Solid‐phase silica‐based extraction leads to underestimation of residual DNA in decellularized tissues

Abstract

Decellularization of animal tissues is a novel route to obtain biomaterials for use in tissue engineering and organ transplantation. Successful decellularization is required as animal DNA causes inflammatory reactions and contains endogenous retroviruses, which could be transmitted to the patient. One of the criteria for successful decellularization is digestion (fragmentation) and elimination (residual quantity) of DNA from the tissue. Quantification of DNA can be done in many ways, but it has recently been shown that silica‐based solid‐phase extraction methods often do not completely purify in particular small DNA fragments. In the context of decellularization, this means that the measured DNA amount is underestimated, which could compromise safety of the processed tissue for in‐patient use. In this article, we review DNA quantification methods used by researchers and assess their influence on the reported DNA contents after decellularization. We find that underestimation of residual DNA amount after silica‐based solid‐phase extraction may be as large as a factor of ten. We therefore recommend a direct assessment of DNA amount in tissue lysate using dsDNA‐specific binding dyes, such as Picogreen, due to their higher accuracy for small fragment detection as well as ease of use and widespread availability.

Porcine Small Intestinal Submucosa (SIS) as a Suitable Scaffold for the Creation of a Tissue-Engineered Urinary Conduit: Decellularization, Biomechanical and Biocompatibility Characterization Using New Approaches

“…The decellularization protocol reported in this study was proven to be effective in the removal of dsDNA: the DNA concentration was found under the threshold of 50 ng/mg dry tissue set by Crapo et al [ 51 ] as necessary to prevent inflammatory reactions caused by animal DNA and the transmission of endogenous retroviruses from animal to patient [ 88 ]. In order to increase both specificity and reproducibility of the DNA quantification, it was decided to apply both UV spectroscopy and fluorescence-based methods.…”

“…Nevertheless, the relationship between residual DNA amount, fragment length, and immunological reactions following xenotransplantation has yet to be clearly defined, as the biological consequences of residual nuclear material or cytoplasmic debris within the scaffold are still unclear. There are no reports showing a direct cause–effect relation between such cellular remnants and an adverse host response to date [ 65 , 88 ]. Even though the U.S. Federal Drug Administration does not set limits for DNA in biologic scaffolds, more systematic studies are needed in order to determine a more specific threshold in terms of acceptable DNA residues.…”

Detergent-Free Decellularization of Notochordal Cell-Derived Matrix Yields a Regenerative, Injectable, and Swellable Biomaterial

“…Extracellular DNA has been implied in the severity of inflammation, 23 − 25 but the amounts found in healthy individuals’ plasma vary greatly depending on the quantification method and lie either below or above the published limit for successful decellularization. 49 As such, this proposed limit may not reflect a universally applicable goal, 38 especially considering the immunoprotected state of the NP within the avascular IVD. 50 Compared to the previously published protocols, we achieved a similar or greater reduction in DNA content when accounting for different normalization methods used (wet/dry weight).…”

“…The DNA concentration was determined using the Qubit DNA assay (Qubit dsDNA HS assay, Thermo-Fisher Scientific, Landsmeer, The Netherlands) following the manufacturer’s instructions. DNA fragment lengths were examined on a 1% agarose gel: 250 μL of digested sample was washed thrice with ultrapure water and then concentrated to 20 μL using ultracentrifugation filters with a 30 kDa molecular weight cutoff for gel electrophoresis 38 (wash/concentration spins: 14 000 g for 10 min, recovery spin: 1000 g for 1 min). To visualize cell nuclei, we reconstituted NCM and dNCM powder to 10% w/v in PBS.…”

Dimercaptosuccinic Acid Functionalized Polystyrene Column for Trace Concentration Determination of Heavy Metal Ions: Experimental and Theoretical Calculation Studies

“…Metal-ligand complex formation is of special significance where a specific ligand can selectively coordinate with the heavy metal ions in order to extract Water 2021, 13, 3056 2 of 12 them from the complex sample matrix [3]. In solid phase extraction (SPE), research has been focused on the preparation of new adsorbent materials including polymeric materials, nano-materials, metal-organic framework, and carbonaceous materials for wastewater remediation [15][16][17][18][19][20]. Polymeric adsorbents has been considered as a promising material, since it has an advantage of column operation over nano-materials, and widely acceptable in water treatment techniques [21][22][23][24][25].…”