Decellularization outcomes by perfusion differ to decellularization outcomes by immersion

Whole organ decellularization is the basis for whole organ tissue engineering. The vascular tree of the organ can be utilized to perfuse the decellularization solutions through the organ.

Porcine kidneys are the ideal basis for kidney tissue engineering for transplantation, due to their similar size compared to human kidneys. Rat kidneys, however, are superior for the generation of human 3D kidney models, as they require less cells and reagents for recellularization.

The best condition identified for decellularization of porcine kidney tissue by immersion and agitation, 1% SDC at 4 °C, was therefore tested in whole organ perfusion-decellularization of rat kidneys. Additionally, an already published protocol for perfusion-decellularization of rat kidneys by Song et al.¹²³, based on perfusion at RT with 1% SDS and 1% TX-100, was applied as comparison. Both protocols were conducted at 4 °C and RT.

113 The experiments showed that SDC is not suitable for perfusion-decellularization of whole rat kidneys. Perfusion with SDC at 4 °C did not completely remove cells and DNA from the ECM. Increasing the temperature to RT led to better, albeit still incomplete decellularization., and to less remnant DNA. Hence, the observations of immersion-decellularization regarding the temperature were confirmed. Perfusion with SDS/TX-100 resulted in complete decellularization and architectural integrity at 4 °C and RT.

Biocompatibility was tested by injecting HUVECs into the renal artery of scaffolds that were decellularized at RT with SDC or SDS/TX-100. SDS/TX-100 RT scaffolds support higher cell viability than SDC scaffolds, as shown by histological and metabolic analysis.

Remaining cell debris inside the SDC scaffold possibly hampered perfusion and therefore the nutrient supply. Moreover, the cell debris could be a reservoir of residual detergents that harmed the reseeded cells, or the cell debris itself had a negative effect on the cells^189,190. The SDS/TX-100 RT protocol was therefore chosen for all subsequent whole organ recellularization experiments.

The question arises, why the immersion-decellularization protocol cannot be transferred to perfusion-decellularization.

Firstly, cell lysis in perfusion-decellularization was not preceded by freezing, thawing and osmotic shock, as it was carried out with the immersion samples¹²⁷.

Secondly, immersion-decellularization was optimized with porcine kidneys, while rat kidneys were used for perfusion-decellularization. As pointed out in 1.3.2, the general architecture of mammalian kidneys is conserved, however, the tubules and glomeruli are smaller in rat than in pig. Rat renal tubules have a mean radius of 29 µm^121,122. The spherical SDS micelles have a radius of only 1,5 nm¹⁹¹. Bile salts form even smaller, disc-shaped micelles^129,192. Hence, tubule size is not the direct problem. However, the fluidics for the transport of decellularization agents and cell debris changed, amplified by the different techniques the decellularization agents were delivered with^193,194.

Lastly, the detergents were applied for 168-240 h in immersion-decellularization, but only 16 h or 120 h for SDS or SDC in perfusion, respectively. Perfusing the detergents through the vascular tree has the advantage of their highly effective distribution. Therefore, the exposure to SDS can be drastically shortened in comparison to immersion-decellularization, which resulted in less damage to the scaffold. In the study by He et al. it was confirmed that shorter SDS incubation leads to improved kidney scaffold characteristics¹⁴⁸, as clearly

114 visualized by the scoring system. For SDC, however, the shorter incubation time limited the clearing of the tissue. To prolong the SDC treatment further is logistically not feasible, as perfusion-decellularization is a low-throughput system.

Conflicting results regarding the effects of SDS and SDC on decellularization have also been described in literature¹²⁶. SDC was successfully applied in the decellularization of heart valves^195,196 or liver177,197,198. For kidney perfusion-decellularization, however, Wang et al.

concluded that SDS is the prefered detergent over SDC¹⁷⁷. Ross et al. decellularized rat kidneys with 4% SDC and 1-10% TX-100 and found that although a higher SDC concentration increases the cell removal efficiency, it still leads to inconsistent decellularization results¹⁶². In this thesis, further inter-study comparisons for kidney perfusion-decellularization data were conducted by applying the scoring system to data from He et al.¹⁴⁸ and Caralt et al.¹⁴⁴. These comparisons confirmed that SDS is necessary for perfusion-decellularization of kidneys. Moreover, it was shown that a low SDS concentration of 0,125% is favorable over higher concentrations.

In conclusion, the optimal conditions for kidney decellularization by immersion and agitation are not transferable to perfusion. As a result of the comparison conducted in this study, SDS appears to be the only detergent that effectively decellularizes organs with a high cell density, such as the kidney, in perfusion conditions. To reduce the inevitable damage to the ECM by SDS, the conditions of SDS exposure should be carefully chosen. Altogether, SDS applied at low concentration, for a relatively short period of time and at a low temperature is advisable.

SDC preserves a more native kidney ECM composition than SDS, but because SDC is less effective in clearing tissues from cellular material, it can only be applied for decellularization of thin tissue slices or tissues with low cell densities. Also, when a decellularization technique is applied that requires long exposure to the decellularization agents, milder detergents, such as SDC, should be preferred.

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5.3 Generation of an in vitro kidney model by recellularization of