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 complexity of ECM is a key aspect in studying kidney-derived cells in vitro.

Previous attempts at replicating ECM microenvironments have focused on decellularizing whole tissue to create scaffolds capable of recellularization. Decellularization has been performed with chemical detergents such as sodium dodecyl sulfate (SDS) or non-ionic detergents, and it utilizes either whole organ perfusion or immersion and agitation methods^{5,6,7,8,9,10,11,12,13}. The scaffolds presented here preserve the structural and biochemical cues found in native tissue ECM; furthermore, recellularization with donor-specific cells has clinical relevance in reconstructive surgery^{14,15,16,17,18,19}. However, these scaffolds lack structural flexibility and are therefore incompatible with many current devices used for in vitro studies. To overcome this limitation, many groups have further processed decellularized ECM into hydrogels^{20,21,22,23,24}. These hydrogels are compatible with injection molding and bioink and circumvent micrometer scale spatial constraints that decellularized scaffolds place on cells. Furthermore, molecular composition and ratios found in native ECM are preserved^3,25. Here we demonstrate a method to fabricate a hydrogel derived from kidney cortex ECM (kECM).

The purpose of this protocol is to produce a hydrogel that replicates the microenvironment of the kidney cortical region. Kidney cortex tissue is decellularized in a 1% SDS solution under constant agitation to remove cellular matter. SDS is commonly used to decellularize tissue because of its ability to quickly remove immunological cellular material^6,7,9,26. The kECM is then subject to mechanical homogenization and lyophilization^5,6,9,11,26. Solubilization in a strong acid with pepsin results in a final hydrogel stock solution^20,27. Native kECM proteins that are important for structural support and signal transduction are preserved^3,25. The hydrogel can also be gelled to within one order of magnitude of native human kidney cortex^28,29,30. This matrix provides a physiological environment that has been used to maintain the quiescence of kidney-specific cells compared to hydrogels from other matrix proteins. Furthermore, matrix composition can be manipulated, for example, through the addition of collagen-I, to model disease environments for the study of renal fibrosis and other kidney diseases^31,32.

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, 2500 mL of 1% SDS solution is sufficient to decellularize a single human kidney.
   3. Transfer the solution to a 500 mL sterile vacuum filter and filter it into sterilized sealable containers.
2. Processing of kidney tissue
   1. Wash and autoclave a pair of forceps, two hemostat clamps, a pair of general service grade scissors, two scalpel blade handles, a 1000 mL beaker covered with aluminum foil, and a 36 x 9 mm stir bar.
   2. Line a tissue culture hood with underpad. Place the beaker, a sterile tissue culture dish (150 x 25 mm), and the whole kidney organ into the hood. Fill the beaker with 500 mL of 1% SDS solution.
      Note: Human kidneys were received on ice from LifeCenter NorthWest.
   3. Place the kidney in the sterile tissue culture dish (Figure 1A). Remove all perirenal fat by lightly shaving around the renal capsule with a scalpel (Figure 1B).
   4. Make a shallow 8-10 cm incision with the scalpel, just deep enough to break open the renal capsule without damaging the underlying cortex tissue, across the superior end of the kidney. Remove the renal capsule by peeling it away from the cortex tissue with two hemostat clamps (Figure 1C).
   5. Bisect the kidney along the coronal plane by using the scalpel along the lateral side of the kidney (Figure 1D). Isolate cortex tissue from both halves by carving out the medullar region with the scalpel (Figure 1E) and dice the cortex tissue into 0.5 cm³ pieces (Figure 1F). Remove any large visible vessels.
3. Isolation of extracellular matrix
   1. In a tissue culture hood, fill a 1000 mL beaker with 500 mL of 1% SDS solution. Place the diced cortex tissue and stir bar into the beaker containing SDS solution. Cover the beaker with autoclaved aluminum foil and place it on a stir plate at approximately 400 rpm outside of the tissue culture hood.
   2. After the cortex tissue has been on the stir plate for 24 h, bring the beaker into a tissue culture hood and add a 40 µm sterile cell strainer made with nylon mesh. Fill a separate 1000 mL beaker with 200 mL of bleach and place it in the tissue culture hood.
   3. Pipette out the SDS solution through the cell strainer into the beaker containing bleach. Pipette out all SDS solution until only decellularized tissue and the cell strainer remain in the beaker.
      Note: The cell strainer should prevent any tissue from being removed during solution aspiration.
   4. Leave the cell strainer in the beaker and fill with 500 mL of fresh SDS solution. Cover the beaker with the same aluminum foil and place onto a stir plate at the same speed as before.
   5. Repeat steps 1.3.1-1.3.3 every 24 hours with fresh SDS solution for a total of five days.
   6. Rinse decellularized tissue with autoclaved DI water every 24 h for 3 days total, following the technique outlined in steps 1.3.1-1.3.3.
   7. Rinse decellularized tissue with cell culture grade water every 24 h for 2 days total, following the technique outlined in steps 1.3.1-1.3.3.
   8. Repeat steps 1.3.1-1.3.2. Transfer the decellularized tissue (referred to as kECM from this point on) into a 30 mL self-standing conical tube and fill it with cell culture grade water until all the tissue is submerged.

2. Fabrication of Hydrogel Stock Solution

1. Mechanical processing of decellularized tissue
   1. In a tissue culture hood, mechanically homogenize the kECM within the conical tube with a tissue homogenizer for 2 min.
      Note: Homogenized kECM should resemble an opaque solution with no visible pieces of ECM.
   2. Submerge the conical tube containing the kECM in liquid nitrogen until boiling surrounding the tube no longer persists. Store the kECM at -4 ˚C overnight.
2. Lyophilization of frozen decellularized tissue
   1. Slightly loosen the conical tube cap to allow for gas exchange and place the tube into a lyophilization machine. Lyophilize the kECM for three days or until it resembles a fine white powder. Store at -4 ˚C.
3. Chemical digestion and solubilization of gel
   1. Autoclave a 20 mL scintillation vial and cap, a 15.9 x 7.9 mm stir bar, and one pair of fine-tip forceps.
   2. Weigh the lyophilized kECM and calculate the volume of HCl and mass of pepsin needed to solubilize the kECM to a 3% (30 mg/mL) solution using the following equations, where m_pepsin is the mass of pepsin, m_tissue is the mass of lyophilized tissue, and V_HCl is the volume of 0.01 N HCl:
      
      
   3. In a tissue culture hood, add porcine gastric pepsin, 0.01 N HCl, and the stir bar to the scintillation vial, and leave it on a stir plate at approximately 500 rpm until all the pepsin has dissolved. Transfer the lyophilized kECM to the scintillation vial and leave the solution on a stir plate at approximately 500 rpm for three days.

3. Hydrogel Gelation

1. Kidney ECM hydrogel preparation
   1. Gel the hydrogel by mixing the kECM hydrogel stock solution with 1 N NaOH, 10x Media Supplement (M199), and cell culture media. Keep all the solutions on ice.
      Note: Final gel concentrations of 7.5 mg/mL were used for cell culture. 1 mL of kECM gel was sufficient for cell culture experiments presented.
   2. Determine the volume of workable kECM gel produced and volume of stock kECM hydrogel needed by using the following equation, where V_final is the volume of gel created, V_{stock kECM} is the volume of stock kECM hydrogel needed, C_{stock kECM} is the concentration of the stock kECM hydrogel, and C_final is the concentration of the final gel:
      
   3. Determine the volume of neutralizing reagents needed by using the following equations, where V_NaOH is the volume of 1 N NaOH, V_10X is the volume of M199 10X media supplement, and V_1X is the volume of cell culture media:
      
      
      
   4. In a tissue culture hood, pipette the neutralizing reagents (NaOH, M199, and cell culture media) into a sterile 30 mL self-standing conical tube. Mix the neutralizing reagent solution with a microspatula.
   5. Use a sterile 1 mL syringe to transfer the appropriate volume of stock kECM hydrogel to the neutralizing reagent solution. Use a microspatula to gently mix the solution until a homogeneous in color hydrogel solution is obtained.
      Note: Avoid introducing air bubbles by stirring slowly and gently.
   6. To incorporate cells into the kECM hydrogel, subtract 10 µL of cell culture media (V_1X) from the neutralizing solution volume calculations in step 3.1.1.3.
      1. Suspend cells into 10 µL of cell culture media. Determine the number of cells to be suspended by using the following equation, where #_cells implies the number of cells to suspend and V_final is the volume of gel created:
         
         Note: 300,000 cells/mL is the concentration of cells used in the kECM gel.
      2. Pipette the 10 µL of cell suspended solution into the final kECM gel after the kECM stock solution has been mixed with neutralizing reagent solution. Stir the solution with a microspatula until the cells are evenly distributed.
2. Use a 1 mL syringe to fill a desired cell culture device with the kECM hydrogel.
3. Allow the gel to set at 37 ˚C for 1 h before transferring or plating cells.

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

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