protocol for mouse ALI organoid culture to establish the culture system of ALI organoids from normal and tumor colorectal tissues of human patients. The current unit presents a protocol for preparation of the ALI organoid culture from normal and tumor colorectal tissues of human patients. ALI organoid culture from human tissues might be useful for examining not only resistance to chemotherapy in a tumor microenvironment but also toxic effects on organoids. © 2018 by John Wiley & Sons, Inc.


Assuntos
Colo/citologia , Organoides/citologia , Técnicas de Cultura de Tecidos/métodos , Células Cultivadas , Colo/anatomia & histologia , Humanos , Mucosa Intestinal/anatomia & histologia , Mucosa Intestinal/citologia , Mesoderma/anatomia & histologia , Mesoderma/citologia , Organoides/anatomia & histologia
10.
Sci Rep ; 7(1): 8591, 2017 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-28819250

RESUMO

Multi-modal three dimensional (3D) optical imaging combining both structural sensitivity and molecular specificity is highly desirable in biomedical research. In this paper, we present a method termed oblique scanning laser microscopy (OSLM) to combine optical coherence tomography (OCT), for simultaneously volumetric structural and molecular imaging with cellular resolution in all three dimensions. Conventional 3D laser scanning fluorescence microscopy requires repeated optical sectioning to create z-stacks in depth. Here, the use of an obliquely scanning laser eliminates the z-stacking process, then allows highly efficient 3D OCT and fluorescence imaging by using only one raster scan. The current setup provides ~3.6 × 4.2 × 6.5 µm resolution in fluorescence imaging, ~7 × 7 × 3.5 µm in OCT in three dimensions, and the current speed of imaging is up to 100 frames per second (fps) over a volume about 0.8 × 1 × 0.5 mm3. We demonstrate several mechanisms for molecular imaging, including intrinsically expressed GFP fluorescence, autofluorescence from Flavin proteins, and exogenous antibody-conjugated dyes. We also demonstrate potential applications in imaging human intestinal organoids (HIOs), colon mucosa, and retina.


Assuntos
Microscopia Confocal/métodos , Imagem Molecular , Animais , Fluorescência , Análise de Fourier , Proteínas de Fluorescência Verde/metabolismo , Humanos , Imageamento Tridimensional , Mucosa Intestinal/anatomia & histologia , Intestinos/anatomia & histologia , Camundongos , Microesferas , Organoides/anatomia & histologia , Imagens de Fantasmas , Ratos , Vasos Retinianos/anatomia & histologia
12.
Gut ; 63(8): 1345-54, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24841573

RESUMO

Limited pools of resident adult stem cells are critical effectors of epithelial renewal in the intestine throughout life. Recently, significant progress has been made regarding the isolation and in vitro propagation of fetal and adult intestinal stem cells in mammals. It is now possible to generate ever-expanding, three-dimensional epithelial structures in culture that closely parallel the in vivo epithelium of the intestine. Growing such organotypic epithelium ex vivo facilitates a detailed description of endogenous niche factors or stem-cell characteristics, as they can be monitored in real time. Accordingly, this technology has already greatly contributed to our understanding of intestinal adult stem-cell renewal and differentiation. Transplanted organoids have also been proven to readily integrate into, and effect the long-term repair of, mouse colonic epithelia in vivo, establishing the organoid culture as a promising tool for adult stem cell/gene therapy. In another exciting development, novel genome-editing techniques have been successfully employed to functionally repair disease loci in cultured intestinal stem cells from human patients with a hereditary defect. It is anticipated that this technology will be instrumental in exploiting the regenerative medicine potential of human intestinal stem cells for treating human disorders in the intestinal tract and for creating near-physiological ex vivo models of human gastrointestinal disease.


Assuntos
Mucosa Intestinal/fisiologia , Organoides/fisiologia , Células-Tronco/fisiologia , Técnicas de Cultura de Tecidos , Animais , Diferenciação Celular , Pesquisa Fetal , Humanos , Mucosa Intestinal/citologia , Intestino Delgado/fisiologia , Organoides/anatomia & histologia , Receptores Acoplados a Proteínas G/análise , Células-Tronco/química , Via de Sinalização Wnt
13.
Transplant Proc ; 40(2): 631-3, 2008 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-18374148

RESUMO

In order to develop articular cartilage grafts, one must control shape and safety. We have developed scaffold-free culture methods in which the cells form multicellular aggregates (organoids). In this study, we applied the organoid culture method to chondrocytes attempting to reconstitute articular cartilage grafts. Primary rat costal chondrocytes and subcultured human articular chondrocytes were immobilized in hollow fibers by centrifugation at a density of 3 x 10(8) cells/cm3 to induce the formation of cylindrical-shaped organoids. To improve convenience, we developed a culture device to form sheet-shaped organoids (organoid-sheet). Primary bovine articular chondrocytes were cultured in this device. These organoids were evaluated by histological and gene expression analyses. In the primary rat culture system, chondrocytes formed cylindrical organoids in hollow fibers. Histochemical analysis revealed the presence of extracellular matrix (collagen and proteoglycan). The organoid maintained cartilage-specific gene expression (type II collagen, aggrecan) for 1 month of culture. In the subcultured human chondrocyte system, the organoid regained the decreased cartilage-specific gene expression. In the primary bovine culture system, the cells formed a 300 microm thickness organoid-sheet including abundant extracellular matrix. In conclusion, our organoid formation method was effective to form cartilage-like tissue. This result suggested that the technique may be applicable for the development of an articular cartilage graft.


Assuntos
Cartilagem Articular/citologia , Cartilagem Articular/transplante , Técnicas de Cultura de Células/métodos , Transplante de Células/métodos , Organoides/anatomia & histologia , Agrecanas/genética , Animais , Cartilagem Articular/anatomia & histologia , Colágeno Tipo II/genética , Meios de Cultura , Marcadores Genéticos , Humanos , Organoides/transplante , Reação em Cadeia da Polimerase , Ratos
14.
Proc Natl Acad Sci U S A ; 103(31): 11461-6, 2006 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-16864785

RESUMO

Tissue engineering is one approach to address the donor-organ shortage, but to attain clinically significant viable cell densities in thick tissues, laboratory-constructed tissues must have an internal vascular supply. We have adopted a biomimetic approach and assembled microscale modular components, consisting of submillimeter-sized collagen gel rods seeded with endothelial cells (ECs) into a (micro)vascularized tissue; in some prototypes the gel contained HepG2 cells to illustrate the possibilities. The EC-covered modules then were assembled into a larger tube and perfused with medium or whole blood. The interstitial spaces among the modules formed interconnected channels that enabled this perfusion. Viable cell densities were high, within an order of magnitude of cell densities within tissues, and the percolating nature of the flow through the construct was evident in microcomputed tomography and Doppler ultrasound measurements. Most importantly, the ECs retained their nonthrombogenic phenotype and delayed clotting times and inhibited the loss of platelets associated with perfusion of whole blood through the construct. Unlike the conventional scaffold and cell-seeding paradigm of other tissue-engineering approaches, this modular construct has the potential to be scalable, uniform, and perfusable with whole blood, circumventing the limitations of other approaches.


Assuntos
Sangue/metabolismo , Técnicas de Cultura , Organoides , Fluxo Sanguíneo Regional , Engenharia Tecidual/métodos , Animais , Linhagem Celular Tumoral , Células Cultivadas , Colágeno/metabolismo , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Humanos , Organoides/anatomia & histologia , Organoides/metabolismo , Porosidade , Distribuição Aleatória , Resistência ao Cisalhamento , Propriedades de Superfície
15.
Int J Artif Organs ; 29(3): 318-28, 2006 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-16685676

RESUMO

In recent years, use of hepatocyte aggregates has led to development of a hybrid artificial liver support system (HALSS) that has high performance. However, in general, their thickness is 100 microm or more, and generation of a dead cell layer due to oxygen exhaustion inside the aggregates has been a universal problem. The present study proposes a novel organoid culture method with better performance than previous organoid culture methods by forming a sheet-shaped organoid (organoid-sheet) with a thickness of approximately 100 microm. The cell number of the organoid-sheet was maintained at approximately 75% of the initial number at 4 days of culture. On the other hand, that of a cylindrical organoid (cylindroid), which formed inside of a plasma separation hollow fiber with 285 microm inner diameter in our previous study, decreased to approximately 50% within 2 days. The ammonia removal rate of the cells in the organoid-sheet was higher than that of the cells in the cylindroid on the first day, but it decreased during the culture time. At day 15, the rate was reduced by almost 50% with respect to the value on the first day. The cells in the cylindroid displayed a lower ammonia removal rate. A significant difference was not observed between the albumin synthesis rates of the two cultures on the first day. However, over a period of time the cells in the organoid-sheet showed a higher albumin synthesis rate than cells in the cylindroid. As this novel organoid maintains these functions for at least 1 month, it is expected to be applied for the development of a HALSS with higher performance.


Assuntos
Hepatócitos , Fígado/fisiologia , Organoides , Técnicas de Cultura de Tecidos/métodos , Animais , Organoides/anatomia & histologia , Ratos , Ratos Wistar , Fatores de Tempo
16.
World J Gastroenterol ; 12(12): 1881-8, 2006 Mar 28.
Artigo em Inglês | MEDLINE | ID: mdl-16609994

RESUMO

AIM: To develop the effective technology for reconstruction of a liver organ in vitro using a bio-artificial liver. METHODS: We previously reported that a radial-flow bioreactor (RFB) could provide a three-dimensional high-density culture system. We presently reconstructed the liver organoid using a functional human hepatocellular carcinoma cell line (FLC-5) as hepatocytes together with mouse immortalized sinusoidal endothelial cell (SEC) line M1 and mouse immortalized hepatic stellate cell (HSC) line A7 as non parenchymal cells in the RFB. Two x 10(7) FLC-5 cells were incubated in the RFB. After 5 d, 2 x 10(7) A7 cells were added in a similar manner followed by another addition of 10(7) M1 cells 5 d later. After three days of perfusion, some cellulose beads with the adherent cells were harvested. The last incubation period included perfusion with 200 nmol/L swinholide A for 2 h and then the remaining cellulose beads along with adherent cells were harvested from the RFB. The cell morphology was observed by transmission electron microscopy (TEM) and scanning electron microscopy (SEM). To assess hepatocyte function, we compared mRNA expression for urea cycle enzymes as well as albumin synthesis by FLC-5 in monolayer cultures compared to those of single-type cultures and cocultures in the RFB. RESULTS: By transmission electron microscopy, FLC-5, M1, and A7 were arranged in relation to the perfusion side in a liver-like organization. Structures resembling bile canaliculi were seen between FCL-5 cells. Scanning electron microscopy demonstrated fenestrae on SEC surfaces. The number of vesiculo-vacuolar organelles (VVO) and fenestrae increased when we introduced the actin-binding agent swinholide-A in the RFB for 2h. With respect to liver function, urea was found in the medium, and expression of mRNAs encoding arginosuccinate synthetase and arginase increased when the three cell types were cocultured in the RFB. However, albumin synthesis decreased. CONCLUSION: Co-culture in the RFB system can dramatically change the structure and function of all cell types, including the functional characteristics of hepatocytes. Our system proves effective for reconstruction of a liver organoid using a bio-artificial liver.


Assuntos
Reatores Biológicos , Biotecnologia/métodos , Fígado Artificial , Organoides/anatomia & histologia , Organoides/fisiologia , Albuminas/análise , Aminoácidos/análise , Animais , Arginase/análise , Argininossuccinato Sintase/análise , Linhagem Celular , Técnicas de Cocultura , Hepatócitos/citologia , Hepatócitos/fisiologia , Humanos , Toxinas Marinhas , Camundongos , Microesferas , RNA Mensageiro , Reação em Cadeia da Polimerase Via Transcriptase Reversa
17.
ASAIO J ; 50(5): 468-72, 2004.
Artigo em Inglês | MEDLINE | ID: mdl-15497387

RESUMO

The objective of this study is to assess the feasibility of creating a tissue engineered stomach using isolated stomach epithelium organoid unit from syngeneic adult donors and a biodegradable polymer scaffold in a rat model. Despite recent advances in reconstruction techniques, total gastrectomy is still accompanied by various complications. As an alternative treatment, a tissue engineered stomach that replaces the mechanical and metabolic functions of a normal stomach is proposed. Stomach epithelium organoid units were isolated from syngeneic adult rats and seeded onto biodegradable polymers. These constructs were implanted into the omenta of recipient adult rats. All constructs were harvested for histologic and immunohistochemical examination at designated time points. Cyst-like structures were formed that showed the development of vascularized tissue with a neomucosa. Immunohistochemical staining for alpha-actin smooth muscle, gastric mucin, and proton pump indicated the presence of a smooth muscle layer and gastric epithelium, as well as the existence of parietal cells of the stomach mucosa, respectively. Epithelium derived stomach organoid units seeded on biodegradable polymers were transplanted in donor rats and have been shown to vascularize, survive, and regenerate into complex tissue resembling a native stomach. These initial results are encouraging, and studies are currently underway to further assess this approach.


Assuntos
Implantes Absorvíveis , Mucosa Gástrica/fisiologia , Estômago/fisiologia , Engenharia Tecidual/métodos , Actinas/metabolismo , Animais , Mucosa Gástrica/anatomia & histologia , Imuno-Histoquímica , Ácido Láctico , Músculo Liso/metabolismo , Organoides/anatomia & histologia , Organoides/fisiologia , Poliésteres , Ácido Poliglicólico , Polímeros , Bombas de Próton/metabolismo , Ratos , Ratos Endogâmicos Lew , Estômago/anatomia & histologia
18.
Invest Ophthalmol Vis Sci ; 18(7): 655-64, 1979 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-109410

RESUMO

The ciliary bodies of five monkey eyes and one human eye were subdivided into five zones. The ciliary epithelium with its bordering stroma was investigated electron microscopically. The number of cell organelles of the nonpigmented (NPE) and pigmented (PE) epithelium (mitochondria, rough endoplasmic reticulum, Golgi complexes); intercellular junctions between NPE and NPE, PE and PE, and NPE and PE (desmosomes, puncta adhaerentia, gap junctions, tight junctions); and fenestrations of the capillary endothelium were quantitatively evaluated. All these types of cell organelles, fenestrations of the capillary endothelium, and gap junctions in the NPE were found in greater numbers at the crests of the ciliary processes than in the valleys between processes. On the other hand, the number of puncta adhaerentia is significantly higher in the valleys than at the crests. In the valleys, the internal limiting membrane performs an elaborate network of electron-dense strands in which many fine zonular fibers terminate. These fibers are believed to belong to the "tension fiber system." Their firm attachment to the ciliary epithelium and the great number of intercellular junctions known as mechanical structures lend further support to our concept that these structures function as a fulcrum in the process of accommodation.


Assuntos
Acomodação Ocular , Corpo Ciliar/ultraestrutura , Animais , Corpo Ciliar/análise , Corpo Ciliar/citologia , Haplorrinos , Humanos , Macaca fascicularis , Microscopia Eletrônica de Varredura , Organoides/anatomia & histologia , Organoides/ultraestrutura , Epitélio Pigmentado Ocular/ultraestrutura
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