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 o....

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....

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.