Name: generation of human kidney tubuloids from tissue and urine
Uploaded: 2021-04-16T13:30:00.000+00:00
Duration: 8 min 34 s
Description: Watch this Scientific Journal Video about Generation of Human Kidney Tubuloids from Tissue and Urine at JoVE.com

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><tbody><tr><td>A83-01</td><td>Tocris</td><td>2939/10</td><td>Stock of 5 mM, final concentration of 5 &amp;micro;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&amp;nbsp; 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>&amp;nbsp;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&amp;nbsp; 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 &amp;micro;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="Tissue digestion and cell culture process diagram; enzyme digestion over time; cell growth phases." class="xfigimg" src="/files/ftp_upload/62404/62404fig01.jpg" data-altupdatedat="1747911167000" data-alt="Figure 1"> 
Figure 1: Tissue-derived kidney tubuloid cultures. (A) Overview of the procedure to mince kidney tissue. Tissue is minced to a size of ~1 mm3 using scalpels. (B) 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 °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 µm. Abbreviations: BME = basement membrane extract. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62404/62404fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Sample washing process and cell growth over 70 days; tube stages, microscopy images for analysis." class="xfigimg" src="/files/ftp_upload/62404/62404fig02.jpg" data-altupdatedat="1747911167000" data-alt="Figure 2"> 
Figure 2: Urine-derived kidney tubuloid cultures. (A) Overview of urine sample processing. 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 µm. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62404/62404fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Microscopy of cell clusters, experimental setup in petri dish, day 0, growth observation diagram." class="xfigimg" src="/files/ftp_upload/62404/62404fig03.jpg" data-altupdatedat="1747911167000" data-alt="Figure 3"> 
Figure 3: Passaging of kidney tubuloid cultures. (A) Representative image of kidney tubuloid cultures after enzymatic digestion. After completing digestion, no more than 10% of intact tubuloid structures should remain. Scale bar = 300µm. (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 µm <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62404/62404fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Kidney tissue and tubuloid histology, HE and PAX8 staining, microscope images, cellular morphology." class="xfigimg" src="/files/ftp_upload/62404/62404fig04.jpg" data-altupdatedat="1747911167000" data-alt="Figure 4"> 
Figure 4: Histological characterization of tubuloid cultures. (A) H&amp;E staining of healthy kidney tissue and tubuloids. Scale bars = 100 µm. (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 µm. Abbreviations: H&amp;E = hematoxylin and eosin; PAX8 = paired box gene 8; MRTK = malignant rhabdoid tumor of the kidney. <a href="https://www-jove-com.remotexs.ntu.edu.sg/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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4.6K 조회수.  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.

비디오: Generation of Human Kidney Tubuloids from Tissue and Urine

Dissection and Culture of Mouse Embryonic Kidney

The goal of this protocol is to describe a method for the dissection, isolation, and culture of mouse metanephric rudiments. 
During mammalian kidney development, the two progenitor tissues, the ureteric bud and the metanephric mesenchyme, communicate and reciprocally induce cellular mechanisms to eventually form the collecting system and the nephrons of the kidney. As mammalian embryos grow intrauterine and therefore are inaccessible to the observer, an organ culture has been developed. With this method, it is possible to study epithelial-mesenchymal interactions and cellular behavior during kidney organogenesis. Furthermore, the origin of congenital kidney and urogenital tract malformations can be investigated. After careful dissection, the metanephric rudiments are transferred onto a filter that floats on culture medium and can be kept in a cell culture incubator for several days. However, one must be aware that the conditions are artificial and could influence the metabolism in the tissue. Also, the penetration of test substances could be limited due to the extracellular matrix and basal membrane present in the explant.
One main advantage of organ culture is that the experimenter can gain direct access to the organ. This technology is cheap, simple, and allows a large number of modifications, such as the addition of biologically active substances, the study of genetic variants, and the application of advanced imaging techniques.

This protocol describes a method for isolating and culturing metanephric rudiments from mouse embryos.

마우스 배아 신장의 해부 및 배양

The goal of this protocol is to describe a method for the dissection, isolation, and culture of mouse metanephric rudiments. 
During mammalian kidney ...

연구

발생학

Epithelial Cell Repopulation and Preparation of Rodent Extracellular Matrix Scaffolds for Renal Tissue Development

This protocol details the generation of acellular, yet biofunctional, renal extracellular matrix (ECM) scaffolds that are useful as small-scale model substrates for organ-scale tissue development. Sprague Dawley rat kidneys are cannulated by inserting a catheter into the renal artery and perfused with a series of low-concentration detergents (Triton X-100 and sodium dodecyl sulfate (SDS)) over 26 hr to derive intact, whole-kidney scaffolds with intact perfusable vasculature, glomeruli, and renal tubules. Following decellularization, the renal scaffold is placed inside a custom-designed perfusion bioreactor vessel, and the catheterized renal artery is connected to a perfusion circuit consisting of: a peristaltic pump; tubing; and optional probes for pH, dissolved oxygen, and pressure. After sterilizing the scaffold with peracetic acid and ethanol, and balancing the pH (7.4), the kidney scaffold is prepared for seeding via perfusion of culture medium within a large-capacity incubator maintained at 37 &#176;C and 5% CO2. Forty million renal cortical tubular epithelial (RCTE) cells are injected through the renal artery, and rapidly perfused through the scaffold under high flow (25 ml/min) and pressure (~230 mmHg) for 15 min before reducing the flow to a physiological rate (4 ml/min). RCTE cells primarily populate the tubular ECM niche within the renal cortex, proliferate, and form tubular epithelial structures over seven days of perfusion culture. A 44 &#181;M resazurin solution in culture medium is perfused through the kidney for 1 hr during medium exchanges to provide a fluorometric, redox-based metabolic assessment of cell viability and proliferation during tubulogenesis. The kidney perfusion bioreactor permits non-invasive sampling of medium for biochemical assessment, and multiple inlet ports allow alternative retrograde seeding through the renal vein or ureter. These protocols can be used to recellularize kidney scaffolds with a variety of cell types, including vascular endothelial, tubular epithelial, and stromal fibroblasts, for rapid evaluation within this system.

This protocol describes decellularization of Sprague Dawley rat kidneys by antegrade perfusion of detergents through the vasculature, producing acellular renal extracellular matrices that serve as templates for repopulation with human renal epithelial cells. Recellularization and use of the resazurin perfusion assay to monitor growth is performed within specially-designed perfusion bioreactors.

상피 세포 다시 채우기 및 신장 조직 개발을위한 설치류 세포 외 기질 비계의 준비

This protocol details the generation of acellular, yet biofunctional, renal extracellular matrix (ECM) scaffolds that are useful as small-scale model ...

생체공학

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.

인간 다능성 줄기 세포로부터 신장 오가노이드를 생성하는 단순화된 방법

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

<meta charset="utf8"></head><body>미세생리학적 시스템을 이용한 신장암과 건강한 조직 공동 배양

Microfluidic Co-culture of Renal Healthy and Tumor Epithelium to Model Kidney Cancer Progression

Microphysiological systems (MPS) have enabled the introduction of more complex and relevant physiological elements into in vitro models, recreating intricate morphological features in three-dimensional environments with dynamic interactions lacking in conventional models. We implemented a renal cell carcinoma (RCC) co-culture model to recreate the cross-talk between healthy and malignant renal tissue. 
This model is based on the referenced multi-organ platform and consists of co-culturing a reconstructed renal proximal tubule with RCC spheroids. Custom-designed 3D-printed chambers were used to culture human renal epithelial proximal tubule cells (RPTEC) and facilitate their self-assembly into a renal tubule contained in a collagen type I matrix. Caki-1 RCC cells were embedded in an agar collagen matrix, subsequently forming cancer spheroids. Both collagen and agar/collagen gels were optimized to maintain their integrity during cyclic perfusion and withstand shear stress during a minimum culture period of 7 days. 
The gels also enable adequate nutrient supply and cell secretions. Moreover, the agar/collagen gels limit the overproliferation of RCC cells, ensuring relatively homogeneous spheroid size. The MPS chip microfluidic circuits comprise two independent culture chambers with the size of a standard 96-microplate well. The renal tubule and RCC gels populate separate chambers and share the same culture media, which is recirculated approximately twice per minute. Under these conditions, we observed upregulation of immune factor expression and secretion in the renal tubules (interleukin-8 and tumor necrosis factor-alpha). The renal tubules also shift their metabolic activity towards glycolysis under the influence of RCC. This novel approach demonstrates that a co-culture-based MPS can amplify the complexity of RCC in vitro and be employed to study the impact of cancer on non-tumor cells.

<meta charset="utf8"></head><body>신장암 진행을 모델링하기 위한 건강한 신장과 종양 상피의 미세유체 공동 배양

Here, we present protocols that detail instructions for implementing 3D cell cultures using collagen and collagen-agarose matrixes in a microphysiological system. These protocols support renal proximal tubule and renal cell carcinoma spheroids co-culture, simulating in vivo conditions and enabling advanced investigation of kidney cancer cell interactions.

신장암 진행을 모델링하기 위한 건강한 신장과 종양 상피의 미세유체 공동 배양

Microphysiological systems (MPS) have enabled the introduction of more complex and relevant physiological elements into in vitro models, recreating ...

암 연구학

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.

닭 배아에서 Intracelomic Transplantation을 통한 신장 오가노이드의 효율적인 혈관 형성

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

Isolation and Culture of Cells from the Nephrogenic Zone of the Embryonic Mouse Kidney

Embryonic development of the kidney has been extensively studied both as a model for epithelial-mesenchymal interaction in organogenesis and to gain understanding of the origins of congenital kidney disease. More recently, the possibility of steering na&iuml;ve embryonic stem cells toward nephrogenic fates has been explored in the emerging field of regenerative medicine. Genetic studies in the mouse have identified several pathways required for kidney development, and a global catalog of gene transcription in the organ has recently been generated <a href="http://www.gudmap.org/">http://www.gudmap.org/</a>, providing numerous candidate regulators of essential developmental functions. Organogenesis of the rodent kidney can be studied in organ culture, and many reports have used this approach to analyze outcomes of either applying candidate proteins or knocking down the expression of candidate genes using siRNA or morpholinos. However, the applicability of organ culture to the study of signaling that regulates stem/progenitor cell differentiation versus renewal in the developing kidney is limited as cultured organs contain a compact extracellular matrix limiting diffusion of macromolecules and virus particles. To study the cell signaling events that influence the stem/progenitor cell niche in the kidney we have developed a primary cell system that establishes the nephrogenic zone or progenitor cell niche of the developing kidney ex vivo in isolation from the epithelial inducer of differentiation. Using limited enzymatic digestion, nephrogenic zone cells can be selectively liberated from developing kidneys at E17.5. Following filtration, these cells can be cultured as an irregular monolayer using optimized conditions. Marker gene analysis demonstrates that these cultures contain a distribution of cell types characteristic of the nephrogenic zone in vivo, and that they maintain appropriate marker gene expression during the culture period. These cells are highly accessible to small molecule and recombinant protein treatment, and importantly also to viral transduction, which greatly facilitates the study of candidate stem/progenitor cell regulator effects. Basic cell biological parameters such as proliferation and cell death as well as changes in expression of molecular markers characteristic of nephron stem/progenitor cells in vivo can be successfully used as experimental outcomes. Ongoing work in our laboratory using this novel primary cell technique aims to uncover basic mechanisms governing the regulation of self-renewal versus differentiation in nephron stem/progenitor cells.

In this report we describe a method for the isolation and culture of the progenitor cell niche from the embryonic mouse kidney that can be used to study signaling pathways regulating stem/progenitor cells of the developing kidney. These cultured cells are highly accessible to small molecule and recombinant protein treatment, and importantly also to viral transduction, which allows efficient manipulation of candidate pathways.

배아 마우스 신장의 Nephrogenic 지대의 세포의 분리 및 문화

Embryonic development of the kidney has been extensively studied both as a model for epithelial-mesenchymal interaction in organogenesis and  to gain ...

생물학

신장 오가노이드 생산을 위한 유도만능줄기세포(Induced Pluripotent Stem Cell) 현탁액 사용

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.

저자 스포트라이트: 신장 오가노이드 생성을 위한 효율적인 생산을 위한 iPSC 분화 최적화 

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.

유도만능줄기세포(Induced Pluripotent Stem Cells)에서 현탁액의 신장 오가노이드 생성

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

Generation of Human Kidney Tubuloids from Tissue and Urine

요약

더 많은 비디오 탐색

이 비디오의 챕터

마우스 배아 신장의 해부 및 배양

상피 세포 다시 채우기 및 신장 조직 개발을위한 설치류 세포 외 기질 비계의 준비

인간 다능성 줄기 세포로부터 신장 오가노이드를 생성하는 단순화된 방법

신장암 진행을 모델링하기 위한 건강한 신장과 종양 상피의 미세유체 공동 배양

신장암 진행을 모델링하기 위한 건강한 신장과 종양 상피의 미세유체 공동 배양

신장암 진행을 모델링하기 위한 건강한 신장과 종양 상피의 미세유체 공동 배양

닭 배아에서 Intracelomic Transplantation을 통한 신장 오가노이드의 효율적인 혈관 형성

배아 마우스 신장의 Nephrogenic 지대의 세포의 분리 및 문화

유도만능줄기세포(Induced Pluripotent Stem Cells)에서 현탁액의 신장 오가노이드 생성

유도만능줄기세포(Induced Pluripotent Stem Cells)에서 현탁액의 신장 오가노이드 생성