Fabricating a Kidney Cortex Extracellular Matrix-Derived Hydrogel

Summary

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.

Abstract

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.

Introduction

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 state^3,4. Recapitulating the complexi....

Protocol

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

1. Preparation of decellularization solution
   1. Sterilize a 5000 mL beaker and a 70 x 10 mm stir bar.
   2. Mix 1:1000 (weight:volume) sodium dodecyl sulfate (SDS) in autoclaved deionized water in the beaker. Leave the solution on a stir plate at approximately 200 rpm for 24 h or until the SDS is completely dissolved.
      Note: Typically, ....

Results

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.......

Discussion

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 responses^33,<.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Preparation of Kidney Tissue
5000 mL Beaker	Sigma-Aldrich	Z740589
Sodium Dodecyl Sulfate (SDS)	Sigma-Aldrich	436143
Sterile H2O			Autoclaved DI H2O
Stir Bar (70 x 10 mm)	Fisher Science	14-512-128
500 mL Vacuum Filter	VWR	97066-202
Stir Plate	Sigma-Aldrich	CLS6795420D
1000 mL Beaker	Sigma-Aldrich	CLS10031L
Forceps	Sigma-Aldrich	F4642	Any similar forceps may be used
Scissor-Handle Hemostat Clamp	Sigma-Aldrich	Z168866
Dissecting Scissors	Sigma-Aldrich	Z265977
Scalpel Handle, No. 4	VWR	25859-000	Any similar scalpel handle may be used
Scalpel Blade, No. 20	VWR	25860-020	Any similar scalpel blade may be used
Stir Bar (38.1 x 9.5 mm)	Fisher Science	14-513-52
Absorbent Underpad	VWR	82020-845
Petri Dish (150 x 25 mm)	Corning	430597
Autoclavable Biohazard Bag	VWR	14220-026
Sterile Cell Strainer (40 um)	Fisher Science	22-363-547
Cell Culture Grade Water	HyClone	SH30529.03
30 mL Freestanding Tube	VWR	89012-778
Fabrication of ECM Gel
Tissue Homogenizer Machine	Polytron	PCU-20110
Freeze Dryer	Labconco	7670520
20 mL Glass Scintillation Vials and Cap	Sigma-Aldrich	V7130
Stir Bar (15.9 x 8 mm)	Fisher Science	14-513-62
Pepsin from Porcine Gastric Mucosa	Sigma-Aldrich	P7012
0.01 N HCl	Sigma-Aldrich	320331	Dilute to 0.01 N HCl with cell culuture water
Kidney ECM Gelation
1 N NaOH (Sterile)	Sigma-Aldrich	415413	Dilute to 1 N in cell culture grade water
Medium 199	Sigma-Aldrich	M4530
15 mL Conical Tube	ThermoFisher	339651
Cell Culture Media	ThermoFisher	11330.032	Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12 (DMEM/F12)
Fetal Bovine Serum (FBS)	Gibco	10082147
Antibiotic-Antimycotic 100X	Life Technologies	15240-062
Insulin, Transferrin, Selenium, Sodium Pyruvate Solution (ITS-A) 100X	Life Technologies	51300-044
1 mL Syringe	Sigma-Aldrich	Z192325
Microspatula	Sigma-Aldrich	Z193208