Published: October 13th, 2018
Here we present a protocol to fabricate a kidney cortex extracellular matrix-derived hydrogel to retain the native kidney extracellular matrix (ECM) structural and biochemical composition. The fabrication process and its applications are described. Finally, a perspective on using this hydrogel to support kidney-specific cellular and tissue regeneration and bioengineering is discussed.
Extracellular matrix (ECM) provides important biophysical and biochemical cues to maintain tissue homeostasis. Current synthetic hydrogels offer robust mechanical support for in vitro cell culture but lack the necessary protein and ligand composition to elicit physiological behavior from cells. This manuscript describes a fabrication method for a kidney cortex ECM-derived hydrogel with proper mechanical robustness and supportive biochemical composition. The hydrogel is fabricated by mechanically homogenizing and solubilizing decellularized human kidney cortex ECM. The matrix preserves native kidney cortex ECM protein ratios while also enabling gelation to physiological mechanical stiffnesses. The hydrogel serves as a substrate upon which kidney cortex-derived cells can be maintained under physiological conditions. Furthermore, the hydrogel composition can be manipulated to model a diseased environment which enables the future study of kidney diseases.
Extracellular matrix (ECM) provides important biophysical and biochemical cues to maintain tissue homeostasis. The complex molecular composition regulates both structural and functional properties of tissue. Structural proteins provide cells with spatial awareness and allow for adhesion and migration1. Bound ligands interact with cell surface receptors to control cell behavior2. Kidney ECM contains a plethora of molecules whose composition and structure varies depending on anatomical location, developmental stage, and disease state3,4. Recapitulating the complexi....
Human kidneys were isolated by LifeCenter Northwest following ethical guidelines set by the Association of Organ Procurement Organizations. This protocol follows animal care and cell culture guidelines outlined by the University of Washington.
1. Preparation of Human Kidney Tissue
The kECM hydrogel provides a matrix for kidney cell culture with similar chemical composition as the native kidney microenvironment. To fabricate the hydrogel, kidney cortex tissue is mechanically isolated from a whole kidney organ and diced (Figure 1). Decellularization with a chemical detergent (Figure 2A.1-A.3) followed by rinses with water to remove detergent particles (Figure 2A.......
Matrices provide important mechanical and chemical cues that govern cell behavior. Synthetic hydrogels are able to support complex 3-dimensional patterning but fail to provide the diverse extracellular cues found in physiological matrix microenvironments. Hydrogels derived from native ECM are ideal materials for both in vivo and in vitro studies. Previous studies have used decellularized ECM hydrogels to coat synthetic biomaterials to prevent host immunological responses33,<.......
The authors would like to acknowledge the Lynn and Mike Garvey Imaging Laboratory at the Institute for Stem Cell and Regenerative Medicine and LifeCenter NorthWest. They would also like to acknowledge the financial support of National Institutes of Health grants, UH2/UH3 TR000504 (to J.H.) and DP2DK102258 (to Y.Z.), NIH T32 training grant DK0007467 (to R.J.N.), and an unrestricted gift from the Northwest Kidney Centers to the Kidney Research Institute.....
|Preparation of Kidney Tissue
|5000 mL Beaker
|Sodium Dodecyl Sulfate (SDS)
|Autoclaved DI H2O
|Stir Bar (70 x 10 mm)
|500 mL Vacuum Filter
|1000 mL Beaker
|Any similar forceps may be used
|Scissor-Handle Hemostat Clamp
|Scalpel Handle, No. 4
|Any similar scalpel handle may be used
|Scalpel Blade, No. 20
|Any similar scalpel blade may be used
|Stir Bar (38.1 x 9.5 mm)
|Petri Dish (150 x 25 mm)
|Autoclavable Biohazard Bag
|Sterile Cell Strainer (40 um)
|Cell Culture Grade Water
|30 mL Freestanding Tube
|Fabrication of ECM Gel
|Tissue Homogenizer Machine
|20 mL Glass Scintillation Vials and Cap
|Stir Bar (15.9 x 8 mm)
|Pepsin from Porcine Gastric Mucosa
|0.01 N HCl
|Dilute to 0.01 N HCl with cell culuture water
|Kidney ECM Gelation
|1 N NaOH (Sterile)
|Dilute to 1 N in cell culture grade water
|15 mL Conical Tube
|Cell Culture Media
|Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12 (DMEM/F12)
|Fetal Bovine Serum (FBS)
|Insulin, Transferrin, Selenium, Sodium Pyruvate Solution (ITS-A) 100X
|1 mL Syringe
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