Organotypic cultures preparation of organotypic cultures another 3d model of tumor microenvironment is the reconstruction of tissue by growing cells in extracellular matrix. To establish an in vitro system which would enable us to observe scn functions over a long period, we developed a new method to culture scn tissues by means of a roller tube. To understand how mammalian organs can be cultured ex vivo, it is useful to consider their constituent parts. Glioblastoma multiforme gbm is one of the most aggressive brain tumors. Gel and sponge techniques, rotating chambers, and hollow fibers are described, and protocols provided for spheroid generation, neuronal aggregates, alginate. This airlift procedure is defined as the start of the culture time of organotypic cocultures. Organotypic cultures as a model to study adult neurogenesis. These co culture models thus offer possibilities for analysis and evaluation that may give valuable insights both in basic research and in the development of innovative cellbased clinical applications. This protocol describes how to dissect, assemble, and cultivate mouse embryonic e brain tissue from age e11. Histotypic tissue culture protocol with the increase use of detached cells since the s, the term tissue culture was substituted by cell culture. Organotypic culture freshney major reference works. The external surfaces of the body and the linings of organs are built from epithelial tissues 8. Characterization of a threedimensional 3d organotypic skin.
Time to time cotton is moistened so that excessive evaporation should not occur. Organotypic slice cultures from embryonic rodent brains are widely used to study brain development. Cns myelination disturbances commonly occur in chronic white matter lesions in neurodevelopmental and adult neurological disorders. I am using these inserts on an everyday basis to culture hypothalamoneurohypophysial hns explants, which are even more finicky then regular e. Organ culture gastrointestinal biopsy dec232007 hello, i was looking in for any protocols for culturing gastrointestinal biopsy tissues, i will be using the tissues for virus culture, hence will also have to monitor the viability of the cells, so as to rule out false results of viral replication. The spinner culture method creates spheroids by preventing cells in suspension from. Threedimensional organotypic culture of stratified epithelia. These preparations can be used for a variety of assays and studies including coculture of different brain regions, cell migration assays, axon guidance assays, and dna electroporation. Development of white matter gliosis in ex vivo slice culture model. Histotypic culture cells have been re aggregated or grown to recreate.
This video introduces histotypic tissue culture, where the growth and propagation of one cell. Adult neurogenesis occurs throughout life, especially from the subgranular zone of hippocampus and the subventricular zone, and can be modulated in physiological and pathological conditions. Ravi vm, joseph k, wurm j, behringer s, garrelfs n, derrico p, naseri y, franco p, meyerluehmann m, sankowski r, shah mj, mader i, delev d, follo m, beck j, schnell o, hofmann ug, heiland dh 2019 human organotypic brain slice culture. Neurodegenerative diseases and a wide variety of inflammatory conditions are linked together and several antiinflammatory compounds considered as having therapeutic potential for example in alzheimers disease ad.
A refined in vitro model to study inflammatory responses in. A read is counted each time someone views a publication summary such as the title, abstract, and list of authors, clicks on a figure, or views or downloads the fulltext. For organ culture care should be taken to handle in such a way that tissue should not be damaged. Let us make indepth study of the definition, history and importance of the organ culture. Organotypic slice cultures to study oligodendrocyte. When we compared our co culture imaging results at 24 h to the co culture colonization data at the same time point fig. Qi xr, verwer rwh, bao am, balesar ra, luchetti s, zhou jn, swaab df 2019 human brain slice culture. Organ culture on plasma clots a plasma clot is prepared by mixing five drops of embryo extract with 15 drops of plasma in a watch glass placed on a cotton wool pad. Organotypic cultures represent tumor microenvironment for. Organotypic slice cultures to study oligodendrocyte dynamics. These cultures are relatively simple to adopt and retain many benefits of primary dissociated cell cultures such as manipulation of the extracellular environment and easy access for repeated longterm live cell imaging and. The traditional approach with the tissue chopper tends to tear and stretch the tissue.
Fef3, fetal human esophageal fibroblasts a standard fibroblast line and other fibroblast lines adultdermalcaf, etc. Organotypic explant culture of glioblastoma multiforme and. Threedimensional organotypic coculture model of intestinal epithelial cells and macrophages to study salmonella enterica colonization patterns jennifer barrila 1, jiseon yang, aurelie crabbe 1,2, shameema f. Numerous techniques and animal models have been developed to demonstrate and observe neural regeneration but, in order to study the. Organotypic culture models \ocms\, when developed and evaluated, will provide information needed to help predict toxicity due to chemical exposures. Experimental models of mammalian biology and disease. The developed ocms will provide the biological understanding and data to help improve, evaluate and ext\ end computational models currently being developed by epa scientists. Hippocampal organotypic culture how is hippocampal organotypic culture abbreviated.
Neural regeneration resides in certain specific regions of adult cns. Finally, organotypic brain slice cultures markedly reduce the number of severe animal experiments contributing to the 3rs reduce, refine, replace. Organotypic culture freshney major reference works wiley. Primary cell organotypic culture pharmacology faculty. The practice of cultivating cells started at the beginning of the 20th century and was developed from a simple exploratory phase to an expansion phase in the s. While there are often advantages to an invivo system, organotypic slice cultures allow one to perform a number of manipulations that are not presently feasible invivo. Therefore, organ culture technique demands more tactful manipulation than tissue culture. Put the plates in the cell culture incubator at 37 c with 5% co 2 at least 2 hr prior to dissection. Untreated samples show less than 2% piincorporation and caspase activity of staurosporinetreated samples, showing that the tissue is viable. Culture medium was changed, and, by lowering the medium level to the lower part of the gels, the cultures were raised to the airliquid interphase thus restricting nourishment to diffusion from below. The development of dynamic macroscale bioreactor systems is regarded as breakthrough in cell culture technology.
There are three main types of three dimensional cultures. A useful tool to study brain disorders and potential therapeutic compounds. The term histotypic culture implies that cells havebeen reaggregated or grown to recreate a threedimensional structure with. For example, cultures may be grown submerged in culture medium and or at an airliquid interface. Organotypic slice culture of the mammalian retina visual. Meaapplications organotypic cultures of hippocampal slice. Materials and methods 1 intestinal organotypic culture experiments animals while the aim of the study was to develop an equine organotypic culture, most of the experimental work used sheep intestine. Traditionally, twodimensional cell culture was used to perform in vitro research, but its efficiency has been called into. Finally, organotypic brain slice cultures markedly reduce the number of severe animal experiments contributing to. When we compared our coculture imaging results at 24 h to the coculture colonization data at the same time point fig.
Organotypic slice cultures from hippocampus were prepared using the modified interface culture method described by stoppini et al. An organotypic culture system ocs allows for the in vitro growth of complex biological tissues in a way that replicates part of their normal physiology and function. Physiological relevance is a key parameter to improve prediction made by cellbased assays. We have devised a culture system that recreates the salient features of the stratified epithelium using primary cell cultures from mouse models. Information and translations of organotypic in the most comprehensive dictionary definitions resource on the web. Development of organotypic liver cell cultures in threedimensional biomaterial hydrogels melina malinen academic dissertation to be publicly discussed, with the permission of the faculty of pharmacy of the university of helsinki, in lecture hall 1, infocenter korona, viikinkaari 11, on september 1 st, 2014, at 12 noon. The extracellular matrix is a network of structural proteins that create a three dimensional environment for cells to occupy, and the cells within it are meant to recapitulate the native physiological processes of the tissue. Organoytpic slice cultures of the central nervous system have proven to be extremely useful for studying neuron and glial cell biology in a semiintact system 1 4. A refined in vitro model to study inflammatory responses. Advances in the formation, use and understanding of multicellular.
Organ culture not whole but pieces of organs can be cultured on artificial medium. Modeling nigrostriatal degeneration in organotypic. Control of culture conditions temperature and ph 4. One of the limitations of conventional tissue culture on flat twodimensional surfaces is the loss of complex interactions between the epithelium and stroma. Mar 11, 2020 iam making organotypic culture with collagen embedded fibroblast. Threedimensional organotypic culture models of human.
Hippocampal organotypic culture how is hippocampal. Sterilize all tools and tissue chopper with 70% ethanol. Modeling nigrostriatal degeneration in organotypic cultures. These gap junctions are much more prevalent in 3d cell culture than 2d and these junctions enable cells to communicate with each other via exchange of ions, small molecules and even electrical currents. An organotypic slice culture model of chronic white matter. Proapoptotic action of corticosterone in hippocampal. The advantage with this culture system is that dispersed monolayer cultures can be used to regenerate tissuelike structures. The development of robust vascular networks in 3d culture 10,11 should enable the generation of larger and more complex tissue constructs 151, improve transplant efficiency 10 and catalyse a transition in 3d culture from the selforganization of simple tissues to the synthetic assembly of multicomponent organs. Tonimarie achilli, julia meyer, and jeffrey r morgan. Changing the file name may cause delays in the processing of your application. In a culture hood, place tissue culture inserts in six well plates and add 1 ml slice culture medium recipe in reagents section. Advances in the formation, use and understanding of multicellular spheroids. For the first time, grow fef3 p5p6 in two 150mm2 dishes until they reach 100%. Primary cell organotypic culture department of pharmacology.
Methods for developing 3d organotypic models differ according to the tissue of interest and the experimental design. Epithelial cells are connected to each other by intercellular junctions and are located within a specialized ecm, which is known as the basement membrane. Organotypic slice culture of the mammalian retina volume 10 issue 2 andreas. Threedimensional 3d culture protocols have been developed for diverse tissues, organs and disease states. In pursuit of this goal, a wide range of techniques has been developed that are referred to as 3d culture, organotypic culture or organoid culture.
Nov 15, 2005 propagated tissue degeneration, especially during aging, has been shown to be enhanced through potentiation of innate immune responses. These coculture models thus offer possibilities for analysis and evaluation that may give valuable insights both in basic research and in the development of innovative cellbased clinical applications. Such a complex brain culture system might provide an excellent model for studying neurodegenerative brain diseases, including e. A versatile prion replication assay in organotypic slices. Primary keratinocytes have finite lifespan in culture, which greatly restricts their use as an invitro cellular model. Thus, data obtained in cell lines are often inapplicable to patient tumors.
Although twodimensional tissue culture has been common for some time, cells behave more. Millicell organotypic insert from millipore biocompare. Organotypic tissue culture for substance evaluation. T1 modeling nigrostriatal degeneration in organotypic cultures, a new ex vivo model of parkinsons disease. Organotypic slice culture of embryonic brain tissue. Characterization of a threedimensional 3d organotypic. Thereafter, a small piece of organ tissue is placed on. Organotypic slice culture of the suprachiasmatic nucleus. L slice culture media scm per well and place cell culture inserts in each well. Definitions the organ culture refers to the in vitro culture and maintenance of an excised organ primordia or whole or part of an organ in a way that may allow differentiation and preservation of the architecture andor function. Our group is focusing on an airliquid interface 3d organotypic model. The constitutive overexpression of the catalytic subunit of telomerase, htert, allows for extended passaging of cells with the benefit of little loss of normal cellular. Threedimensional organotypic coculture model of intestinal.
Growth and propagation of cell lines in three dimensional matrix to high cell density represent histotypic cultures. Recent studies support that myelination failure can involve a disrupted cellular repair mechanism where oligodendrocyte ol progenitor cells opcs proliferate in lesions with diffuse astrogliosis, but fail to fully differentiate to mature myelinating ols. Gbm cell lines used in laboratory studies are frequently passaged in various culture media at high proliferation rates, resulting in significant genetic and molecular alterations. It challenges the problem of multiorgan in vitro systems and tries to estimate, how far in vitro organs are from being valid systems for industrial high content substance testing. Cultures formed from reaggregated cells are always called organotypic cultures, whereas tissue explants are called both organandorganotypic culture interchangeably. Because epidermis and other skin components, such as hair follicles, can be readily maintained in vitro, ocss have gained broad popularity in dermatological research. Owing to their inherent complexity, most adult tissues cannot be easily maintained in vitro.