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

Summary

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.

Abstract

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.

Introduction

Advancements in 3D cell culture systems have revolutionized tissue engineering and regenerative medicine by offering more physiologically relevant models compared to traditional 2D cultures^1,2. 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 activ....

Protocol

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

1. 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).
   1. Design the chambers using computer-assisted design software. The chamber ....

Discussion

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.......

Materials

Name	Company	Catalog Number	Comments
1.5-2.0 mL  tubes	Eppendorf	003012/10237-20205
24-well plates	Sarstedt	83.3922.500
3D printer	Prusa
Agarose	Carl Roth	3810.3
AutoDesk Tinker CAD software			computer-assisted design software
Caki-1 cells	ATCC	HTB-46
Caspase activity	Promega	G8090	Caspase 3/7 assay
Cell viability	Promega	G7570	Cell Titer Glo assay
Collagen Type I – rat tail, 3.0 mg/mL	Corning	354236
DMEM/12F Medium	PAN Biotech	PO4-41650
DPBS solution	PAN Biotech	P04-53500
Epidermal Growth Factor	Merck	E4127
Fetal Calf Serum	PAN Biotech	P30-3033
Genipin (30 mM)	Merck	G4796
HUMIMIC chips 2	TissUse		multi-organ chips
HUMIMIC control unit	TissUse		multi-organ chip control unit
Hydrocortisone	Merck	H6909
Incubator (37 °C, 5% CO2)	nd
Insulin,Sodium selenite,Transferrin (IST)	Merck	I1884
LDH release	Promega	J2380
Metal spatula	nd
NaOH (1 M)	Carl Roth	P031.2
Petri dishes	Sarstedt	82.1135.500
Polypropylene (PP) filament	Verbatin	55952
RNA-easy extraction kit	Qiagen	74104
RPTEC/TERT cells	ATCC	CRL - 4031
TNF-alfa ELISA	R&D Systems	DY210-05
Triiodothyronine (T3 )	Merck	709611
Trypsin-EDTA	PAN Biotech	P10-021100