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Experimental Dermatology Mar 2010The organ culture of human scalp hair follicles (HFs) is the best currently available assay for hair research in the human system. In order to determine the hair...
The organ culture of human scalp hair follicles (HFs) is the best currently available assay for hair research in the human system. In order to determine the hair growth-modulatory effects of agents in this assay, one critical read-out parameter is the assessment of whether the test agent has prolonged anagen duration or induced catagen in vitro. However, objective criteria to distinguish between anagen VI HFs and early catagen in human HF organ culture, two hair cycle stages with a deceptively similar morphology, remain to be established. Here, we develop, document and test an objective classification system that allows to distinguish between anagen VI and early catagen in organ-cultured human HFs, using both qualitative and quantitative parameters that can be generated by light microscopy or immunofluorescence. Seven qualitative classification criteria are defined that are based on assessing the morphology of the hair matrix, the dermal papilla and the distribution of pigmentary markers (melanin, gp100). These are complemented by ten quantitative parameters. We have tested this classification system by employing the clinically used topical hair growth inhibitor, eflornithine, and show that eflornithine indeed produces the expected premature catagen induction, as identified by the novel classification criteria reported here. Therefore, this classification system offers a standardized, objective and reproducible new experimental method to reliably distinguish between human anagen VI and early catagen HFs in organ culture.
Topics: Apoptosis; Cell Proliferation; Eflornithine; Female; Hair; Hair Follicle; Humans; Ki-67 Antigen; Organ Culture Techniques
PubMed: 19725870
DOI: 10.1111/j.1600-0625.2009.00939.x -
The American Journal of Pathology Jan 1995Proteinase levels were assessed in organ culture fluids from human neonatal foreskin maintained under growth factor-free conditions and in the presence of a combination...
Proteinase levels were assessed in organ culture fluids from human neonatal foreskin maintained under growth factor-free conditions and in the presence of a combination of growth factors (ie, epidermal growth factor, insulin, hydrocortisone, pituitary extract, and all-trans-retinoic acid). Analysis of culture fluids by gelatin zymography revealed the presence of 92-kd and 72-kd gelatinases. There was a greater amount of 92-kd gelatinase activity in the presence of growth factors whereas the levels of 72-kd gelatinase were similar in growth factor-free and growth factor-containing media. Experiments with keratinocytes and fibroblasts in monolayer culture and with isolated dermal tissue in organ culture indicated that the epithelial component was responsible for most of the 92-kd gelatinase activity whereas fibroblasts were primarily responsible for the 72-kd gelatinase activity. Activation with aminophenyl mercuric acetate, requirement for divalent cations, inhibition with EDTA, and insensitivity to inhibition with phenylmethyl sulfonyl fluoride indicated that both gelatinases were metalloproteinases. In additional studies, culture fluids were examined for the presence of plasminogen activator activity. This was detected in culture fluids from tissues maintained under both conditions but was increased in the growth factor-containing medium. The increased amount seen in the growth factor-containing medium appeared to be due almost entirely to a single factor, ie, all-trans-retinoic acid. In monolayer culture, both keratinocytes and fibroblasts produced plasminogen activator; the level was higher in keratinocyte culture fluids than in culture fluids from fibroblasts.
Topics: Cells, Cultured; Fibroblasts; Growth Substances; Humans; Keratinocytes; Metalloendopeptidases; Organ Culture Techniques; Serine Endopeptidases; Skin
PubMed: 7856729
DOI: No ID Found -
Der Ophthalmologe : Zeitschrift Der... Jul 2020The appearance of the novel severe acute respiratory syndrome-coronavirus-2 (SARS-CoV‑2) poses challenges in ophthalmology particularly for eye banks. A valid risk... (Review)
Review
The appearance of the novel severe acute respiratory syndrome-coronavirus-2 (SARS-CoV‑2) poses challenges in ophthalmology particularly for eye banks. A valid risk assessment for the removal and processing of donor corneas is difficult due to the lack of data. The risk to infect transplant recipients with SARS-CoV‑2 still appears very unlikely due to the experience with severe acute respiratory syndrome -coronavirus(‑1) (SARS-CoV(‑1)) and Middle East respiratory syndrome-coronavirus (MERS-CoV); however, due to the occurrence of angiotensin-converting enzyme 2 (ACE2) receptors in the cornea an infection of this tissue with SARS-CoV‑2 cannot be completely excluded. Therefore, routine testing of the organ culture medium used for donor corneas for SARS-CoV‑2 prior to transplantation during the coronavirus disease 2019 (COVID‑19) pandemic should be considered.
Topics: Betacoronavirus; COVID-19; Cornea; Coronavirus Infections; Humans; Organ Culture Techniques; Pandemics; Pneumonia, Viral; SARS-CoV-2
PubMed: 32556552
DOI: 10.1007/s00347-020-01152-z -
Annals of Biomedical Engineering Nov 2010Previous research in our lab suggested that heart valve tissues cultured without mechanical stimulation do not retain their in vivo microstructure, i.e., cell density...
Previous research in our lab suggested that heart valve tissues cultured without mechanical stimulation do not retain their in vivo microstructure, i.e., cell density decreased within the deep tissue layers and increased at the periphery. In this study, a splashing rotating bioreactor was designed to apply mechanical stimulation to a mitral valve leaflet segment. Porcine valve segments (n = 9-10 per group) were cultured in the bioreactor for 2 weeks (dynamic culture), negative controls were cultured without mechanical stimulation (static culture), and baseline controls were fresh uncultured samples. Overall changes in cellularity and extracellular matrix (ECM) structure were assessed by H&E and Movat pentachrome stains. Tissues were also immunostained for multiple ECM components and turnover mediators. After 2 weeks of culture, proliferating cells were distributed throughout the tissue in segments cultured in the bioreactor, in contrast to segments cultured without mechanical stimulation. Most ECM components, especially collagen types I and III, better maintained normal expression patterns and magnitudes (as found in baseline controls) over 2 weeks of dynamic organ culture compared to static culture. Lack of mechanical stimulation changed several aspects of the tissue microstructure, including the cell distribution and ECM locations. In conclusion, mechanical stimulation by the bioreactor maintained tissue integrity, which will enable future in vitro investigation of mitral valve remodeling.
Topics: Animals; Bioreactors; Cell Proliferation; Extracellular Matrix; Organ Culture Techniques; Swine; Tricuspid Valve
PubMed: 20661646
DOI: 10.1007/s10439-010-0129-9 -
International Journal of Experimental... Oct 2004Angiogenesis, the development of new blood vessels from an existing vasculature, is essential in normal developmental processes and in numerous pathologies, including... (Review)
Review
Angiogenesis, the development of new blood vessels from an existing vasculature, is essential in normal developmental processes and in numerous pathologies, including diabetic retinopathy, psoriasis and tumour growth and metastases. One of the problems faced by angiogenesis researchers has been the difficulty of finding suitable methods for assessing the effects of regulators of the angiogenic response. The ideal assay would be reliable, technically straightforward, easily quantifiable and, most importantly, physiologically relevant. Here, we review the advantages and limitations of the principal assays in use, including those for the proliferation, migration and differentiation of endothelial cells in vitro, vessel outgrowth from organ cultures and in vivo assays such as sponge implantation, corneal, chamber, zebrafish, chick chorioallantoic membrane (CAM) and tumour angiogenesis models.
Topics: Angiogenesis Modulating Agents; Animals; Biological Assay; Cell Differentiation; Cell Division; Cell Movement; Endothelial Cells; Endothelium, Vascular; Extraembryonic Membranes; Humans; Neovascularization, Pathologic; Neovascularization, Physiologic; Organ Culture Techniques
PubMed: 15379956
DOI: 10.1111/j.0959-9673.2004.00396.x -
Archives of Dermatological Research 1994Human dermal fibroblast and human epidermal keratinocyte survival was examined under various conditions in organ culture. Using cell recovery from organ-cultured tissue...
Human dermal fibroblast and human epidermal keratinocyte survival was examined under various conditions in organ culture. Using cell recovery from organ-cultured tissue as the criterion, it was observed that no keratinocytes and few fibroblasts survived incubation for 10-12 days in serum-free basal medium containing a low level (0.15 mM) of extracellular Ca2+. Increasing the extracellular Ca2+ concentration to 1.4 mM or treating the tissue with 3 microM retinoic acid (RA) under low Ca2+ conditions resulted in increased keratinocyte and fibroblast survival; the two treatments together were more effective than either treatment alone. The same treatments preserved fibroblast survival when pieces of isolated dermal tissue were incubated in organ culture and also supported fibroblast survival in monolayer culture. These findings indicate that recovery of keratinocytes and fibroblasts from skin after maintenance in organ culture provides a simple but definitive measure of the viability of the major cellular elements present in the tissue. These findings suggest that RA treatment enhances survival of both fibroblasts and keratinocytes and that these effects of RA can be seen at physiological Ca2+ concentrations as well as at suboptimal levels of extracellular Ca2+. Finally, these results indicate that the dermis is a direct target of RA.
Topics: Adult; Calcium; Cell Survival; Fibroblasts; Humans; Keratinocytes; Organ Culture Techniques; Skin; Tretinoin
PubMed: 7864657
DOI: 10.1007/BF00371569 -
Journal of Cellular and Molecular... Mar 2019Organotypic slice culture is a living cell research technique which blends features of both in vivo and in vitro techniques. While organotypic brain slice culture...
Organotypic slice culture is a living cell research technique which blends features of both in vivo and in vitro techniques. While organotypic brain slice culture techniques have been well established in rodents, there are few reports on the study of organotypic slice culture, especially of the central nervous system (CNS), in chicken embryos. We established a combined in ovo electroporation and organotypic slice culture method to study exogenous genes functions in the CNS during chicken embryo development. We performed in ovo electroporation in the spinal cord or optic tectum prior to slice culture. When embryonic development reached a specific stage, green fluorescent protein (GFP)-positive embryos were selected and fluorescent expression sites were cut under stereo fluorescence microscopy. Selected tissues were embedded in 4% agar. Tissues were sectioned on a vibratory microtome and 300 μm thick sections were mounted on a membrane of millicell cell culture insert. The insert was placed in a 30-mm culture dish and 1 ml of slice culture media was added. We show that during serum-free medium culture, the slice loses its original structure and propensity to be strictly regulated, which are the characteristics of the CNS. However, after adding serum, the histological structure of cultured-tissue slices was able to be well maintained and neuronal axons were significantly longer than that those of serum-free medium cultured-tissue slices. As the structure of a complete single neuron can be observed from a slice culture, this is a suitable way of studying single neuronal dynamics. As such, we present an effective method to study axon formation and migration of single neurons in vitro.
Topics: Animals; Central Nervous System; Chick Embryo; Chickens; Electroporation; Embryonic Development; Green Fluorescent Proteins; Nerve Tissue Proteins; Neurons; Organ Culture Techniques
PubMed: 30565384
DOI: 10.1111/jcmm.14080 -
BioTechniques Mar 2017Tissue engineering approaches using growth factors and various materials for repairing chronic perforations of the tympanic membrane are being developed, but there are...
Tissue engineering approaches using growth factors and various materials for repairing chronic perforations of the tympanic membrane are being developed, but there are surprisingly few relevant tissue culture models available to test new treatments. Here, we present a simple three-dimensional model system based on micro-dissecting the rat tympanic membrane umbo and grafting it into the membrane of a cell culture well insert. Cell outgrowth from the graft produced sufficient cells to populate a membrane of similar surface area to the human tympanic membrane within 2 weeks. Tissue grafts from the annulus region also showed cell outgrowth but were not as productive. The umbo organoid supported substantial cell proliferation and migration under the influence of keratinocyte growth medium. Cells from umbo grafts were enzymatically harvested from the polyethylene terephthalate (PET) membrane for expansion in routine culture and cells could be harvested consecutively from the same graft over multiple cycles. We used harvested cells to test cell migration properties and to engraft a porous silk scaffold material as proof-of-principle for tissue engineering applications. This model is simple enough to be widely adopted for tympanic membrane regeneration studies and has promise as a tissue-equivalent model alternative to animal testing.
Topics: Animals; Cell Culture Techniques; Cell Movement; Cells, Cultured; Humans; Organ Culture Techniques; Rats; Tissue Engineering; Tissue Scaffolds; Tympanic Membrane
PubMed: 28298177
DOI: 10.2144/000114523 -
Cells, Tissues, Organs 2016The intervertebral disc (IVD) is a fibrocartilaginous joint between two vertebral bodies. An IVD unit consists of a gelatinous central nucleus pulposus, encased by the...
The intervertebral disc (IVD) is a fibrocartilaginous joint between two vertebral bodies. An IVD unit consists of a gelatinous central nucleus pulposus, encased by the annulus fibrosus, which is sandwiched between cartilaginous endplates (EPs). The IVD homeostasis can be disrupted by injuries, ageing and/or genetic predispositions, leading to degenerative disc disorders and subsequent lower back pain. The complex structure and distinct characteristics of IVDs warrant the establishment of robust in vitro IVD organ culture for studying the etiology and treatment of disc degeneration. Here, we isolate mouse lumbar IVDs and culture the minimal IVD units in submersion or suspension medium supplemented with 2% bovine serum or 10% fetal bovine serum (FBS). We find the minimal IVD units remain healthy for up to 14 days when cultured in submersion culture supplemented with 10% FBS. New bone formation in the EPs of the cultured IVDs can be assessed with calcein labeling. Furthermore, the cultured IVDs can be effectively transduced by recombinant adenovirus, and transgene expression lasts for 2 weeks. Thus, our findings demonstrate that the optimized IVD organ culture system can be used to study IVD biology and screen for biological factors that may prevent, alleviate and/or treat disc degeneration.
Topics: Adenoviridae; Animals; Cell Line; Cell Proliferation; HEK293 Cells; Humans; Intervertebral Disc; Intervertebral Disc Degeneration; Lumbosacral Region; Male; Mice; Organ Culture Techniques; Proliferating Cell Nuclear Antigen; Transduction, Genetic
PubMed: 26447649
DOI: 10.1159/000439268 -
Experimental Dermatology Dec 2015
Topics: Dermatitis; Humans; Microscopy, Confocal; Models, Biological; Organ Culture Techniques; Permeability; Skin
PubMed: 26268366
DOI: 10.1111/exd.12823