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://www.jove.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://www.jove.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://www.jove.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

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	<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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1.5-2.0 mL&#160; tubes&#160;</td>
			<td>Eppendorf</td>
			<td>003012/10237-20205</td>
			<td></td>
		</tr>
		<tr>
			<td>24-well plates&#160;</td>
			<td>Sarstedt</td>
			<td>83.3922.500</td>
			<td></td>
		</tr>
		<tr>
			<td>3D printer&#160;</td>
			<td>Prusa</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose&#160;</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&#160;</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 &#8211; rat tail, 3.0 mg/mL</td>
			<td>Corning</td>
			<td>354236</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/12F Medium&#160;</td>
			<td>PAN Biotech</td>
			<td>PO4-41650</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS solution&#160;</td>
			<td>PAN Biotech</td>
			<td>P04-53500</td>
			<td></td>
		</tr>
		<tr>
			<td>Epidermal Growth Factor</td>
			<td>Merck&#160;</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)&#160;</td>
			<td>Merck&#160;</td>
			<td>G4796</td>
			<td></td>
		</tr>
		<tr>
			<td>HUMIMIC chips 2&#160;</td>
			<td>TissUse</td>
			<td></td>
			<td>multi-organ chips</td>
		</tr>
		<tr>
			<td>HUMIMIC control unit&#160;</td>
			<td>TissUse</td>
			<td></td>
			<td>multi-organ chip control unit&#160;</td>
		</tr>
		<tr>
			<td>Hydrocortisone&#160;</td>
			<td>Merck&#160;</td>
			<td>H6909</td>
			<td></td>
		</tr>
		<tr>
			<td>Incubator (37 &#176;C, 5% CO2)&#160;</td>
			<td>nd</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin,Sodium selenite,Transferrin (IST)</td>
			<td>Merck&#160;</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&#160;</td>
			<td>nd</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NaOH (1 M)&#160;</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&#160;</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&#160;</td>
			<td>ATCC</td>
			<td>CRL - 4031</td>
			<td></td>
		</tr>
		<tr>
			<td>TNF-alfa ELISA</td>
			<td>R&#38;D Systems</td>
			<td>DY210-05</td>
			<td></td>
		</tr>
		<tr>
			<td>Triiodothyronine (T3 )</td>
			<td>Merck&#160;</td>
			<td>709611</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA</td>
			<td>PAN Biotech</td>
			<td>P10-021100</td>
			<td></td>
		</tr>
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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

Research

JoVE Journal

Cancer Research

456 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

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 ...

Radionuclide-fluorescence Reporter Gene Imaging to Track Tumor Progression in Rodent Tumor Models

Metastasis is responsible for most cancer deaths. Despite extensive research, the mechanistic understanding of the complex processes governing metastasis remains incomplete. In vivo models are paramount for metastasis research, but require refinement. Tracking spontaneous metastasis by non-invasive in vivo imaging is now possible, but remains challenging as it requires long-time observation and high sensitivity. We describe a longitudinal combined radionuclide and fluorescence whole-body in vivo imaging approach for tracking tumor progression and spontaneous metastasis. This reporter gene methodology employs the sodium iodide symporter (NIS) fused to a fluorescent protein (FP). Cancer cells are engineered to stably express NIS-FP followed by selection based on fluorescence-activated cell sorting. Corresponding tumor models are established in mice. NIS-FP expressing cancer cells are tracked non-invasively in vivo at the whole-body level by positron emission tomography (PET) using the NIS radiotracer [18F]BF4-. PET is currently the most sensitive in vivo imaging technology available at this scale and enables reliable and absolute quantification. Current methods either rely on large cohorts of animals that are euthanized for metastasis assessment at varying time points, or rely on barely quantifiable 2D imaging. The advantages of the described method are: (i) highly sensitive non-invasive in vivo 3D PET imaging and quantification, (ii) automated PET tracer production, (iii) a significant reduction in required animal numbers due to repeat imaging options, (iv) the acquisition of paired data from subsequent imaging sessions providing better statistical data, and (v) the intrinsic option for ex vivo confirmation of cancer cells in tissues by fluorescence microscopy or cytometry. In this protocol, we describe all steps required for routine NIS-FP-afforded non-invasive in vivo cancer cell tracking using PET/CT and ex vivo confirmation of in vivo results. This protocol has applications beyond cancer research whenever in vivo localization, expansion and long-time monitoring of a cell population is of interest.

We describe a protocol for preclinical in vivo tracking of cancer metastasis. It is based on a radionuclide-fluorescence reporter combining the sodium iodide symporter, detected by non-invasive [18F]tetrafluoroborate-PET, and a fluorescent protein for streamlined ex vivo confirmation. The method is applicable for preclinical in vivo cell tracking beyond tumor biology.

Metastasis is responsible for most cancer deaths. Despite extensive research, the mechanistic understanding of the complex processes governing metastasis ...

Acellular and Cellular Lung Model to Study Tumor Metastasis

It is difficult to isolate tumor cells at different points of tumor progression. We created an ex vivo lung model that can show the interaction of tumor cells with a natural matrix and continual flow of nutrients, as well as a model that shows the interaction of tumor cells with normal cellular components and a natural matrix. The acellular ex vivo lung model is created by isolating a rat heart-lung block and removing all the cells using the decellularization process. The right main bronchus is tied off and tumor cells are placed in the trachea by a syringe. The cells move and populate the left lung. The lung is then placed in a bioreactor where the pulmonary artery receives a continual flow of media in a closed circuit. The tumor grown on the left lung is the primary tumor. The tumor cells that are isolated in the circulating media are circulating tumor cells and the tumor cells in the right lung are metastatic lesions. The cellular ex vivo lung model is created by skipping the decellularization process. Each model can be used to answer different research questions.

Here, we present a protocol for an ex vivo lung cancer model that mimics the steps of tumor progression and helps to isolate a primary tumor, circulating tumor cells, and metastatic lesions.

It is difficult to isolate tumor cells at different points of tumor progression. We created an ex vivo lung model that can show the interaction of tumor ...

Measuring Bone Remodeling and Recreating the Tumor-Bone Microenvironment Using Calvaria Co-culture and Histomorphometry

Bone is a connective tissue constituted of osteoblasts, osteocytes, and osteoclasts and a mineralized extracellular matrix, which gives it its strength and flexibility and allows it to fulfill its functions. Bone is continuously exposed to a variety of stimuli, which in pathological conditions can deregulate bone remodeling. To study bone biology and diseases and evaluate potential therapeutic agents, it has been necessary to develop in vitro and in vivo models.
This manuscript describes the dissection process and culture conditions of calvarias isolated from neonatal mice to study bone formation and the bone tumor microenvironment. In contrast to in vitro and in vivo models, this ex vivo model allows preservation of the three-dimensional environment of the tissue as well as the cellular diversity of the bone while culturing under defined conditions to simulate the desired microenvironment. Therefore, it is possible to investigate bone remodeling and its mechanisms, as well as the interactions with other cell types, such as the interactions between cancer cells and bone.
The assays reported here use calvarias from 5-7 day old BALB/C mice. The hemi-calvarias obtained are cultured in the presence of insulin, breast cancer cells (MDA-MB-231), or conditioned medium from breast cancer cell cultures. After analysis, it was established that insulin induced new bone formation, while cancer cells and their conditioned medium induced bone resorption. The calvarial model has been successfully used in basic and applied research to study bone development and cancer-induced bone diseases. Overall, it is an excellent option for an easy, informative, and low-cost assay.

The ex vivo culture of bone explants can be a valuable tool for the study of bone physiology and the potential evaluation of drugs in bone remodeling and bone diseases. The presented protocol describes the preparation and culture of calvarias isolated from newborn mice skulls, as well as its applications.

Bone is a connective tissue constituted of osteoblasts, osteocytes, and osteoclasts and a mineralized extracellular matrix, which gives it its strength ...

Comparing Metastatic Clear Cell Renal Cell Carcinoma Model Established in Mouse Kidney and on Chicken Chorioallantoic Membrane

Metastatic clear cell renal cell carcinoma (ccRCC) is the most common subtype of kidney cancer. Localized ccRCC has a favorable surgical outcome. However, one third of ccRCC patients will develop metastases to the lung, which is related to a very poor outcome for patients. Unfortunately, no therapy is available for this deadly stage, because the molecular mechanism of metastasis remains unknown. It has been known for 25 years that the loss of function of the von Hippel-Lindau (VHL) tumor suppressor gene is pathognomonic of ccRCC. However, no clinically relevant transgenic mouse model of ccRCC has been generated. The purpose of this protocol is to introduce and compare two newly established animal models for metastatic ccRCC. The first is renal implantation in the mouse model. In our laboratory, the CRISPR gene editing system was utilized to knock out the VHL gene in several RCC cell lines. Orthotopic implantation of heterogeneous ccRCC populations to the renal capsule created novel ccRCC models that develop robust lung metastases in immunocompetent mice. The second model is the chicken chorioallantoic membrane (CAM) system. In comparison to the mouse model, this model is more time, labor, and cost-efficient. This model also supported robust tumor formation and intravasation. Due to the short 10 day period of tumor growth in CAM, no overt metastasis was observed by immunohistochemistry (IHC) in the collected embryo tissues. However, when tumor growth was extended by two weeks in the hatched chicken, micrometastatic ccRCC lesions were observed by IHC in the lungs. These two novel preclinical models will be useful to further study the molecular mechanism behind metastasis, as well as to establish new, patient-derived xenografts (PDXs) toward the development of novel treatments for metastatic ccRCC.

Metastatic clear cell renal cell carcinoma is a disease without a comprehensive animal model for thorough preclinical investigation. This protocol illustrates two novel animal models for the disease: the orthotopically implanted mouse model and the chicken chorioallantoic membrane model, both of which demonstrate lung metastasis resembling clinical cases.

Metastatic clear cell renal cell carcinoma (ccRCC) is the most common subtype of kidney cancer. Localized ccRCC has a favorable surgical outcome. However, ...

Studying the Effects of Tumor-Secreted Paracrine Ligands on Macrophage Activation using Co-Culture with Permeable Membrane Supports

Tumor-derived paracrine signaling is an overlooked component of local immunosuppression and can lead to a permissive environment for continued cancer growth and metastasis. Paracrine signals can involve cell-cell contact between different cell types, such as PD-L1 expressed on the surface of tumors interacting directly with PD-1 on the surface of T cells, or the secretion of ligands by a tumor cell to affect an immune cell. Here we describe a co-culture method to interrogate the effects of tumor-secreted ligands on immune cell (macrophage) activation. This straightforward procedure utilizes commercially available 0.4 &#181;m polycarbonate membrane permeable supports and standard tissue culture plates. In the process described, macrophages are cultured in the upper chamber and tumor cells in the lower chamber. The presence of the 0.4 &#181;m barrier allows for the study of intercellular signaling without the confounding variable of physical contact, because the two cell types share the same medium and exposure to paracrine ligands. This approach can be combined with others, such as genetic alterations of the macrophage (e.g., isolation from genetic knock-out mice) or tumor (e.g., CRISPR-mediated alterations) to study the role of specific secreted factors and receptors. The approach also lends itself to standard molecular biological analyses such as quantitative reverse transcription polymerase chain reaction (qRT-PCR) or Western blot analysis, without the need for flow sorting to separate the two cell populations. Enzyme-linked immunosorbent assays (ELISAs) can similarly be utilized to measure secreted ligands to better understand the dynamic interaction of cell signaling in the multiple cell type context. Duration of co-culture can also be varied for the study of temporally regulated events. This co-culture method is a robust tool that facilitates the study of tumor-secreted signals in the immune context.

Here, we present a method using permeable membrane supports to facilitate the study of non-contact paracrine signaling used by tumor cells to suppress the immune response. The system is amenable to studying the role of tumor-secreted factors in dampening macrophage activation.

Tumor-derived paracrine signaling is an overlooked component of local immunosuppression and can lead to a permissive environment for continued cancer ...

Detection of Lung Tumor Progression in Mice by Ultrasound Imaging

With ~1.6 million victims per year, lung cancer contributes tremendously to the worldwide burden of cancer. Lung cancer is partly driven by genetic alterations in oncogenes such as the KRAS oncogene, which constitutes ~25% of lung cancer cases. The difficulty in therapeutically targeting KRAS-driven lung cancer partly stems from having poor models that can mimic the progression of the disease in the lab. We describe a method that permits the relative quantification of primary KRAS lung tumors in a Cre-inducible LSL-KRAS G12D mouse model via ultrasound imaging. This method relies on brightness (B)-mode acquisition of the lung parenchyma. Tumors that are initially formed in this model are visualized as B-lines and can be quantified by counting the number of B-lines present in the acquired images. These would represent the relative tumor number formed on the surface of the mouse lung. As the formed tumors develop with time, they are perceived as deep clefts within the lung parenchyma. Since the circumference of the formed tumor is well-defined, calculating the relative tumor volume is achieved by measuring the length and width of the tumor and applying them in the formula used for tumor caliper measurements. Ultrasound imaging is a non-invasive, fast and user-friendly technique that is often used for tumor quantifications in mice. Although artifacts may appear when obtaining ultrasound images, it has been shown that this imaging technique is more advantageous for tumor quantifications in mice compared to other imaging techniques such as computed tomography (CT) imaging and bioluminescence imaging (BLI). Researchers can investigate novel therapeutic targets using this technique by comparing lung tumor initiation and progression between different groups of mice.

This protocol describes the steps taken to induce KRAS lung tumors in mice as well as the quantification of formed tumors by ultrasound imaging. Small tumors are visualized in early timepoints as B-lines. At later timepoints, relative tumor volume measurements are achieved by the measurement tool in the ultrasound software.

With ~1.6 million victims per year, lung cancer contributes tremendously to the worldwide burden of cancer. Lung cancer is partly driven by genetic ...

Multidimensional Coculture System to Model Lung Squamous Carcinoma Progression

Tumor-stroma interactions play a critical role in the development of lung squamous carcinoma (LUSC). However, understanding how these dynamic interactions contribute to tissue architectural changes observed during tumorigenesis remains challenging due to the lack of appropriate models. In this protocol, we describe the generation of a 3D coculture model using a LUSC primary cell culture known as TUM622. TUM622 cells were established from a LUSC patient-derived xenograft (PDX) and have the unique property to form acinar-like structures when seeded in a basement membrane matrix. We demonstrate that TUM622 acini in 3D coculture recapitulate key features of tissue architecture during LUSC progression as well as the dynamic interactions between LUSC cells and components of the tumor microenvironment (TME), including the extracellular matrix (ECM) and cancer-associated fibroblasts (CAFs). We further adapt our principal 3D culturing protocol to demonstrate how this system could be utilized for various downstream analyses. Overall, this organoid model creates a biologically rich and adaptable platform that enables one to gain insight into the cell-intrinsic and extrinsic mechanisms that promote the disruption of epithelial architectures during carcinoma progression and will aid the search for new therapeutic targets and diagnostic markers.

An in vitro model system was developed to capture tissue architectural changes during lung squamous carcinoma (LUSC) progression in a 3-dimensional (3D) co-culture with cancer-associated fibroblasts (CAFs). This organoid system provides a unique platform to investigate the roles of diverse tumor cell-intrinsic and extrinsic changes that modulate the tumor phenotype.

Tumor-stroma interactions play a critical role in the development of lung squamous carcinoma (LUSC). However, understanding how these dynamic interactions ...

Co-culture of Glioblastoma Stem-like Cells on Patterned Neurons to Study Migration and Cellular Interactions

Glioblastomas (GBMs), grade IV malignant gliomas, are one of the deadliest types of human cancer because of their aggressive characteristics. Despite significant advances in the genetics of these tumors, how GBM cells invade the healthy brain parenchyma is not well understood. Notably, it has been shown that GBM cells invade the peritumoral space via different routes; the main interest of this paper is the route along white matter tracts (WMTs). The interactions of tumor cells with the peritumoral nervous cell components are not well characterized. Herein, a method has been described that evaluates the impact of neurons on GBM cell invasion. This paper presents an advanced co-culture in vitro assay that mimics WMT invasion by analyzing the migration of GBM stem-like cells on neurons. The behavior of GBM cells in the presence of neurons is monitored by using an automated tracking procedure with open-source and free-access software. This method is useful for many applications, in particular, for functional and mechanistic studies as well as for analyzing the effects of pharmacological agents that can block GBM cell migration on neurons.

Here, we present an easy-to-use co-culture assay to analyze glioblastoma (GBM) migration on patterned neurons. We developed a macro in FiJi software for easy quantification of GBM cell migration on neurons, and observed that neurons modify GBM cell invasive capacity.

Glioblastomas (GBMs), grade IV malignant gliomas, are one of the deadliest types of human cancer because of their aggressive characteristics. Despite ...