We thank all the patients and their families for participating in the study. We thank the clinical team who facilitated our research. We are grateful for support of the European Research Council (ERC) starting grant 850571 (J.D.), the Dutch Cancer Society (KWF)/Alpe d&#39;HuZes Bas Mulder Award (no. 10218, J.D.), Oncode Institute, and Foundation Children Cancer Free (KiKa no. 292, C.C.)

NOTE: The experiments described herein were approved by the medical ethical committee of the Erasmus Medical Center (Rotterdam, the Netherlands) and Princess M&#225;xima Center for Pediatric Oncology (Utrecht, the Netherlands).
1. Generation of human kidney tubuloids from tissue
<ol>
	<li>Materials
	<ol>
		<li>Prewarm multiwell tissue culture plates (6, 12 and 24 wells) overnight at 37 &#176;C.</li>
		<li>Prepare basal medium (AdDF+++) by adding 1x L-alanine/L-glutamine supplement, 1% w/v 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid (HEPES, 10 mM), and 1% Penicillin/Streptomycin antibiotics to Advanced DMEM/F12.</li>
		<li>Prepare culture medium by adding 1.5% B27 supplement, 10% R-spondin-conditioned medium, epidermal growth factor (50 ng/mL), fibroblast growth factor-10 (100 ng/mL), N-acetylcysteine (1.25 mM), Rho-kinase inhibitor Y-27632 (10 &#181;M), broad-spectrum antibiotics (0.1 mg/mL), and A83-01 (5 &#181;M) to AdDF+++. Warm to 37 &#176;C before use.</li>
		<li>Prepare the basement membrane extract (BME) by thawing an aliquot overnight at 4 &#176;C. Keep the BME on ice during the procedure.</li>
		<li>Prepare collagenase solution by diluting collagenase to a final concentration of 1 mg/mL in AdDF+++, with the addition of 10 &#181;M Y-27632. Warm up to 37 &#176;C.</li>
		<li>Prepare 10 cm Petri dishes, scalpels, and tweezers. Disinfect the utensils by applying an ultraviolet lamp for 20 min, followed by washings with disinfectant, demi water, and 70% v/v ethanol. Air-dry the utensils.</li>
		<li>Prewarm a horizontal shaker to 37 &#176;C.</li>
	</ol>
	</li>
	<li>Procedure
	<ol>
		<li>Collect kidney tissue in a 50 mL tube filled with 30-40 mL of AdDF+++ medium. Place the piece of tissue in ~1 mL of medium in a 10 cm Petri dish. Mince the tissue into pieces of ~1 mm3 size using scalpels (Figure 1A).</li>
		<li>Transfer the minced tissue to a 15 mL tube using forceps and scalpels. Add 5 mL of AdDF+++ to the Petri dish, wash, and collect the medium with a 10 mL sterile pipette. Transfer all these contents to the same tube.</li>
		<li>Centrifuge at 300 &#215; g for 5 min at room temperature, and remove the supernatant. Add 3-4 mL of collagenase solution to the 15 mL tube. Move the tube to a horizontal shaker set at 37 &#176;C and a speed of 250 rpm.</li>
		<li>After the first 15 min of incubation, check the sample and vigorously shake the tube. Repeat until the suspension is homogeneous and most pieces of tissue have disappeared, with a maximum incubation time of 45-60 min. (Figure 1B).</li>
		<li>Fill up the tube with AdDF+++, and mix by inverting the tube 5-10x. Centrifuge at 300 &#215; g for 5 min at 4 &#176;C, and remove the supernatant. If the pellet is red, proceed with step 1.2.6. Otherwise, go to step 1.2.8.</li>
		<li>Add 1 mL of red blood cell lysis buffer (see the Table of Materials) on top of the cell pellet. Gently tap the tube to resuspend the pellet (do not resuspend with pipette tip). Incubate at room temperature for 5 min.</li>
		<li>Fill up the tube with AdDF+++. Centrifuge at 300 &#215; g for 5 min at 4 &#176;C, and remove the supernatant. If blood cells are still visible, repeat the procedure once more, starting from step 1.2.6. Otherwise, proceed with step 1.2.8.</li>
		<li>If chunks of tissue are visible at this point of the protocol, proceed with step 1.2.8. Otherwise, proceed with the measurement of the pellet volume in step 1.2.9. Add 5 mL of AdDF+++ to the tube, and resuspend with a 10 mL sterile pipette. Filter the suspension through a 100 &#181;m nylon cell strainer placed on a 50 mL tube.</li>
		<li>Transfer the filtered suspension to a new 15 mL tube. Centrifuge at 300 &#215; g for 5 min at 4 &#176;C, and remove the supernatant. Measure the volume of the cell pellet using a p1000 pipette set to a known volume. Carefully pipette up and down to resuspend without creating air bubbles. Transfer the tube to ice for 1 min.</li>
		<li>Add 70-75% volume of BME to the pellet. Resuspend using a p1000 or p200 pipette, and plate 15 &#181;L droplets in a prewarmed, multiwell cell culture plate (6, 12, or 24 wells, based on total plating volume (&#60;100 &#181;L, 24 wells; 100-200 &#181;L, 12 wells; &#62;200 &#181;L, 6 wells)).</li>
		<li>Turn the plate upside down, and let the plate rest in the incubator for 15-20 min at 37 &#176;C (Figure 1C). Add prewarmed culture medium, and inspect the cultures (Figure 1C,D).</li>
	</ol>
	</li>
</ol>
2. Generation of human kidney tubuloids from urine
NOTE: Urine is a hostile environment for cells. It is important for a successful execution of this protocol that urine samples are processed as soon as possible, preferably within 4 h from excretion. In the meantime, urine samples should be stored at 4 &#176;C.
<ol>
	<li>Materials
	<ol>
		<li>Prewarm multiwell tissue culture plates (6, 12 and 24 wells) overnight at 37 &#176;C.</li>
		<li>Prepare washing medium: AdDF+++ supplemented with broad-spectrum antibiotics (0.1 mg/mL) and 10 &#181;M Y-27632.</li>
		<li>Prepare culture medium as described above. Warm up at 37 &#176;C before use.</li>
		<li>Prepare BME matrix. Keep on ice during the procedure.</li>
	</ol>
	</li>
	<li>Procedure
	<ol>
		<li>Collect a urine sample, and divide it equally into 50 mL tubes (Figure 2A-I). Add 10-20 mL of washing medium to each of the tubes. Centrifuge at 300 &#215; g for 5 min at 4 &#176;C (Figure 2A-II), and remove the supernatant carefully.</li>
		<li>Add 10 mL of washing medium to each of the tubes. Carefully resuspend the pellets using a 10 mL sterile pipette. Centrifuge at 300 x g for 5 min at 4 &#176;C (Figure 2A-III), and remove the supernatant carefully.</li>
		<li>Resuspend the pellets, and transfer the contents of all the tubes into one 15 mL tube. Fill the tube with washing medium. Centrifuge at 300 x g for 5 min at 4 &#176;C (Figure 2A-IV), and remove the supernatant.</li>
		<li>Measure the volume of the cell pellet using a p1000 pipette set to a known volume. Carefully pipette up and down to resuspend without creating air bubbles. Transfer the tube to ice for 1 min.</li>
		<li>Add 70-75% volume of the BME to the pellet. Resuspend using a p1000 or p200 pipette, and plate 15 &#181;L droplets in a prewarmed, multiwell cell culture plate (6, 12, or 24 wells, based on total plating volume (&#60;100 &#181;L, 24 wells; 100-200 &#181;L, 12 wells; &#62;200 &#181;L, 6 wells)).</li>
		<li>Turn the plates upside down in the incubator for 15-20 min at 37 &#176;C. Add prewarmed culture medium, and inspect the cultures (Figure 2B).</li>
	</ol>
	</li>
</ol>
3. Expansion of tubuloid cultures
NOTE: Tubuloid cultures can be passaged approximately every 1-2 weeks with a split ratio of 1:2-1:3. They can be typically expanded for approximately 15 passages, with line-specific variations.
<ol>
	<li>Materials
	<ol>
		<li>Prewarm multiwell tissue culture plates (6, 12 and 24 wells) overnight at 37 &#176;C.</li>
		<li>Prepare basal medium (AdDF+++), and keep it on ice during the procedure.</li>
		<li>Prepare culture medium as previously described. Warm up to 37 &#176;C before use.</li>
		<li>Prepare BME matrix, and keep on ice during the procedure.</li>
		<li>Prepare trypsin replacement agent with the addition of Y-27632 to a concentration of 10 &#181;M. Warm up to 37 &#176;C before use.</li>
		<li>Autoclave non-filtered p10 tips.</li>
		<li>Cool the centrifuge to 4 &#176;C.</li>
	</ol>
	</li>
	<li>Procedure
	<ol>
		<li>Disrupt the droplets containing the tubuloids by pipetting up and down with a p1000 pipette using the medium present in the well. Use the tip to scrape the bottom of the well, making sure to collect all cells attached to the bottom.</li>
		<li>Collect the contents in a 15 mL tube. Add 10 mL of AdDF+++. Centrifuge at 300 x g for 5 min at 4 &#176;C, and remove the supernatant.</li>
		<li>Based on the size of the pellet, add trypsin replacement agent supplemented with 10 &#181;M Y-27632. Use 1 mL of trypsin replacement agent for 200 &#181;L of BME droplets containing tubuloids. Incubate at 37 &#176;C for 5 min.</li>
		<li>Use a p1000 pipette with tip, and insert a non-filtered, sterile p10 tip over the 1000 &#181;L tip. Pipette up and down for 20-30x to mechanically disrupt the organoids.</li>
		<li>Check under the microscope to see if many intact organoids are still present in the tube (Figure 3A). If more than 10% of intact organoids are present at this point, repeat from the 5 min incubation in step 3.2.3 to the microscopic observation for intact organoids in step 3.2.5&#160;once more. Otherwise, proceed with step 3.2.6.</li>
		<li>Fill up the tube with AdDF+++. Centrifuge at 300 &#215; g for 5 min at 4 &#176;C, and remove the supernatant. Measure the volume of the cell pellet using a p1000 pipette set to a known volume. Carefully pipette up and down to resuspend without creating air bubbles. Transfer the tube to ice for 1 min.</li>
		<li>Add 70-75% volume of BME to the pellet. Resuspend using a p1000 or p200 pipette, and plate 15 &#181;L droplets in a prewarmed, multiwell cell culture plate (6, 12, or 24 wells, based on total plating volume (&#60;100 &#181;L, 24 wells; 100-200 &#181;L, 12 wells; &#62;200 &#181;L, 6 wells)).</li>
		<li>Turn the plates upside down, and let the plate rest in the incubator for 15-20 min at 37 &#176;C. Add prewarmed culture medium, and inspect the cultures (Figure 3B).</li>
	</ol>
	</li>
</ol>

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The authors declare no conflict of interest.

Organoids are considered avatars of the tissue from which they are derived. They allow for rapid expansion of patient material while retaining the genotypic and phenotypic characteristics of the tissue of origin15. Organoid technology has recently opened the doors for the development of more representative preclinical models, which can be used as important tools to translate findings from the bench to the bedside. Kidney tubuloids are promising in vitro models for testing drug-induced nephrotoxicity, a common side effect of many chemotherapeutic drugs2,8,12. As such, patient-derived tumor organoid cultures were demonstrated to be predictive for patient response to treatment16,17,18. Testing drugs in a high-throughput manner on tubuloids therefore potentially allows better definition of therapeutic windows and decreases the risk of drug-induced nephrotoxicity in patients.
Commonly used antibiotics have been described to exert a toxic effect on the kidneys19. Although the presence of broad-spectrum antibiotics is necessary for the successful establishment of the cultures by preventing contamination, it is important to consider their potential nephrotoxicity. Although no negative effects of antibiotics have been observed on the establishment of tubuloid cultures, further investigation is needed to thoroughly evaluate their effects. Tubuloids can be exploited for studying and modeling diseases8. Ciliopathies (pathological dysfunction of cilia) as well as other genetic syndromes affecting the kidney could be studied by either generating tubuloid lines directly from affected subjects, or by using healthy cultures in which disease-specific driver mutations can be introduced via CRISPR/Cas9 genome editing20.
Although tubuloids are multicellular kidney cultures, they lack several renal cell types, including podocytes and endothelial cells8. Moreover, in contrast to some other ASC-derived organoid models, tubuloids have a limited replicative potential as they can be cultured for approximately 15 passages. This limited lifespan can, however, be significantly extended by the addition of Wnt to the culture medium21. Further optimization of the tubuloid culture protocol is required to make them even more representative&#160;of the kidney, including more differentiated cell types. Although the efficiency of tubuloid establishment from tissue samples is very high (&#62;95%), it can, in rare cases, fail. There can be different causes including: 1) poor quality of the starting material (e.g., necrotic tissue as a consequence of drug treatment), 2) overdigestion of the tissue sample, or 3) contamination of the primary sample.
To make sure that the quality of the tissue samples received is sufficient to proceed with the protocol, it is important to maintain close contact with the pathology staff performing the evaluation of the tissue upon surgery. If sufficient material is available, its viability must be confirmed by histological examination (e.g., hematoxylin and eosin staining). Furthermore, to prevent cell lysis during enzymatic digestion, it is important that the incubation procedure is no longer than 1 h. Lastly, to prevent contamination, antibiotics and antifungal agents should be added to the washing and culture media.
Urine can contain exfoliated epithelial cells that are not derived from the kidney22, which can contaminate the tubuloid cultures. These include, for instance, urothelium cells that are, in contrast to renal tubular cells, positive for tumor protein P63 and negative for PAX88. It is therefore recommended to test the cultures for PAX8 positivity to confirm the purity of established kidney tubuloid lines before proceeding with follow-up experiments (Figure 4B).
Urine represents a hostile environment for cells due to high osmotic pressure and low pH. It is therefore crucial for the success of the protocol that samples are processed as soon as possible upon urine collection. As such, the collected urine should be diluted and extensively washed with buffered solution as soon as possible to ensure presence of viable cells at the time of seeding. The success rate of tubuloid establishment will significantly decrease if urine is stored for several hours before processing. Lastly, although sterile, urine has a high risk of contamination associated with the collection process. It is therefore important to supplement washing and growth medium with antibiotics and antifungal agents. When taking all the above into account, a success rate of approximately 50%&#160;can be achieved.

Kidneys perform the function of systemically controlling the balance of body fluids. The impairment of their physiological function can be caused by different factors, including diabetes, hypertension, and drug-induced toxicity1. For a better understanding of normal kidney physiology as well as the development of renal diseases, the use of representative preclinical models is crucial. In recent years, several in vitro kidney models have been generated based on the so-called organoid technology2. Organoids are three-dimensional, multicellular structures resembling the morphology and physiology of the tissue (normal or diseased) from which they originate. They can be generated from pluripotent (PSCs) or adult stem cells (ASCs), each with their own characteristics and applications.
PSC-derived kidney organoids mimic nephrogenesis3,4,5. They can also be established from patient-derived committed cells by forced dedifferentiation (induced pluripotency or iPSC). iPSCs can subsequently be differentiated into the different cell types of the nephron, the functional unit of the kidney, by timely exposure to specific growth factor cocktails. While creating a rather complete mini-organ in a dish, their establishment remains time-consuming, and due to the reprogramming protocol, iPSCs can be susceptible to undesired genetic instability6. Furthermore, iPSC organoids are not able to fully mature into adult kidney cells, revealing a transcriptome profile that resembles the fetal kidney at an early development stage7.
ASC-derived human kidney tubuloids have been shown to recapitulate the renewal of adult kidney epithelium. They primarily represent the proximal tubule, loop of Henle, distal tubules, and collecting duct, as confirmed by the expression of different transporter proteins8,9,10. The tubuloid culture protocol allows for the rapid expansion of patient-derived kidney tissue, while retaining a stable genome. Research applications include studying normal kidney physiology, nephrotoxicity, drug testing, as well as disease modelling8,10,11,12. A potential limitation of the establishment of patient-derived organoid cultures, including tubuloids, is the availability of fresh tissue. However, several reports have shown that urine can serve as a source for kidney epithelial cells, thereby providing a much simpler, less invasive strategy to obtain patient material for tubuloid cultures8,13,14. Indeed, it has been recently shown that tubuloids can be grown from urine8. This article describes the establishment and maintenance of tubuloid cultures from kidney tissue and urine.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>A83-01</td>
			<td>Tocris</td>
			<td>2939/10</td>
			<td>Stock of 5 mM, final concentration of 5 &#181;M, activin receptor-like kinase 5 inhibitor</td>
		</tr>
		<tr>
			<td>Advanced DMEM/F12</td>
			<td>ThermoFisher Scientific</td>
			<td>12634010</td>
			<td></td>
		</tr>
		<tr>
			<td>antiPAX8 antibody</td>
			<td>LSBio</td>
			<td>LS-B13466</td>
			<td></td>
		</tr>
		<tr>
			<td>B27 supplement</td>
			<td>ThermoFisher Scientific</td>
			<td>17504044</td>
			<td>Stock of 50x, final concentration of&#160; 1x</td>
		</tr>
		<tr>
			<td>BME</td>
			<td>Trevigen</td>
			<td>3533-010-02</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase</td>
			<td>Sigma Aldrich</td>
			<td>&#160;C9407</td>
			<td>Stock of 10 mg/mL, final concentration of 1 mg/mL</td>
		</tr>
		<tr>
			<td>EGF</td>
			<td>Peprotech</td>
			<td>AF-100-15</td>
			<td>Stock of 0.5 mg/mL, final concentration of 50 ng/mL</td>
		</tr>
		<tr>
			<td>FGF10</td>
			<td>Peprotech</td>
			<td>100-26</td>
			<td>Stock of 0.1 mg/mL, final concentration of 100 ng/mL</td>
		</tr>
		<tr>
			<td>GlutaMAX - L-alanine/L-glutamine</td>
			<td>Gibco</td>
			<td>35050061</td>
			<td>Stock of 100x, final concentration of 1x</td>
		</tr>
		<tr>
			<td>Hepes</td>
			<td>Gibco</td>
			<td>15630106</td>
			<td>Stock of 1 M, final concentration of 10 mM</td>
		</tr>
		<tr>
			<td>Multiwell tissue culture plates 12 wells</td>
			<td>CELLSTAR</td>
			<td>665180</td>
			<td></td>
		</tr>
		<tr>
			<td>Multiwell tissue culture plates 24 wells</td>
			<td>CELLSTAR</td>
			<td>662160</td>
			<td></td>
		</tr>
		<tr>
			<td>Multiwell tissue culture plates 6 wells</td>
			<td>CELLSTAR</td>
			<td>657160</td>
			<td></td>
		</tr>
		<tr>
			<td>N-acetylcysteine</td>
			<td>Sigma Aldrich</td>
			<td>A9165</td>
			<td>Stock of 500 mM, final concentration of 1.25 mM</td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Gibco</td>
			<td>15140163</td>
			<td>Stock of 10.000 U/mL, final concentration of&#160; 100 U/mL</td>
		</tr>
		<tr>
			<td>Primocin - broad-range antibiotics</td>
			<td>Invivogen</td>
			<td>ant-pm-1</td>
			<td>Stock of 50 mg/mL, final concentration of 0.1 mg/mL</td>
		</tr>
		<tr>
			<td>Red blood cells lysis buffer</td>
			<td>Roche</td>
			<td>11814389001</td>
			<td></td>
		</tr>
		<tr>
			<td>RhoKinase inhibitor Y-27632</td>
			<td>Abmole Bioscience</td>
			<td>M1817</td>
			<td>Stock of 100 mM, final concentration of 10 &#181;M</td>
		</tr>
		<tr>
			<td>R-spondin conditioned medium</td>
			<td></td>
			<td></td>
			<td>Produced in house with the use of stable cell lines generated by Calvin Kuo lab. Final concentration is 10%</td>
		</tr>
		<tr>
			<td>TrypLe Express/ trypsin replacement agent</td>
			<td>ThermoFisher Scientific</td>
			<td>12605010</td>
			<td></td>
		</tr>
	</tbody>
</table>

generation of human kidney tubuloids from tissue and urine

Adult stem cell (ASC)-derived human kidney epithelial organoids, or tubuloids, can be established from healthy and diseased kidney epithelium with high efficiency. Normal kidney tubuloids recapitulate many aspects of their tissue of origin. They represent distinct nephron segments - most notably of the proximal tubule, loop of Henle, distal tubules, and collecting duct - and can be used to study normal kidney physiology. Furthermore, tubuloid technology facilitates disease modeling, e.g., for infectious diseases as well as for cancer. Obtaining kidney epithelial cells for tubuloid generation is, however, dependent on leftover surgical material (such as partial) nephrectomies) or needle biopsies. The ability to grow tubuloids from urine would provide an alternative, less invasive source of healthy kidney epithelial cells. It has been previously shown that tubuloid cultures can be successfully generated from only a few milliliters of freshly collected urine. This article describes the protocols to generate and propagate ASC-derived human kidney tubuloid cultures from tissue and urine samples.

For kidney tissue, tubuloid structures typically appear within 7 days after establishment (Figure 1D). Lack of apparent growth within the first 7 days indicates an unsuccessful outcome of the protocol. Generally, tubuloid cultures need to be passaged within 1-2 weeks after first plating. For urine, cell growth becomes apparent approximately 14-21 days after establishment, with the appearance of compact tubuloid structures and/or adherent cells on the bottom of the plate (Figure 2B). Culture establishment most likely has failed if no growth can be detected with a brightfield microscope within 4 weeks after plating. For urine-derived cultures, the first passaging generally occurs between 3 and 4 weeks. While in the first passages kidney tubuloid cultures consist mainly of compact structures, the presence of cystic epithelial tubuloids increases with the increase of passage number (Figure 1D and Figure 2B). The successful generation of human kidney tubuloid cultures can be assessed by performing immunohistochemical staining for markers expressed in tubular kidney epithelium, such as paired box gene 8 protein (PAX8) (Figure 4A,B).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62404/62404fig01.jpg" /> 
Figure 1: Tissue-derived kidney tubuloid cultures. (A)&#160;Overview of the procedure to mince kidney tissue. Tissue is minced to a size of ~1 mm3 using scalpels. (B)&#160;Example of correct enzymatic digestion of healthy kidney tissue. Tissue is shown before (left) and after (right) 45 min of enzymatic digestion with collagenase. Few pieces of tissue are still visible at the bottom of the tube, and the solution should become cloudy, indicating the presence of cells in the suspension. (C) Representative image of cell culture plates after plating of BME droplets containing the processed kidney tissue. After plating the droplets, culture plates are turned upside down (left) and placed in the incubator at 37 &#176;C. After 15-20 min, prewarmed culture medium is added to the well (right). (D) Representative brightfield images of tissue-derived tubuloid cultures. The first tubuloid structures become visible 2-3 days after first seeding. With increasing passage numbers, tubuloids typically change morphology to a more cystic phenotype. Scale bars = 300 &#181;m. Abbreviations: BME = basement membrane extract. <a href="https://www.jove.com/files/ftp_upload/62404/62404fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62404/62404fig02.jpg" /> 
Figure 2: Urine-derived kidney tubuloid cultures. (A)&#160;Overview of urine sample processing.&#160;As soon as possible after collection (I), urine samples are divided into 50 mL tubes and diluted in washing medium (II). After a second washing step (III), the content of the tubes is pooled and a third and final wash step (IV) is performed before plating. (B) Representative brightfield images of urine-derived tubuloid cultures. The first tubuloid structures and adherent cells should be visible within 21 days after first plating. Scale bars = 300 &#181;m. <a href="https://www.jove.com/files/ftp_upload/62404/62404fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/62404/62404fig03.jpg" /> 
Figure 3: Passaging of kidney tubuloid cultures. (A)&#160;Representative image of kidney tubuloid cultures after enzymatic digestion. After completing digestion, no more than 10% of intact tubuloid structures should remain. Scale bar = 300&#181;m.&#160;(B) Representative image of kidney tubuloid cultures after plating. Inspection of the cultures confirms that the majority of the tubuloids have been disrupted. Scale bar = 300 &#181;m <a href="https://www.jove.com/files/ftp_upload/62404/62404fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/62404/62404fig04.jpg" /> 
Figure 4: Histological characterization of tubuloid cultures. (A)&#160;H&#38;E staining of healthy kidney tissue and tubuloids. Scale bars = 100 &#181;m. (B) Immunohistochemistry of PAX8 in normal kidney tissue, tubuloids, MRTK tissue, and MRTK organoids. PAX8 positivity of the organoid structures confirms their kidney epithelial origin. Healthy kidney tissue shows both positive (tubules) and negative structures (glomeruli). MRTK tissue and organoids were included as negative control. Scale bars = 100 &#181;m. Abbreviations: H&#38;E = hematoxylin and eosin; PAX8 = paired box gene 8; MRTK = malignant rhabdoid tumor of the kidney. <a href="https://www.jove.com/files/ftp_upload/62404/62404fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Generation of Human Kidney Tubuloids from Tissue and Urine at JoVE.com

Generation of Human Kidney Tubuloids from Tissue and Urine

Human kidney tubuloid cultures represent a valuable in vitro model to study kidney physiology and disease. Tubuloids can be established from kidney tissue (healthy and diseased) as well as urine, the latter representing an easily obtainable and less invasive source of research material.

Adult stem cell (ASC)-derived human kidney epithelial organoids, or tubuloids, can be established from healthy and diseased kidney epithelium with high ...

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4.4K Views.  Princess Máxima Center for Pediatric Oncology. Human kidney tubuloid cultures represent a valuable in vitro model to study kidney physiology and disease. Tubuloids can be established from kidney tissue (healthy and diseased) as well as urine, the latter representing an easily obtainable and less invasive source of research material.

Video: Generation of Human Kidney Tubuloids from Tissue and Urine

Neuronal Nuclei Isolation from Human Postmortem Brain Tissue

Neurons in the human brain become postmitotic largely during prenatal development, and thus maintain their nuclei throughout the full lifespan. However, little is known about changes in neuronal chromatin and nuclear organization during the course of development and aging, or in chronic neuropsychiatric disease. However, to date most chromatin and DNA based assays (other than FISH) lack single cell resolution. To this end, the considerable cellular heterogeneity of brain tissue poses a significant limitation, because typically various subpopulations of neurons are intermingled with different types of glia and other non-neuronal cells. One possible solution would be to grow cell-type specific cultures, but most CNS cells, including neurons, are ex vivo sustainable, at best, for only a few weeks and thus would provide an incomplete model for epigenetic mechanisms potentially operating across the full lifespan.  Here, we provide a protocol to extract and purify nuclei from frozen (never fixed) human postmortem brain. The method involves extraction of nuclei in hypotonic lysis buffer, followed by ultracentrifugation and immunotagging with anti-NeuN antibody. Labeled neuronal nuclei are then collected separately using fluorescence-activated sorting. This method should be applicable to any brain region in a wide range of species and suitable for chromatin immunoprecipitation studies with site- and modification-specific anti-histone antibodies, and for DNA methylation and other assays. 

The cellular heterogeneity of brain tissue poses a significant limitation for the study of epigenetic markings in chromatin because most assays lack single cell resolution. Neurons typically are intermingled with glia and other non-neuronal cells. We provide a protocol to extract and collect neuronal nuclei from human brain.

Neurons in the human brain become postmitotic largely during prenatal development, and thus maintain their nuclei throughout the full lifespan. However, ...

Generation of Single-Cell Suspensions from Mouse Neural Tissue

Within the nervous system, hundreds of neuronal and glial cell types have been described. Each specific cell type in the brain or spinal cord has a repertoire of cell surface molecules, or molecular determinants, through which it can be identified and characterized. Currently, robust cell identification and separation technologies require single-cell preparations to be generated while simultaneously limiting cell death and destruction of characteristic surface protein. The gentleMACS Dissociator, when used in combination with trypsin or papain-based dissociation kits, can effectively and gently dissociate brain tissue while preserving antigen epitopes and limiting cell loss. Standardized preparation of single-cell suspensions is achieved using C Tubes and optimized, preset gentleMACS Programs. Once generated, single-cell suspensions can be treated with monoclonal conjugates like Anti-Prominin-1 MicroBeads, which identify neural progenitors, or purified further using Myelin Removal Beads.

Dissociating cells from specific tissue types requires specific parameters for tissue aggitation to obtain a high volume of viable, culturable cells. The Miltenyi gentleMACS Dissociator optimizes this task with a simple, practical protocol. In this publication the use of this apparatus on nerual tissue is explained.

Within the nervous system, hundreds of neuronal and glial cell types have been described. Each specific cell type in the brain or spinal cord has a ...

Generation of Human CD40-activated B cells

CD40-activated B cells (CD40-B cells) have been identified as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cancer immunotherapy 1-3. Compared to Dendritic cells (DCs), the best characterized APC, CD40-B cells have several distinct biological and technical properties. Similar to DCs, B cells show an increased expression of MHC and co-stimulatory molecules (Fig.1b), exhibit a strong migratory capacity and present antigen presentation efficiently to T cells, after stimulation with interleukin-4 and CD40 ligand (CD40L). However, in contrast to immature or mature DCs, CD40-B cells express the full lymph node homing triad consisting of CD62L, CCR7/CXCR4, and leukocyte function antigen-1 (LFA1, CD11a/CD18), necessary for homing to secondary lymphoid organs (Fig.1a) 3. CD40-B cells can be generated without difficulties from very small amounts of peripheral blood which can be further expanded in vitro to very large amounts of highly-pure CD40-B cells (&gt;109 cells per patient) from healthy donors as well as cancer patients (Fig.1c,d) 1,4. 
In this protocol we demonstrate how to obtain fully activated CD40-B cells from human PBMC. Key molecules for the cell culture are CD40 ligand, interleukin-4 (IL-4) and cyclosporin A (CsA), which are replenished in a 3-4 day culture cycle. For laboratory purposes CD40-stimulation is provided by NIH/3T3 cells expressing recombinant human CD40 ligand (tCD40L NIH/3T3) 5. To avoid contamination with non-transfected cells, expression of the human CD40 ligand on the transfectants has to be checked regularly (Fig.2). 
After 14 days CD40-B cell cultures consist of more than 95% pure B cells and an expansion of CD40-B cells over 65 days is frequently possible without any loss of function 1, 4. CD40-B cells efficiently take up, process and present antigens to T cells 6. They do not only prime na&#970;ve, but also expand memory T cells 7,8. CD40-activated B cells can be used to study B-cell activation, differentiation and function. Moreover, they represent a promising tool for therapeutic or preventive vaccination against tumors 9.

In this video we present the ex vivo generation and expansion of human CD40-activated B cells (CD40-B) from peripheral blood mononuclear cells (PBMC) by stimulation with CD40 ligand and interleukin-4.

CD40-activated B cells (CD40-B cells) have been identified as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cancer ...

Generation of Human Cardiomyocytes: A Differentiation Protocol from Feeder-free Human Induced Pluripotent Stem Cells

In order to investigate the events driving heart development and to determine the molecular mechanisms leading to myocardial diseases in humans, it is essential first to generate functional human cardiomyocytes (CMs). The use of these cells in drug discovery and toxicology studies would also be highly beneficial, allowing new pharmacological molecules for the treatment of cardiac disorders to be validated pre-clinically on cells of human origin. Of the possible sources of CMs, induced pluripotent stem (iPS) cells are among the most promising, as they can be derived directly from readily accessible patient tissue and possess an intrinsic capacity to give rise to all cell types of the body 1. Several methods have been proposed for differentiating iPS cells into CMs, ranging from the classical embryoid bodies (EBs) aggregation approach to chemically defined protocols 2,3. In this article we propose an EBs-based protocol and show how this method can be employed to efficiently generate functional CM-like cells from feeder-free iPS cells.

Pluripotent stem cells, either embryonic or induced pluripotent stem (iPS) cells, constitute a valuable source of human differentiated cells, including cardiomyocytes. Here, we will focus on cardiac induction of iPS cells, showing how to use them to obtain functional human cardiomyocytes through an embryoid bodies-based protocol.

In order to investigate the events driving heart development and to determine the molecular mechanisms leading to myocardial diseases in humans, it is ...

Isolation of Primary Myofibroblasts from Mouse and Human Colon Tissue

The myofibroblast is a stromal cell of the gastrointestinal (GI) tract that has been gaining considerable attention for its critical role in many GI functions. While several myofibroblast cell lines are commercially available to study these cells in vitro, research results from a cell line exposed to experimental cell culture conditions have inherent limitations due to the overly reductionist nature of the work. Use of primary myofibroblasts offers a great advantage in terms of confirming experimental findings identified in a cell line. Isolation of primary myofibroblasts from an animal model allows for the study of myofibroblasts under conditions that more closely mimic the disease state being studied. Isolation of primary myofibroblasts from human colon tissue provides arguably the most relevant experimental data, since the cells come directly from patients with the underlying disease. We describe a well-established technique that can be utilized to isolate primary myofibroblasts from both mouse and human colon tissue. These isolated cells have been characterized to be alpha-smooth muscle actin and vimentin-positive, and desmin-negative, consistent with subepithelial intestinal myofibroblasts. Primary myofibroblast cells can be grown in cell culture and used for experimental purposes over a limited number of passages.

The myofibroblast is an influential stromal cell of the gastrointestinal tract that regulates important physiologic processes in both normal and disease states. We describe a technique that allows for the isolation of primary myofibroblasts from both mouse and human colon tissue, which can be utilized for in vitro experimentation.

The myofibroblast is  a stromal cell of the gastrointestinal (GI) tract that has been gaining  considerable attention for its critical role in many GI ...

Efficient Generation Human Induced Pluripotent Stem Cells from Human Somatic Cells with Sendai-virus

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs include modeling pathogenesis of human genetic diseases, autologous cell therapy after gene correction, and personalized drug screening by providing a source of patient-specific and symptom relevant cells. However, there are several hurdles to overcome, such as eliminating the remaining reprogramming factor transgene expression after human iPSCs production. More importantly, residual transgene expression in undifferentiated human iPSCs could hamper proper differentiations and misguide the interpretation of disease-relevant in vitro phenotypes. With this reason, integration-free and/or transgene-free human iPSCs have been developed using several methods, such as adenovirus, the piggyBac system, minicircle vector, episomal vectors, direct protein delivery and synthesized mRNA. However, efficiency of reprogramming using integration-free methods is quite low in most cases.
Here, we present a method to isolate human iPSCs by using Sendai-virus (RNA virus) based reprogramming system. This reprogramming method shows consistent results and high efficiency in cost-effective manner.

Here, we present our established method to reprogram human somatic cells into transgene-free human iPSCs with Sendai virus, which shows consistent outcome and enhanced efficiency.

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs ...

Generation and Culture of Blood Outgrowth Endothelial Cells from Human Peripheral Blood

Historically, the limited availability of primary endothelial cells from patients with vascular disorders has hindered the study of the molecular mechanisms underlying endothelial dysfunction in these individuals. However, the recent identification of blood outgrowth endothelial cells (BOECs), generated from circulating endothelial progenitors in adult peripheral blood, may circumvent this limitation by offering an endothelial-like, primary cell surrogate for patient-derived endothelial cells. Beyond their value to understanding endothelial biology and disease modeling, BOECs have potential uses in endothelial cell transplantation therapies. They are also a suitable cellular substrate for the generation of induced pluripotent stem cells (iPSCs) via nuclear reprogramming, offering a number of advantages over other cell types. We describe a method for the reliable generation, culture and characterization of BOECs from adult peripheral blood for use in these and other applications. This approach (i) allows for the generation of patient-specific endothelial cells from a relatively small volume of adult peripheral blood and (ii) produces cells that are highly similar to primary endothelial cells in morphology, cell signaling and gene expression.

This protocol allows for the reliable generation and characterization of blood outgrowth endothelial cells (BOECs) from a small volume of adult peripheral blood. BOECs can be used as a surrogate for endothelial cells from patients with vascular disorders and as a substrate for the generation of induced pluripotent stem cells.

Historically, the limited availability of primary endothelial cells from patients with vascular disorders has hindered the study of the molecular ...

Y-27632 Enriches the Yield of Human Melanocytes from Adult Skin Tissues

The isolation and culture of primary melanocytes from skin tissues is very important for biological research and has been widely used for clinical applications. Isolating primary melanocytes from skin tissues by the conventional method usually takes about 3 to 4 weeks to passage sufficiently. More importantly, the tissues used are usually newborn foreskins and it is still a challenge to efficiently isolate primary melanocytes from adult tissues. We recently developed a new isolation method for melanocytes that adds Y-27632, a Rho kinase inhibitor, to the initial culture medium for 48 h. Compared with the conventional protocol, this new method dramatically increases the yield of melanocytes and shortens the time required to isolate melanocytes from foreskin tissues. We now describe this new method in more detail using adult epidermis to efficiently culture primary melanocytes. Importantly, we show that melanocytes obtained from adult tissues prepared by this new method can function normally. This new protocol will significantly benefit studies of pigmentation defects and melanomas using primary melanocytes prepared from easily accessed adult skin tissues.

This paper reports that the addition of Y-27632 to TIVA medium can significantly increase the yield of melanocytes from adult skin tissues.

The isolation and culture of primary melanocytes from skin tissues is very important for biological research and has been widely used for clinical ...

Application of Laser Microdissection to Uncover Regional Transcriptomics in Human Kidney Tissue

Gene expression analysis of human kidney tissue is an important tool to understand homeostasis and disease pathophysiology. Increasing the resolution and depth of this technology and extending it to the level of cells within the tissue is needed. Although the use of single nuclear and single cell RNA sequencing has become widespread, the expression signatures of cells obtained from tissue dissociation do not maintain spatial context. Laser microdissection (LMD) based on specific fluorescent markers would allow the isolation of specific structures and cell groups of interest with known localization, thereby enabling the acquisition of spatially-anchored transcriptomic signatures in kidney tissue. We have optimized an LMD methodology, guided by a rapid fluorescence-based stain, to isolate five distinct compartments within the human kidney and conduct subsequent RNA sequencing from valuable human kidney tissue specimens. We also present quality control parameters to enable the assessment of adequacy of the collected specimens. The workflow outlined in this manuscript shows the feasibility of this approach to isolate sub-segmental transcriptomic signatures with high confidence. The methodological approach presented here may also be applied to other tissue types with substitution of relevant antibody markers.

We describe a protocol for laser microdissection of sub-segments of the human kidney, including the glomerulus, proximal tubule, thick ascending limb, collecting duct and interstitium. The RNA is then isolated from the obtained compartments and RNA sequencing is carried out to determine changes in the transcriptomic signature within each sub-segment.

Gene expression analysis of human kidney tissue is an important tool to understand homeostasis and disease pathophysiology. Increasing the resolution and ...

Tissue Collection and RNA Extraction from the Human Osteoarthritic Knee Joint

Osteoarthritis (OA) is a chronic and degenerative joint disease most often affecting the knee. As there is currently no cure, total knee arthroplasty (TKA) is a common surgical intervention. Experiments using primary human OA tissues obtained from TKA provide the capability to investigate disease mechanisms ex vivo. While OA was previously thought to impact mainly the cartilage, it is now known to impact multiple tissues in the joint. This protocol describes patient selection, sample processing, tissue homogenization, RNA extraction, and quality control (based on RNA purity, integrity, and yield) from each of seven unique tissues to support disease mechanism investigation in the knee joint. With informed consent, samples were obtained from patients undergoing TKA for OA. Tissues were dissected, washed, and stored within 4 h of surgery by flash freezing for RNA or formalin fixation for histology. Collected tissues included articular cartilage, subchondral bone, meniscus, infrapatellar fat pad, anterior cruciate ligament, synovium, and vastus medialis oblique muscle. RNA extraction protocols were tested for each tissue type. The most significant modification involved the method of disintegration used for low-cell, high-matrix, hard tissues (considered as cartilage, bone, and meniscus) versus relatively high-cell, low-matrix, soft tissues (considered as fat pad, ligament, synovium, and muscle). It was found that pulverization was appropriate for hard tissues, and homogenization was appropriate for soft tissues. A proclivity for some subjects to yield higher RNA integrity number (RIN) values than other subjects consistently across multiple tissues was observed, suggesting that underlying factors such as disease severity may impact RNA quality. The ability to isolate high-quality RNA from primary human OA tissues provides a physiologically relevant model for sophisticated gene expression experiments, including sequencing, that can lead to clinical insights that are more readily translated to patients.

Primary tissues obtained from patients following total knee arthroplasty provide an experimental model for osteoarthritis research with maximal clinical translatability. This protocol describes how to identify, process, and isolate RNA from seven unique knee tissues to support mechanistic investigation in human osteoarthritis.

Osteoarthritis (OA) is a chronic and degenerative joint disease most often affecting the knee. As there is currently no cure, total knee arthroplasty ...