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. 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=Global+Quantification+of+Tissue+Dynamics+in+the+Developing+Mouse+Kidney.">Global Quantification of Tissue Dynamics in the Developing Mouse Kidney.</a> Developmental Cell. 29, 188-202 (2014).</li><li>Saarela, U., 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=Novel+fixed+z-direction+(FiZD)+kidney+primordia+and+an+organoid+culture+system+for+time-lapse+confocal+imaging.">Novel fixed z-direction (FiZD) kidney primordia and an organoid culture system for time-lapse confocal imaging.</a> Development. 144 (6), 1113-1117 (2017).</li><li>Yalcin, H. C., Shekhar, A., Rane, A. A., Butcher, J. 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. 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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 border="0" cellpadding="0" cellspacing="0" style="width:1179px;" width="1178">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:429px;">Adjustable Spade Drill Bit</td>
			<td style="width:289px;">Bosch</td>
			<td style="width:277px;">2609255277</td>
			<td style="width:183px;">For drilling 20 mm diameter holes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">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 height="25">
			<td height="25" style="height:25px;">Cell Incubator</td>
			<td>Panasonic</td>
			<td>13090543</td>
			<td>Temperature set at 37&#176; C, 90% humidity, 5% CO2</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Cell Incubator</td>
			<td>SANYO</td>
			<td>10070347</td>
			<td>Chicken CAM-culture incubator. Temperature set at 37&#176; C, 90% humidity, 5% CO2</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">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 height="21">
			<td height="21" style="height:21px;">Circular Saw Blade for Dremel Rotary Tool</td>
			<td>Dremel</td>
			<td>SC690</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Confocal Microscope</td>
			<td>Zeiss</td>
			<td>LSM780</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Corning Transwell Multiple Well Plate with Permeable Polycarbonate Membrane Inserts</td>
			<td>Corning</td>
			<td>10301031</td>
			<td>For 6-well plates. 40 &#181;m pore size</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Countersink Drill Bit</td>
			<td>Craftomat, Bauhaus</td>
			<td>22377902</td>
			<td>For polishing (20.5 mm, 1/4&#34;, HSS)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Disposable Glass Capillary Tube</td>
			<td>Blaubrand</td>
			<td>7087 33</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Disposable Scalpel</td>
			<td>Swann-Morton</td>
			<td>0501</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dissecting Microscope</td>
			<td>Olympus</td>
			<td>SZ61</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Drilling Machine</td>
			<td>Bosch</td>
			<td>GSR 18 V-EC Professional</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:429px;">Dulbecco&#39;s Modified Eagle&#39;s Medium</td>
			<td>Sigma</td>
			<td>D777</td>
			<td>High glucose</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">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&#176; C with relative humidity set above 60%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ethanol (70%)</td>
			<td>VWR</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fertilized Eggs</td>
			<td style="width:289px;">Haaviston Siitoskanala, Panelia, Finland</td>
			<td></td>
			<td>Hy-Line White and Nick Chick</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fetal Bovine Serum</td>
			<td>HyClone</td>
			<td>SH3007003HI Thermo Scientific</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Forceps DUMONT #5</td>
			<td>Dumont</td>
			<td>#5SF</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glass Coverslips</td>
			<td>Menzel-Gl&#228;ser</td>
			<td>Menzel BBAD02200220#A1TBCMNZ#0##</td>
			<td>22x22 mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Histoacryl Glue</td>
			<td>Braun</td>
			<td>1050052</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Matrigel</td>
			<td>Corning</td>
			<td>356230</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">On-stage Incubator</td>
			<td>Okolab</td>
			<td></td>
			<td>Boldline, custom made</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PBS -/-</td>
			<td>Corning</td>
			<td>20-031-CV</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PBS +/+</td>
			<td>Biowest</td>
			<td>X0520-500</td>
			<td>Washing the mini reservoirs.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Penicillin and Streptomycin</td>
			<td>Sigma</td>
			<td>P4333</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Polystyrene Beads</td>
			<td>Corpuscular</td>
			<td>1000263-10</td>
			<td>70 &#181;m in diameter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Rotary Multi Tool System</td>
			<td>Dremel</td>
			<td>4000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Soldering Iron</td>
			<td>Weller</td>
			<td>TCP S</td>
			<td>Heated glass capillary can also be used</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:429px;">Thincert 12-well Cell Culture Inserts With 0.4 &#181;m-pore Polystyrene Membrane</td>
			<td>Greiner Bio-One</td>
			<td>665641</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:429px;">Thincert 6-well Cell Culture Inserts With 0.4 &#181;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 ...

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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 ...

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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

Culture and Co-Culture of Mouse Ovaries and Ovarian Follicles

The mammalian ovary is composed of ovarian follicles, each follicle consisting of a single oocyte surrounded by somatic granulosa cells, enclosed together within a basement membrane. A finite pool of follicles is laid down during embryonic development, when oocytes in meiotic arrest form a close association with flattened granulosa cells, forming primordial follicles. By or shortly after birth, mammalian ovaries contain their lifetime&#8217;s supply of primordial follicles, from which point onwards there is a steady release of follicles into the growing follicular pool.
The ovary is particularly amenable to development in vitro, with follicles growing in a highly physiological manner in culture. This work describes the culture of whole neonatal ovaries containing primordial follicles, and the culture of individual ovarian follicles, a method which can support the development of follicles from an immature through to the preovulatory stage, after which their oocytes are able to undergo fertilization in vitro. The work outlined here uses culture systems to determine how the ovary is affected by exposure to external compounds. We also describe a co-culture system, which allows investigation of the interactions that occur between growing follicles and the non-growing pool of primordial follicles.

This protocol describes the primary culture/co-culture of mouse ovarian tissue, using ovaries from neonatal mice and individual ovarian follicles from prepubertal mice. The culture techniques support development in a highly physiological manner, allowing investigation of the effect of extrinsic agents on the ovary, and of interactions between ovarian follicles.

The mammalian ovary is composed of ovarian follicles, each follicle consisting of a single oocyte surrounded by somatic granulosa cells, enclosed together ...

Ex Utero Electroporation and Organotypic Slice Culture of Mouse Hippocampal Tissue

Mouse genetics offers a powerful tool determining the role of specific genes during development. Analyzing the resulting phenotypes by immunohistochemical and molecular methods provides information of potential target genes and signaling pathways. To further elucidate specific regulatory mechanisms requires a system allowing the manipulation of only a small number of cells of a specific tissue by either overexpression, ablation or re-introduction of specific genes and follow their fate during development. To achieve this ex utero electroporation of hippocampal structures, especially the dentate gyrus, followed by organotypic slice culture provides such a tool. Using this system to generate mosaic deletions allows determining whether the gene of interest regulates cell-autonomously developmental processes like progenitor cell proliferation or neuronal differentiation. Furthermore it facilitates the rescue of phenotypes by re-introducing the deleted gene or its target genes. In contrast to in utero electroporation the ex utero approach improves the rate of successfully targeting deeper layers of the brain like the dentate gyrus. Overall ex utero electroporation and organotypic slice culture provide a potent tool to study regulatory mechanisms in a semi-native environment mirroring endogenous conditions.

Ex Utero Electroporation and Organotypic Slice Culture of Mouse Hippocampal Tissue

Here we present a protocol providing a tool to examine regulatory mechanisms of specific genes during hippocampal development. Employing ex utero electroporation and organotypic slice culture allows the up- and down-regulation of the expression of genes of interest in single cells and follow their fate during development.

Mouse genetics offers a powerful tool determining the role of specific genes during development. Analyzing the resulting phenotypes by immunohistochemical ...

Analysis of Zebrafish Kidney Development with Time-lapse Imaging Using a Dissecting Microscope Equipped for Optical Sectioning

In order to understand organogenesis, the spatial and temporal alterations that occur during development of tissues need to be recorded. The method described here allows time-lapse analysis of normal and impaired kidney development in zebrafish embryos by using a fluorescence dissecting microscope equipped for structured illumination and z-stack acquisition. To visualize nephrogenesis, transgenic zebrafish (Tg(wt1b:GFP)) with fluorescently labeled kidney structures were used. Renal defects were triggered by injection of an antisense morpholino oligonucleotide against the Wilms tumor gene wt1a, a factor known to be crucial for kidney development.
The advantage of the experimental setup is the combination of a zoom microscope with simple strategies for re-adjusting movements in x, y or z direction without additional equipment. To circumvent focal drift that is induced by temperature variations and mechanical vibrations, an autofocus strategy was applied instead of utilizing a usually required environmental chamber. In order to re-adjust the positional changes due to a xy-drift, imaging chambers with imprinted relocation grids were employed.
In comparison to more complex setups for time-lapse recording with optical sectioning such as confocal laser scanning&#160;or light sheet microscopes, a zoom microscope is easy to handle. Besides, it offers dissecting microscope-specific benefits such as high depth of field and an extended working distance.
The method to study organogenesis presented here can also be used with fluorescence stereo microscopes not capable of optical sectioning. Although limited for high-throughput, this technique offers an alternative to more complex equipment that is normally used for time-lapse recording of developing tissues and organ dynamics.

The method described here allows time-lapse analysis of organ development in zebrafish embryos by using a fluorescence dissecting microscope capable of performing optical sectioning and simple strategies of readjustment to correct focal and planar drift.

In order to understand organogenesis, the spatial and temporal alterations that occur during development of tissues need to be recorded. The method ...

A Simple Chamber for Long-term Confocal Imaging of Root and Hypocotyl Development

Several aspects of plant development, such as lateral root morphogenesis, occur on time spans of several days. To study underlying cellular and subcellular processes, high resolution time-lapse microscopy strategies that preserve physiological conditions are required. Plant tissues must have adequate nutrient and water supply with sustained gaseous exchange but, when submerged and immobilized under a coverslip, they are particularly susceptible to anoxia. One strategy that has been successfully employed is the use of a perfusion system to maintain a constant supply of oxygen and nutrients. However, such arrangements can be complicated, cumbersome, and require specialized equipment. Presented here is an alternative strategy for a simple imaging system using perfluorodecalin as an immersion medium. This system is easy to set up, requires minimal equipment, and is easily mounted on a microscope stage, allowing several imaging chambers to be set up and imaged in parallel. In this system, lateral root growth rates are indistinguishable from growth rates under standard conditions on agar plates for the first two days, and lateral root growth continues at reduced rates for at least another day. Plant tissues are supplied with nutrients via an agar slab that can be used also to administer a range of pharmacological compounds. The system was established to monitor lateral root development but is readily adaptable to image other plant organs such as hypocotyls and primary roots.

Presented here is a simple technique for high-resolution confocal time-lapse imaging of root and hypocotyl development for up to 3 days using high numerical-aperture objectives and perfluorodecalin as an immersion medium.

Several aspects of plant development, such as lateral root morphogenesis, occur on time spans of several days. To study underlying cellular and ...

Culture of Adult Transgenic Zebrafish Retinal Explants for Live-cell Imaging by Multiphoton Microscopy

An endogenous regeneration program is initiated by M&#252;ller glia in the adult zebrafish (Danio rerio) retina following neuronal damage and death. The M&#252;ller glia re-enter the cell cycle and produce neuronal progenitor cells that undergo subsequent rounds of cell divisions and differentiate into the lost neuronal cell types. Both M&#252;ller glia and neuronal progenitor cell nuclei replicate their DNA and undergo mitosis in distinct locations of the retina, i.e. they migrate between the basal Inner Nuclear Layer (INL) and the Outer Nuclear Layer (ONL), respectively, in a process described as Interkinetic Nuclear Migration (INM). INM has predominantly been studied in the developing retina. To examine the dynamics of INM in the adult regenerating zebrafish retina in detail, live-cell imaging of fluorescently-labeled M&#252;ller glia/neuronal progenitor cells is required. Here, we provide the conditions to isolate and culture dorsal retinas from Tg[gfap:nGFP]mi2004 zebrafish that were exposed to constant intense light for 35 h. We also show that these retinal cultures are viable to perform live-cell imaging experiments, continuously acquiring z-stack images throughout the thickness of the retinal explant for up to 8 h using multiphoton microscopy to monitor the migratory behavior of gfap:nGFP-positive cells. In addition, we describe the details to perform post-imaging analysis to determine the velocity of apical and basal INM. To summarize, we established conditions to study the dynamics of INM in an adult model of neuronal regeneration. This will advance our understanding of this crucial cellular process and allow us to determine the mechanisms that control INM.

Zebrafish retinal regeneration has mostly been studied using fixed retinas. However, dynamic processes such as interkinetic nuclear migration occur during the regenerative response and require live-cell imaging to investigate the underlying mechanisms. Here, we describe culture and imaging conditions to monitor Interkinetic Nuclear Migration (INM) in real-time using multiphoton microscopy.

An endogenous regeneration program is initiated by M&#252;ller glia in the adult zebrafish (Danio rerio) retina following neuronal damage and death. The ...

Multimodal Hierarchical Imaging of Serial Sections for Finding Specific Cellular Targets within Large Volumes

Targeting specific cells at ultrastructural resolution within a mixed cell population or a tissue can be achieved by hierarchical imaging using a combination of light and electron microscopy. Samples embedded in resin are sectioned into arrays consisting of ribbons of hundreds of ultrathin sections and deposited on pieces of silicon wafer or conductively coated coverslips. Arrays are imaged at low resolution using a digital consumer like smartphone camera or light microscope (LM) for a rapid large area overview, or a wide field fluorescence microscope (fluorescence light microscopy (FLM)) after labeling with fluorophores. After post-staining with heavy metals, arrays are imaged in a scanning electron microscope (SEM). Selection of targets is possible from 3D reconstructions generated by FLM or from 3D reconstructions made from the SEM image stacks at intermediate resolution if no fluorescent markers are available. For ultrastructural analysis, selected targets are finally recorded in the SEM at high-resolution (a few nanometer image pixels). A ribbon-handling tool that can be retrofitted to any ultramicrotome is demonstrated. It helps with array production and substrate removal from the sectioning knife boat. A software platform that allows automated imaging of arrays in the SEM is discussed. Compared to other methods generating large volume EM data, such as serial block-face SEM (SBF-SEM) or focused ion beam SEM (FIB-SEM), this approach has two major advantages: (1) The resin-embedded sample is conserved, albeit in a sliced-up version. It can be stained in different ways and imaged with different resolutions. (2) As the sections can be post-stained, it is not necessary to use samples strongly block-stained with heavy metals to introduce contrast for SEM imaging or render the tissue blocks conductive. This makes the method applicable to a wide variety of materials and biological questions. Particularly prefixed materials e.g., from biopsy banks and pathology labs, can directly be embedded and reconstructed in 3D.

This protocol targets specific cells in tissue for imaging at nanoscale resolution using a scanning electron microscope (SEM). Large numbers of serial sections from resin-embedded biological material are first imaged in a light microscope to identify the target and then in a hierarchical manner in the SEM.

Targeting specific cells at ultrastructural resolution within a mixed cell population or a tissue can be achieved by hierarchical imaging using a ...

Live Cell Imaging of Chromosome Segregation During Mitosis

Chromosomes must be reliably and uniformly segregated into daughter cells during mitotic cell division. Fidelity of chromosomal segregation is controlled by multiple mechanisms that include the Spindle Assembly Checkpoint (SAC). The SAC is part of a complex feedback system that is responsible for prevention of a cell progress through mitosis unless all chromosomal kinetochores have attached to spindle microtubules. Chromosomal lagging and abnormal chromosome segregation is an indicator of dysfunctional cell cycle control checkpoints and can be used to measure the genomic stability of dividing cells. Deregulation of the SAC can result in the transformation of a normal cell into a malignant cell through the accumulation of errors during chromosomal segregation. Implementation of the SAC and the formation of the kinetochore complex are tightly regulated by interactions between kinases and phosphatase such as Protein Phosphatase 2A (PP2A). This protocol describes live cell imaging of lagging chromosomes in mouse embryonic fibroblasts isolated from mice that had a knockout of the PP2A-B56&#947; regulatory subunit. This method overcomes the shortcomings of other cell cycle control imaging techniques such as flow cytometry or immunocytochemistry that only provide a snapshot of a cell cytokinesis status, instead of a dynamic spatiotemporal visualization of chromosomes during mitosis.

This protocol describes an easy and convenient method to label and visualize live chromosomes in mitotic cells using Histone2B-GFP BacMam 2.0 label and a spinning disc confocal microscopy system.

Chromosomes must be reliably and uniformly segregated into daughter cells during mitotic cell division. Fidelity of chromosomal segregation is controlled ...

Organotypic Culture Method to Study the Development Of Embryonic Chicken Tissues

The embryonic chicken is commonly used as a reliable model organism for vertebrate development. Its accessibility and short incubation period makes it ideal for experimentation. Currently, the study of these developmental pathways in the chicken embryo is conducted by applying inhibitors and drugs at localized sites and at low concentrations using a variety of methods. In vitro tissue&#160;culturing is a technique that enables the study of tissues separated from the host organism, while simultaneously bypassing many of the physical limitations present when working with whole embryos, such as the susceptibility of embryos to high doses of potentially lethal chemicals. Here, we present an organotypic culturing protocol for culturing the embryonic chicken half head in vitro, which presents new opportunities for the examination of developmental processes beyond the currently established methods.

Here, we present an organotypic culturing protocol to grow embryonic chicken organs in vitro. Using this method, the development of embryonic chicken tissue can be studied, while maintaining a high degree of control over the culture environment.

The embryonic chicken is commonly used as a reliable model organism for vertebrate development. Its accessibility and short incubation period makes it ...

A Layered Mounting Method for Extended Time-Lapse Confocal Microscopy of Whole Zebrafish Embryos

Dynamics of development can be followed by confocal time-lapse microscopy of live transgenic zebrafish embryos expressing fluorescence in specific tissues or cells. A difficulty with imaging whole embryo development is that zebrafish embryos grow substantially in length. When mounted as regularly done in 0.3-1% low melt agarose, the agarose imposes growth restriction, leading to distortions in the soft embryo body. Yet, to perform confocal time-lapse microscopy, the embryo must be immobilized. This article describes a layered mounting method for zebrafish embryos that restrict the motility of the embryos while allowing for the unrestricted growth. The mounting is performed in layers of agarose at different concentrations. To demonstrate the usability of this method, whole embryo vascular, neuronal and muscle development was imaged in transgenic fish for 55 consecutive hours. This mounting method can be used for easy, low-cost imaging of whole zebrafish embryos using inverted microscopes without requirements of molds or special equipment.

This article describes a method to mount fragile zebrafish embryos for extended time-lapse confocal microscopy. This low-cost method is easy to perform using regular glass-bottom microscopy dishes for imaging on any inverted microscope. The mounting is performed in layers of agarose at different concentrations.

Dynamics of development can be followed by confocal time-lapse microscopy of live transgenic zebrafish embryos expressing fluorescence in specific tissues ...

4D Microscopy: Unraveling Caenorhabditis elegans Embryonic Development Using Nomarski Microscopy

4D microscopy is an invaluable tool for unraveling the embryonic developmental process in different animals. Over the last decades, Caenorhabditis elegans has emerged as one of the best models for studying development. From an optical point of view, its size and transparent body make this nematode an ideal specimen for DIC (Differential Interference Contrast or Nomarski) microscopy. This article illustrates a protocol for growing C. elegans nematodes, preparing and mounting their embryos, performing 4D microscopy and cell lineage tracing. The method is based on multifocal time-lapse records of Nomarski images and analysis with specific software. This technique reveals embryonic developmental dynamics at the cellular level. Any embryonic defect in mutants, such as problems in spindle orientation, cell migration, apoptosis or cell fate specification, can be efficiently detected and scored. Virtually every single cell of the embryo can be followed up to the moment the embryo begins to move. Tracing the complete cell lineage of a C. elegans embryo by 4D DIC microscopy is laborious, but the use of specific software greatly facilitates this task. In addition, this technique is easy to implement in the lab. 4D microscopy is a versatile tool and opens the possibility of performing an unparalleled analysis of embryonic development.

4D Microscopy: Unraveling Caenorhabditis elegans Embryonic Development Using Nomarski Microscopy

Here, we present a protocol for preparing and mounting Caenorhabditis elegans embryos, recording development under a 4D microscope and tracing cell lineage.

4D microscopy is an invaluable tool for unraveling the embryonic developmental process in different animals. Over the last decades, Caenorhabditis elegans ...