The effect of the detergent on kidney decellularization by immersion

compare decellularization protocols. This method was applied to porcine kidney tissue to compare the isolated effects of the detergents SDC, SDS and TX-100. Theoretically, mild, non-ionic detergents, such as TX-100, are the most desirable detergents to preserve the ECM during decellularization. However, especially for decellularization of dense organs like kidneys, non-ionic detergents might not be strong enough to remove the cells. Mild, ionic detergents, such as SDC, might provide a solution but have not been investigated thoroughly^126,127.

Cell lysis was provoked in 0,125 cm³ porcine kidney cubes by freezing and thawing and subsequent osmotic shock by diH20 treatment. The samples were then immersed in 1% SDC, 1% SDS or 1% TX-100 for 7 to 10 days under constant agitation to solubilize and remove cellular debris. Lastly, the samples were extensively washed to remove any residual detergent and analyzed for cell removal and architectural integrity by histology.

Histological analysis revealed that TX-100 only removed a fraction of the cellular material;

it is therefore not suitable as sole detergent for kidney decellularization. TX-100 is a non-ionic detergent. With an HLB of 13,5 its uncharged hydrophilic head group and

109 hydrophobic tail, TX-100 can break lipid–lipid and lipid–protein interactions. It dissolves cell membranes and membrane proteins by association with their hydrophobic parts, a micelle-like interaction that mimics the lipid environment of the protein and thereby even supports its continued activity. However, TX-100 cannot dissolve protein-protein interactions. It neither penetrates into proteins nor denatures them^127,129. Therefore, it is clear that TX-100 hardly penetrates through the porcine kidney cube, as it was confirmed in other studies144,156,177. Surprisingly, three other studies on kidney immersion-decellularization reported successful decellularization with TX-100. In these studies, however, the immersed tissue pieces were smaller and the detergent was applied in higher concentration, or for up to two weeks149,175,178. In general, TX-100 is only suitable, when applied during decellularization of thin tissues, such as heart valves¹⁷⁹.

Immersion with SDS at 4 °C resulted in completely decellularized tissue. SDS is a strong ionic detergent with an HLB of 40. It not only disrupts cell membranes forming mixed micelles, its monomers also bind cooperatively to proteins, which are thereby forced into drastic conformational changes and denaturation. Hence, SDS is effective in dissociating protein-protein interactions and solubilizing cytoplasmic compounds^127,129. SDS is widely used as a decellularization agent, due to its excellent cell clearing properties.

Also, the SDC 4 °C samples were predominantly cleared of cellular material. However, small zones with DAPI stained cellular debris were still observable. A DNase digest was added after the detergent treatment that reduced the total DNA amount drastically. Although SDC is categorized as an ionic detergent, it has an HLB of 16. Its hydrophilic properties and behavior are therefore much closer to TX-100 than to SDS. Comparable to TX-100, SDC does not induce protein denaturation. This complements its natural function as a bile acid in the gut, where it dissolves lipids to assist fat digestion^129,180. However, the ionic head group explains the better cell removal efficiency of SDC than TX-100.

The composition analysis of decellularized ECM scaffolds is challenging. ECM proteins are essentially insoluble in standard detergent based lysis buffers as they have a high molecular mass and are abundantly covalently cross-linked. Exactly this property is utilized in decellularization but hinders the proteomic analysis. Dot blot and western blot analyses (data not shown), did not result in robust quantifications. Mass spectrometry approaches face the same hurdles. Only very recently, a chemical digestion with hydroxylamine for insoluble extracellular matrix characterization was identified¹⁸¹. Hence, in this study the composition

110 was analyzed by immunofluorescent staining of ECM proteins, bulk collagen and GAG quantifications, as well as enzyme-linked immunosorbent assay (ELISA) for bFGF and VEGF.

Decellularization with SDS resulted in slightly higher collagen content per dry weight than with SDC, though the difference was not significant. This finding is probably due to a higher fraction of cellular remnants in the dry weight of SDC-decellularized scaffolds and not due to the removal of collagen by SDC, since SDC as a mild detergent cannot dissolve highly crosslinked collagen molecules. The normalization to dry weight after decellularization impedes the clear interpretation of the quantification results if the samples still contain a considerable amount of cellular debris. However, it is the gold standard in decellularization studies to date123,149,156. In future, it would be advisable to normalize quantification results to wet weight of the tissue samples before decellularization instead, to facilitate the comparison of absolute quantification results.

Also, decellularization with SDS resulted in a higher GAG per dry weight ratio than SDC treatment. On the contrary, the cytokines bFGF and VEGF are much better preserved with SDC than with SDS. Both cytokines are mainly bound to the ECM by GAGs. Therefore, it seems that although SDC removes GAG chains from the scaffold, it does not strip the cytokines from the remaining chains. On the other hand, SDS might not deplete the GAG chains, but denature or remove the bound cytokines.

The data from recellularization with human iPSC-derived intermediate mesoderm cells¹¹³ suggest that the cytokines retain their biological activity after SDC treatment, since the SDC treated samples harbor the highest levels of growth factors and achieved the best results for cell attachment and viability. No negative effect of minor cellular remnants was observed in the recellularization experiment. Moreover, the recellularization data indicate that high GAG and total collagen contents do not sufficiently predict cell performance in decellularized ECM. A test for biocompatibility should therefore always be included in the evaluation of decellularization strategies.

SDS-decellularized scaffolds have an increased stiffness in comparison to SDC-decellularized scaffolds, quantified by measuring the E modulus, which again is an indication of alterations and denaturation in the SDS-treated ECM.

111 In conclusion, the initial hypothesis that SDS treatment produces ECM scaffolds of minor quality was confirmed for decellularization of porcine kidneys by immersion.

SDC-decellularization leads to more native, biologically active porcine kidney scaffolds.

5.2.3 The effect of the temperature on kidney decellularization by immersion