Image-Based Method to Quantify Decellularization of Tissue Sections.

Maria Narciso,Ramon Farré,Daniel Navajas,Isaac Almendros,Jorge Otero,Núria Gavara

doi:10.3390/ijms22168399

Abstract

Tissue decellularization is typically assessed through absorbance-based DNA quantification after tissue digestion. This method has several disadvantages, namely its destructive nature and inadequacy in experimental situations where tissue is scarce. Here, we present an image processing algorithm for quantitative analysis of DNA content in (de)cellularized tissues as a faster, simpler and more comprehensive alternative. Our method uses local entropy measurements of a phase contrast image to create a mask, which is then applied to corresponding nuclei labelled (UV) images to extract average fluorescence intensities as an estimate of DNA content. The method can be used on native or decellularized tissue to quantify DNA content, thus allowing quantitative assessment of decellularization procedures. We confirm that our new method yields results in line with those obtained using the standard DNA quantification method and that it is successful for both lung and heart tissues. We are also able to accurately obtain a timeline of decreasing DNA content with increased incubation time with a decellularizing agent. Finally, the identified masks can also be applied to additional fluorescence images of immunostained proteins such as collagen or elastin, thus allowing further image-based tissue characterization.

Highlights

Tissue decellularization has become a very relevant method in recent years
To assess the decellularization of the tissue, the standard approach is to use absorbancebased DNA quantification [4,5,6,7]. This step can be performed with commercially available kits that typically work by digesting a piece of the decellularized tissue, isolating and purifying its DNA and using a spectrophotometer to measure the amount of DNA content per mg of dry tissue
Content, the tissue is conventionally classified as either successfully decellularized or not, depending on whether it passes the gold standard set by Crapo et al in 2011 [8]: tissues are considered decellularized if their DNA content is below 50 ng per mg of dry tissue

Summary

Introduction

Tissue decellularization has become a very relevant method in recent years. On the one hand, the production of acellular scaffolds has applications in cell culture, tissue repair and regenerative medicine, while on the other hand, the study of the extracellular matrix (ECM) and its interactions with their homing cells can yield further understanding of multiple diseases and pathologies [1]. To assess the decellularization of the tissue, the standard approach is to use absorbancebased DNA quantification [4,5,6,7] This step can be performed with commercially available kits that typically work by digesting a piece of the decellularized tissue, isolating and purifying its DNA and using a spectrophotometer to measure the amount of DNA content per mg of dry tissue. While this method provides an initial quantitative estimate of DNA content, the tissue is conventionally classified as either successfully decellularized or not, depending on whether it passes the gold standard set by Crapo et al in 2011 [8]: tissues are considered decellularized if their DNA content is below 50 ng per mg of dry tissue. Crapo et al suggested that the DNA fragment length should be below 200 bp; this metric is rarely assessed

Methods

Results

Conclusion