Us epa, ord, chemical safety for sustainability subject. T o demonstrate the suitability of the culture system for physiological analyses we performed apoptotic cell death analyses and veri wed photoreceptor viability. Organotypic hippocampal slice cultures ohsc represent an in vitro model that simulates the in vivo situation very well. Since preclinical data obtained from animal models in particular can often not be extrapolated to the human condition, it is expedient to validate at least fundamental biological parameters in human cells with organotypic systems. An organotypic slice culture to study the formation of. Organotypic culture models for predictive toxicology star. Organotypic cultures as tools for functional screening in the cns. New platform for high throughput preclinical human trials. Reiner, bernd heimrichl, flavio keller 2 and helmut l. However, it has been a difficult challenge to culture adult rodent retina. Experimental models of mammalian biology and disease. Changing the file name may cause delays in the processing of your application.
Organotypic cultures as tools for testing neuroactive drugs. Organotypic culture basically involves the combination of cells from different lineages in a determined ratio to create a component of an organ. Even though the synaptic organization is not exactly the same as in native tissue, the main characteristics and functions are preserved. The developed ocms will provide the biological understanding and data to help improve, evaluate and exte\ nd computational models currently being developed by epa scientists. The dual s1pr1s1pr5 drug baf312 siponimod attenuates. Culture of organotypic tissues is an area of much interest. Clearly, test systems of intermediate complexity would alleviate this inefficiency.
Assembly of a functional 3d primary cardiac construct. Further, soluble factors and cytokines can be studied in this system either by the exogenous addition of neutralization or by specific immunoglobulins. The initial steps in the preparation of slice cultures are similar to those used to prepare acute slices. Organotypic culture to assess cell adhesion, growth and. Preparation of organotypic hippocampal slice cultures using. The field of organotypic cultures seeks ultimately to support. Haas 1 departments of tneurosurgerv and 2pharmacology, university of ziirich, ziirich switzerland and 3kinsmen laboratory of. Dec 23, 2019 histotypic culture pdf although twodimensional tissue culture has been common for some time, cells behave more realistically in a threedimensional culture.
Colonoids represent a primary tissue source acting as a potential starting material for development of an in vitro model of the colon. Environmental protection agency presents organotypic culture models for predictive toxicology star research centers grantee kicko. Organotypic culture models for predictive toxicology research centers author. Tubular organotypic culture model of human kidney plos. Here we describe a vibratomebased improved slicing protocol to obtain high quality slices for use in assessing the neuroprotective potential of novel substances or the biological behavior of tumor cells.
Organotypic hippocampal slice cultures as a model to study. Organotypic slice cultures to study oligodendrocyte. Printable nanofibrous scaffolds for 3d organotypic cultures yihua loo1, anupama lakshmanan1, ming ni1, lai ling toh1, shi wang2, charlotte a. There is an increasing demand for better and more meaningful invitro test and model systems in biomedical research. Alteration of transcriptomic profiles in organotypic. Clsm b slices improved immunohistochemical protocol for. Key features of colonic crypt isolation and subsequent colonoid culture have not been systematically optimized compromising. Algimatrix tm 3d culture system 6well plate life technologies was used for the organotypic cell culture. Because epidermis and other skin components, such as hair follicles, can be readily maintained in vitro, ocss have gained broad popularity in dermatological research. In a culture hood, place tissue culture inserts in six well plates and add 1 ml slice culture medium recipe in reagents section. Do not use bleach on the stlv machine or parts at any point use of bleach may damage the center membrane of the stlv andor leave residues that affect cell viability.
A technique for repeated recordings in cortical organotypic. Methods for developing 3d organotypic models differ according to the tissue of interest and the experimental design. Systems biology for organotypic cell cultures altex. Our results show that the 3d organotypic heparg cultures maintain high liverspecific functionality during 3 weeks of culture and exhibit higher sensitivity for acetaminophen and rosiglitazone toxicity. Organotypic slice preparation organotypic slice cultures were prepared on hydrophilic cell culture membranes picmorg50, millicell according to the general procedures of stoppini et al. Scientific protocols organotypic explant culture of. This is the sixth edition of the leading text in the basic methodology of cell culture, worldwide. Regarding organotypic brain slice cultures, the complex threedimensional architecture is partly maintained, while pathways are largely disconnected but could also be reestablished. In vitro tissue constructs with faithful representation of in vivo tissue characteristics are needed for screening and characteristic assessment. New advances enable longterm organotypic culture of colonic epithelial stem cells that develop into structures known as colonoids.
Similarly, stromalepithelial interactions can be dissected easily in organotypic culture okawa et al. Organotypic cultures of hippocampal slices preparation, recording and data analysis. I practiced with pork liver from the butchery, but i failed in doing the slices. This chapter will concentrate on the culturing of hippocampal slices. Download file creating multiple organotypic models on a single 3d cell culture platform, biotechniques 62. Current drug targetscns and neurological disorders 44. The purpose of this present study was to develop organotypic tissue culture of adult. Sterilize all tools and tissue chopper with 70% ethanol. Generation of genetically modified organotypic skin cultures. Here we describe a simple method for repeated recordings and stimulating the same organotypic slices different neurons over a 24h window. Coculture system with an organotypic brain slice and 3d. Preparation of organotypic brain slice cultures for. The small intestine model showed columnar epithelial cell morphology similar to in vivo human small intestine, and showed expression of tight junctions and specific drug transporters.
All following steps were performed in a laminar flow hood for cell culture under semisterile conditions. Organotypic culture 26 questions with answers science. Organotypic culture implies the interaction of two or more cell types from a complex tissue or organ 10. K, the dual s1pr1s1pr5 drug baf312 siponimod attenuates demyelina tion in org anotypic slice cultures, journal of neuroinflammation, 1, 2016, 31. We here describe the kidney organotypic culture method that uses adult whole kidney. Generation of genetically modified organotypic skin. No part of this document may be reproduced or transmitted without the express written permission of multi channel systems mcs gmbh. Background organotypic tissue culture of adult rodent retina with an acute gene transfer that enables the efficient introduction of variable transgenes would greatly facilitate studies into retinas of adult rodents as animal models. Information in this document is subject to change without notice. Organotypic culture models developed from experimental animals for chemical toxicity screening r43r44 clinical trial not allowed rfaes20005. Our group is focusing on an airliquid interface 3d organotypic model. This concept suggests that if you want to study the biology of isolated cells, or use the cells as a substrate, conventional monolayer or suspension cultures may be.
Jun 30, 2016 human organotypic cultured cardiac slices. The culture was performed in a humidified atmosphere containing 5% co 2 at 34c. Once a basic culture technique is in place, the behavior of human epidermis can be studied and compared. For example, cultures may be grown submerged in culture medium and or at an airliquid interface. We use the conventional interface organotypic culture method together with a custom chamber, which allows recordings on the intact. In vitro tissue constructs with faithful representation of in vivo tissue characteristics are needed for screening and characteristic assessment of a variety of tissue types.
Organotypic brain slices are prepared from whole postnatal or adult brains a, and 100to 400lmthick sections are cut with a vibratome b and placed in an insert with 0. Thus, as compared to primary singlecell cultures, such an organotypic slice culture model is at least closest to an in vivo situation. To better understand the cellular and molecular signals responsible for growth and pattern formation during regeneration, i have adapted the use organotypic slice culture for axolotl blastemas. Organotypic culture to assess cell adhesion, growth and alignment of different organs on silk fibroin. A populationbased organotypic human in vitro model for cardiotoxicity testing. Organotypic cultures as a model to study adult neurogenesis. Ian freshney, phd, is an honorary senior research fellow in the centre for oncology and applied pharmacology, part of the cancer research uk beatson laboratories at the university of glasgow.
Organotypic culture is an effective tool for cell therapies of diseases in central nervous. The culture was established for up to 16 days, as preliminary experiments indicated important germ cell loss and seminiferous tubules disorganization beyond that stage. Assembly of a functional 3d primary cardiac construct using. An onchip micropump enabled metabolite transport between the 2 organotypic cultures and added physiological shear stress. The traditional approach with the tissue chopper tends to tear and stretch the tissue. Product selection guidedata sheet cell culture solutions. Cellculture methods that simplify the inherent complexities of the kidney have not sufficiently. The slice remains welloxygenated without drying out and adequate nutrition is provided through the membrane. The membrane inserts are placed into standard tissue culture dishes for longterm culture. I tried few times in vitro mouse skeletal muscle culture, overall i test soleo and edl muscles in order to guarantee edium diffusion in whole tissue avoiding necrosis deeplu in the organotypic. This book provides an overview of established 3d cell culture assays from leaders in the field. Here we describe a vibratomebased improved slicing protocol to obtain high quality slices for use in assessing the neuroprotective potential.
Organotypic hippocampal slice cultures for studies of brain damage, neuroprotection and neurorepair. Organotypic culture of central histamine neurons peter b. After aseptic removal, the tissue is sliced into sections of 100400 mm in thickness by means of a tissue chopper or a vibratome and subsequently washed in a balanced salt solution to allow tissue debris and potentially toxic substances, such as excitatory amino acids, to diffuse away. Genetically manipulated fibroblasts can also be used.
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. Slices from neonatal animals can also be kept in prolonged culture, over several weeks or even months, as so called organotypic cultures otc. Substance exposure to organotypic human liver and small. At all stages of organotypic retina culture, the outer segments of rod and scone photoreceptors are immunopositive for their respective photopigment. How should i go about editing immunofluorescence images of. Organotypic cultures, including but not limited to whole. Prepare an organotypic adult retinal explant culture and maintain this culture. The great variety in 3d culture techniques reflects both the various requirements of the different organs and the distinct goals of the investigators. Cardiac tissue analogs are particularly sought after due to the phenotypic. Rigorously revised, it features updates on specialized techniques in stem cell research and tissue engineering.
Organotypic culture the key to performing the best organotypic tissue culture is the preservation of not only the cell, but also the health of the subcellular structures within each cell. The data suggest that the 3d organotypic heparg cultures provide a suitable in vitro tool for the assessment of druginduced hepatotoxicity. Organotypic cultures best recapitulate the 3d organization of the cns and can be explored taking advantage of many techniques. Threedimensional organotypic culture of stratified epithelia. Nih funding opportunities and notices in the nih guide for grants and contracts.
Meaapplications organotypic cultures of hippocampal slice. One million cells were seeded to each well and cultured in the. Slices from a variety of brain regions have been successfully cultured. Sep 08, 2019 histotypic culture pdf although twodimensional tissue culture has been common for some time, cells behave more realistically in a threedimensional culture. Various subfields use these terms either interchangeably or distinctly. With the advances in the organotypic culture techniques, it is now possible to develop certain tissues or tissue models. Microdissection and organotypic culture of neonatal mouse. Photoreceptor vitality in organotypic cultures of mature. Their contributions cover a wide spectrum of techniques and approaches for 3d cell culture, from organoid cultures through organotypic models to microfluidic approaches and emerging 3d bioprinting techniques, which are used in developmental, stem cell, cancer, and pharmacological studies, among many. The alginate scaffoldbased organotypic culture system is a promising, reliable, and easy system that can be configured into a high throughput fashion for the identification of critical signaling pathways and screening of molecular drug targets specific for hcc.
Organotypic raft culture of phks the open lab book v1. Fef3, fetal human esophageal fibroblasts a standard fibroblast line and other fibroblast lines adultdermalcaf, etc. Jeanluc duval1, tony dinis1,2, guillaume vidal1, pascale. Use sterile tissue culture technique throughout all aspects of this protocol. Organotypic culture models for predictive toxicology. Short communication evaluation of epidermal neural crest. Precisionary organotypic culture experiments precisionary. Organotypic culture 26 questions with answers science topic. Organotypic culture models for predictive toxicology center. Alteration of transcriptomic profiles in organotypic culture of bronchial tissues indirectly exposed to cigarette smoke and novel tobacco product vapor kazushi matsumura, shinkichi ishikawa, shigeaki ito scientific product assessment center, japan tobacco inc. Organotypic culture of bonelike structures using composite. The organotypic retina cultures of both model organisms are created as cocultures of the retina and the pigment epithelium, still attached to outer segments of the photoreceptor cells. Jun 26, 2019 histotypic culture pdf although twodimensional tissue culture has been common for some time, cells behave more realistically in a threedimensional culture.
Jun 27, 2018 we have developed a novel organotypic brain slice culture model to study alzheimers disease using 3xtgad mice which brings the potential of substantially reducing the number of rodents used in dementia research from an estimated 20,000 per year. Organotypic cultures preparation of organotypic cultures another 3d model of tumor microenvironment is the reconstruction of tissue by growing cells in extracellular matrix. They are manufactured from clear virgin polystyrene, and are supplied sterile and nonpyrogenic. For the first time, grow fef3 p5p6 in two 150mm2 dishes until they reach 100%. Optimization of 3d organotypic primary colonic cultures. No part of this document may be reproduced or transmitted without the express written permission of. 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. Short communication evaluation of epidermal neural crest stem cells in organotypic spinal cord slice culture platform spinal cord injury epidermal neural crest stem cells organotypic slice culture s. Organotypic culture models developed from experimental animals for chemical toxicity screening r43 rfaes17008. This document includes full details for performing the following steps.
Regenerated human skin can be used in downstream applications such as gene expression profiling, immunostaining, and chromatin immunoprecipitations followed by high throughput sequencing. Cultures formed from reaggregated cells are always called organotypic cultures, whereas tissue explants are called both organandorganotypic culture interchangeably. Do not use general stock glassware, even if it has been autoclaved. Organotypic cultures represent tumor microenvironment for. Organotypic tissue culture of adult rodent retina followed by. How should i go about editing immunofluorescence images of full thickness skin and organotypic skin models with figi image j.
In this study, a co culture of 3d human liver organoids and differentiated primary human small intestine epithelial tissues were combined in a dynamicallyperfused multiorganchip moc system. I would like to work with organotypic culture of liver slices, but i have no experience. Multiwell receiver plates tissue culture treated solid bottom receiver plates designed to work with our millicell single well inserts are available in 6, 12, and 24well formats. Remove the spacer and allow the 3d tumor spheroid to coculture with the organotypic slice for 2496 hr. The liver and primary intestine equivalents were kept viable in a co culture for 14 days. Trypsinize the phks off the flask and resuspend in say 2ml raft medium. Organotypic hippocampal cultures offer the possibility for easy gene manipulation and precise pharmacological intervention but maintain synaptic organization that is critical to understanding synapse function in a more naturalistic context than routine culture dissociated neurons methods. Organotypic culture models \ocms\, when developed and evaluated, will provide information needed to help predict toxicity due to chemical exposures.
Organotypic culture of bonelike structures using composite ceramic fibrin scaffolds. A major question in regeneration biology is how the nerve exerts its growth promoting effect on blastema cells. Thus, generation of these genetically modified organotypic skin cultures will allow the determination of genes that are critical for maintaining skin homeostasis. 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. He is the author or editor of numerous books and a worldrenowned expert on cell culture technique. Immunofluorescence staining of astrocytes and microglial in the organotypic brain slice coculture. Demonstrate the potential of organotypic culture systems to fill crucial needs in.
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