Direct expenses of renal transplant.

<p>All values in US$.</p

Showing the source of funding (the total exceeds 50 as some patients had more than one funding source).

<p>Showing the source of funding (the total exceeds 50 as some patients had more than one funding source).</p

Responses to the questions asked the day before surgery.

<p>Responses to the questions asked the day before surgery.</p

Pie chart shows the extent of financial crisis suffered by the families of the patients.

<p>Pie chart shows the extent of financial crisis suffered by the families of the patients.</p

Indirect expenses.

<p>All values in US$.</p

Decellularized renal scaffold from cryostored kidneys retained the cells growth characteristics.

<p>Photomicrographs following phase contrast microscopy represented <b>a)</b> control kidney from group I, <b>b)</b> decellularized kidney from freshly isolated kidneys, group II and <b>c)</b> decellularized kidney from cryostored kidneys, group III. As shown the acellular matrix appeared to be biocompatible when murine C₃H₁₀T½ cells were seeded <i>in vitro</i>. The cells showed potential for the attachment and underwent proliferation and remained viable when cultured with the decellularized structures whether kidneys were obtained following 3 months of cryostorage or freshly isolated kidneys. Black arrows represent kidney scaffold, white arrows represent attached cells.</p

A. Perfusion with 1% SDS completely decellularized the kidney structures.

<p>The collateral kidneys from rats were isolated and perfused with normal saline prior to perfusion with 1% SDS diluted in distilled water at room temperature for a period of 48 hours. The kidneys were then washed with a solution of deoxyribonuclease I (0.2 mg/ml) and 10 mM MgCl₂ in PBS at room temperature for a period of 16 h to ensure complete removal of detergents and nuclear material. Finally the structures were rinsed with PBS to remove deoxyribonuclease 1 and MgCl₂ The photomicrographs in <b>Panel-A</b> represents <b>a)</b> control freshly isolated kidney representing group I, <b>b)</b> decellularized freshly isolated kidney representing group II, and <b>c)</b> decellularized kidney from 3 months cryopreserved kidneys representing group III. White transparent appearance of both kidneys from groups I & II shows that kidneys have undergone complete loss of parenchyma in comparison to freshly isolated kidneys group I. <b>Panel B</b> represents a graph of total DNA content analyzed in control kidneys, group I, following decellularization of freshly isolated kidneys, group II and cryostroed kidneys, group III. Based on the DNA quantification it was observed that 1% SDS was sufficient to decellularize the kidneys to similar extent whether the kidneys were freshly isolated or cryostored. Data are expressed as mean ±SEM. Based on ANOVA, significant differences among groups II, III w.r.t. control group I are indicated at *p<0.05.</p

Collagen content of basement membrane is well retained whether or not the kidneys were cryostored prior to decellularization.

<p>Both Masson’s trichrome staining <b>(photomicrogrpahs a-c)</b> and immunohistochemistry using anti collagen IV antisera <b>(photomicrograph d-f)</b> were used to analyze the effect of cryostorage on collagen status of the decellularized structures. Masson’s trichrome stains collagen blue, cellular component red and nuclei black. The blue stained structures (black arrows) showed well retained collagen component of the ECM from <b>a)</b> represented control kidney from group I, <b>b)</b> decellularized kidney from group II, <b>c)</b> decellularized kidney from group III. Almost similar extent of staining patterns among group II and III demonstrated that decellularization process of kidneys did not affect the collagen content whether the tissues were cryopreserved or not. Absence of red and black stain re-confirms the absence of any cellular material and nucleus in these decellularized kidneys. With respect to immunohistochemistry <b>(fig d-f)</b> green fluorescence represented collagen IV and the red fluorescence indicated cell nucleus (Propidium iodide, PI stain). Photomicrographs represented <b>d)</b> control kidney from group I, <b>e)</b> decellularized structures from freshly isolated kidney, group II and <b>f)</b> decellularized structure from cryostored kidney, group III at 200X. Where-ever shown, the white arrows represent renal cells (red fluorescence) and black arrows represent collagen 1V (green fluorescence). It is clear from the Figs that after decellularization overall presence of ECM component collagen IV in glomerulus and kidney structure (black arrow) is preserved and cells are completely removed as no red fluorescence observed when compared with control.</p

The decellularized structures from cryostored kidneys retained the recellularization potential similar to that of freshly decellularized kidneys.

<p>Approximately 2 million C₃H₁₀T½ cells were used for the recellularization. The recellularization of the scaffolds was analyzed at different time intervals Photomicrographs <b>a), d), g)</b> represented decellularized kidney scaffolds without recellularization as control, photomicrographs <b>b), e), h)</b> represented recellularized kidney scaffolds from group II and photomicrographs <b>c), f), i)</b> represented recellularized kidney scaffolds from group III for day 2, 4 and 6, respectively. The cells movement was found to be increased to more deeper areas at different time points following recellularization. Black arrows represented cells repopulating in glomerulus and white arrows represented cells repopulating in different renal tissue areas.</p

Decellularized scaffold of cryopreserved rat kidney retains its recellularization potential

<div><p>The multi-cellular nature of renal tissue makes it the most challenging organ for regeneration. Therefore, till date whole organ transplantations remain the definitive treatment for the end stage renal disease (ESRD). The shortage of available organs for the transplantation has, thus, remained a major concern as well as an unsolved problem. In this regard generation of whole organ scaffold through decellularization followed by regeneration of the whole organ by recellularization is being viewed as a potential alternative for generating functional tissues. Despite its growing interest, the optimal processing to achieve functional organ still remains unsolved. The biggest challenge remains is the time line for obtaining kidney. Keeping these facts in mind, we have assessed the effects of cryostorage (3 months) on renal tissue architecture and its potential for decellularization and recellularization in comparison to the freshly isolated kidneys. The light microscopy exploiting different microscopic stains as well as immuno-histochemistry and Scanning electron microscopy (SEM) demonstrated that ECM framework is well retained following kidney cryopreservation. The strength of these structures was reinforced by calculating mechanical stress which confirmed the similarity between the freshly isolated and cryopreserved tissue. The recellularization of these bio-scaffolds, with mesenchymal stem cells quickly repopulated the decellularized structures irrespective of the kidneys status, i.e. freshly isolated or the cryopreserved. The growth pattern employing mesenchymal stem cells demonstrated their equivalent recellularization potential. Based on these observations, it may be concluded that cryopreserved kidneys can be exploited as scaffolds for future development of functional organ.</p></div