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Stem Cells and Development Jan 2010Recent advances in the biology of stem cells has resulted in significant interest in the development of normal epithelial cell lines from the intestinal mucosa, both to... (Review)
Review
Recent advances in the biology of stem cells has resulted in significant interest in the development of normal epithelial cell lines from the intestinal mucosa, both to exploit the therapeutic potential of stem cells in tissue regeneration and to develop treatment models of degenerative disorders of the digestive tract. However, the difficulty of propagating cell lines of normal intestinal epithelium has impeded research into the molecular mechanisms underlying differentiation of stem/progenitor cells into the various intestinal lineages. Several short-term organ/organoid and epithelial cell culture models have been described. There is a dearth of long-term epithelial and/or stem cell cultures of intestine. With an expanding role of stem cells in the treatment of degenerative disorders, there is a critical need for additional efforts to develop in vitro models of stem/progenitor epithelial cells of intestine. The objective of this review is to recapitulate the current status of technologies and knowledge for in vitro propagation of intestinal epithelial cells, markers of the intestinal stem cells, and gene and protein expression profiles of the intestinal cellular differentiation.
Topics: Algorithms; Animals; Cell Differentiation; Cells, Cultured; Epithelial Cells; Gene Expression Profiling; Humans; Intestinal Mucosa; Models, Biological; Organ Culture Techniques; Tissue Engineering
PubMed: 19580443
DOI: 10.1089/scd.2009.0109 -
The Journal of Investigative Dermatology Nov 2013
Review
Topics: Biomedical Research; Dermatology; Education, Medical, Continuing; Humans; Keratinocytes; Organ Culture Techniques; Skin
PubMed: 24129782
DOI: 10.1038/jid.2013.387 -
European Cells & Materials Feb 2019Priming towards a discogenic phenotype and subsequent cryopreservation of microencapsulated bone marrow stromal cells (BMSCs) may offer an attractive therapeutic...
Priming towards a discogenic phenotype and subsequent cryopreservation of microencapsulated bone marrow stromal cells (BMSCs) may offer an attractive therapeutic approach for disc repair. It potentially obviates the need for in vivo administration of exogenous growth factors, otherwise required to promote matrix synthesis, in addition to providing 'off-the-shelf' availability. Cryopreserved and primed BMSC microcapsules were evaluated in an in vitro surrogate co-culture model system with nucleus pulposus (NP) cells under intervertebral disc (IVD)-like culture conditions and in an ex vivo bovine organ culture disc model. BMSCs were microencapsulated in alginate microcapsules and primed for 14 d with transforming growth factor beta-3 (TGF-β3) under low oxygen conditions prior to cryopreservation. For the in vitro phase, BMSC microcapsules (unprimed or primed) were cultured for 28 d in a surrogate co-culture model system mimicking that of the IVD. For the ex vivo phase, microcapsules (unprimed or primed) were injected into the NP of bovine discs that underwent nucleotomy. In vitro results revealed that although NP cells produced significantly more matrix components in co-culture with BMSC microcapsules regardless of the differentiation state, unprimed microcapsules were inadequate at synthesising matrix as compared to primed microcapsules. However, this difference was diminished when evaluated in the ex vivo organ culture model,withboth unprimed and primed BMSC microcapsules accumulating large amounts of sulphated glycosaminoglycan (sGAG) and collagen and filling the defect cavity. Both models demonstrated that cryopreservation of BMSC microcapsules may offer a feasible strategy for predesigned delivery through cryobanking for on-demand regeneration of the IVD.
Topics: Animals; Cattle; Cell Proliferation; Cell Separation; Cell Survival; Coculture Techniques; Collagen; Cryopreservation; DNA; Glycosaminoglycans; Intervertebral Disc; Microspheres; Organ Culture Techniques; Regeneration; Stromal Cells; Swine
PubMed: 30768674
DOI: 10.22203/eCM.v037a09 -
Journal of Visualized Experiments : JoVE Feb 2010In all mammals, the sensory epithelium for audition is located along the spiraling organ of Corti that resides within the conch shaped cochlea of the inner ear (fig 1)....
In all mammals, the sensory epithelium for audition is located along the spiraling organ of Corti that resides within the conch shaped cochlea of the inner ear (fig 1). Hair cells in the developing cochlea, which are the mechanosensory cells of the auditory system, are aligned in one row of inner hair cells and three (in the base and mid-turns) to four (in the apical turn) rows of outer hair cells that span the length of the organ of Corti. Hair cells transduce sound-induced mechanical vibrations of the basilar membrane into neural impulses that the brain can interpret. Most cases of sensorineural hearing loss are caused by death or dysfunction of cochlear hair cells. An increasingly essential tool in auditory research is the isolation and in vitro culture of the organ explant. Once isolated, the explants may be utilized in several ways to provide information regarding normative, anomalous, or therapeutic physiology. Gene expression, stereocilia motility, cell and molecular biology, as well as biological approaches for hair cell regeneration are examples of experimental applications of organ of Corti explants. This protocol describes a method for the isolation and culture of the organ of Corti from neonatal mice. The accompanying video includes stepwise directions for the isolation of the temporal bone from mouse pups, and subsequent isolation of the cochlea, spiral ligament, and organ of Corti. Once isolated, the sensory epithelium can be plated and cultured in vitro in its entirety, or as a further dissected micro-isolate that lacks the spiral limbus and spiral ganglion neurons. Using this method, primary explants can be maintained for 7-10 days. As an example of the utility of this procedure, organ of Corti explants will be electroporated with an exogenous DsRed reporter gene. This method provides an improvement over other published methods because it provides reproducible, unambiguous, and stepwise directions for the isolation, microdissection, and primary culture of the organ of Corti.
Topics: Animals; Dissection; Electroporation; Fluorescent Dyes; Luminescent Proteins; Mice; Organ Culture Techniques; Organ of Corti; Plasmids
PubMed: 20134402
DOI: 10.3791/1685 -
The Journal of Cell Biology Jan 2018The biology of aging is challenging to study, particularly in humans. As a result, model organisms are used to approximate the physiological context of aging in humans.... (Review)
Review
The biology of aging is challenging to study, particularly in humans. As a result, model organisms are used to approximate the physiological context of aging in humans. However, the best model organisms remain expensive and time-consuming to use. More importantly, they may not reflect directly on the process of aging in people. Human cell culture provides an alternative, but many functional signs of aging occur at the level of tissues rather than cells and are therefore not readily apparent in traditional cell culture models. Organoids have the potential to effectively balance between the strengths and weaknesses of traditional models of aging. They have sufficient complexity to capture relevant signs of aging at the molecular, cellular, and tissue levels, while presenting an experimentally tractable alternative to animal studies. Organoid systems have been developed to model many human tissues and diseases. Here we provide a perspective on the potential for organoids to serve as models for aging and describe how current organoid techniques could be applied to aging research.
Topics: Aging; Animals; Cell Culture Techniques; Cell Proliferation; Humans; Models, Theoretical; Organ Culture Techniques; Organoids; Pluripotent Stem Cells
PubMed: 29263081
DOI: 10.1083/jcb.201709054 -
In Vitro Cellular & Developmental... Jan 2011We have recently shown that a multi-mineral extract from the marine red algae, Lithothamnion calcareum, suppresses colon polyp formation and inflammation in mice. In the...
We have recently shown that a multi-mineral extract from the marine red algae, Lithothamnion calcareum, suppresses colon polyp formation and inflammation in mice. In the present study, we used intact human colon tissue in organ culture to compare responses initiated by Ca(2+) supplementation versus the multi-mineral extract. Normal human colon tissue was treated for 2 d in culture with various concentrations of calcium or the mineral-rich extract. The tissue was then prepared for histology/immunohistochemistry, and the culture supernatants were assayed for levels of type I procollagen and type I collagen. At higher Ca(2+) concentrations or with the mineral-rich extract, proliferation of epithelial cells at the base and walls of the mucosal crypts was suppressed, as visualized by reduced Ki67 staining. E-cadherin, a marker of differentiation, was more strongly expressed at the upper third of the crypt and at the luminal surface. Treatment with Ca(2+) or with the multi-mineral extract influenced collagen turnover, with decreased procollagen and increased type I collagen. These data suggest that calcium or mineral-rich extract has the capacity to (1) promote differentiation in human colon tissue in organ culture and (2) modulate stromal function as assessed by increased levels of type I collagen. Taken together, these data suggest that human colon tissue in organ culture (supporting in vivo finding in mice) will provide a valuable model for the preclinical assessment of agents that regulate growth and differentiation in the colonic mucosa.
Topics: Biomarkers; Cadherins; Calcium; Cell Differentiation; Collagen Type I; Colon; Colonic Polyps; Complex Mixtures; Histological Techniques; Humans; Immunohistochemistry; Models, Biological; Organ Culture Techniques; Rhodophyta
PubMed: 21104039
DOI: 10.1007/s11626-010-9358-3 -
Microcirculation (New York, N.Y. : 1994) Nov 2019Immense progress in microscale engineering technologies has significantly expanded the capabilities of in vitro cell culture systems for reconstituting physiological... (Review)
Review
Immense progress in microscale engineering technologies has significantly expanded the capabilities of in vitro cell culture systems for reconstituting physiological microenvironments that are mediated by biomolecular gradients, fluid transport, and mechanical forces. Here, we examine the innovative approaches based on microfabricated vessels for studying lymphatic biology. To help understand the necessary design requirements for microfluidic models, we first summarize lymphatic vessel structure and function. Next, we provide an overview of the molecular and biomechanical mediators of lymphatic vessel function. Then we discuss the past achievements and new opportunities for microfluidic culture models to a broad range of applications pertaining to lymphatic vessel physiology. We emphasize the unique attributes of microfluidic systems that enable the recapitulation of multiple physicochemical cues in vitro for studying lymphatic pathophysiology. Current challenges and future outlooks of microscale technology for studying lymphatics are also discussed. Collectively, we make the assertion that further progress in the development of microscale models will continue to enrich our mechanistic understanding of lymphatic biology and physiology to help realize the promise of the lymphatic vasculature as a therapeutic target for a broad spectrum of diseases.
Topics: Animals; Humans; Lab-On-A-Chip Devices; Lymphatic Vessels; Microfluidic Analytical Techniques; Organ Culture Techniques
PubMed: 30946511
DOI: 10.1111/micc.12547 -
Japanese Journal of Pharmacology Jun 2002The benefit of organ culture is to retain the original structural relationship between various cell species and their interactions and enable us to study the long-term... (Review)
Review
The benefit of organ culture is to retain the original structural relationship between various cell species and their interactions and enable us to study the long-term effects of exogenous stimuli. Organ culture methods have been used especially in the studies of the proliferative vascular diseases, such as atherosclerosis and restenosis. We describe here that organ culture is a useful in vitro method to study the biology of vascular and other smooth muscle organs.
Topics: Animals; Blood Vessels; Humans; Muscle, Smooth; Organ Culture Techniques
PubMed: 12120766
DOI: 10.1254/jjp.89.93 -
ACS Infectious Diseases Mar 2018Each year there are more than 15 000 cases of human disease caused by infections with tick-borne viruses (TBVs). These illnesses occur worldwide and can range from... (Review)
Review
Each year there are more than 15 000 cases of human disease caused by infections with tick-borne viruses (TBVs). These illnesses occur worldwide and can range from very mild illness to severe encephalitis and hemorrhagic fever. Although TBVs are currently identified as neglected vector-borne pathogens and receive less attention than mosquito-borne viruses, TBVs are expanding into new regions, and infection rates are increasing. Furthermore, effective vaccines, diagnostic tools, and other countermeasures are limited. The application of contemporary technologies to TBV infections presents an excellent opportunity to develop improved, effective countermeasures. Experimental tick and mammal models of infection can be used to characterize determinants of infection, transmission, and virulence and to test candidate countermeasures. The use of ex vivo tick cultures in TBV research provides a unique way to look at infection in specific tick organs. Mammal ex vivo organ slice and, more recently, organoid cultures are additional models that can be used to elucidate direct tissue-specific responses to infection. These ex vivo model systems are convenient for testing methods involving transcript knockdown and small molecules under tightly controlled conditions. They can also be combined with in vitro and in vivo studies to tease out possible host factors and potential vaccine or therapeutic candidates. In this brief perspective, we describe how ex vivo cultures can be combined with modern technologies to advance research on TBV infections.
Topics: Animals; Encephalitis Viruses, Tick-Borne; Mammals; Models, Theoretical; Organ Culture Techniques; Virology
PubMed: 29473735
DOI: 10.1021/acsinfecdis.7b00274 -
Journal of Visualized Experiments : JoVE Jun 2011Ovarian cancer is the fifth leading cause of cancer deaths in women and has a 63% mortality rate in the United States(1). The cell type of origin for ovarian cancers is...
Ovarian cancer is the fifth leading cause of cancer deaths in women and has a 63% mortality rate in the United States(1). The cell type of origin for ovarian cancers is still in question and might be either the ovarian surface epithelium (OSE) or the distal epithelium of the fallopian tube fimbriae(2,3). Culturing the normal cells as a primary culture in vitro will enable scientists to model specific changes that might lead to ovarian cancer in the distinct epithelium, thereby definitively determining the cell type of origin. This will allow development of more accurate biomarkers, animal models with tissue-specific gene changes, and better prevention strategies targeted to this disease. Maintaining normal cells in alginate hydrogels promotes short term in vitro culture of cells in their three-dimensional context and permits introduction of plasmid DNA, siRNA, and small molecules. By culturing organs in pieces that are derived from strategic cuts using a scalpel, several cultures from a single organ can be generated, increasing the number of experiments from a single animal. These cuts model aspects of ovulation leading to proliferation of the OSE, which is associated with ovarian cancer formation. Cell types such as the OSE that do not grow well on plastic surfaces can be cultured using this method and facilitate investigation into normal cellular processes or the earliest events in cancer formation(4). Alginate hydrogels can be used to support the growth of many types of tissues(5). Alginate is a linear polysaccharide composed of repeating units of β-D-mannuronic acid and α-L-guluronic acid that can be crosslinked with calcium ions, resulting in a gentle gelling action that does not damage tissues(6,7). Like other three-dimensional cell culture matrices such as Matrigel, alginate provides mechanical support for tissues; however, proteins are not reactive with the alginate matrix, and therefore alginate functions as a synthetic extracellular matrix that does not initiate cell signaling(5). The alginate hydrogel floats in standard cell culture medium and supports the architecture of the tissue growth in vitro. A method is presented for the preparation, separation, and embedding of ovarian and oviductal organ pieces into alginate hydrogels, which can be maintained in culture for up to two weeks. The enzymatic release of cells for analysis of proteins and RNA samples from the organ culture is also described. Finally, the growth of primary cell types is possible without genetic immortalization from mice and permits investigators to use knockout and transgenic mice.
Topics: Alginates; Animals; Female; Hydrogels; Mice; Organ Culture Techniques; Ovary; Oviducts
PubMed: 21712801
DOI: 10.3791/2804