Name: Development of Human Renal Tubular Epithelial Cell Primary Cultures in Monolayers and Three-Dimensional Conditions (Video) | JoVE Journal
Uploaded: 2025-06-13T14:10:00.000+00:00
Duration: 6 min 32 s
Description: Learn to establish human renal tubular epithelial cell primary cultures in monolayer and 3D conditions for studying Shiga toxin effects on kidney cells. This method enables investigation of cytotoxic mechanisms and potential therapeutic interventions.

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We are grateful to the Pediatric Urology team at the &#34;Secci&#243;n de Cirugi&#769;a Pedi&#225;trica, Hospital General de Pediatr&#237;a Pedro de Elizalde&#34;, Buenos Aires, Argentina, for providing kidney samples. This work was supported by grants to C. Silberstein from the University of Buenos Aires (UBACYT20020190100072BA) and to C Ibarra and C Silberstein from the National Scientific and Technical Research Council (CONICET: PUE 0041). This work was partially supported by the Programa Iberoamericano de Ciencia y Tecnolog&#237;a para el Desarrollo (CYTED) (through Red STEC/SUH N&#186; 224RT0152).

The Ethics Committee of the Hospital General de Ni&#241;os Pedro de Elizalde approved the use of human renal tissues for the development of primary cell cultures for research purposes. Written informed consent from the next of kin or guardians on behalf of the children was obtained for the use of kidney samples for research.
NOTE: The primary cultures of human renal tubular epithelial cells (HRTEC) are derived from pediatric patients undergoing nephrectomies, indicated for the correction of urological conditions or tumor resections, at Hospital General de Pediatr&#237;a Pedro de Elizalde in Buenos Aires, Argentina.
1. Development of primary cultures of human renal tubular epithelial cells (HRTEC)
NOTE: The cortical renal fragments are dissected from kidneys removed from pediatric patients undergoing nephrectomies at the hospital. The primary culture of the HRTEC is performed according to the method described previously11.
The protocol is described in the following steps:
<ol>
	<li>Keep the kidney fragment refrigerated in sterile phosphate buffered saline (PBS) or Hanks&#39; Balanced Salt solution (Hanks) until it is processed in the laboratory.</li>
	<li>Dissect the renal capsule with forceps. Separate the renal cortex from the medulla and cut it into small fragments of around 1 mm.</li>
	<li>To perform the digestion, place the kidney fragments in sterile Hanks solution without Ca2+ and Mg2+, supplemented with 0.1% collagenase type I.
	<ol>
		<li>Incubate the renal fragments for 30 min at 37 &#176;C with shaking.</li>
	</ol>
	</li>
	<li>To stop digestion, add one volume of PBS containing 10% fetal bovine serum (FBS) and wash the sample three times by centrifugation at 160 &#215; g for 10 min each, in a centrifuge refrigerated at 4 &#176;C. In each centrifugation, discard the supernatant and resuspend the pellet in the same solution.</li>
	<li>Pass the product of digestion through a 70 &#181;m pore mesh to filter the tubules and discard the glomeruli that remain retained in the filter.</li>
	<li>Centrifuge the filtered tubules at 160 &#215; g for 10 min at 4 &#176;C, discard the supernatant, and resuspend the pellet in sterile Hanks solution, without Ca2+ and Mg2+, supplemented with 0.2% collagenase type I.
	<ol>
		<li>Incubate the sample for 30 min at 37 &#176;C with shaking.</li>
	</ol>
	</li>
	<li>To stop digestion, add one volume of PBS containing 10% fetal bovine serum (FBS).
	<ol>
		<li>Wash the sample three times by centrifuging at 160 &#215; g, 10 min each, in a centrifuge refrigerated at 4 &#176;C. In each centrifugation, discard the supernatant and resuspend the pellet in the same solution.</li>
	</ol>
	</li>
	<li>After the last centrifugation, resuspend the cell pellet in 5 mL of complete culture medium (RPMI 1640 supplemented with 5% FBS, L-glutamine [2 mmol/L], penicillin [100 U/mL], streptomycin [100 &#181;g/mL], and endothelial cell growth supplement [ECGS, 1%]).
	<ol>
		<li>Seed the cell suspension in a 25 cm2 culture flask with a vented lid.</li>
		<li>Incubate the cells at 37 &#176;C in a humid atmosphere and 5% CO2.</li>
	</ol>
	</li>
	<li>The next day, wash the cell culture 3 times, discarding the liquid from the flask, adding 5 mL of PBS each, and adding 5 mL of complete culture medium to the flask. These washes eliminate red blood cells and tubular fragments that were not fully digested and not attached to the culture flask.</li>
	<li>Incubate the epithelial cells in the cell culture incubator at 37 &#176;C, allowing them to grow in monolayer to confluence.</li>
	<li>Change the medium with a fresh complete culture medium every 3 days and observe the cell culture daily under an inverted microscope.</li>
</ol>
2. Development of three-dimensional HRTEC cultures (3D-HRTEC)
NOTE: This section provides the steps to develop three-dimensional cultures (3D-HRTEC). The 3D-HRTEC cultures are performed according to the method described previously12.
<ol>
	<li>Use a BM matrix from Engelbreth-Holm-Swarm murine sarcoma. This BM matrix is delivered frozen, and in this state, it remains solid. Thaw the BM matrix solution overnight in an ice bath in the refrigerator at 4 &#176;C, as indicated by the manufacturer.</li>
	<li>Cool the tips in the freezer before placing the BM matrix and leave the multi-well plate on ice to prevent solidification of the matrix during the process.</li>
	<li>Take an aliquot of liquid BM matrix (150 mL/cm2) and place it into each well of the plate to cover the bottom of the well. Incubate the plate in the cell incubator at 37 &#176;C for 1 h to turn the BM matrix into a gel state.</li>
	<li>Meanwhile, to detach confluent HRTEC primary cultures, discard the culture medium from the flask and add 1 mL of 0.25% trypsin and 0.02% EDTA solution.
	<ol>
		<li>Spread the liquid over the entire surface of the flask and incubate for 5 min at 37 &#176;C in the cell culture incubator.</li>
		<li>Observe under an inverted microscope whether all cells have detached.</li>
	</ol>
	</li>
	<li>Resuspend the cells in 10 mL of culture medium or PBS, pass them to a 15 mL tube, and centrifuge at 160 &#215; g for 10 min at 4 &#176;C.
	<ol>
		<li>Discard the supernatant and resuspend the cell pellet in a complete culture medium containing the same supplements used for HRTEC primary cultures.</li>
		<li>Count the number of cells in a Neubauer chamber.</li>
	</ol>
	</li>
	<li>Seed 4 &#215; 104&#160;cells/150 &#181;L per well onto the BM matrix. Incubate the cell culture for several days in a cell culture incubator at 37 &#176;C, in a humid atmosphere and 5% CO2.</li>
	<li>Change the medium every 2 days with fresh medium.</li>
	<li>Monitor cell aggregation and formation of tubular shape structures under an inverted optical microscope.</li>
</ol>

<ol><li>Rivas, M., Chinen, I., Miliwebsky, E., Masana, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Risk+factors+for+Shiga+toxin-producing+Escherichia+coli-associated+human+diseases%5BTitle%5D)+AND+%22Microbiol+Spectr%22%5BJournal%5D)">Risk factors for Shiga toxin-producing Escherichia coli-associated human diseases.</a> Microbiol Spectr. 2 (5), (2014).</li>
<li>Gianantonio, C. A., Vitacco, M., Mendilaharzu, F., Gallo, G. E., Sojo, E. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+hemolytic-uremic+syndrome%5BTitle%5D)+AND+%22Nephron%22%5BJournal%5D)">The hemolytic-uremic syndrome.</a> Nephron. 11 (2), 174-192 (1973).</li>
<li>Alconcher, L. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Hemolytic+uremic+syndrome+associated+with+Shiga+toxin-producing+Escherichia+coli+infection+in+Argentina%3A+update+of+serotypes+and+genotypes+and+their+relationship+with+severity+of+the+disease%5BTitle%5D)+AND+%22Pediatr+Nephrol%22%5BJournal%5D)">Hemolytic uremic syndrome associated with Shiga toxin-producing Escherichia coli infection in Argentina: update of serotypes and genotypes and their relationship with severity of the disease.</a> Pediatr Nephrol. 36 (9), 2811-2817 (2021).</li>
<li>Alconcher, L. F., Lucarelli, L. I., Bronfen, S., Meni Battaglia, L., Balestracci, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dynamic+evolution+of+kidney+function+in+patients+with+STEC-hemolytic+uremic+syndrome+followed+for+more+than+15+years%3A+unexpected+changes%5BTitle%5D)+AND+%22Pediatr+Nephrol%22%5BJournal%5D)">Dynamic evolution of kidney function in patients with STEC-hemolytic uremic syndrome followed for more than 15 years: unexpected changes.</a> Pediatr Nephrol. 40 (1), 143-150 (2025).</li>
<li>Monteverde, M. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+decrease+in+the+incidence+of+Shiga+toxin-related+hemolytic+uremic+syndrome+as+a+cause+of+kidney+transplantation+at+an+Argentine+Referral+Center%5BTitle%5D)+AND+%22Pediatr+Transplant%22%5BJournal%5D)">A decrease in the incidence of Shiga toxin-related hemolytic uremic syndrome as a cause of kidney transplantation at an Argentine Referral Center.</a> Pediatr Transplant. 27 (4), e14489(2023).</li>
<li>Sandvig, K., van Deurs, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Entry+of+ricin+and+Shiga+toxin+into+cells%3A+molecular+mechanisms+and+medical+perspectives%5BTitle%5D)+AND+%22EMBO+J%22%5BJournal%5D)">Entry of ricin and Shiga toxin into cells: molecular mechanisms and medical perspectives.</a> EMBO J. 19 (22), 5943-5950 (2000).</li>
<li>Obrig, T. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Escherichia+coli+Shiga+toxin+mechanisms+of+action+in+renal+disease%5BTitle%5D)+AND+%22Toxins+%28Basel%29%22%5BJournal%5D)">Escherichia coli Shiga toxin mechanisms of action in renal disease.</a> Toxins (Basel). 2 (12), 2769-2794 (2010).</li>
<li>Karpman, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Apoptosis+of+renal+cortical+cells+in+the+hemolytic-uremic+syndrome%3A+in+vivo+and+in+vitro+studies%5BTitle%5D)+AND+%22Infect+Immun%22%5BJournal%5D)">Apoptosis of renal cortical cells in the hemolytic-uremic syndrome: in vivo and in vitro studies.</a> Infect Immun. 66 (2), 636-644 (1998).</li>
<li>Pistone Creydt, v, Silberstein, C., Zotta, E., Ibarra, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cytotoxic+effect+of+Shiga+toxin-2+holotoxin+and+its+B+subunit+on+human+renal+tubular+epithelial+cells%5BTitle%5D)+AND+%22Microbes+Infect%22%5BJournal%5D)">Cytotoxic effect of Shiga toxin-2 holotoxin and its B subunit on human renal tubular epithelial cells.</a> Microbes Infect. 8, 410-419 (2006).</li>
<li>Silberstein, C., Pistone Creydt, V., Gerhardt, E., Nuñez, P., Ibarra, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Inhibition+of+water+absorption+in+human+proximal+tubular+epithelial+cells+in+response+to+Shiga+toxin-2%5BTitle%5D)+AND+%22Pediatr+Nephrol%22%5BJournal%5D)">Inhibition of water absorption in human proximal tubular epithelial cells in response to Shiga toxin-2.</a> Pediatr Nephrol. 23, 1981-1990 (2008).</li>
<li>Márquez, L. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effects+of+Escherichia+coli+subtilase+cytotoxin+and+Shiga+toxin+2+on+primary+cultures+of+human+renal+tubular+epithelial+cells%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Effects of Escherichia coli subtilase cytotoxin and Shiga toxin 2 on primary cultures of human renal tubular epithelial cells.</a> PLoS One. 9, e87022(2014).</li>
<li>Márquez, L. B., Araoz, A., Repetto, H. A., Ibarra, F. R., Silberstein, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effects+of+Shiga+toxin+2+on+cellular+regeneration+mechanisms+in+primary+and+three-dimensional+cultures+of+human+renal+tubular+epithelial+cells%5BTitle%5D)+AND+%22Microb+Pathog%22%5BJournal%5D)">Effects of Shiga toxin 2 on cellular regeneration mechanisms in primary and three-dimensional cultures of human renal tubular epithelial cells.</a> Microb Pathog. 99, 87-94 (2016).</li>
<li>Sánchez, D. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Eliglustat+prevents+Shiga+toxin+2+cytotoxic+effects+in+human+renal+tubular+epithelial+cells%5BTitle%5D)+AND+%22Pediatr+Res%22%5BJournal%5D)">Eliglustat prevents Shiga toxin 2 cytotoxic effects in human renal tubular epithelial cells.</a> Pediatr Res. 91 (5), 1121-1129 (2022).</li>
<li>Sánchez, D. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Estradiol+stimulates+cell+proliferation+through+classic+estrogen+receptors+and+GPER-1+in+human+renal+tubular+epithelial+cell+primary+cultures%5BTitle%5D)+AND+%22Biochem+Biophys+Res+Commun%22%5BJournal%5D)">Estradiol stimulates cell proliferation through classic estrogen receptors and GPER-1 in human renal tubular epithelial cell primary cultures.</a> Biochem Biophys Res Commun. 512, 170-175 (2019).</li>
<li>Fernandez Miyakawa, M., Zabal, O., Silberstein, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Clostridium+perfringens+epsilon+toxin+is+cytotoxic+for+human+renal+tubular+epithelial+cells%5BTitle%5D)+AND+%22Hum+Exp+Toxicol%22%5BJournal%5D)">Clostridium perfringens epsilon toxin is cytotoxic for human renal tubular epithelial cells.</a> Hum Exp Toxicol. 30, 275-282 (2011).</li>
<li>Chen, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cooperative+application+of+transcriptomics+and+ceRNA+hypothesis%3A+ln+cRNA-00742%2FmiR-116+targets+CD74+to+mediate+vanadium-induced+mitochondrial+apoptosis+in+duck+liver%5BTitle%5D)+AND+%22J+Hazard+Mater%22%5BJournal%5D)">Cooperative application of transcriptomics and ceRNA hypothesis: ln cRNA-00742/miR-116 targets CD74 to mediate vanadium-induced mitochondrial apoptosis in duck liver.</a> J Hazard Mater. 480, 135904(2024).</li>
<li>Chen, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Copper+toxicity+in+the+liver+of+broiler+chicken%3A+insights+from+metabolomics+and+AMPK-mTOR+mediated+autophagy+perspective%5BTitle%5D)+AND+%22Poult+Sci%22%5BJournal%5D)">Copper toxicity in the liver of broiler chicken: insights from metabolomics and AMPK-mTOR mediated autophagy perspective.</a> Poult Sci. 103 (9), 104011(2024).</li>
<li>Zhang, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Role+of+Caveolin-1+on+the+molybdenum+and+cadmium+exposure+induces+pulmonary+ferroptosis+and+fibrosis+in+the+sheep%5BTitle%5D)+AND+%22Environ+Pollut%22%5BJournal%5D)">Role of Caveolin-1 on the molybdenum and cadmium exposure induces pulmonary ferroptosis and fibrosis in the sheep.</a> Environ Pollut. 334, 122207(2023).</li>
<li>Fujiki, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Blockage+of+Akt+activation+suppresses+cadmium-induced+renal+tubular+cellular+damages+through+aggrephagy+in+HK-2+cells%5BTitle%5D)+AND+%22Sci+Rep%22%5BJournal%5D)">Blockage of Akt activation suppresses cadmium-induced renal tubular cellular damages through aggrephagy in HK-2 cells.</a> Sci Rep. 14, 14552(2024).</li>
<li>Joun, J. I. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Antioxidative+effects+of+molybdenum+and+its+association+with+reduced+prevalence+of+hyperuricemia+in+the+adult+population%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Antioxidative effects of molybdenum and its association with reduced prevalence of hyperuricemia in the adult population.</a> PLoS One. 19 (8), e0306025(2024).</li>
</ol>

The authors declare no conflict of interest.

Primary cultures should be performed on the same day of nephrectomy. HRTEC primary cultures can be used between 1-5 passages to guarantee the morphogenetic stability of the cells. After the fifth passage, cells begin to change their morphology, showing fibroblast-like appearance or increase in vacuoles, and can become quiescent.
If a larger kidney fragment is used, the DNA released by cell lysis after digestion may interfere with the washing process. In this case, it is recommended to use a la...

In our group, primary cultures of human renal tubular epithelial cells (HRTEC) developed in monolayers and in three-dimensional (3D-HRTEC) conditions were used to study in the pediatric kidney the cytotoxic action of Shiga toxin type 2 (Stx2) caused after gastrointestinal infection with Shiga toxin-producing Escherichia coli (STEC).
Stx1 and Stx2 (Stxs) are the main virulence factors of STEC able to cause post-diarrheal hemolytic uremic syndrome (HUS)1. HUS is a systemic complication that affects around 5-10% of STEC-infected children and consists of hemolytic anemia, thrombocytopenia, and pediatric acute kidney injury (AKI), requiring supportive treatment2. In Argentina, STEC-HUS is an endemic disease with an annual incidence of 6.52 cases per 100,000 children under 5 years of age (the highest worldwide), representing one of the leading causes of acute kidney injury3. Although the mortality is less than 4%3, approximately 50% of patients develop renal sequels, constituting the second most frequent cause of kidney transplant in our country4,5.
Prior to internalization and exerting its cytotoxic action, Stx2 binds specifically to the glycolipid globotriaosylceramide (Gb3) receptor expressed on the cell surface of target organs6. The kidney is the main organ damaged by Stxs due to the presence of Stx-sensitive cells, which express high amounts of biologically active Gb3 receptors in renal epithelial and endothelial cells7,8.
Previous works carried out in HRTEC cultured as monolayers showed that Stx2 inhibited protein synthesis, decreased cell viability and cell proliferation, and induced apoptosis and necrosis9,10,11,12. Moreover, the exposure of 3D-HRTEC to Stx2 inhibited cell migration and the development of tubular structures with characteristics of the renal proximal tubules12,13.
The primary cultures of HRTEC and 3D-HRTEC cultures were also used to evaluate the effect of estradiol on cell proliferation by measuring the incorporation of 5-bromo-2-deoxyuridine (BrdU) into the DNA of cells in the S-phase of the cell cycle14.
To isolate the epithelial cells, we start from renal fragments obtained from pediatric nephrectomies indicated for the correction of urological conditions.

<table><tbody><tr><td>Collagenase type I</td><td>Sigma Merck, St Louis, MO, USA</td><td>637958</td><td></td></tr><tr><td>Endothelial cell growth supplement</td><td>Sigma Merck, St Louis, MO, USA</td><td>E2759</td><td></td></tr><tr><td>Matrigel basement membrane matrix</td><td>BD Biosciences, USA; Sigma Merck, St Louis, MO, USA</td><td>356234</td><td></td></tr><tr><td>70 &amp;mu;m pore mesh filter</td><td>BD Bioscience, MA, USA</td><td>css010070</td><td></td></tr><tr><td>RPMI 1640 medium with L-glutamine&amp;nbsp;</td><td>Sigma Merck, St Louis, MO, USA</td><td>R8758-500ml</td><td></td></tr><tr><td>Hanks&amp;rsquo; Balanced salt Solution&amp;nbsp; Ca2+ and Mg2+ free</td><td>Gibco Invitrogen, NY, USA</td><td>14175046</td><td></td></tr><tr><td>Trypsin-EDTA solution</td><td>Sigma Merck, St Louis, MO, USA</td><td>T4049</td><td></td></tr></tbody></table>

development of human renal tubular epithelial cell primary cultures in monolayers and three-dimensional conditions

Development of Human Renal Tubular Epithelial Cell Primary Cultures in Monolayers and Three-Dimensional Conditions

Human renal tubular epithelial cell (HRTEC) primary cultures are isolated from kidneys removed from pediatric patients undergoing nephrectomies indicated by the Pediatric Nephrology Unit of the Hospital General de Pediatr&#237;a Pedro de Elizalde, Buenos Aires, Argentina. Macroscopically, the normal kidney cortex is dissected from the renal medulla and cut into fragments. Cortical fragments are then digested in Hank&#39;s solution supplemented with 0.1% collagenase type 1 and filtered through a 70 &#181;m pore size mesh to separate the renal tubules from glomeruli. Renal tubules undergo a second digestion with collagenase to isolate epithelial cells. Finally, the enzyme digestion is stopped, and the epithelial cells are grown in flasks containing RPMI 1640 with supplements under 5% CO2at 37 &#176;C until confluence. HRTEC cells can be grown as monolayers on 96-well plates, glass coverslips into 24-well plates, or cell-culture holders coated with collagen. Also, three-dimensional human renal tubular epithelial cell (3D-HRTEC) cultures are obtained from HRTEC cells seeded on a basement membrane (BM) matrix. Cell aggregation and formation of tubular shape structures are monitored. We have used HRTEC primary cultures for many years to study the cytotoxic action caused by Shiga toxin in the pediatric kidney, which leads to pathophysiological alterations resulting in hemolytic uremic syndrome (HUS). Both HRTEC and 3D-HRTEC cultures can be used to evaluate the effects of different toxins, synthetic drugs, biological factors, hormones, etc, specifically on the human renal proximal tubule epithelium.

Confluent HRTEC primary cultures showed a similar morphology observed under light microscopy (Figure 1). Cells were characterized by immunofluorescence as described in our previous publications using antibodies against aquaporin 1 (AQP1) and PECAM CD319,12,13. Immunofluorescence studies showed that more than 90% of HRTEC were positive for AQP1, which demonstrates that their origin is in the human re...

Here, we describe the protocols for obtaining primary cultures of human renal tubular epithelial cells (HRTEC) from the kidney cortex to develop monolayers and three-dimensional cultures on an extracellular matrix (3D-HRTEC).

Human renal tubular epithelial cell (HRTEC) primary cultures are isolated from kidneys removed from pediatric patients undergoing nephrectomies indicated ...

Human renal tubular epithelial cells established in both primary and three dimensional cultures, were developed as a model to investigate the cytotoxic effects of Shiga toxin, which induces pathophysiological alterations, culminating in hemolytic uremic syndrome. No specific treatment exists for hemolytic uremic syndrome. Therefore, the current challenge lies in evaluating drugs in HRTEC primary cultures to inhibit the effects of Shiga toxin on the human kidney.Shiga toxin has been shown to inhibit cell viability and proliferation, induce apoptosis in HRTEC primary cultures and suppress the development of tubular structures in three dimensional HRTEC cultures. The protocols offer the advantage of enabling the study of epithelial cell functional mechanisms in cell cultures that retain the original characteristics of the human renal proximal tubule. To begin, dissect the renal capsule using forceps.Separate the renal cortex from the medulla and cut the cortex into small fragments of approximately one millimeter. Place the kidney fragments in sterile hanks solution without calcium and magnesium supplemented with 0.1%collagenase type one to perform the digestion. Incubate the renal fragments for 30 minutes at 37 degrees Celsius with shaking.To stop digestion, add one volume of PBS, containing 10%fetal bovine serum or FBS. Wash the sample three times by centrifuging at 160G for 10 minutes each in a centrifuge refrigerated at four degrees Celsius, discarding supernatant and resuspending the pellet in the same solution each time. Pass the digested product through a 70 micrometer poor mesh to filter the tubules and discard the glomeruli that remained retained in the filter.Centrifuge the filtered tubules at 160G for 10 minutes at four degrees Celsius. After discarding the supernatant, resuspend the pellet in sterile hanks solution without calcium and magnesium supplemented with 0.2%collagenase type one. Incubate the sample for 30 minutes at 37 degrees Celsius with shaking.To stop digestion, add one volume of PBS containing 10%FBS before washing the sample by centrifugation as shown previously. After the last centrifugation, resuspend the cell pellet in five milliliters of complete culture medium in a 25 square centimeter culture flask with a vented lid. Close the lid of the culture flask and incubate the cells at 37 degrees Celsius in a humid atmosphere with 5%carbon dioxide.After adding the culture medium and incubating the cells again at 37 degrees Celsius, observe the cells under an inverted microscope. Take an aliquot of liquid basement membrane matrix at a volume of 150 microliters per square centimeter and dispense it into each well of a multi-well plate to cover the bottom. Incubate the plate in a cell culture incubator at 37 degrees Celsius for one hour to allow the matrix to solidify into a gel.To detach confluent primary HRTEC cultures after discarding the culture medium from the flask, add one milliliter of 0.25%trypsin with 0.02%EDTA solution. Spread the solution evenly across the surface of the flask before incubating it for five minutes at 37 degrees Celsius. Observe the cells under an inverted microscope to confirm complete cell detachment.After resuspending the detached cells in 10 milliliters of PBS or culture medium, centrifuge at 160G for 10 minutes at four degrees Celsius. Once the supernatant is discarded and the pellet is resuspended in complete culture medium with the same supplements used for primary HRTEC cultures, count the resuspended cells using a Neubauer chamber. Seed 40, 000 cells in 150 microliters of medium per well onto the solidified basement membrane matrix and incubate the cell cultures for several days at 37 degrees Celsius in a humid atmosphere with 5%carbon dioxide.Monitor the cultures regularly using an inverted optical microscope to observe cell aggregation and formation of tubular structures. HRTEC seeded on the basement membrane matrix initiated tubular genesis by migrating and organizing into multicellular cords within the first four to 10 hours of culture. By one day after seeding, cellular aggregates emerged from the cords and began forming early tubular structures.The 3D tubular structures continued to elongate and mature progressively by three days and eight days of culture. Hematoxylin and eosin staining of the 3D HRTEC structures revealed a central lumen surrounded by epithelial cells. Immunofluorescence confirmed the expression of aquaporin one protein in both apical and basolateral membranes of the 3D HRTEC epithelial cells mirroring the expression pattern observed in proximal tubules of the human kidney.

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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.

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

Presented here is a chemically defined protocol for the derivation of human kidney podocytes from induced pluripotent stem cells with high efficiency (&gt;90%) and independent of genetic manipulations or subpopulation selection. This protocol produces the desired cell type within 26 days and could be useful for nephrotoxicity testing and disease modeling.

Guided Differentiation of Mature Kidney Podocytes from Human Induced Pluripotent Stem Cells Under Chemically Defined Conditions

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.

Generation of Human Kidney Tubuloids from Tissue and Urine

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.

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

We present an optimized protocol for processing whole human kidneys to isolate and culture primary renal proximal tubule epithelial cells and the application of these cells in a three-dimensional,  microfluidic, microphysiological platform to recapitulate the renal proximal tubule.

Isolation of Primary Human Proximal Tubule Epithelial Cells and Their Use in Creating a Microphysiological Model of the Renal Proximal Tubule

Learn to establish human renal tubular epithelial cell primary cultures in monolayer and 3D conditions for studying Shiga toxin effects on kidney cells. This method enables investigation of cytotoxic mechanisms and potential therapeutic interventions.

Development of Human Renal Tubular Epithelial Cell Primary Cultures in Monolayers and Three-Dimensional Conditions

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