Maryna Somova was supported by the University of Greifswald Doctoral Scholarship - Landesgraduiertenf&#246;rderungsgesetzes (Act on State Graduate Funding) of Mecklenburg-Vorpommern. The authors would like to thank Dr. Janosh Schoon and Dirk Stobe from the Center for Orthopedics, Trauma Surgery and Rehabilitation Medicine, University Medicine Greifswald for their insights in 3D cell culture and sample preparation.

NOTE: These protocols outline the comprehensive steps for preparing 3D collagen-agarose gels, injecting cells, perfusing the samples, and extracting them for further analysis. Adjust incubation times and conditions based on specific experimental requirements.
1. Preparation of collagen and agarose gel matrix
<ol>
	<li>3D printing 
	NOTE: To create the appropriate shape for the gel matrix, we developed a unique chamber design that shapes the gel into a physiologically relevant organoid form (see Supplemental File 1).
	<ol>
		<li>Design the chambers using computer-assisted design software. The chamber comprises four parallel compartments: two cell compartments, one matrix compartment, and a connecting canal (Figure 1 and Figure 2).</li>
		<li>Build the chambers by 3D printing; a heating nozzle&#160;melts a bioplastic filament and deposits layer by layer. To follow this protocol, use Polypropylene (PP), a non-toxic bioplastic filament, and a Cartesian-type 3D printer, named after the XYZ coordinate system.</li>
		<li>Add a reverse-cone shape feature to the cell compartments to direct cells into the canal.</li>
		<li>Give a round shape to the matrix compartment, which is crucial for forming collagen gel.</li>
		<li>Connect the cell and matrix compartments with a canal (diameter 0.04 mm, Figure 3B), allowing cells to flow from the cell compartment into the matrix compartment and attach to the tube surface.</li>
	</ol>
	</li>
	<li>Preparation of collagen matrix for the cultivation of RPTEC-TERT1 cells 
	NOTE: This procedure ensures that the collagen matrix is prepared correctly and loaded into the chamber, creating a suitable environment for the subsequent injection and cultivation of RPTEC/TERT1 cells and the growth of the 3D cell structure. Ensure all steps are performed under sterile conditions to avoid contamination.
	<ol>
		<li>Prepare the crosslinking solution by mixing 50 &#181;L of Genipin (30 mM) with 50 &#181;L of NaOH (1 M) using a vortex to ensure thorough mixing.</li>
		<li>Add 1,000 &#181;L of collagen type I to the prepared Genipin and NaOH solution while keeping the mixture on ice to prevent premature gelation. Mix the solution again using a vortex to achieve a homogeneous mixture. 
		&#8203;NOTE: Keep the mixed solution on ice continuously to prevent premature gelation.</li>
		<li>Using a 1,000 &#181;L pipette tip, collect the entire prepared solution and add 150-200 &#181;L of the solution into the matrix compartment of the 3D printed chamber.</li>
		<li>Let the matrix polymerize by keeping the chamber in the CO2 incubator at 37 &#7506;C for 60-90 min. Add 150 &#181;L of culture medium on the top of the matrix and leave it overnight in the incubator.</li>
	</ol>
	</li>
	<li>Preparation of collagen-agarose matrix and embedding of Caki-1 cells 
	NOTE: Both RPTEC/TERT1 and Caki-1 cells were cultured using the same culture medium, consisting of DMEM F-12 high glucose media supplemented with ITS (10 &#181;g/mL of insulin, 5.5 &#181;g/mL of transferrin, and 5 ng/mL of sodium selenite), 10 ng/mL of epidermal growth factor (EGF), 36 ng/mL of hydrocortisone, 100 U/mL of penicillin, 100 &#181;g/mL of streptomycin (5% v/v), and 10% (v/v) Fetal Calf Serum (FCS).
	<ol>
		<li>Dissolve agarose powder in either sterile water or DPBS to achieve a final concentration of 2%. Place the agarose solution on a heating block set to 85 &#7506;C to ensure complete dissolution. 
		NOTE: Handle the agarose solution carefully due to its temperature sensitivity.</li>
	</ol>
	</li>
	<li>Caki-1 cells embedding
	<ol>
		<li>Detach Caki-1 cells from their culture flasks by adding 1x trypsin-EDTA and incubating&#160;at 37 &#176;C until the cells detach. Neutralize the 1x trypsin by adding an equal volume of culture medium.</li>
		<li>Collect the cell suspension, count cells, and centrifuge at 2,500 &#215; g for 5 min. Discard the supernatant and resuspend the cell pellets in fresh culture medium to a final concentration of 1 &#215; 103 cells/mL (Caki-1). 
		NOTE: Ensure the cell suspension is thoroughly mixed and free of clumps before loading into the chamber.</li>
		<li>Add 25 &#181;L of cell suspension into a 1.5 mL microcentrifuge tube. 
		NOTE: Ensure the syringe or pipette is sterilized and appropriate for the handling of cells.</li>
		<li>Add 75 &#181;L of collagen type I into the tube with the cell suspension and vortex. 
		NOTE: Keep the mixture on ice continuously to prevent premature gelation.</li>
		<li>Add 100 &#181;L of melted agarose gel 2% and mix carefully on vortex until the gel becomes homogeneous. 
		NOTE: Ensure even distribution of the cells within the gel. Avoid introducing air bubbles during injection, as they can disrupt cell distribution and gel integrity!</li>
		<li>After 1 h, add 150 &#181;L of cell media at the top of the matrix and remove it from the chamber using a spatula. Place the 3D matrixes with cells in a 24-well plate with 1,000 &#181;L of culture medium and incubate at 37 &#176;C, 5% CO2.</li>
	</ol>
	</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/67456/67456fig01.jpg" /> 
Figure 1: 3D printed chamber design for the reconstruction of renal tubules. (A,B) Representation of the 3D printed chamber designed to accommodate a crosslinked collagen matrix to reconstruct renal tubules. Each chamber consists of four individual compartments, that are used to produce an equal amount of gels. 1-insertion point for the filament, 2-pre-chambers to be filled with agar only to prevent leaks, 3-collagen chamber, 4-cell chamber. (C) Dimensions of the crosslinked collagen gel and embedded renal tubule. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/67456/67456fig01large.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/67456/67456fig02v2.jpg" /> 
Figure 2: Preparation of collagen and agar-collagen gels. (A) 3D printed chamber with filaments inserted. (B) The crosslinked collagen solution is dispensed in the wells of the chamber (C) Chamber filled with collagen solution is placed in the incubator to facilitate the matrix polymerization (D) Collection of RPTEC-TERT1 cells before injection into the collagen matrix (E) Adding cell suspension the cell compartment of the chamber (F) Removing the filament with the aid of forceps, enabling the cells to populate the hollow tubular structure inside the matrix (G) Suspension of RCC cells mixed with collagen in a 1.5mL tube (H) Adding 2% agar solution the cells to generate the RCC gel. (I) The gels are placed in the incubator to facilitate matrix gelation (J,K,L) Removing the polymerized collagen gel with the renal tubules from the 3D printed chamber with the aid of a spatula, and placing them in a 24-well plate before further processing (M,N,O) Removing the polymerized collagen-agar gel with the RCC cells with the aid of a spatula, and placing them in a 24-well plate before further processing <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/67456/67456fig02large.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/67456/67456fig03.jpg" /> 
Figure 3: Assembly of the microfluidic culture system. (A,B) Opening the&#160;culture compartments of the Humimic chip2 using a dedicated tool (C) After removing the&#160;culture media used to wash the chip, add 400 &#956;L of fresh culture media. (D,E) After removing the renal and RCC gels from the respective 24 well plates place each gel in the culture compartment of a single perfusion circuit of the Humimic chip using spatulas to handle the gels. (F,G) After positioning the gels in the chip, the culture chambers are sealed, and the chip is linked to the perfusion unit through three micro-pumps in each microfluidic circuit, connected by specialized tubing. (H) The chips are placed in the incubator for the duration of the culture period (I) Perfusion parameters set in the control unit. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/67456/67456fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
2. Injection of RPTEC/TERT1 cells
<ol>
	<li>RPTEC/TERT1 cells injection
	<ol>
		<li>Detach RPTEC/TERT1 cells from their culture flasks by adding 1x trypsin-EDTA and incubating at 37 &#176;C until the cells detach.</li>
		<li>Neutralize the 1x trypsin by adding an equal volume of culture medium with supplements. Collect the cell suspension and centrifuge at 2,500 &#215; g for 5 min.</li>
		<li>Discard the supernatant and resuspend the cell pellets in fresh culture medium to a final concentration of 1 &#215; 106 cells/mL (RPTEC/TERT1). Ensure the cell suspension is thoroughly mixed and free of clumps before loading into the chamber.</li>
		<li>Load 10 &#181;L of the prepared cell suspension into a micro-pipette with a 10&#181;L tip. Ensure the&#160;pipette is sterilized and appropriate for the handling of cells. Carefully inject the cell suspension into the solidified collagen (step 1.2.4) through the cell compartment within the chamber.&#160;</li>
		<li>Gently remove the filament using forceps, allowing the cells to populate the hollow tubular structure within the matrix 
		NOTE: Ensure even distribution of the cells within the gel. Avoid introducing air bubbles during injection, as they can disrupt cell distribution and gel integrity.</li>
		<li>Place the chambers in a 37 &#176;C, 5% CO2 incubator. After 60 min, add 150 &#181;L of cell media on the top of the matrix to avoid drying. Incubate for 24 h to allow cells to acclimate and integrate into the gel matrix.</li>
		<li>On the next day, remove the matrix from the chamber using a spatula. Place the 3D matrixes with cells in a 24-well plate with 1,000 &#181;L of culture medium and incubate at 37 &#176;C with 5% CO2. 
		NOTE: The gels prepared with either the reconstructed renal tubule and the embedded RCC spheroids will be added to the microfluid system for co-culture</li>
	</ol>
	</li>
</ol>
3. Perfusion of 3D cell samples
<ol>
	<li>3D cell perfusion in the multi-organ chip system
	<ol>
		<li>Connect the chip with the 3D cell to the perfusion system with tubing.</li>
		<li>Set the system: Frequency: 0.25 Hz, Vacuum: -220 mbar, Pressure: 800 mbar, and connect chip 2 to the system (Figure 3).</li>
		<li>Cultivate the dynamic 3D cell culture for 5 days in an incubator at 37 &#176;C with 5% CO2.</li>
	</ol>
	</li>
</ol>
4. 3D cell samples post perfusion collection
<ol>
	<li>Post perfusion collection and analysis
	<ol>
		<li>Remove the gels from the perfusion system and rinse them with PBS.</li>
		<li>Fix the gels by placing the samples in 2% paraformaldehyde for 2-3 h at room temperature. Store fixed samples at 4 &#186;C.</li>
		<li>Stain the gels with dyes (e.g., Hoechst33342, phalloidin, ZO1, tubulin).</li>
		<li>After 5 days of perfusion using the multi-organ chip system, various assays, and methods were performed on the 3D cell cultures and collected media to assess cell viability, morphology, gene expression levels, and functional adjustments. The RPTEC/TERT1 cells in the collagen gel matrix and the Caki-1 cells in the collagen-agarose matrix maintain high viability; confirm this using viability assays such as live/dead staining.</li>
	</ol>
	</li>
</ol>

Renal Cancer and Healthy Tissue Co-Culture Using Microphysiological Systems

<ol>
	<li>Ainslie, G. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microphysiological+lung+models+to+evaluate+the+safety+of+new+pharmaceutical+modalities:+A+biopharmaceutical+perspective.">Microphysiological lung models to evaluate the safety of new pharmaceutical modalities: A biopharmaceutical perspective.</a> Lab Chip. 19 (19), 3152-3161 (2019).</li><li>Van Ness, K. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microphysiological+systems+to+assess+nonclinical+toxicity.">Microphysiological systems to assess nonclinical toxicity.</a> Curr Protoc Toxicol. 73, 14.18.1-14.18.28 (2017).</li><li>Majo, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+extracellular+matrix+components+to+renal+cell+carcinoma+behavior.">Impact of extracellular matrix components to renal cell carcinoma behavior.</a> Front Oncol. 10, 625 (2020).</li><li>Caetano-Pinto, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidermal+growth+factor+receptor+mediates+the+basolateral+uptake+of+phosphorothioate-modified+antisense+oligonucleotides+in+the+kidney.">Epidermal growth factor receptor mediates the basolateral uptake of phosphorothioate-modified antisense oligonucleotides in the kidney.</a> Organs-on-a-Chip. 4, 100022 (2022).</li><li>Manduca, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D+cancer+models:+One+step+closer+to+in+vitro+human+studies.">3D cancer models: One step closer to in vitro human studies.</a> Front Immunol. 14, 1175503 (2023).</li><li>Simon, B. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+RPTEC/TERT1+cell+line+models+key+renal+cell+responses+to+the+environmental+toxicants,+benzo[a]pyrene+and+cadmium.">The RPTEC/TERT1 cell line models key renal cell responses to the environmental toxicants, benzo[a]pyrene and cadmium.</a> Toxicol Rep. 1, 231-242 (2014).</li><li>Ogura, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+of+high+integrity+RNA+extraction+from+cell/agarose+construct.">Methods of high integrity RNA extraction from cell/agarose construct.</a> BMC Res Notes. 8, 644 (2015).</li><li>Glube, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Caki-1+cells+represent+an+in+vitro+model+system+for+studying+the+human+proximal+tubule+epithelium.">Caki-1 cells represent an in vitro model system for studying the human proximal tubule epithelium.</a> Nephron Exp Nephrol. 107 (2), e47-e56 (2007).</li><li>K&#252;hnl, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+application+scenario-dependent+pharmacokinetics+and+pharmacodynamic+properties+of+permethrin+and+hyperforin+in+a+dynamic+skin+and+liver+multi-organ-chip+model.">Characterization of application scenario-dependent pharmacokinetics and pharmacodynamic properties of permethrin and hyperforin in a dynamic skin and liver multi-organ-chip model.</a> Toxicology. 448, 152637 (2021).</li><li>Padmyastuti, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic-based+prostate+cancer+model+for+investigating+the+secretion+of+prostate-specific+antigen+and+microRNAs+in+vitro.">Microfluidic-based prostate cancer model for investigating the secretion of prostate-specific antigen and microRNAs in vitro.</a> Sci Rep. 13 (1), 11623 (2023).</li><li>Zhang, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer-on-a-Chip:+Models+for+studying+metastasis.">Cancer-on-a-Chip: Models for studying metastasis.</a> Cancers (Basel). 14 (3), 648 (2022).</li><li>Sobczuk, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Choosing+the+right+animal+model+for+renal+cancer+research.">Choosing the right animal model for renal cancer research.</a> Transl Oncol. 13 (3), 100745 (2020).</li><li>Ewart, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Performance+assessment+and+economic+analysis+of+a+human+Liver-Chip+for+predictive+toxicology.">Performance assessment and economic analysis of a human Liver-Chip for predictive toxicology.</a> Commun Med. 2 (1), 154 (2022).</li><li>Caetano-Pinto, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kidney+cancer+and+chronic+kidney+disease:+Too+close+for+comfort.">Kidney cancer and chronic kidney disease: Too close for comfort.</a> Biomedicines. 9 (12), 1761 (2021).</li><li>Del Piccolo, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor-on-chip+modeling+of+organ-specific+cancer+and+metastasis.">Tumor-on-chip modeling of organ-specific cancer and metastasis.</a> Adv Drug Deliv Rev. 175, 113798 (2021).</li><li>Soo, J. Y. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+predictive+in+vitro+models+of+drug-induced+nephrotoxicity.">Advances in predictive in vitro models of drug-induced nephrotoxicity.</a> Nat Rev Nephrol. 14 (6), 378-393 (2018).</li><li>Chapron, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+improved+vascularized,+dual-channel+microphysiological+system+facilitates+modeling+of+proximal+tubular+solute+secretion.">An improved vascularized, dual-channel microphysiological system facilitates modeling of proximal tubular solute secretion.</a> ACS Pharmacol Transl Sci. 3 (3), 496-508 (2020).</li></ol>

The authors have no conflicts of interest to declare.

The protocol described in this study represents the development of a complex kidney cancer model, leveraging the integration of two distinct cell types-renal proximal tubular epithelial cells (RPTEC/TERT1) and renal cell carcinoma (Caki-1) cells-within specific collagen and collagen-agarose gel matrixes in a microfluidic system. The preparation of collagen gels is critical to the success of this model. The precise concentration of collagen and agarose is necessary to maintain the structural integrity of the matrix throug...

Advancements in 3D cell culture systems have revolutionized tissue engineering and regenerative medicine by offering more physiologically relevant models compared to traditional 2D cultures1,2. In this study, we used collagen and collagen-agarose gel matrixes, given their ability to mimic the extracellular matrix (ECM) environment, promoting more accurate cellular behavior and function, while being compatible with the dynamic culture conditions employed. 
Collagen, the most abundant protein in the ECM, plays a crucial role in maintaining the structural integrity and biological activity of tissues3. Type I collagen, commonly sourced from rat tail, was favored for its biocompatibility and ability to be tailored to meet different matrix rigidity conditions, as well as providing a proven substrate for epithelial cells4. In the 3D collagen matrix, renal cells are provided with a scaffold that supports their adhesion, proliferation, and differentiation. This environment enables cells to exhibit more natural morphologies and behaviors, including gene expression patterns and cellular interactions that reflect in vivo conditions5. Consequently, collagen gels have been extensively utilized in applications ranging from cancer research to tissue engineering due to their capacity to replicate the ECM's supportive and regulatory roles. 
The immortalized renal proximal tubular epithelial cell line RPTEC/TERT1 was used to represent healthy kidney epithelium. This cell line overexpresses the human telomerase reverse transcriptase, enabling it to proliferate in culture, while maintaining a representative RPTEC phenotype. Rat tail collagen type I gel matrix was employed to recreate the renal microenvironment. This collagen gel matrix supports RPTEC/TERT16 cells in maintaining their native characteristics and physiological functions. In contrast, a hybrid collagen-agarose gel matrix is utilized for culturing renal cell carcinoma (Caki-1) cells. Agarose, a polysaccharide derived from seaweed, adds another dimension to 3D cell culture systems. It is thermally reversible, allowing easy handling and manipulation during the preparation process7. Agarose gels provide a neutral supportive matrix that maintains cell viability and promotes the formation of complex structures by proliferating cancer cells. When combined with collagen, the resulting hybrid gel leverages the biochemical cues from collagen and the structural support from agarose. This combination creates a suitable microenvironment for the renal cell carcinoma (RCC) Caki-1 cells8 for growth into spheroids and is used as the kidney cancer model. 
The use of these different 3D matrixes for RPTEC/TERT1 and Caki-1 cells underscores the importance of tailoring the ECM environment to the specific needs of different cell types. The gels comprising either RPTEC, representing healthy renal epithelium, or Caki-1 cells, representing RCC, were combined in a microfluidic system that recirculates culture media between the cells, effectively exposing the healthy renal cell model to the secretions of the RCC, without direct contact. The TissUse HUMUMIC platform9,10(henceforth referred to as the multi-organ chip platform) employed in this study consists of a microphysiology chip with two independent fluidic circuits, with flow driven by an external perfusion unit.

<table><tbody><tr><td>1.5-2.0 mL&amp;nbsp; tubes&amp;nbsp;</td><td>Eppendorf</td><td>003012/10237-20205</td><td></td></tr><tr><td>24-well plates&amp;nbsp;</td><td>Sarstedt</td><td>83.3922.500</td><td></td></tr><tr><td>3D printer&amp;nbsp;</td><td>Prusa</td><td></td><td></td></tr><tr><td>Agarose&amp;nbsp;</td><td>Carl Roth</td><td>3810.3</td><td></td></tr><tr><td>AutoDesk Tinker CAD software</td><td></td><td></td><td>computer-assisted design software</td></tr><tr><td>Caki-1 cells&amp;nbsp;</td><td>ATCC</td><td>HTB-46</td><td></td></tr><tr><td>Caspase activity</td><td>Promega</td><td>G8090</td><td>Caspase 3/7 assay</td></tr><tr><td>Cell viability</td><td>Promega</td><td>G7570</td><td>Cell Titer Glo assay</td></tr><tr><td>Collagen Type I &amp;ndash; rat tail, 3.0 mg/mL</td><td>Corning</td><td>354236</td><td></td></tr><tr><td>DMEM/12F Medium&amp;nbsp;</td><td>PAN Biotech</td><td>PO4-41650</td><td></td></tr><tr><td>DPBS solution&amp;nbsp;</td><td>PAN Biotech</td><td>P04-53500</td><td></td></tr><tr><td>Epidermal Growth Factor</td><td>Merck&amp;nbsp;</td><td>E4127</td><td></td></tr><tr><td>Fetal Calf Serum</td><td>PAN Biotech</td><td>P30-3033</td><td></td></tr><tr><td>Genipin (30 mM)&amp;nbsp;</td><td>Merck&amp;nbsp;</td><td>G4796</td><td></td></tr><tr><td>HUMIMIC chips 2&amp;nbsp;</td><td>TissUse</td><td></td><td>multi-organ chips</td></tr><tr><td>HUMIMIC control unit&amp;nbsp;</td><td>TissUse</td><td></td><td>multi-organ chip control unit&amp;nbsp;</td></tr><tr><td>Hydrocortisone&amp;nbsp;</td><td>Merck&amp;nbsp;</td><td>H6909</td><td></td></tr><tr><td>Incubator (37 &amp;deg;C, 5% CO&lt;sub&gt;2&lt;/sub&gt;)&amp;nbsp;</td><td>nd</td><td></td><td></td></tr><tr><td>Insulin,Sodium selenite,Transferrin (IST)</td><td>Merck&amp;nbsp;</td><td>I1884</td><td></td></tr><tr><td>LDH release</td><td>Promega</td><td>J2380</td><td></td></tr><tr><td>Metal spatula&amp;nbsp;</td><td>nd</td><td></td><td></td></tr><tr><td>NaOH (1 M)&amp;nbsp;</td><td>Carl Roth</td><td>P031.2</td><td></td></tr><tr><td>Petri dishes</td><td>Sarstedt</td><td>82.1135.500</td><td></td></tr><tr><td>Polypropylene (PP) filament&amp;nbsp;</td><td>Verbatin</td><td>55952</td><td></td></tr><tr><td>RNA-easy extraction kit</td><td>Qiagen</td><td>74104</td><td></td></tr><tr><td>RPTEC/TERT cells&amp;nbsp;</td><td>ATCC</td><td>CRL - 4031</td><td></td></tr><tr><td>TNF-alfa ELISA</td><td>R&amp;amp;D Systems</td><td>DY210-05</td><td></td></tr><tr><td>Triiodothyronine (T3 )</td><td>Merck&amp;nbsp;</td><td>709611</td><td></td></tr><tr><td>Trypsin-EDTA</td><td>PAN Biotech</td><td>P10-021100</td><td></td></tr></tbody></table>

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.

The HUMIMIC system provides a dynamic environment that enables continuous nutrient and oxygen supply while removing metabolic waste, thus maintaining cell viability and function over extended periods. These systems are particularly beneficial for creating complex, organ-on-a-chip models that replicate the microenvironment of specific tissues. It is specifically designed for organ-on-a-chip applications and allows for the precise control of fluid flow and shear stress, which are critical factors in simulating the physiolo...

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

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.

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

microfluidic-co-culture-renal-healthy-tumor-epithelium-to-model

Universidad de Cartagena UDEC

Research

JoVE Journal

Cancer Research

538 Views.  University Medicine Greifswald. 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.

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

Microfluidic Device for Recreating a Tumor Microenvironment in Vitro

We have developed a microfluidic device that mimics the delivery and systemic clearance of drugs to heterogeneous three-dimensional tumor tissues in vitro. Nutrients delivered by vasculature fail to reach all parts of tumors, giving rise to heterogeneous microenvironments consisting of viable, quiescent and necrotic cell types. Many cancer drugs fail to effectively penetrate and treat all types of cells because of this heterogeneity. Monolayers of cancer cells do not mimic this heterogeneity, making it difficult to test cancer drugs with a suitable in vitro model. Our microfluidic devices were fabricated out of PDMS using soft lithography. Multicellular tumor spheroids, formed by the hanging drop method, were inserted and constrained into rectangular chambers on the device and maintained with continuous medium perfusion on one side. The rectangular shape of chambers on the device created linear gradients within tissue. Fluorescent stains were used to quantify the variability in apoptosis within tissue. Tumors on the device were treated with the fluorescent chemotherapeutic drug doxorubicin, time-lapse microscopy was used to monitor its diffusion into tissue, and the effective diffusion coefficient was estimated. The hanging drop method allowed quick formation of uniform spheroids from several cancer cell lines. The device enabled growth of spheroids for up to 3 days. Cells in proximity of flowing medium were minimally apoptotic and those far from the channel were more apoptotic, thereby accurately mimicking regions in tumors adjacent to blood vessels. The estimated value of the doxorubicin diffusion coefficient agreed with a previously reported value in human breast cancer. Because the penetration and retention of drugs in solid tumors affects their efficacy, we believe that this device is an important tool in understanding the behavior of drugs, and developing new cancer therapeutics.

Microfluidic Device for Recreating a Tumor Microenvironment in Vitro

We present the procedure for fabrication and operation of a microfluidic device that recreates heterogeneous tumor microenvironments in vitro. The variability in apoptosis within tumor tissue was quantified using fluorescent stains and the effective diffusion coefficient of the chemotherapeutic drug doxorubicin into tumor tissue was evaluated.

We have developed a microfluidic device that mimics the delivery and systemic clearance of drugs to heterogeneous three-dimensional tumor tissues in ...

Bioengineering

A Murine Orthotopic Bladder Tumor Model and Tumor Detection System

This protocol describes the generation of bladder tumors in female C57BL/6J mice using the murine bladder cancer cell line MB49, which has been modified to secrete human Prostate Specific Antigen (PSA), and the procedure for the confirmation of tumor implantation. In brief, mice are anesthetized using injectable drugs and are made to lay in the dorsal position. Urine is vacated from the bladder and 50 &#181;L of poly-L-lysine (PLL) is slowly instilled at a rate of 10 &#181;L/20 s using a 24 G IV catheter. It is left in the bladder for 20 min by stoppering the catheter. The catheter is removed and PLL is vacated by gentle pressure on the bladder. This is followed by instillation of the murine bladder cancer cell line (1 x 105 cells/50 &#181;L) at a rate of 10 &#181;L/20 s. The catheter is stoppered to prevent premature evacuation. After 1 h, the mice are revived with a reversal drug, and the bladder is vacated. The slow instillation rate is important, as it reduces vesico-ureteral reflux, which can cause tumors to occur in the upper urinary tract and in the kidneys. The cell line should be well re-suspended to reduce clumping of cells, as this can lead to uneven tumor sizes after implantation.
This technique induces tumors with high efficiency. Tumor growth is monitored by urinary PSA secretion. PSA marker monitoring is more reliable than ultrasound or fluorescence imaging for the detection of the presence of tumors in the bladder. Tumors in mice generally reach a maximum size that negatively impacts health by about 3 - 4 weeks if left untreated. By monitoring tumor growth, it is possible to differentiate mice that were cured from those that were not successfully implanted with tumors. With only end-point analysis, the latter may be mistakenly assumed to have been cured by therapy.

This protocol describes the generation of murine orthotopic bladder tumors in female C57BL/6J mice and the monitoring of tumor growth.

This protocol describes the generation of bladder tumors in female C57BL/6J mice using the murine bladder cancer cell line MB49, which has been modified ...

A Syngeneic Mouse Model of Metastatic Renal Cell Carcinoma for Quantitative and Longitudinal Assessment of Preclinical Therapies

Renal cell carcinoma (RCC) affects &#62; 60,000 people in the United States annually, and ~ 30% of RCC patients have multiple metastases at the time of diagnosis. Metastatic RCC (mRCC) is incurable, with a median survival time of only 18 months. Immune-based interventions (e.g., interferon (IFN) and interleukin (IL)-2) induce durable responses in a fraction of mRCC patients, and multikinase inhibitors (e.g., sunitinib or sorafenib) or anti-VEGF receptor monoclonal antibodies (mAb) are largely palliative, as complete remissions are rare. Such shortcomings in current therapies for mRCC patients provide the rationale for the development of novel treatment protocols. A key component in the preclinical testing of new therapies for mRCC is a suitable animal model. Beneficial features that recapitulate the human condition include a primary renal tumor, renal tumor metastases, and an intact immune system to investigate any therapy-driven immune effector responses and the formation of tumor-induced immunosuppressive factors. This report describes an orthotopic mRCC mouse model that has all of these features. We describe an intrarenal implantation technique using the mouse renal adenocarcinoma cell line Renca, followed by the assessment of tumor growth in the kidney (primary site) and lungs (metastatic site).

Implementation of an orthotopic model of renal cell carcinoma in immunocompetent mice affords the investigator a clinically-relevant system defined by the presence of a primary renal tumor and lung metastases in the same animal. This system can be used to preclinically test a variety of treatments in vivo.

Renal cell carcinoma (RCC) affects &#62; 60,000 people in the United States annually, and ~ 30% of RCC patients have multiple metastases at the time of ...

Monitoring Cancer Cell Invasion and T-Cell Cytotoxicity in 3D Culture

Significant progress has been made in treating cancer with immunotherapy, although a large number of cancers remain resistant to treatment. A limited number of assays allow for direct monitoring and mechanistic insights into the interactions between tumor and immune cells, amongst which, T-cells play a significant role in executing the cytotoxic response of the adaptive immune system to cancer cells. Most assays are based on two-dimensional (2D) co-culture of cells due to the relative ease of use but with limited representation of the invasive growth phenotype, one of the hallmarks of cancer cells. Current three-dimensional (3D) co-culture systems either require special equipment or separate monitoring for invasion of co-cultured cancer cells and interacting T-cells.
Here we describe an approach to simultaneously monitor the invasive behavior in 3D of cancer cell spheroids and T-cell cytotoxicity in co-culture. Spheroid formation is driven by enhanced cell-cell interactions in scaffold-free agarose microwell casts with U-shaped bottoms. Both T-cell co-culture and cancer cell invasion into type I collagen matrix are performed within the microwells of the agarose casts without the need to transfer the cells, thus maintaining an intact 3D co-culture system throughout the assay. The collagen matrix can be separated from the agarose cast, allowing for immunofluorescence (IF) staining and for confocal imaging of cells. Also, cells can be isolated for further growth or subjected to analyses such as for gene expression or fluorescence activated cell sorting (FACS). Finally, the 3D co-culture can be analyzed by immunohistochemistry (IHC) after embedding and sectioning. Possible modifications of the assay include altered compositions of the extracellular matrix (ECM) as well as the inclusion of different stromal or immune cells with the cancer cells.

The presented approach simultaneously evaluates cancer cell invasion in 3D spheroid assays and T-cell cytotoxicity. Spheroids are generated in a scaffold-free agarose multi-microwell cast. Co-culture and embedding in type I collagen matrix are performed within the same device which allows to monitor cancer cell invasion and T-cell mediated cytotoxicity.

Significant progress has been made in treating cancer with immunotherapy, although a large number of cancers remain resistant to treatment. A limited ...

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

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

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

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

Developmental Biology

Modeling Ovarian Cancer Multicellular Spheroid Behavior in a Dynamic 3D Peritoneal Microdevice

Ovarian cancer is characterized by extensive peritoneal metastasis, with tumor spheres commonly found in the malignant ascites. This is associated with poor clinical outcomes and currently lacks effective treatment. Both the three-dimensional (3D) environment and the dynamic mechanical forces are very important factors in this metastatic cascade. However, traditional cell cultures fail to recapitulate this natural tumor microenvironment. Thus, in vivo-like models that can emulate the intraperitoneal environment are of obvious importance. In this study, a new microfluidic platform of the peritoneum was set up to mimic the situation of ovarian cancer spheroids in the peritoneal cavity during metastasis. Ovarian cancer spheroids generated under a non-adherent condition were cultured in microfluidic channels coated with peritoneal mesothelial cells subjected to physiologically relevant shear stress. In summary, this dynamic 3D ovarian cancer-mesothelium microfluidic platform can provide new knowledge on basic cancer biology and serve as a platform for potential drug screening and development.

To study ovarian tumor progression in a physiologically relevant model, multicellular spheroids were cultured in a microdevice under simulated fluid flow. This dynamic 3D model emulates the intraperitoneal environment with the cellular and mechanical components where ovarian cancer metastasis occurs.

Ovarian cancer is characterized by extensive peritoneal metastasis, with tumor spheres commonly found in the malignant ascites. This is associated with ...

Generation of Heterogeneous Drug Gradients Across Cancer Populations on a Microfluidic Evolution Accelerator for Real-Time Observation

Conventional cell culture remains the most frequently used preclinical model, despite its proven limited ability to predict clinical results in cancer. Microfluidic cancer-on-chip models have been proposed to bridge the gap between the oversimplified conventional 2D cultures and more complicated animal models, which have limited ability to produce reliable and reproducible quantitative results. Here, we present a microfluidic cancer-on-chip model that reproduces key components of a complex tumor microenvironment in a comprehensive manner, yet is simple enough to provide robust quantitative descriptions of cancer dynamics. This microfluidic cancer-on-chip model, the &#34;Evolution Accelerator,&#34; breaks down a large population of cancer cells into an interconnected array of tumor microenvironments while generating a heterogeneous chemotherapeutic stress landscape. The progression and the evolutionary dynamics of cancer in response to drug gradient can be monitored for weeks in real time, and numerous downstream experiments can be performed complementary to the time-lapse images taken through the course of the experiments.

We present a microfluidic cancer-on-chip model, the &#34;Evolution Accelerator&#34; technology, which provides a controllable platform for long-term real-time quantitative studies of cancer dynamics within well-defined environmental conditions at the single-cell level. This technology is expected to work as an in vitro model for fundamental research or pre-clinical drug development.

Conventional cell culture remains the most frequently used preclinical model, despite its proven limited ability to predict clinical results in cancer. ...

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

Kidney disease affects more than 10% of the global population and costs billions of dollars in federal expenditures. The most severe forms of kidney disease and eventual end-stage renal failure are often caused by the damage to the glomerular podocytes, which are the highly specialized epithelial cells that function together with endothelial cells and the glomerular basement membrane to form the kidney&#8217;s filtration barrier. Advances in renal medicine have been hindered by the limited availability of primary tissues and the lack of robust methods for the derivation of functional human kidney cells, such as podocytes. The ability to derive podocytes from renewable sources, such as stem cells, could help advance current understanding of the mechanisms of human kidney development and disease, as well as provide new tools for therapeutic discovery. The goal of this protocol was to develop a method to derive mature, post-mitotic podocytes from human induced pluripotent stem (hiPS) cells with high efficiency and specificity, and under chemically defined conditions. The hiPS cell-derived podocytes produced by this method express lineage-specific markers (including nephrin, podocin, and Wilm&#8217;s Tumor 1) and exhibit the specialized morphological characteristics (including primary and secondary foot processes) associated with mature and functional podocytes. Intriguingly, these specialized features are notably absent in the immortalized podocyte cell line widely used in the field, which suggests that the protocol described herein produces human kidney podocytes that have a developmentally more mature phenotype than the existing podocyte cell lines typically used to study human kidney biology.

Presented here is a chemically defined protocol for the derivation of human kidney podocytes from induced pluripotent stem cells with high efficiency (&#62;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.

Kidney disease affects more than 10% of the global population and costs billions of dollars in federal expenditures. The most severe forms of kidney ...

Isogenic Kidney Glomerulus Chip Engineered from Human Induced Pluripotent Stem Cells

Chronic kidney disease (CKD) affects 15% of the U.S. adult population, but the establishment of targeted therapies has been limited by the lack of functional models that can accurately predict human biological responses and nephrotoxicity. Advancements in kidney precision medicine could help overcome these limitations. However, previously established in vitro models of the human kidney glomerulus-the primary site for blood filtration and a key target of many diseases and drug toxicities-typically employ heterogeneous cell populations with limited functional characteristics and unmatched genetic backgrounds. These characteristics significantly limit their application for patient-specific disease modeling and therapeutic discovery. 
This paper presents a protocol that integrates human induced pluripotent stem (iPS) cell-derived glomerular epithelium (podocytes) and vascular endothelium from a single patient to engineer an isogenic and vascularized microfluidic kidney glomerulus chip. The resulting glomerulus chip is comprised of stem cell-derived endothelial and epithelial cell layers that express lineage-specific markers, produce basement membrane proteins, and form a tissue-tissue interface resembling the kidney's glomerular filtration barrier. The engineered glomerulus chip selectively filters molecules and recapitulates drug-induced kidney injury. The ability to reconstitute the structure and function of the kidney glomerulus using isogenic cell types creates the opportunity to model kidney disease with patient specificity and advance the utility of organs-on-chips for kidney precision medicine and related applications.

Presented here is a protocol to engineer a personalized organ-on-a-chip system that recapitulates the structure and function of the kidney glomerular filtration barrier by integrating genetically matched epithelial and vascular endothelial cells differentiated from human induced pluripotent stem cells. This bioengineered system can advance kidney precision medicine and related applications.

Chronic kidney disease (CKD) affects 15% of the U.S. adult population, but the establishment of targeted therapies has been limited by the lack of ...

Microfluidic Co-Culture Models for Dissecting the Immune Response in in vitro Tumor Microenvironments

Complex disease models demand cutting-edge tools able to deliver physiologically and pathologically relevant, actionable insights, and unveil otherwise invisible processes. Advanced cell assays closely mimicking in vivo scenery are establishing themselves as novel ways to visualize and measure the bidirectional tumor-host interplay influencing the progression of cancer. Here we describe two versatile protocols to recreate highly controllable 2D and 3D co-cultures in microdevices, mimicking the complexity of the tumor microenvironment (TME), under natural and therapy-induced immunosurveillance. In section 1, an experimental setting is provided to monitor crosstalk between adherent tumor cells and floating immune populations, by bright field time-lapse microscopy. As an applicative scenario, we analyze the effects of anti-cancer treatments, such as the so-called immunogenic cancer cell death inducers on the recruitment and activation of immune cells. In section 2, 3D tumor-immune microenvironments are assembled in a competitive layout. Differential immune infiltration is monitored by fluorescence snapshots up to 72 h, to evaluate combination therapeutic strategies. In both settings, image processing steps are illustrated to extract a plethora of immune cell parameters (e.g., immune cell migration and interaction, response to therapeutic agents). These simple and powerful methods can be further tailored to simulate the complexity of the TME encompassing the heterogeneity and plasticity of cancer, stromal and immune cells subtypes, as well as their reciprocal interactions as drivers of cancer evolution. The compliance of these rapidly evolving technologies with live-cell high-content imaging can lead to the generation of large informative datasets, bringing forth new challenges. Indeed, the triangle &#39;&#39;co-cultures/microscopy/advanced data analysis&#34; sets the path towards a precise problem parametrization that may assist tailor-made therapeutic protocols. We expect that future integration of cancer-immune on-a-chip with artificial intelligence for high-throughput processing will synergize a large step forward in leveraging the capabilities as predictive and preclinical tools for precision and personalized oncology.

In the age of immunotherapy and single-cell genomic profiling, cancer biology requires novel in vitro and computational tools for investigating the tumor-immune interface in a proper spatiotemporal context. We describe protocols to exploit tumor-immune microfluidic co-cultures in 2D and 3D settings, compatible with dynamic, multiparametric monitoring of cellular functions.

Complex disease models demand cutting-edge tools able to deliver physiologically and pathologically relevant, actionable insights, and unveil otherwise ...

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

Summary

Explore More Videos

Microfluidic Device for Recreating a Tumor Microenvironment in Vitro

A Murine Orthotopic Bladder Tumor Model and Tumor Detection System

A Syngeneic Mouse Model of Metastatic Renal Cell Carcinoma for Quantitative and Longitudinal Assessment of Preclinical Therapies

Monitoring Cancer Cell Invasion and T-Cell Cytotoxicity in 3D Culture

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

Modeling Ovarian Cancer Multicellular Spheroid Behavior in a Dynamic 3D Peritoneal Microdevice

Generation of Heterogeneous Drug Gradients Across Cancer Populations on a Microfluidic Evolution Accelerator for Real-Time Observation

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

Isogenic Kidney Glomerulus Chip Engineered from Human Induced Pluripotent Stem Cells

Microfluidic Co-Culture Models for Dissecting the Immune Response in in vitro Tumor Microenvironments