This work was supported financially by the Suomen Akatemia (Academy of Finland) (206038, 121647, 250900, 260056; Centre of Excellence grant 2012-2017 251314), Munuaiss&#228;&#228;ti&#246; - Finnish Kidney and Liver Association, the Sigrid Juseliuksen S&#228;&#228;ti&#246;, Victoriastiftelsen, The Swedish Cultural Foundation in Finland, Novo Nordisk, Sy&#246;p&#228;j&#228;rjest&#246;t (Cancer Society of Finland), the European Community&#8217;s Seventh Framework Programme (FP7/2007-2013; grant FP7-HEALTH-F5-2012-INNOVATION-1 EURenOmics 305608), and H2020 Marie Sklodowska-Curie Actions Innovative Training Network &#8220;RENALTRACT&#8221; Project ID 642937. The authors thank Paula Haipus, Johanna Kekolahti-Liias and Hannele H&#228;rkman for technical assistance.
Figures 3, 4, and Movie 2 are reprinted with permission from Development.

Animal care and procedures were in accordance with Finnish national legislation for the use of laboratory animals, the European Convention for the protection of vertebrate animals used for experimental and other scientific purposes (ETS 123), and the EU Directive 86/609/EEC.
1. Fabrication of minireservoirs for cultivation of mouse embryonic kidneys and renal organoids on chicken CAM and setting up xenotransplantation experiments
<ol>
	<li>Use transwell cell culture inserts designed for 6 well or 12 well plates. 
	NOTE: Depending on the particular experiment, large or small minireservoirs can be used. It is best to use small minireservoirs for xenografting up to eight embryonic kidneys or when several minireservoirs will be placed on same chicken embryo (e.g., experiment and control samples on same chicken CAM).</li>
	<li>Cut down the sides of the inserts using a rotary multitool with a circular saw blade or a hand saw to create a 2 mm high plastic ring with a permeable membrane attached to it.</li>
	<li>Polish the edges of these minireservoirs with a scalpel or a sharp knife.</li>
	<li>Sterilize the minireservoirs in 70% ethanol for at least 1 h.</li>
	<li>Wash the minireservoirs in autoclaved double-distilled water and let them dry in a laminar hood.</li>
	<li>Rinse the minireservoirs in PBS and culture medium (high-glucose DMEM/10% FBS/1% penicillin-streptomycin).</li>
	<li>Place the reservoir in a Petri dish, on top of a drop (600 &#181;L/400 &#181;L) of culture medium with the membrane facing up.</li>
	<li>Arrange dissected ex vivo embryonic kidneys or renal organoids evenly on the membrane with the help of a pipette or a glass capillary tube. Avoid leaving a lot of liquid around the kidneys.</li>
	<li>Let the samples attach to the membrane for 2&#8211;24 h in a cell culture incubator at 37 &#176;C and 5% CO2. 
	NOTE: Up to eight E11.5 embryonic mouse kidneys or kidney organoids can be arranged on the small insert. The large insert is recommended if either bigger pieces of embryonic tissue will be used for xenotransplant or a cell-hydrogel mixture on a larger area of the CAM.</li>
	<li>Take 8-day-old ex ovo cultured embryonic chickens prepared according to previously published methods out of the cell culture incubator12,13.</li>
	<li>Transfer the minireservoirs to the CAM so that the grafts are facing the CAM, and the membrane overlays them. Place the minireservoirs in the periphery of the CAM, so that they are not covering the chicken embryo. 
	NOTE: When using the smaller minireservoirs fabricated from transwell inserts for 12 well plates, up to three minireservoirs can be placed on one CAM.</li>
	<li>Add 500 &#181;L (6 well insert) or 300 &#181;L (12 well insert) of culture medium to the minireservoirs.</li>
	<li>Cultivate the samples on chicken CAM for a maximum of 9 days. Replace the culture medium in the minireservoirs daily. 
	NOTE: Vascularization of the samples can be observed as soon as 24&#8211;48 h after transplantation.</li>
</ol>
2. Fabrication of custom-designed plates and setting up FiZD cultures
<ol>
	<li>Drill 20 mm diameter holes in the bottom of a 6 well plate. 
	NOTE: For FiZD experiments, use 6 well plates with a 16 mm well depth (specified in Table of Materials).</li>
	<li>Polish the rims of the holes (especially on the upper side) using an electric drill with a countersink drill bit, a scalpel, or a sharp knife.</li>
	<li>Sterilize the plates in 70% ethanol for at least 1 h.</li>
	<li>Wash the plates in autoclaved double-distilled water and let them dry in sterile conditions.</li>
	<li>Thoroughly wash 22 mm x 22 mm glass coverslips with 70% ethanol or clean them according to the published protocol by Saarela et al.11.</li>
	<li>Glue the coverslips to the upper side of the holes in 6 well plates using a nontoxic tissue glue. Apply glue around the hole and gently place the coverslip to cover it. Let the glue dry.</li>
	<li>Inspect the plates under a stereomicroscope and remove excess dried glue from the surface of the coverslips if needed. 
	NOTE: Check that there is no glue above the coverslip to ensure even compression of the samples.</li>
	<li>Mix polystyrene beads (70 &#181;m particle size) with an equal volume of hydrogel. A volume of 50&#8211;100 &#181;L per well is sufficient. 
	NOTE: Keep the mixture of hydrogel and polystyrene beads on ice.</li>
	<li>Place a transwell insert under a dissecting microscope with the membrane up.</li>
	<li>Arrange the samples (e.g., ex vivo embryonic kidneys or renal organoids) evenly on the membrane.</li>
	<li>Add the hydrogel/polystyrene bead mixture next to the samples carefully to avoid damage to fragile tissues.</li>
	<li>Flip the transwell insert so that the membrane with the samples assembled on it is facing down.</li>
	<li>Gently place the insert into a well of the modified 6 well plate and gently press it down so the samples are compressed to the level of the beads. 
	NOTE: Follow the progress of the compression using a microscope.</li>
	<li>Keep the insert slightly pressed to the well with one hand and fix it to the plate by melting plastic with a soldering iron at three points at the periphery of the insert.</li>
	<li>When the insert is fixed to the plate, add 2 mL of culture medium (DMEM/10% FBS/1% penicillin-streptomycin) to the well and continue assembling the remaining wells in the plate in the same way.</li>
	<li>Transfer the finished plate to an on-stage incubator of an inverted microscope for time-lapse imaging.</li>
	<li>Perform time-lapse imaging of the samples using appropriate experimental settings. 
	NOTE: Changing the culture medium in the wells of the plate during time-lapse experiments is not required but can be easily done through the holes on the sides of the insert.</li>
</ol>

<ol>
	<li>Saxen, L., Wartiovaara, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+contact+and+cell+adhesion+during+tissue+organization.">Cell contact and cell adhesion during tissue organization.</a> International Journal of Cancer. 1 (3), 271-290 (1966).</li><li>Saxen, L., Toivonen, S., Vainio, T., Korhonen, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Untersuchungen+&#252;ber+die+tubulogenese+der+niere.">Untersuchungen &#252;ber die tubulogenese der niere.</a> Zeitschrift F&#252;r Naturforschung. 20 (4), (1965).</li><li>Saxen, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Organogenesis of the Kidney. 1st ed. , (1987).</li><li>Takasato, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kidney+organoids+from+human+iPS+cells+contain+multiple+lineages+and+model+human+nephrogenesis.">Kidney organoids from human iPS cells contain multiple lineages and model human nephrogenesis.</a> Nature. 536 (7615), 238 (2016).</li><li>Halt, K. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD146++cells+are+essential+for+kidney+vasculature+development.">CD146+ cells are essential for kidney vasculature development.</a> Kidney International. 90, 311-324 (2016).</li><li>van den Berg, C. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Renal+Subcapsular+Transplantation+of+PSC-Derived+Kidney+Organoids+Induces+Neo-vasculogenesis+and+Significant+Glomerular+and+Tubular+Maturation+In+Vivo.">Renal Subcapsular Transplantation of PSC-Derived Kidney Organoids Induces Neo-vasculogenesis and Significant Glomerular and Tubular Maturation In Vivo.</a> Stem Cell Reports. 10 (3), 751-765 (2018).</li><li>Sariola, H., Ekblom, P., Lehtonen, E., Saxen, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiation+and+vascularization+of+the+metanephric+kidney+grafted+on+the+chorioallantoic+membrane.">Differentiation and vascularization of the metanephric kidney grafted on the chorioallantoic membrane.</a> Developmental Biology. 96 (2), 427-435 (1983).</li><li>Sariola, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Incomplete+fusion+of+the+epithelial+and+endothelial+basement+membranes+in+interspecies+hybrid+glomeruli.">Incomplete fusion of the epithelial and endothelial basement membranes in interspecies hybrid glomeruli.</a> Cell Differentiation. 14 (3), 189-195 (1984).</li><li>Ekblom, P., Sariola, H., Karkinen-J&#228;&#228;skel&#228;inen, M., Sax&#233;n, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+origin+of+the+glomerular+endothelium.">The origin of the glomerular endothelium.</a> Cell Differentiation. 11 (1), 35-39 (1982).</li><li>Short, K. 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T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+ex-ovo+Chicken+Embryo+Culture+System+Suitable+for+Imaging+and+Microsurgery+Applications.">An ex-ovo Chicken Embryo Culture System Suitable for Imaging and Microsurgery Applications.</a> Journal of Visualized Experiments. 44, e2154 (2010).</li><li>Schomann, T., Qunneis, F., Widera, D., Kaltschmidt, C., Kaltschmidt, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+method+for+ex+ovo-cultivation+of+developing+chicken+embryos+for+human+stem+cell+xenografts.">Improved method for ex ovo-cultivation of developing chicken embryos for human stem cell xenografts.</a> Stem Cells International. 2013, 960958 (2013).</li><li>Prunskaite-Hyyryl&#228;inen, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wnt4+coordinates+directional+cell+migration+and+extension+of+the+M&#252;llerian+duct+essential+for+ontogenesis+of+the+female+reproductive+tract.">Wnt4 coordinates directional cell migration and extension of the M&#252;llerian duct essential for ontogenesis of the female reproductive tract.</a> Human Molecular Genetics. 25 (6), 1059-1073 (2016).</li><li>Gustafsson, E., Brakebusch, C., Hietanen, K., F&#228;ssler, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tie-1-directed+expression+of+Cre+recombinase+in+endothelial+cells+of+embryoid+bodies+and+transgenic+mice.">Tie-1-directed expression of Cre recombinase in endothelial cells of embryoid bodies and transgenic mice.</a> Journal of Cell Science. 114, 671-676 (2001).</li><li>Homan, K. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow-enhanced+vascularization+and+maturation+of+kidney+organoids+in+vitro.">Flow-enhanced vascularization and maturation of kidney organoids in vitro.</a> Nature Methods. 16 (3), 255-262 (2019).</li><li>Trowell, O. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+modified+technique+for+organ+culture+in+vitro.">A modified technique for organ culture in vitro.</a> Experimental Cell Research. 6 (1), 246-248 (1954).</li></ol>

The authors have nothing to disclose.

Two detailed protocols are presented that refine the classical renal organotypic culture method, and enable vascularization, extended development, and optimal 4D (i.e., 3D image and time) imaging of ex vivo embryonic kidneys and organoids. This section highlights the critical steps in the methods and discusses troubleshooting.
The significant difference between other CAM culture methods and this improved chicken CAM culture method is the use of custom-made minireservoirs in the xenografting of...

Organotypic culture of embryonic kidneys became an important model to study nephrogenesis decades ago1,2,3. Renal organoids represent an advanced model system for studying development of healthy and diseased kidneys4. The main drawback for both methods, however, is that neither method recapitulates the main function of the kidney: blood filtration. Nephrons and renal vasculature develop in renal organoids and organotypic cultures similarly to early stage in vivo development; however, the glomeruli formed in vitro remain avascular5. Vascularization of ex vivo embryonic kidneys and renal organoids was previously demonstrated in transplantation experiments only under in vivo conditions. For example, transplantation of human pluripotent stem cell-derived renal organoids under a mouse kidney capsule allows development of the nephrons in the organoid to a functional stage6.
An intermediate approach between purely in vitro cultures and in vivo transplantation methods is xenotransplantation to the CAM of avian embryos. Vascularization of intact mouse kidney primordia has been demonstrated previously using this system7,8. However, it was also shown that the renal vasculature in the xenotransplanted murine kidney was derived from the host endothelium, not the graft9. This observation significantly reduced the potential of chimeric (avian-mammalian) models of embryonic kidney to study development of the renal vasculature, because the experimental conditions were nonpermissive for the survival of donor-derived endothelial cells.
Presented in the first part of this protocol is an improved method for cultivation of mouse embryonic kidneys on CAM of avian eggs, combining microenvironmental conditions of organotypic culture and xenotransplantation. The main improvement to previous methods is that instead of placing the mouse embryonic kidneys and renal organoids directly on the CAM, the implantation area is overlaid with permeable minireservoirs filled with culture medium that supply the transplanted tissue with nutrients and protect it from drying. The success rate of the experiments significantly increases and the conditions for development of donor-derived vasculature improve. Application of this method to xenotransplant cultures results in the development of glomerular vasculature comprised of endogenous endothelial cells from donor kidneys.
Detailed analysis of cellular morphogenesis is another important application of kidney culture models. Previously reported methods of time-lapse image acquisition of kidney cultures are sufficient only for analysis of overall morphology and patterning of embryonic kidney, but not for tracking individual cells10. Recently, a novel Fixed Z-Direction (FiZD) method aimed for high-resolution confocal 3D time-lapse imaging of renal organoids and organotypic cultures was described11. In this method, the organoids and embryonic organs are gently compressed between a glass coverslip and a permeable membrane of a transwell insert in a custom-designed plate until the thickness of the sample reaches 70 &#181;m, providing optimal optical conditions for imaging. In the second part of the methods, a detailed protocol for the fabrication of a custom-designed plate and the setup of FiZD experiments for long-term organoid imaging is described.

<table><tbody><tr><td>Adjustable Spade Drill Bit</td><td>Bosch</td><td>2609255277</td><td>For drilling 20 mm diameter holes</td></tr><tr><td>Automatic Egg Turner</td><td>OLBA B.V., Netherlands</td><td>AT-42</td><td>For incubation of eggs before ex ovo setup.</td></tr><tr><td>Cell Incubator</td><td>Panasonic</td><td>13090543</td><td>Temperature set at 37&amp;deg; C, 90% humidity, 5% CO&lt;sub&gt;2&lt;/sub&gt;</td></tr><tr><td>Cell Incubator</td><td>SANYO</td><td>10070347</td><td>Chicken CAM-culture incubator. Temperature set at 37&amp;deg; C, 90% humidity, 5% CO&lt;sub&gt;2&lt;/sub&gt;</td></tr><tr><td>Cellstar 6-well Plate</td><td>Greiner</td><td>M9062</td><td>16 mm depth of wells to match with the inserts</td></tr><tr><td>Circular Saw Blade for Dremel Rotary Tool</td><td>Dremel</td><td>SC690</td><td></td></tr><tr><td>Confocal Microscope</td><td>Zeiss</td><td>LSM780</td><td></td></tr><tr><td>Corning Transwell Multiple Well Plate with Permeable Polycarbonate Membrane Inserts</td><td>Corning</td><td>10301031</td><td>For 6-well plates. 40 &amp;micro;m pore size</td></tr><tr><td>Countersink Drill Bit</td><td>Craftomat, Bauhaus</td><td>22377902</td><td>For polishing (20.5 mm, 1/4&quot;, HSS)</td></tr><tr><td>Disposable Glass Capillary Tube</td><td>Blaubrand</td><td>7087 33</td><td></td></tr><tr><td>Disposable Scalpel</td><td>Swann-Morton</td><td>0501</td><td></td></tr><tr><td>Dissecting Microscope</td><td>Olympus</td><td>SZ61</td><td></td></tr><tr><td>Drilling Machine</td><td>Bosch</td><td>GSR 18 V-EC Professional</td><td></td></tr><tr><td>Dulbecco&#039;s Modified Eagle&#039;s Medium</td><td>Sigma</td><td>D777</td><td>High glucose</td></tr><tr><td>Egg Incubator Compact S84</td><td>Grumbach, Germany</td><td>8012</td><td>For incubation of eggs before ex ovo setup. Temperature set at 38&amp;deg; C with relative humidity set above 60%</td></tr><tr><td>Ethanol (70%)</td><td>VWR</td><td></td><td></td></tr><tr><td>Fertilized Eggs</td><td>Haaviston Siitoskanala, Panelia, Finland</td><td></td><td>Hy-Line White and Nick Chick</td></tr><tr><td>Fetal Bovine Serum</td><td>HyClone</td><td>SH3007003HI Thermo Scientific</td><td></td></tr><tr><td>Forceps DUMONT #5</td><td>Dumont</td><td>#5SF</td><td></td></tr><tr><td>Glass Coverslips</td><td>Menzel-Gl&amp;auml;ser</td><td>Menzel BBAD02200220#A1TBCMNZ#0##</td><td>22x22 mm</td></tr><tr><td>Histoacryl Glue</td><td>Braun</td><td>1050052</td><td></td></tr><tr><td>Matrigel</td><td>Corning</td><td>356230</td><td></td></tr><tr><td>On-stage Incubator</td><td>Okolab</td><td></td><td>Boldline, custom made</td></tr><tr><td>PBS -/-</td><td>Corning</td><td>20-031-CV</td><td></td></tr><tr><td>PBS +/+</td><td>Biowest</td><td>X0520-500</td><td>Washing the mini reservoirs.</td></tr><tr><td>Penicillin and Streptomycin</td><td>Sigma</td><td>P4333</td><td></td></tr><tr><td>Polystyrene Beads</td><td>Corpuscular</td><td>1000263-10</td><td>70 &amp;micro;m in diameter</td></tr><tr><td>Rotary Multi Tool System</td><td>Dremel</td><td>4000</td><td></td></tr><tr><td>Soldering Iron</td><td>Weller</td><td>TCP S</td><td>Heated glass capillary can also be used</td></tr><tr><td>Thincert 12-well Cell Culture Inserts With 0.4 &amp;micro;m-pore Polystyrene Membrane</td><td>Greiner Bio-One</td><td>665641</td><td></td></tr><tr><td>Thincert 6-well Cell Culture Inserts With 0.4 &amp;micro;m-pore Polystyrene Membrane</td><td>Greiner Bio-One</td><td>657610</td><td></td></tr></tbody></table>

optimization of renal organoid and organotypic culture for vascularization, extended development, and improved microscopy imaging

Embryonic kidney organotypic cultures, and especially pluripotent stem cell-derived kidney organoids, are excellent tools for following developmental processes and modelling kidney disease. However, the models are limited by a lack of vascularization and functionality. To address this, an improved protocol for the method of xenografting cells and tissues to the chorioallantoic membrane (CAM) of an avian embryo to gain vascularization and restoration of blood flow was developed. The grafts are overlaid with custom-made minireservoirs that fix the samples to the CAM and supply them with culture medium that protects the grafts from drying. The improved culture method allows xenografts to grow for up to 9 days. The manuscript also describes how to provide optimal conditions for long-term confocal imaging of renal organoids and organotypic cultures using the previously published Fixed Z-Direction (FiZD) method. This method gently compresses an embryonic organ or organoid between a glass coverslip and membrane in a large amount of medium and provides excellent conditions for imaging for up to 12 days. Together, these methods allow vascularization and blood flow to renal organoids and organotypic kidney cultures with improved confocal imaging. The methods described here are highly beneficial for studying fundamental and applied functions of kidneys ex vivo. Both methods are applicable to various types of tissues and organoids.

The CAM culture protocol presented here enabled highly efficient vascularization of renal organoids and embryonic kidneys as a result of xenotransplantation on chicken CAM (Figure 1, Movie 1). Minireservoirs containing culture medium supplied nutrients to donor tissue and protected it from drying during the time period preceding proper vascularization. This method provided permissive conditions for donor-derived endothelial cells to grow. Therefore, renal vasculature in the ...

Watch this Scientific Journal Video about Optimization of Renal Organoid and Organotypic Culture for Vascularization, Extended Development, and Improved Microscopy Imaging at JoVE.com

Optimization of Renal Organoid and Organotypic Culture for Vascularization, Extended Development, and Improved Microscopy Imaging

This work describes two methods for studying organ development, an improved xenotransplantation setup on chorioallantoic membrane (CAM) from avian embryos that allows for vascularization of cultured embryonic organs and organoids and a novel fixed z-direction organ culture method with modified experimental conditions that allows for high-resolution time-lapse confocal imaging.

Embryonic kidney organotypic cultures, and especially pluripotent stem cell-derived kidney organoids, are excellent tools for following developmental ...

optimization-renal-organoid-organotypic-culture-for-vascularization

Research

JoVE Journal

Developmental Biology

7.1K Views.  University of Oulu. This work describes two methods for studying organ development, an improved xenotransplantation setup on chorioallantoic membrane (CAM) from avian embryos that allows for vascularization of cultured embryonic organs and organoids and a novel fixed z-direction organ culture method with modified experimental conditions that allows for high-resolution time-lapse confocal imaging.

Video: Optimization of Renal Organoid and Organotypic Culture for Vascularization, Extended Development, and Improved Microscopy Imaging

Enhanced Viability for Ex vivo 3D Hydrogel Cultures of Patient-Derived Xenografts in a Perfused Microfluidic Platform

Patient-derived xenografts (PDX), generated when resected patient tumor tissue is engrafted directly into immunocompromised mice, remain biologically stable, thereby preserving molecular, genetic, and histological features, as well as heterogeneity of the original tumor. However, using these models to perform a multitude of experiments, including drug screening, is prohibitive both in terms of cost and time. Three-dimensional (3D) culture systems are widely viewed as platforms in which cancer cells retain their biological integrity through biochemical interactions, morphology, and architecture. Our team has extensive experience culturing PDX cells in vitro using 3D matrices composed of hyaluronic acid (HA). In order to separate mouse fibroblast stromal cells associated with PDXs, we use rotation culture, where stromal cells adhere to the surface of tissue culture-treated plates while dissociated PDX tumor cells float and self-associate into multicellular clusters. Also floating in the supernatant are single, often dead cells, which present a challenge in collecting viable PDX clusters for downstream encapsulation into hydrogels for 3D cell culture. In order to separate these single cells from live cell clusters, we have employed density step gradient centrifugation. The protocol described here allows for the depletion of non-viable single cells from the healthy population of cell clusters that will be used for further in vitro experimentation. In our studies, we incorporate the 3D cultures in microfluidic plates which allow for media perfusion during culture. After assessing the resultant cultures using a fluorescent image-based viability assay of purified versus non-purified cells, our results show that this additional separation step substantially reduced the number of non-viable cells from our cultures.

This protocol demonstrates methods to enable extended in vitro culture of patient-derived xenografts (PDX). One step enhances overall viability of multicellular cluster cultures in 3D hydrogels, through straightforward removal of non-viable single cells. A secondary step demonstrates best practices for PDX culture in a perfused microfluidic platform.

Patient-derived xenografts (PDX), generated when resected patient tumor tissue is engrafted directly into immunocompromised mice, remain biologically ...

Cancer Research

Three-Dimensional Imaging of Organoids to Study Primary Ciliogenesis During ex vivo Organogenesis

Organoids are stem cell-derived three-dimensional structures that reproduce ex vivo the complex architecture and physiology of organs. Thus, organoids represent useful models to study the mechanisms that control stem cell self-renewal and differentiation in mammals, including primary ciliogenesis and ciliary signaling. Primary ciliogenesis is the dynamic process of assembling the primary cilium, a key cell signaling center that controls stem cell self-renewal and/or differentiation in various tissues. Here we present a comprehensive protocol for the immunofluorescence staining of cell lineage and primary cilia markers, in whole-mount mouse mammary organoids, for light sheet microscopy. We describe the microscopy imaging method and an image processing technique for the quantitative analysis of primary cilium assembly and length in organoids. This protocol enables a precise analysis of primary cilia in complex three-dimensional structures at the single cell level. This method is applicable for immunofluorescence staining and imaging of primary cilia and ciliary signaling in mammary organoids derived from normal and genetically modified stem cells, from healthy and pathological tissues, to study the biology of the primary cilium in health and disease.

Three-Dimensional Imaging of Organoids to Study Primary Ciliogenesis During ex vivo Organogenesis

Stem cell-derived organoids facilitate the analysis of molecular and cellular processes that regulate stem cell self-renewal and differentiation during organogenesis in mammalian tissues. Here we present a protocol for the analysis of the biology of the primary cilium in mouse mammary organoids.

Organoids are stem cell-derived three-dimensional structures that reproduce ex vivo the complex architecture and physiology of organs. Thus, organoids ...

Genetics

A Simplified Method for Generating Kidney Organoids from Human Pluripotent Stem Cells

Kidney organoids generated from hPSCs have provided an unlimited source of renal tissue. Human kidney organoids are an invaluable tool for studying kidney disease and injury, developing cell-based therapies, and testing new therapeutics. For such applications, large numbers of uniform organoids and highly reproducible assays are needed. We have built upon our previously published kidney organoid protocol to improve the overall health of the organoids. This simple, robust 3D protocol involves the formation of uniform embryoid bodies in minimum component medium containing lipids, insulin-transferrin-selenium-ethanolamine supplement and polyvinyl alcohol with GSK3 inhibitor (CHIR99021) for 3 days, followed by culture in knock-out serum replacement (KOSR)-containing medium. In addition, agitating assays allows for reduction in clumping of the embryoid bodies and maintaining a uniform size, which is important for reducing variability between organoids. Overall, the protocol provides a fast, efficient, and cost-effective method for generating large quantities of kidney organoids.

Here we describe a protocol to generate kidney organoids from human pluripotent stem cells (hPSCs). This protocol generates kidney organoids within two weeks. The resulting kidney organoids can be cultured in large-scale spinner flasks or multi-well magnetic stir plates for parallel drug-testing approaches.

Kidney organoids generated from hPSCs have provided an unlimited source of renal tissue. Human kidney organoids are an invaluable tool for studying kidney ...

Efficient Vascularization of Kidney Organoids through Intracelomic Transplantation in Chicken Embryos

Kidney organoids derived from human induced pluripotent stem cells contain nephron-like structures that resemble those in the adult kidney to a certain degree. Unfortunately, their clinical applicability is hampered by the lack of a functional vasculature and consequently limited maturation in vitro. The transplantation of kidney organoids in the celomic cavity of chicken embryos induces vascularization by perfused blood vessels, including the formation of glomerular capillaries, and enhances their maturation. This technique is very efficient, allowing for the transplantation and analysis of large numbers of organoids. This paper describes a detailed protocol for the intracelomic transplantation of kidney organoids in chicken embryos, followed by the injection of fluorescently labeled lectin to stain the perfused vasculature, and the collection of transplanted organoids for imaging analysis. This method can be used to induce and study organoid vascularization and maturation to find clues for enhancing these processes in vitro and improve disease modeling.

Here, we present a detailed protocol for the transplantation of kidney organoids in the celomic cavity of chicken embryos. This method induces vascularization and enhanced maturation of the organoids within 8 days and can be used to study these processes in an efficient manner.

Kidney organoids derived from human induced pluripotent stem cells contain nephron-like structures that resemble those in the adult kidney to a certain ...

Using Induced Pluripotent Stem Cell Suspension for Kidney Organoid Production

Generating Kidney Organoids in Suspension from Induced Pluripotent Stem Cells

Kidney organoids can be generated from induced pluripotent stem cells (iPSCs) through various approaches. These organoids hold great promise for disease modeling, drug screening, and potential therapeutic applications. This article presents a step-by-step procedure to create kidney organoids from iPSCs, starting from the posterior primitive streak (PS) to the intermediate mesoderm (IM). The approach relies on the APEL 2 medium, which is a defined, animal component-free medium. It is supplemented with a high concentration of WNT agonist (CHIR99021) for a duration of 4 days, followed by fibroblast growth factor 9 (FGF9)/heparin and a low concentration of CHIR99021 for an additional 3 days. During this process, emphasis is given to selecting the optimal cell density and CHIR99021 concentration at the start of iPSCs, as these factors are critical for successful kidney organoid generation. An important aspect of this protocol is the suspension culture in a low adherent plate, allowing the IM to gradually develop into nephron structures, encompassing glomerular, proximal tubular, and distal tubular structures, all presented in a visually comprehensible format. Overall, this detailed protocol offers an efficient and specific technique to produce kidney organoids from diverse iPSCs, ensuring successful and consistent results.

Author Spotlight: Optimizing iPSC Differentiation for Efficient Production to Generate Kidney Organoids 

This protocol presents a comprehensive and efficient method for producing kidney organoids from induced pluripotent stem cells (iPSCs) using suspension culture conditions. The primary emphasis of this study lies in the determination of the initial cell density and the WNT agonist concentration, thereby benefiting investigators interested in kidney organoid research.

Kidney organoids can be generated from induced pluripotent stem cells (iPSCs) through various approaches. These organoids hold great promise for disease ...

Grafting Human Brain Organoids into the Chorioallantoic Membrane of the Chick Embryo

Early Unguided Human Brain Organoid Neurovascular Niche Modeling into the Permissive Chick Embryo Chorioallantoic Membrane

Engrafting organoids into vascularized tissues in model animals, such as the immunodeficient mouse or chick embryo chorioallantoic membrane (CAM), has proven efficient for neovascularization modeling. The CAM is a richly vascularized extraembryonic membrane, which shows limited immunoreactivity, thus becoming an excellent hosting model for human origin cell transplants.
This paper describes the strategy to engraft human brain organoids differentiated at multiple maturation stages into the CAM. The cellular composition of brain organoids changes with time, reflecting the milestones of human brain development. We grafted brain organoids at relevant maturation stages: neuroepithelial expansion (18 DIV), early neurogenesis (60 DIV), and early gliogenesis (180 DIV) into the CAM of embryonic day (E)7 chicken embryos. Engrafted brain organoids were harvested 5 days later and their histological features were analyzed.
No histological signs of neovascularization in the grafted organoids or abnormal blood vessels adjacent to the graftings were detected. Moreover, remarkable changes were observed in the cellular composition of the grafted organoids, namely, an increase in the number of glial fibrillary acidic protein-positive-reactive astrocytes. However, the cytoarchitectural changes were dependent on the organoid maturation stage. Altogether, these results suggest that brain organoids can grow in the CAM, and they show differences in the cytoarchitecture depending on their maturation stage at grafting.

Author Spotlight: Optimizing the Neurovascular Development of Human Brain Organoid in Chick Embryo

Here, we present a protocol to engraft human brain organoids at multiple maturation stages into the chick chorioallantoic membrane (CAM). Brain organoids were grown following unguided standardized protocols.

Engrafting organoids into vascularized tissues in model animals, such as the immunodeficient mouse or chick embryo chorioallantoic membrane (CAM), has ...

In Vitro Pancreas Organogenesis from Dispersed Mouse Embryonic Progenitors

The pancreas is an essential organ that regulates glucose homeostasis and secretes digestive enzymes. Research on pancreas embryogenesis has led to the development of protocols to produce pancreatic cells from stem cells 1. The whole embryonic organ can be cultured at multiple stages of development 2-4. These culture methods have been useful to test drugs and to image developmental processes. However the expansion of the organ is very limited and morphogenesis is not faithfully recapitulated since the organ flattens. 
We propose three-dimensional (3D) culture conditions that enable the efficient expansion of dissociated mouse embryonic pancreatic progenitors. By manipulating the composition of the culture medium it is possible to generate either hollow spheres, mainly composed of pancreatic progenitors expanding in their initial state, or, complex organoids which progress to more mature expanding progenitors and differentiate into endocrine, acinar and ductal cells and which spontaneously self-organize to resemble the embryonic pancreas. 
We show here that the in vitro process recapitulates many aspects of natural pancreas development. This culture system is suitable to investigate how cells cooperate to form an organ by reducing its initial complexity to few progenitors. It is a model that reproduces the 3D architecture of the pancreas and that is therefore useful to study morphogenesis, including polarization of epithelial structures and branching. It is also appropriate to assess the response to mechanical cues of the niche such as stiffness and the effects on cell&#180;s tensegrity.

In Vitro Pancreas Organogenesis from Dispersed Mouse Embryonic Progenitors

The three-dimensional culture method described in this protocol recapitulates pancreas development from dispersed embryonic mouse pancreas progenitors, including their substantial expansion, differentiation and morphogenesis into a branched organ. This method is amenable to imaging, functional interference and manipulation of the niche.

The pancreas is an essential organ that regulates glucose homeostasis and secretes digestive enzymes. Research on pancreas embryogenesis has led to the ...

Biology

hiPSC-Derived Vascular Organoids for Drug Screening and Disease Modeling

Vascular Organoid Generation from Human-Induced Pluripotent Stem Cells

Vascular organoids derived from human induced pluripotent stem cells (hiPSCs) recapitulate the cell type diversity and complex architecture of human vascular networks. This three-dimensional (3D) model holds substantial potential for vascular pathology modeling and in vitro drug screening. Despite recent advances, a key technical challenge remains in reproducibly generating organoids with consistent quality, which is crucial for downstream assays and applications. Here, a modified protocol is presented that improves both the homogeneity and reproducibility of vascular organoid generation. The modified protocol incorporates the use of microwells and the CEPT cocktail (chroman 1, emricasan, polyamines, and the integrated stress response inhibitor, trans-ISRIB) to improve embryoid body formation and cell survival. Differentiated, mature vascular organoids generated using this protocol are characterized by whole-mount 3D immunofluorescence microscopy to analyze their morphology and complex vasculature. This protocol enables the production of high-quality vascular organoids in a scalable manner, potentially facilitating their use in disease modeling and drug screening applications.

Author Spotlight: Improving Reproducibility in Vascular Organoids Using ROCK Inhibitors and Microwell Confinement

This protocol presents a method for generating vascular organoids using human pluripotent stem cells.

Vascular organoids derived from human induced pluripotent stem cells (hiPSCs) recapitulate the cell type diversity and complex architecture of human ...

Bioengineering

Single-Cell Resolution Three-Dimensional Imaging of Intact Organoids

Organoid technology, in vitro 3D culturing of miniature tissue, has opened a new experimental window for cellular processes that govern organ development and function as well as disease. Fluorescence microscopy has played a major role in characterizing their cellular composition in detail and demonstrating their similarity to the tissue they originate from. In this article, we present a comprehensive protocol for high-resolution 3D imaging of whole organoids upon immunofluorescent labeling. This method is widely applicable for imaging of organoids differing in origin, size and shape. Thus far we have applied the method to airway, colon, kidney, and liver organoids derived from healthy human tissue, as well as human breast tumor organoids and mouse mammary gland organoids. We use an optical clearing agent, FUnGI, which enables the acquisition of whole 3D organoids with the opportunity for single-cell quantification of markers. This three-day protocol from organoid harvesting to image analysis is optimized for 3D imaging using confocal microscopy.

The entire 3D structure and cellular content of organoids, as well as their phenotypic resemblance to the original tissue can be captured using the single-cell resolution 3D imaging protocol described here. This protocol can be applied to a wide range of organoids varying in origin, size and shape.

Organoid technology, in vitro 3D culturing of miniature tissue, has opened a new experimental window for cellular processes that govern organ development ...

A High-Throughput Platform for Culture and 3D Imaging of Organoids

The characterization of a large number of three-dimensional (3D) organotypic cultures (organoids) at different resolution scales is currently limited by standard imaging approaches. This protocol describes a way to prepare microfabricated organoid culture chips, which enable multiscale, 3D live imaging on a user-friendly instrument requiring minimal manipulations and capable of up to 300 organoids/h imaging throughput. These culture chips are compatible with both air and immersion objectives (air, water, oil, and silicone) and a wide range of common microscopes (e.g., spinning disk, point scanner confocal, wide field, and brightfield). Moreover, they can be used with light-sheet modalities such as the single-objective, single-plane illumination microscopy (SPIM) technology (soSPIM). 
The protocol described here gives detailed steps for the preparation of the microfabricated culture chips and the culture and staining of organoids. Only a short length of time is required to become familiar with, and consumables and equipment can be easily found in normal biolabs. Here, the 3D imaging capabilities will be demonstrated only with commercial standard microscopes (e.g., spinning disk for 3D reconstruction and wide field microscopy for routine monitoring).

This paper presents a fabrication protocol for a new type of culture substrate with hundreds of microcontainers per mm2, in which organoids can be cultured and observed using high-resolution microscopy. The cell seeding and immunostaining protocols are also detailed.

The characterization of a large number of three-dimensional (3D) organotypic cultures (organoids) at different resolution scales is currently limited by ...

Optimization of Renal Organoid and Organotypic Culture for Vascularization, Extended Development, and Improved Microscopy Imaging

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Enhanced Viability for Ex vivo 3D Hydrogel Cultures of Patient-Derived Xenografts in a Perfused Microfluidic Platform

Three-Dimensional Imaging of Organoids to Study Primary Ciliogenesis During ex vivo Organogenesis

A Simplified Method for Generating Kidney Organoids from Human Pluripotent Stem Cells

Efficient Vascularization of Kidney Organoids through Intracelomic Transplantation in Chicken Embryos

Generating Kidney Organoids in Suspension from Induced Pluripotent Stem Cells

Early Unguided Human Brain Organoid Neurovascular Niche Modeling into the Permissive Chick Embryo Chorioallantoic Membrane

In Vitro Pancreas Organogenesis from Dispersed Mouse Embryonic Progenitors

Vascular Organoid Generation from Human-Induced Pluripotent Stem Cells

Single-Cell Resolution Three-Dimensional Imaging of Intact Organoids

A High-Throughput Platform for Culture and 3D Imaging of Organoids