Tunable Hydrogels from Pulmonary Extracellular Matrix for 3D Cell Culture

Summary

This is a method to create a 3-dimensional cell culture scaffold from pulmonary extracellular matrix. Intact lung is processed into hydrogels that can support the growth of cells in three-dimensions.

Abstract

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung tissue from porcine, rat, or mouse, the tissue is perfused and submerged in subsequent chemical detergents to remove the cellular debris. Histological comparison of the tissue before and after processing confirms removal of over 95% of double stranded DNA and alpha galactosidase staining suggests the majority of cellular debris is removed. After decellularization, the tissue is lyophilized and then cryomilled into a powder. The matrix powder is digested for 48 hr in an acidic pepsin digestion solution and then neutralized to form the pregel solution. Gelation of the pregel solution can be induced by incubation at 37 °C and can be used immediately following neutralization or stored at 4 °C for up to two weeks. Coatings can be formed using the pregel solution on a non-treated plate for cell attachment. Cells can be suspended in the pregel prior to self-assembly to achieve a 3D culture, plated on the surface of a formed gel from which the cells can migrate through the scaffold, or plated on the coatings. Alterations to the strategy presented can impact gelation temperature, strength, or protein fragment sizes. Beyond hydrogel formation, the hydrogel stiffness may be increased using genipin.

Introduction

Translating in vitro results to the clinic is one of the most challenging issues facing biomedical researchers. In vitro research on tissue culture plastic is easier, more convenient, and maintains high cell viability.¹ This approach is a reasonable starting point, but the results have limited clinical translation. Increasingly, laboratories are incorporating three-dimensional constructs to replace the traditional two-dimensional methods. Reviews are available for many three-dimensional environments, from biological scaffolds to polymeric scaffolds.^2,3

Biological frameworks can mimic characteristics of in vivo environments as they contain many of the protein and glycosaminoglycan components of the native matrix and provide familiar binding sites for cells to attach to and recognize. Extracellular matrix (ECM) derived materials have been shown to be capable scaffolds for cell attachment and proliferation.⁴ One challenge that limits the application of ECM hydrogel platforms stems from their inherently weak mechanical properties following gelation. Native tissue often has mechanical properties that are magnitudes higher than hydrogels. Non-toxic crosslinking agents can increase the mechanical properties of hydrogels to better mimic the native tissue environment. Genipin is a non-toxic, natural crosslinker derived from Gardenia plants with the ability to closely tailor mechanical properties of ECM with changes in genipin concentration^5,6.

Nearly all cells in the body exist in, and organize on, ECM that they either produce or maintain. New focus on the universal importance of ECM in the organization, condition, and function in every organ or system has sparked the production of matrix based platforms for in vitro investigation. Porcine small intestine submucosa is the most extensively studied naturally-derived scaffold, and it has been used to regenerate tendons, ligaments, skeletal muscle⁴, and even bone⁷. Matrices from other organs and donor species have also demonstrated good tissue regeneration potential. The use of foreign ECM components causes minimal issues with immunomodulation. After elimination of host cellular matter, the remaining ECM will be similar in amino acid content and organization to all other mammalian species⁸. There is a growing line of thinking that the best way to examine cell-ECM interactions in vitro is to utilize organ-specific ECM scaffolds. Each organ provides a unique composition of proteins and proteoglycans to create cellular niches. Niches provide structural, functional and even the enzymatic breakdown of the extracellular matrix contributing to biophysical signaling. To attain an in vitro microenvironment most similar to the in vivo microenvironment, use of tissue specific ECM would optimize the cellular niches for research.

The goal of this protocol is to provide a method for establishing a hydrogel scaffold unique to the lung ECM. This method provides a platform for in vitro research on lung cell-ECM interactions.

Protocol

Solution	Sterile Filter	Directions
DiH2O	Yes	DiH2O; Sterile filtered
0.1% Triton X-100 Solution	Yes	Under fume hood add 100 µl Triton-X 100 Solution to 100 ml DiH2O and agitate until dissolved; sterile filter.
2% Deoxycholate Solution	Yes	Under fume hood add 2 g Sodium Deoxycholate solution per 100 ml DiH2O and agitate until dissolved; Sterile filter
1 M NaCl	Yes	Add 58.44 g NaCl to 1 L of DiH2O; agitate until dissolved; sterile filter
DNase solution	Yes	Add 12,000 units DNase to 1 L DiH2O; Add 0.156 g MgSO4 (Anhydrous), and 0.222 g CaCl2; agitate until dissolved; sterile filter
PBS	Yes	Combine 27.2 g Na2HPO4 · 7H2O (dibasic heptahydrate), 80 g NaCl, and 2 g KCl with 10 L DiH2O; agitate until dissolved; adjust pH to 7.4; sterile filter

Table 1: Solutions Required for Tissue Decellularization. Make the solutions above for the decellularization process. Store at 4 °C. Approximately 2.5 L will be needed for one porcine lung.

1. Hydrogel Formation

1. Porcine Lung Decellularization (adapted from references^9,10)
   1. Obtain, an en bloc normal porcine lung, from a slaughterhouse, with heart and vasculature intact.
   2. Use tissue scissors or a scalpel to dissect away the heart, cutting vasculature as close as possible to the heart, remaining vasculature will be used in later steps for perfusion.
   3. Carefully dissect away the connective tissue surrounding the trachea, bronchi, and vasculature using a scalpel or scissors.
      NOTE: Determine the best lung to use for the rest of the procedure by choosing a lung without large cuts or punctures through the pleura and large amounts of atelectasis or vascular occlusion that may impede the effectiveness of the decellularization process.
   4. Cut away the suboptimal lung leaving as much bronchus attached to the trachea as possible (heart, connective tissue and the lobes of the removed lung may be disposed of). There should be one lung remaining attached to the trachea and vasculature.
      NOTE: Retain sections for histology if desired.⁹
   5. Close disconnected bronchi with clamps or suture to prevent excess backflow.
   6. Prepare the decellularization solutions and refrigerate at 4 °C until needed (Table 1).
   7. Using a hand pump cannulated to fit the pulmonary artery, perfuse the lung tissue 3 times with DI water through both pulmonary artery and trachea. Perfuse vasculature first each time. Begin with around 1 L into vasculature and 1.5 L into trachea for each perfusion, but as cellular debris is removed more liquid can be perfused through the system.
   8. Perfuse both vasculature and trachea with Triton X-100 solution.
   9. Submerge lung tissue in Triton X-100 solution for 24 hr at 4 °C.
   10. Perfuse vasculature and trachea 3 times with DI water to rinse.
   11. Perfuse both vasculature and trachea with deoxycholate solution.
   12. Submerge the tissue sections in deoxycholate for 24 hr at 4°C.
   13. Perfuse vasculature and trachea 3 times with DI water to rinse.
   14. Perfuse both vasculature and trachea with NaCl solution.
   15. Submerge tissue in filtered NaCl solution for 1 hr at 4 °C.
   16. Perfuse vasculature and trachea 3 times with DI water to rinse.
   17. Perfuse both vasculature and trachea with DNase solution.
   18. Submerge tissue in filtered DNase solution for 1 hr at 4 °C.
   19. Perfuse both vasculature and trachea 5 times with PBS.
   20. Dissect away noticeable cartilaginous tissue, trachea and all tubules 2 mm or larger in diameter (primarily found around the hilum and medial portions of the lungs) from conducting airways, leaving only respiratory zones (primarily the peripheral areas).
   21. Dissect tissue into 1 inch sections or smaller. Orientation of the tissue does not matter for this step.
   22. Pour off excess liquid and place tissue in 50 ml conical tubes. Freeze the tissue at -80 °C.
       NOTE: Retain sections for histology to ensure removal of cells and cellular debris, if desired. We use H&E, α-galactosidase, picogreen, hydroxyproline, ninhydrin, alcian blue, SDS-PAGE, and mass spectrometry to characterize.⁹
2. Lung Processing
   1. Remove lids from frozen tubes containing decellularized lung tissue.
   2. Place filter paper over the tube opening and secure with rubber band.
      NOTE: Tube and contents should still be frozen otherwise place in -80 °C until frozen.
   3. Lyophilize the tissue until all excess liquid is gone, using a freeze dryer according to manufacturer directions. Store at -80 °C until ready to mill.
   4. Before beginning add liquid nitrogen to freezer mill to cool insulation and internal components to working conditions.
   5. Remove magnetic mill bar from mill tube and add tissue to cover bottom.
   6. Replace mill bar and add loosely packed tissue. The mill bar should still move freely.
   7. Close the mill tube and place in freezer mill.
   8. Fill liquid nitrogen to max fill line.
   9. Freezer mill all tissue into fine powder (approximately 5 min, impaction rate of ~600 min^-1), in a polycarbonate cylinder with a stainless steel impactor as well as stainless steel end plugs using the freezer mill according to manufacturer directions. Store at -80 °C until ready for use.
3. Micro-porous gel formation (8 mg/ml) (adapted from references^9,11)
   1. Add 1% (w/v) of the decellularized powder and 0.1% (w/v) of pepsin to 0.01 M HCl, under constant agitation (should be able to see flow at the top level of liquid), at room temperature, for 48 hr.
      NOTE: The powder is statically charged, so adding the HCl after the powder affords the opportunity to wash the excess off the tube walls.
   2. After digestion for 48 h, place the solution and reagents on ice for 5 min.
   3. Using refrigerated 10% (v/v) 0.1 M NaOH, and 11.11% (v/v) 10x PBS (to bring the entire solution to 1x concentration), bring the digested protein solution to physiologic pH of 7.4.
      NOTE: The solution can now be stored at 4 °C for up to one week. Perform gelation kinetics using rheometry⁹ if desired.

2. Cross-linking Hydrogels to Improve Mechanical Strength

1. Solutions of 1%, 0.1%, and 0.01% w/v genipin crosslinking solution were prepared by dissolving the necessary amount of genipin powder in 10% DMSO.
2. Vortex the solution every 15 min for 1 hr.
3. Add genipin solution to cover ECM hydrogels (100 µL for each well of a 96-well plate) that have previously been assembled in part 1.3.4.
4. Leave each ECM hydrogel to crosslink for 24 hr at 37 °C.
5. Rinse the ECM hydrogel 3 times with PBS until the washing solution is no longer blue. The crosslinked hydrogels will then be ready for further characterization and cell culture studies.

3. Cell Culture with Microporous Gel

1. Two-dimensional coating
   1. Using the solution from 1.3.3, add 20 µl of pregel solution to each well of a 96-well non-treated tissue culture plate.
   2. Refrigerate overnight at 4 °C to allow protein adsorption to plate.
   3. Aspirate pregel solution and rinse with PBS.
   4. Passage cells⁹.
   5. Add 10,000 cells/cm² to wells.
   6. Increase media to 100 µl per well and incubate at 37 °C.
2. Three dimensional cell culture⁹
   1. Using solution from 1.3.3, resuspend pelleted cells to a concentration of 1,000,000 cells/ml of pregel solution.
   2. Quickly dispense 16 µl of pregel-cell suspension into each well of a 96-well plate, to form a ~500 µm thick hydrogel for 3D cell culture.
   3. Incubate at 37 °C for 30 min for gel to form.
   4. Add 100 µl of media to the top of formed hydrogels and incubate at 37 °C.
3. Three dimensional cell culture on variable stiffness gels
   1. Using solution from 1.3.3, pipette 100 µl of pregel solution into each well of a 96-well plate.
   2. Incubate at 37 °C for 30 minutes for gel to form.
      Note: Cross-linking steps from part 2 can be used here.
   3. Passage cells⁹.
   4. Add 10,000 cells/cm² to wells.
   5. Increase media to 100 µl per well and incubate at 37 °C.

Results

Using this method, we have produced hydrogels from normal pig, rat, and mouse lungs (Figure 1). Processed lungs provide an estimated 5 mg, 40 mg, and 10 g of ECM powder respectively. An overview of the process is shown in Figure 2. Key visualizations during the process include: white appearance of the lungs after rinsing deoxycholate; after the pregel formation, the solution should be opaque and the solution should appear homogenous for months if stored a...

Discussion

One of the integral aspects of biology is the self-organization of molecules into hierarchal structures that perform a specific task.¹³ In the lab, self-assembly depends on numerous factors such as salt concentration, pH, and digestion duration. As shown, a self-organizing hydrogel forms when solubilized proteins return to a physiological temperature. The hydrogel formed is capable of promoting cellular attachment and proliferation in vitro.

Cellular response to biophysical...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Triton X-100	Fisher Scientific	BP151-100	Use in fume hood with eye protection and gloves.
Sodium Deoxycholate	Sigma-Aldrich	D6750-100g	Use in hood with eye protection and gloves.
Magnesium Sulfate	Sigma-Aldrich	M7506-500g	None
Calcium Chloride	Sigma-Aldrich	C1016-500g	None
DNase	Sigma-Aldrich	D5025-150KU	None
HCl	Sigma-Aldrich	258148-500ML	Use with eye protection and gloves.
Pepsin	Sigma-Aldrich	P6887-5G	Use in fume hood with eye protection and gloves.
Sodium Hydroxide	Fisher Scientific	BP359-500	Use with eye protection and gloves.
Genipin	Wako Chemicals	078-03021	Use in fume hood with eye protection and gloves.
PBS 10x	Quality Biological	119-069-151	None
PBS	VWR	45000-448	None
Filter Paper	Whatman	8519	N/A
Hand pump	Fisher Scientific	10-239-1	N/A
Graduate Beaker	VitLab	445941	N/A
Cryomill	SPEX	6700	Use cryogloves and eye protection.
Lyophilizer	FTS FlexiDry		Use gloves.
Rheometer	Discovery	HR-2	Use gloves and eye protection.