
==== Front
Sci Rep
Sci Rep
Scientific Reports
2045-2322
Nature Publishing Group UK London

39278961
71973
10.1038/s41598-024-71973-4
Article
Efficient decellularization of human fetal kidneys through optimized SDS exposure
Khosropanah Mohamad Hossein
Torabinavid Parham
Azimzadeh Ashkan
Tanourlouee Saman Behboodi
Kajbafzadeh Abdol-Mohammad kajbafzd@sina.tums.ac.ir

grid.414206.5 Pediatric Urology and Regenerative Medicine Research Center, Gene, Cell and Tissue Research Institute, Pediatric Center of Excellence, Children’s Medical Center, Tehran University of Medical Sciences, No. 62, Dr. Qarib’s St, Keshavarz Blvd, Tehran, 14194 33151 Iran
16 9 2024
16 9 2024
2024
14 2154521 5 2024
2 9 2024
© The Author(s) 2024
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.
Chronic kidney disease poses a significant threat to public health. Renal replacement therapy is the primary treatment option for end-stage kidney disease. However, there is a promising and relatively new method in regenerative medicine for creating a functional organ known as whole kidney decellularization. This method uses the intrinsic vasculature to perfuse the decellularizing agent into the tissue, effectively penetrating and removing cellular material. The regenerated bioscaffolds could serve as a source of organ donation. This study is focused on evaluating the effectiveness of various SDS exposures in decellularizing human fetal kidneys. The study included human fetal kidneys harvested from fetuses terminated before 14 weeks of gestational age. Kidneys were divided into six treatment groups based on SDS concentration and duration of perfusion. Decellularization, scanning electron microscopy, histopathological staining, immunofluorescent staining, and immunohistochemistry staining were performed to evaluate the adequacy of the process. The statistical analysis revealed that the SDS 0.1% treatment group had the highest collagen deposition after 24 h, significantly greater than the SDS 0.5% treatment group at 24 and 48 h. No significant differences were observed among the other treatment groups. The study concludes that the SDS 0.1% treatment group for 24 h was the most effective in terms of ECM content preservation and effective cell removal. This treatment showed better results than the other treatment groups and can be considered for future whole kidney decellularization studies.

Keywords

Decellularized extracellular matrix
Tissue engineering
Renal replacement therapy
Regenerative medicine
Fetus
Kidney transplantation
Subject terms

Regenerative medicine
Tissue engineering
Kidney diseases
Vice-Chancellor for Research of Tehran University of Medical Sciences1401-4-468-64142). issue-copyright-statement© Springer Nature Limited 2024
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pmcIntroduction

Chronic kidney disease (CKD) is a significant public health issue affecting a vast number of individuals in the USA, estimated to be as many as 37 million. In 2019, treating individuals with CKD incurred expenses of $87.2 billion, while treating those with end-stage renal disease (ESRD) resulted in additional costs of $37.3 billion. Costs can be mainly reduced by preventing high-risk individuals from progressing to CKD and subsequently to the ESRD stage1,2. Prioritizing the identification, monitoring, and treatment of CKD is paramount. The optimal treatment for ESRD is renal replacement therapy (RRT), which can significantly improve the patient's quality of life. Therefore, it is essential to implement proactive and therapeutic measures to address CKD, reducing its prevalence and impact on society and the economy3. RRT is primarily performed through transplantation (54%), dialysis (40%), or hemodialysis. In 2021, 25,000 kidney transplants were conducted, with roughly 76% being from deceased donors and 24% from living donors. Between 2011 and 2017, the number of patients added to the kidney transplant waitlist was relatively stable, with an annual average of about 24,000 to 26,000. The number gradually increased in the following two years, peaking at 28,556 in 20192.

Hence, introducing new treatments that are as efficient as RRT is mandatory due to discrepancies between the rates of organ donors and recipients. A relatively novel method for creating functional organs is using regenerative medicine and tissue engineering approaches. One such technique, called decellularization, involves perfusing a decellularizing agent into the organ's intrinsic vasculature, effectively removing cellular materials while leaving the extracellular matrix (ECM) intact. This method is gaining popularity due to its ability to preserve the ECM, which is crucial for maintaining the organ's structure and supporting the growth of new cells4. Decellularizing donor organs and recellularizing the resulting ECM has led to significant advancements in whole organ engineering methods. This progress indicates that it may be possible to use this approach for transplantation purposes in the future. This method has been effectively used to generate functional kidney constructs, which have been implanted in vivo with partial success5,6.

Our previous decellularization protocols effectively removed cellular components from sheep and rabbit kidneys7,8. A notable difference exists between the decellularization procedures outlined in various studies, even for identical organs or species. This highlights the importance of adopting a more comprehensive, evidence-based methodology9. Numerous factors must be taken into account when considering decellularization, including the selection of appropriate decellularizing agents based on the tissue type, the concentration and duration of exposure to the agent during perfusion, the use of additional protocol steps such as freezing or biological agents, as well as factors related to tissue harvesting4. Optimizing the decellularization protocol is a rational strategy for ensuring long-term patency following transplantation.

Major efforts have been made in recent years to improve methods of decellularization, allowing for the preservation of the native ECM composition as well as the appropriate three-dimensional architecture and region-specific cues required for further cellular adhesion and differentiation9. The detergent type and concentration can impact the ECM composition and disrupt microarchitecture10. Commonly used detergents for kidney decellularization are SDS, TritonX-100, 3-([3-cholamidopropyl]dimethylammonio)-1-propanesulfonate (CHAPS), and sodium deoxycholate (SDC), which are most commonly employed in tissue engineering approaches. Cell properties that dictate the decellularization strategy are resistance to the detergent, retention of extracellular matrix (ECM) proteins as a scaffold for future reseeded cells, decellularization of ECM networks giving the direction of signals to the seeding cell, and biocompatible cleaning of cellular membranes. Detergents, including SDS, Triton X-100, SDC, and CHAPS, have been used to solubilize cell membranes by disrupting lipid-lipid hydrophobicity or lipid-protein amphipathic interaction and cross-linking with protein in the ECM9. Although SDS disrupts the lipid bilayer and solubilizes the hydrophobic aspect, leading to a reduced decellularization time during the chemical decellularization process of human embryonic kidneys, applying high concentrations of SDS (1%) disrupts the ECM growth factors11.

Our previous review on decellularizing agents shows that SDS is effective at cellular removal and biocompatible for several tissue engineering applications. Despite the problem of potential denaturation of growth factor proteins, SDS is the detergent that has been most frequently used for decellularization of employed washes, resulting in efficient clearance of anionic SDS molecules from the decellularized tissue compared, for example, with a nonionic detergent alternative4. In the kidney, even though SDS efficacy can be enhanced by modification of features such as perfusion flow rate and sonication. This work aimed to develop a method to decellularize human fetal kidneys as efficiently and quickly as possible. Our goal was to optimize two key parameters, the concentration of the decellularizing agent and the duration of perfusion, in order to assess the impact of varying exposure to the decellularization agent on whole human fetal kidney ECM bioscaffolds.

While numerous review articles compare the superiority of the detergents for kidney decellularization, and because each detergent has specific merits and disadvantages, we employed a well-known decellularizing agent with promising outcomes advocated in previous studies, SDS4,9. However, our study did not delve into alternative methods and their impacts, which may present opportunities for further investigation. To determine optimization, we assessed the structural and functional characteristics using techniques such as histology, immunofluorescent staining, quantitative immunohistochemistry (IHC) assays of collagen (I, IV), fibronectin, elastin, laminin, and glycosaminoglycan (GAG) content. Hence, to the best of our knowledge, we optimized acellular kidney scaffolds harvested from human fetal kidneys for the first time by comparing the outcomes following various SDS concentrations, each at either 24 or 48-h duration of perfusion.

Results

The kidneys were prepared, cannulated, and divided into six groups. Groups one, three, and five were connected to a peristaltic pump for 24 h, while groups two, four, and six were perfused for 48 h. Figure 1 shows the kidneys' color change after being connected to the peristaltic pump. Subsequently, the acellular kidneys were subjected to CT-angiography (with and without contrast); two different conditions of the acellular kidneys are shown in Fig. 2.Fig. 1 The appearance of the fetal kidney undergoes a transformation from the initial stage A to the final stage C as it passes through the detergent treatment and decellularization process. The completion of the decellularization process is indicative of the final stage C.

Fig. 2 CT angiography demonstrates high-resolution images of the blood vessels and tissues. Following fetal kidney decellularization, we performed CT angiography to visualize the renal system's blood vessels and organs, providing crucial insights into blood flow and potential irregularities. During the procedure, a cannula, indicated by an arrow, is inserted to administer the contrast medium. This noninvasive approach enables a comprehensive evaluation of the acellular renal system, aiding in assessing vascular integrity and identifying possible vascular leaks.

Morphologic analysis

In the study, SEM was utilized to assess how the morphology of fetal acellular kidneys was impacted by varying concentrations and perfusion durations of SDS (Fig. 3). Six samples were meticulously prepared and DAPI-stained after the acellularization process, with images taken via a fluorescent microscope (Fig. 4). Subsequently, specimens underwent H&E, MT, AB, laminin, fibronectin, elastin, collagen I, and IV staining. Primary results revealed that samples sent to the laboratory in formalin were devoid of viable cells.Fig. 3 Scanning electron microscopy (SEM) images were utilized to examine the extracellular matrix (ECM) surface of a decellularized fetal kidney using six distinct protocols labeled from A to F. Preserving the structural integrity of the scaffold is imperative during the decellularization process to ensure its reliability for subsequent in vivo studies. The SEM images provide detailed visual representations of the ECM surface, revealing consistent structural characteristics post-decellularization in protocol A. However, a gradual alteration in the ECM structure was observed across protocols B to F, indicating a progressive change from the initial state. Protocol A maintained the integrity of collagen, fibronectin, and elastin filaments without any disruption, whereas protocols E and F exhibited destructive effects, leading to the rupture and dissociation of these essential components. These findings offer valuable insights for further exploration of the structural and functional attributes of decellularized fetal kidneys.

Fig. 4 The DAPI staining of decellularized fetal kidney sections, following protocols one through six (denoted as A through F, respectively), was visualized at a magnification of 100×. The DAPI results indicated remarkable cell removal in all protocols, although structural variations were observed between protocols A, B, C, and D compared to E and F. The stroma's coherence from protocols A to D was notably consistent. However, in protocols E and F, despite successful cell removal, the stromal structure was extensively disrupted, leading to artifacts in the fluorescent imaging.

Quantitative analysis

The statistical results of histopathologic evaluations in the six groups are depicted in Fig. 5. The findings indicated that the outcomes in kidneys underwent decellularization using SDS at 0.1% and 24 h were superior to those of the other groups concerning their contents of collagens (type I, IV), GAGs, laminin, fibronectin, and elastin. Further analysis indicated that the highest collagen deposition occurred in the SDS 0.1% treatment group after 24 h, which was significantly greater compared to the SDS 0.5% treatment group at 24 and 48 h; however, no significant differences were observed among the other treatment groups. When evaluating the GAG content, a gradual decrease in content was noted with increasing SDS concentration and exposure time. The SDS 0.5% treatment group observed the lowest GAG content after 48 h of exposure. Except for the GAG content in the SDS 0.1% group compared to the SDS 0.5% group (P < 0.05), no significant differences were detected among the other groups. Our assessments revealed a subtle variation in laminin content, with the highest observed in the SDS 0.1% group after 24 h of exposure, surpassing the 2% laminin exposure area. However, no significant differences in laminin content were observed among the treatment groups. Evaluating fibronectin content also revealed the highest levels in the SDS 0.1% group after 24 h of exposure. This was significantly greater compared to the SDS 0.3% group after 48 h of exposure, as well as the SDS 0.5% group after both 24 and 48 h of exposure (P < 0.05, P < 0.01, P < 0.0001, respectively). Furthermore, a significant decrease in fibronectin content was observed in the SDS 0.5% group after 48 h of exposure, compared to the SDS 0.3% group after 24 h and the SDS 0.1% group after 48 h of exposure time. Elastin content exhibited minimal variation among the groups, with the lowest amount found in the SDS 0.5% treatment group. A significant difference was detected between the SDS 0.5% group after 48 h of exposure and the SDS 0.1% and SDS 0.3% groups after 24 h of exposure (P < 0.05). In the evaluation of collagen I content in the groups, the highest amount of this protein was detected in the SDS 0.1% treatment group after 24 h, which was statistically significant when compared to the SDS 0.1% group after 48 h, as well as the SDS 0.3% and SDS 0.5% treatment groups (P < 0.0001). SDS 0.1% for 48 h of exposure also exhibited significantly higher collagen I content than the SDS 0.5% treatment group (P < 0.05). Similar patterns were observed in collagen IV content, with the highest levels found in the SDS 0.1% treatment groups, significantly exceeding the levels in the SDS 0.5% treatment groups. Significant differences were also observed between the SDS 0.3% treatment groups and the SDS 0.5% group (P < 0.05). A summary of the above data is shown in Fig. 6.Fig. 5 Histological illustration of decellularized fetal kidney (HE, MT, and AB staining) and IHC for ECM components (Laminin, fibronectin, elastin, collagen I, collagen IV) in decellularized whole fetal kidney bioscaffolds. Selected representative SDS decellularization protocols (groups 1–6) are shown.

Fig. 6 Decellularized fetal kidneys were used to quantify IHC markers using protocols one to six. The data are represented as mean ± SD (n = 4/group).

Discussion

Decellularizing kidneys is complex due to the vast number of cells and intricate structure. Detergents or enzymes remove cells while preserving the glomerulus and tubule to create acellular kidney scaffolds. Promising methods include decellularizing organ-specific ECM and using 3D bioprinting technology4,9.

The physical dimensions of pigs and sheep bear similarities to those of humans. These animals allow for frequent blood sampling due to their blood volume. The use of male subjects significantly aids in the collection of 24-h urine samples for more accurate monitoring of renal function. Pigs and sheep serve as valuable tools for research on renal transplantation. Their anesthesia and surgical procedures closely resemble those used in human conditions, making them an excellent training model for aspiring surgeons. Their renal anatomy closely resembles that of humans, with multipapillary and multilobular kidneys, setting them apart from other species. However, their size poses limitations, requiring dedicated space and incurring costs for food, drugs, and surgical materials. Considering the diversity of vasculature, sheep, rabbits, and pigs each have a single renal artery. Unlike dogs and rodents, which lack multiple medullary pyramids and therefore bypass segmental arteries, humans, pigs, and sheep possess a complex system of interlobar and segmental arteries that supply the numerous kidney lobes12. Sheep kidneys differ from human kidneys in the distribution and volume of renal artery segments and arterial injury rates following cranial pole resection. Sheep kidneys possess anterior and posterior segments, a similarity not found in pigs13. Therefore, it is imperative to consider these anatomical variances when utilizing sheep as a model for renal experimental or training protocols. The branching pattern of the renal artery into segmental arteries in pigs differs significantly from that in human kidneys. Moreover, the correlation between the posterior segmental artery and cranial infundibulum exhibits notable distinctions between the two species. Notably, the healing process of porcine kidneys after partial nephrectomies diverges from that of human kidneys. Given these distinctive anatomical variations, the porcine model proves unsuitable for investigating hemostatic techniques in renal procedures14–16. Simoes examined the internal anatomy and histological characteristics of pig kidneys, noting both similarities and distinctions from human kidneys17. While cross-species immunological differences have historically hindered xenotransplantation, recent advancements in porcine genome engineering have led to successful initial experiments. However, there is still limited understanding of the immune response when pig kidneys are transplanted into human recipients. Meanwhile, the transplantation of organs harvested from animal models to humans is highly debatable18. Hence, the technique of decellularization provides the only viable option for developing a stable, three-dimensional substrate that safeguards the fundamental structural, biological, and organizational features of an organ. While each species has many advantages and disadvantages, we still need more extensive research, including clinical trials, to fully understand their outcomes after transplantation.

Human discarded kidneys are the preferred source for kidney scaffold decellularization. They were first decellularized in 2013 using SDS 0.5% for 48 h, which resulted in successful cell removal while preserving ECM integrity and biomechanical properties19. Another study used a mixed infusion of SDS 0.5% and DNase into the renal artery and ureters. This process preserved the vasculature well and retained crucial growth factors that are necessary for future angiogenesis and promoting cell differentiation20. The ultimate goal of tissue engineering and regenerative medicine might include utilizing acellular organs in humans. Decellularization and tissue engineering techniques offer a less invasive approach to regenerate tissues and organs as an alternative to address diseases when living organ donors are insufficient. Various research groups have employed different decellularization protocols, leading to disparities in the results and approaches within the same species and organ studies6. Despite the shortage of organ donors, kidneys from aborted fetuses under 16 weeks may be a viable substitute due to better adaptation post-transplant. Establishing a standardized decellularization protocol is crucial for enabling precise comparisons of outcomes and fostering progress in the field across research groups.

An essential step in decellularization is identifying desirable ECM components such as GAGs, collagens, fibronectin, and laminin still present in the tissue. A satisfactory amount of these molecules in the ECM supports the tissue's functionality and mechanical characteristics4. It is crucial to ensure sufficient oxygen supply to the tissues when using decellularized scaffolds. The preservation of the ECM components and the vascular structure of the tissue are critical factors in achieving this. For instance, laminin and fibronectin found in the basement can serve as examples in this regard21. Using advanced imaging techniques, CT-angiography can showcase the remaining vasculature of tissue following the decellularization process22. Hence, we evaluated the mentioned factors in acellular kidney scaffolds to evaluate the adequacy of our bioengineered scaffolds.

Our team advocates for an evidence-based methodology to create a meticulously evaluated protocol for whole kidney decellularization. This approach employs SDS with continuous perfusion to maintain crucial ECM elements within the bioscaffold. Our decision to utilize SDS was grounded in a thorough narrative review and meta-analysis of kidney decellularization, which revealed its prominence as the primary decellularizing agent, frequently paired with Triton X-1004,23. SDS is an ionic detergent that dissolves DNA and cell membranes, disrupts the structure of proteins, and eliminates growth factors and GAGs. This can ultimately lead to the breakdown of the ECM, resulting in a loss of its integrity and function4. In an experiment, kidneys were agitated for 3–4 weeks in either SDS 1% or Triton X-100 1% solution. The Triton-treated scaffold exhibited increased porosity, leading to a noteworthy enhancement in water uptake capacity. This improvement allowed preserving vital ECM proteins and growth factors while ensuring exceptional biocompatibility. Conversely, the SDS-treated scaffolds exhibited optimal biomechanical properties24. Using an SDS 1% solution achieved the utmost level of ECM integrity preservation, cell removal, and elimination of xenoantigens25. Numerous concentrations of SDS are utilized for kidney decellularization, from 0.1%18,26,27 to 3%28. Indeed, Manalastas aimed to determine the most effective concentration of SDS for decellularization. They demonstrated that increasing the concentration of SDS beyond 0.245% or 0.0085 mol/L did not result in a higher reaction rate. In fact, lowering the rate and raising the concentration beyond the determined limit could lead to the formation of detergent micelles rather than mixed detergent-lipid micelles, which are necessary for eliminating cells by penetrating the cell membrane. Hence, investigating the optimal concentration of SDS is essential to ensure successful decellularization29.

Decellularization relies heavily on factors like route, flow rate, temperature, and detergent contact duration. Perfusion and immersion/agitation are pivotal for physical disruption. The perfusion method via organ vasculature is revolutionizing the process. Taylor investigated the effectiveness of whole organ decellularization using perfusion decellularization, demonstrating optimal biomechanical properties30. Manalastas studied how flow rate and sonication affect decellularization of porcine kidneys. Sonication damages cell membranes, reducing decellularization time. Higher flow rates and sonication powers initially speed up decellularization. However, minimal flow rate at 15 ml/min reached a point where increasing sonication no longer impacted total time. High sonication power disrupts detergent micelle formation by breaking down monomers and lengthening decellularization. Combining sonication with perfusion decreased decellularization time from 19 to 2 h but affected ECM integrity29. Fischer highlighted the importance of ambient temperature in kidney decellularization. The research identified 4 °C as the optimal temperature and SDS 1% as the most effective detergent for porcine scaffolds. Ambient temperature notably affected ECM integrity, while detergent choice and concentration affected molecular characteristics such as GAGs, collagens, and cytokines31.

Eliminating ionic detergents like SDS from ECM is challenging due to their polarity. To remove the remaining ionic detergents from the tissue, one typically needs to perform a thorough wash with non-ionic detergents like Triton X-1004. Moreover, due to the detrimental effects induced by residual amounts of SDS in acellular scaffolds, adjustment of the SDS concentration used for decellularization is of great importance. Hence, Kajbafzadeh used a relatively novel colorimetric method using methylene blue to detect the amount of residual SDS in acellular scaffolds32.

Our findings (SDS 0.1% for 24 h) were relatively far from those advocated in the literature (0.245%), showing promising outcomes regarding ECM content preservation and cell removal. Our study also yielded results closer to He et al.'s, supporting the feasibility of using lower SDS concentrations (SDS 0.125% for 4 h)11. Our findings could have a broader application in kidney tissue engineering, particularly for human studies, due to structural differences between rat and human fetal kidneys, as indicated by He et al. Therefore, in the case of human kidney decellularization, Orlando and colleagues were among the first to utilize human discarded kidneys. They developed human acellular kidney scaffolds using SDS 0.5% perfusion for 48 h19. Eight other studies used human kidneys as the tissue sample for decellularization (Table 1). Peloso also perfused SDS 0.5% (+ DNase I) through cannulated ureters and arteries20. Bongolan advocated promising outcomes following submicellar administration of SDS with various concentrations (0.05%, 0.075%, 0.1%, 1%)18. Leuning decellularized kidneys via agent perfusion through the renal arteries, veins, and ureters33. We also performed decellularization via cannulated renal arteries and veins.Table 1 Studies decellularized harvested human kidneys.

Study	Year	Author	No. kidneys	Initial preparation	Decellularizing agents	
1	2022	Shahraki53	8	PBS	1-TX 1% or SDS 1%	+	DNase I	
2-TX 1% or SDS 0.5%	–	
2	2022	Bongolan18	–	–	1-SDS 0.05%	24 h	PBS	
2-SDS 0.075%	24 h	
3-SDS 0.1%	24 h	
4-SDS 1%	24 h	
3	2020	Ullah54	–	Distilled water	SDS 1%	3 h	DNase I	
4	2019	Ullah55	13	FTC	SDS 0.1%	1 h	DNase I	
5	2019	Leuning33	–	Heparin + SDS + PBS + DNase I	TX 1%	24 h	SDS 1%	
6	2018	Bombelli56	18	PBS	Trypsin 0.02% + TWEEN-20 + SDC 4%	
7	2016	Nagao57	–	–	SDS 1%	5 days		
8	2015	Peloso20	40	PBS	SDS 0.5%	48 h	DNase I	
9	2013	Orlando19	10	Distilled water	SDS 0.5%	48 h		
SDS Sodium dodecyl sulfate, TX Triton X-100, PBS Phosphate-buffered saline, FTC Freeze–thaw cycle.

Although numerous studies used single-agent therapy with various outcomes, Peloso suggested advantages over simultaneous administration of Triton X-100 1% with SDS 1% for enhancing cell removal and preserving ECM components34. Hence, we used simultaneous SDS and Triton X-100 administration for fetal kidney decellularization. Our study explored six decellularization treatment protocols to extract ECM from kidneys, building on established methods.

The scarcity of uncontaminated and free-of-by-products animal sources constraints using naturally sourced ECM. Besides, due to the annual reported rates (73 million worldwide)35, considered ethical guidelines, harvesting organs from aborted fetuses might be possible as a promising source of organs for decellularization. This is the first study to develop acellular kidney scaffolds derived from human aborted fetuses. We optimized SDS concentration and exposure duration for optimal cell removal and ECM component preservation. Our analysis revealed that utilizing a lower concentration of SDS and a shorter exposure time of 24 h yielded superior results. This observation can be explained by the threshold effect of SDS concentration introduced by Manalastas, indicating that higher concentrations and longer exposure times do not necessarily translate into better outcomes, supported in our prior study, showing significant loss of mechanical properties, including GAG reduction, impaired collagen integrity, and altered ECM interactions after a gradual increase in SDS concentrations36. Similarly, to determine the optimal concentration and decellularization time for SDS, He and colleagues compared various SDS concentrations (0.125%, 0.25%, 0.5%, 1%) at different durations, concluding that the best preservation of ECM components and growth factors with decreasing concentration of SDS; however, no more reduction lesser than 0.125% could yield in better results11.

We have analyzed two crucial factors (detergent concentration and perfusion duration) in the decellularization procedure of fetal kidneys. However, several other significant parameters also require consideration9. To fully characterize bio-scaffolds, functional tests of bioactivity, biomechanical tests, and in-vitro/ in-vivo studies are necessary. Comparative studies on recellularization with different decellularization parameters must be conducted for future improvement. Safe, reliable, and consistent whole-organ bioscaffolds are critical for regenerating organ replacements.

Tissue engineering (TE) holds promise for enhancing patient outcomes by presenting alternative approaches for organ transplants, managing diseases lacking curative treatments, and offering personalized medical solutions. Transitioning TE interventions to patient care commonly involves participation in clinical trials to gather empirical data or integration as a novel therapy to improve patient accessibility via strategies like off-label use and compassionate applications. The current TE discourse centers on establishing ethical advancement guidelines and anticipating forthcoming implications. Hence, a concise exploration of ethical considerations, scalability, and clinical applicability is imperative in this evaluation. Furthermore, the repopulation of decellularized matrices with cells and the utilization of recellularized structures have the potential to serve as an alternative method to support renal replacement therapy9. Utilizing human fetal kidneys enables the regeneration of human-relevant scaled tissues for personalized therapeutic interventions. The recellularized matrices are anticipated to be transplanted into immunocompetent animal models to showcase their in vivo differentiation towards functional progeny. Significant advancements have been achieved in the past two decades in producing living replacement tissues, organs, and systems by integrating 3D biological scaffolds with self-organizing cellular populations4. Various TE strategies involve fabricating 3D biological scaffolds for cell seeding, commonly used with heart valves37,38, dermal regenerative templates39,40, synthetic bladders41,42, and airways43. The use of decellularized whole organs is typically reserved for rare cases where natural growth and developmental signaling preservation are crucial. We anticipate significant progress in the availability of appropriate cells for TE procedures and in promoting intricate 3D organization and vascularization in reconstructed tissues and organs. The clinical applications of human fetal kidneys and their decellularized biological scaffolds hold immense potential.

Newborns with kidney dysplasia, hypoplasia, cystic dysplasia, and renal tubular developmental and cystic malformations are at a heightened risk of impaired overall body growth44,45. Early investment in reconstructing a replacement kidney for those with a missing or non-functional kidney, particularly in infancy, may be highly beneficial. Bilateral kidney hypoplasia can result in oligo-anuric and anuric hypoplastic kidney syndromes, often accompanied by abnormal kidney function, hypertension, weak muscle tone, amnion defects, oligo-hydramnion, persistently low urine output, renal arterial hypoplasia, and lung hypoplasia. Consequently, pulmonary underdevelopment may lead to neonatal hypoxemia, pulmonary hypertension, and postnatal mortality45. Enhancing or replacing the non-functional kidney presents an opportunity to alleviate suffering, reduce the need for long-term peritoneal dialysis and transplantation, and minimize the requirement for invasive artificial kidney support in perinatal end-of-life care. Although the utilization of fetal ECM for cell reseeding appears beneficial, this study also elucidated the detrimental effects of SDS exposure. Our results offer important insights. Long-term exposure causes gradual cell settlement and erosion. Interestingly, short-term SDS exposure seems relatively harmless to the surrounding ECM.

Moreover, our awareness stems from our previous investigations on the architecture of the human fetal lung, identifying it as structurally vulnerable46. Additionally, the kidneys exhibit metabolic activity and intricate fluid-based circuitry. Conversely, during to this study, the fragility of kidney tissue was noted, limiting the initial formation of ECM constructs to small-scale punches. The study of kidney research involving perinatal organs complements investigations on acellular scaffolds and drug toxicity tests conducted on rat or primate embryos using ECM organs. Given the intricate nature of fetal kidneys, it is conceivable that human decellularized organs could be utilized to expose the fetus to substances or gene editing during the 8th to the 12th week of gestation46,47. This hypothesis could be verified by applying a transcriptome analysis of human developmental kidneys and then comparing the human and animal models in an exposome study. The individualized and unique attributes of TE present challenges to conventional clinical trial routes, underscoring the significance of offering guidance on advancing clinical translation in this field. The necessity for appropriate comparators and the patient-specific nuances of TE interventions pose challenges in conducting traditional randomized controlled trials (RCTs) or extrapolating findings across patients. Thus, it is imperative to establish comprehensive guidelines for the deployment of recellularized organs and to discern suitable candidates for transplantation to optimize the utilization of regenerated kidneys in clinical settings. Moreover, the societal implications of integrating recellularized kidneys into clinical practice should not be underestimated, given that the social perception of such interventions carries profound implications for the acceptance of this innovative approach. Selecting appropriate patients and considering a comprehensive social context are crucial aspects. Hence, the inclusion of healthy patients with promising prognoses in clinical trials lacks justification. Rather, focusing on terminally ill patients who stand to benefit the most, with minimal risks involved, should form the primary basis for patient recruitment. The collection of extended data from clinical trials and subsequent follow-up studies to evaluate the safety and efficacy of TE interventions holds significant importance. The preservation of clinical trial data is vital for monitoring adverse events and establishing a robust evidence base. By integrating unbiased and transparent publication practices and employing scientifically rigorous research methodologies, we can enhance reproducibility and facilitate the clinical translation of tissue engineering products. Establishing clear guidelines and robust oversight mechanisms for the use of human tissues is essential for effective management. Additionally, maintaining consistent standards for TE approaches is crucial.

Further research is imperative to enhance the widespread availability and accessibility of bioengineered kidneys, given the escalating incidence of CKDs. Kidney TE has emerged as a promising approach for renal replacement therapy. The utilization of acellular-ECM models through recellularization techniques offers numerous advantages, including natural scaffolds, biochemistrical properties, vasculature, architecture, and the facilitation of stem cell differentiation into organ-specific phenotypes. Significant strides have been made in addressing renal failure through TE and regenerative medicine, encompassing strategies such as utilizing endogenous renal cells and niches for in situ renal regeneration, cell therapy utilizing autologous and allogeneic cell sources, and the development of cell-based renal structures9. These innovative research endeavors have introduced novel strategies for tackling kidney diseases and hold practical implications. The development and reconstitution of acellular scaffolds play a crucial role in creating bioengineered organs. While promising results have been observed in the recellularization of these scaffolds, further preclinical and clinical evaluations are necessary to optimize all parameters and address any potential limitations. Moreover, ethical oversight and regulatory mechanisms are essential to assess and manage potential risks. Lastly, the establishment of global regulations is imperative to govern the utilization of experimental TE and regenerative medicine therapies as innovative treatments beyond clinical trial boundaries.

The current investigation involves the retrieval of human fetal kidneys from elective pregnancy terminations. Utilizing these organs poses intricate cultural, ethical, and legal challenges due to the illegality of inducing stillbirth in many nations to obtain an OPT. Furthermore, in countries where it is permissible, policies often mandate ethical exclusion criteria for selection and research limitations before the first trimester or after the point of medical viability (typically 24 weeks gestation), albeit subject to variation across jurisdictions. These discrepancies create numerous logistical hurdles for procurement, including variations in local or regional laws. Despite these complexities, the perception of pregnancy termination and the utilization of human organs for research and tissue engineering remains largely positive. Over the years, the scientific community and the general public have endorsed using human fetal tissue in fundamental research endeavors to enhance human health and explore potential remedies48,49.

Ethicists, policy advisors, and society have extensively debated these morally contentious issues in relative isolation, with little tangible progress in real-world applications50. Over the course of several decades, researchers worldwide have persistently dedicated themselves to the exploration and advancement of these underserved areas. Their daily endeavors involve navigating complex ethical considerations, securing approval, and adhering to stringent regulations governing the use of human tissue for research purposes. Notably, efforts have been made to provide parents who have encountered pregnancy loss with the opportunity to explore experimental treatments. Scholarly literature often proposes opt-out or automated consent mechanisms to streamline donor movements and reduce waiting lists, thereby enhancing the likelihood of achieving positive outcomes for patients. Ultimately, adherence to ethical frameworks is a crucial cornerstone in the advancement of human regenerative medicine48,51. A recent review has tackled ethical challenges and emphasized the many barriers to applying laboratory research in real medical settings52.

The widespread use of animal organs for developing acellular scaffolds and conducting xenotransplantation have raised numerous religious, cultural, moral, and technical conflicts51. Studies have indicated that the use of computational and/or in-vitro models, whenever feasible, can significantly reduce the reliance on animal testing. In cases where animal models are unavoidable, it is recommended to use a single large animal instead of multiple small animals and conduct all experiments on the same animal, continuously monitoring its well-being52. Using human tissue and organs also raises similar ethical questions, emphasizing the importance of providing a completely detailed informed consent form to the patients, informing them about the process, further uses, storage of the tissue, and data privacy. Furthermore, patients who are about to receive the organ need more detailed informed consent, including the advantages and possible risks, the importance of long-term follow-up, and alternative treatments in case of transplant failure49,50. Human fetal kidneys derived from miscarriage or pregnancy termination could emerge as a novel organ reservoir for transplantation in the Netherlands52. A dearth of research exists on the ethical dimensions of utilizing human fetal kidneys and engaging pertinent stakeholders49,50,52. Consequently, we conducted an ethical appraisal to assess the feasibility of employing these kidneys. Our ethical deliberation utilized a three-step framework to structure and guide organizational considerations regarding the exploration and utilization of human fetal kidneys. This methodical approach encompasses the entire donation process, including identification, communication, and collaboration with relevant stakeholders to delineate the necessary procedures for researching and utilizing these organs. Engaging in the initial phase of this ethical decision-making process is a pivotal step in establishing a conscientious research agenda. Concerns surrounding the use of human fetal kidneys from miscarriage or pregnancy termination as a new organ resource do not stem from unfounded fears of stigma or disgust towards human fetal material. Maintaining proper disposal practices like burial or cremation after pregnancy termination is crucial. However, using human fetal kidneys for experimentation poses ethical challenges. Implementing strict safeguards and procedures is essential to ensure ethical decision-making and avoid unnecessary actions.

While our primary objective was to assess the feasibility of utilizing human fetal kidneys for future clinical applications, we concede that our study is constrained by several limitations. These limitations encompass the imperative requirement for additional scrutiny of other influential variables, such as ambient temperature and adjustments in perfusion flow rates. Furthermore, conducting a more comprehensive array of supplementary examinations is essential, including assessments of biomechanical properties, biological compatibility, and the potential for subsequent recellularization. Hence, further research involving in-vivo investigations is imperative. Through a comprehensive examination of perfusion decellularization parameters using SDS on fetal kidneys, this study has demonstrated enhanced preservation of GAG and ECM contents while minimizing the deleterious effects of the decellularizing agent. Based on the results, utilizing 0.1% SDS for 24 h yields the most advantageous outcomes compared to higher concentrations and more extended perfusion periods. This approach is particularly recommended for acellularizing the kidney of a fetus terminated before 14 weeks. The implications of these findings extend beyond fetal kidneys and could be applied to the decellularization of whole organs from other species. This breakthrough can potentially set a new standard for developing regenerated organ replacements for transplantation.

Materials and methods

SDS and Triton X-100 were procured from Sigma-Aldrich©. The CD56, CD133, and CD326 antibodies were obtained from Biorbyt©.

Kidney retrieval and preparation

Human fetal kidneys (n = 12) from six aborted fetuses at 14 weeks of gestation were used in research after informed consent was obtained from parents and/or legal guardians and ethical approval from the Tehran University of Medical Sciences was achieved. First, we dissected six aborted fetuses below 16 weeks of gestation, and both fetal kidneys were removed surgically under sterile conditions using standard techniques for tissue removal. Each kidney was collected with an intact aorta and inferior vena cava to preserve the entire renal artery and vein length. The surrounding tissues were meticulously removed to cause no rupture or injury. Kidneys were assigned into six groups according to the SDS concentration and perfusion duration. In all groups, kidneys were rinsed in sterile deionized water to remove surface blood and excess tissues. The main renal artery as an afferent and inferior vena cava as an efferent were used to provide an antegrade perfusion (artery to vein). Kidneys were cannulated with an 18G catheter and fixed with surgical knots for further adaption to the perfusion-decellularization system as described previously7. The peristaltic pump was used to attach the afferent cannula. A thorough washout of the inside blood was performed by perfusing phosphate-buffered saline (PBS) for one hour. All the decellularizing solutions were fortified with 100 U/mL penicillin, 0.1 mg/mL streptomycin, and 0.25 mg/mL amphotericin B.

Kidney decellularization

The process started at room temperature, at a 6 mL/min perfusion rate. Total decellularization time was 24 or 48 h. SDS in various concentrations (0.1%, 0.3%, and 0.5%) were perfused via cannulation into the kidney using the pump. Our primary goal was to investigate fetal kidney decellularization's optimal concentration and perfusion time. Decontamination was finally performed using antimicrobial penicillin, streptomycin, and antifungal amphotericin to achieve completely sterilized scaffolds. All steps and experimental groups are illustrated in Tables 2 and 3. Lastly, as previously described, the adequacy of remnant SDS removal from scaffolds was confirmed with colorimetric assay32.Table 2 Various decellularization protocols for perfused renal scaffolds of the human fetuses.

Group	Detergent concentration	Duration (h)		Detergent concentration	Duration (min)	
1	SDS 0.1%	24	Then	Triton X-100 2%	30	
2	SDS 0.1%	48	Triton X-100 2%	30	
3	SDS 0.3%	24	Triton X-100 2%	30	
4	SDS 0.3%	48	Triton X-100 2%	30	
5	SDS 0.5%	24	Triton X-100 2%	30	
6	SDS 0.5%	48	Triton X-100 2%	30	

Table 3 Sequential steps of the decellularization procedure.

Step	Perfused solution	
1	Deionized water + PBS	
2	SDS	
3	Deionized water	
4	Triton X-100	
5	Deionized water	

CT-angiography and SEM analysis

After the human fetal kidneys were acellularized, CT-angiography was performed with the help of dye at the radiology department of the Children's Medical Center. Different tissue samples were fixed with glutaraldehyde for 24 h at 4 °C and then dehydrated in ethanol at various concentrations (30%, 50%, 70%, 90%, and 100%). These samples were then dried using a CO2 critical point dryer. Finally, the samples were mounted on aluminum stubs, coated with gold, and examined under Scanning electron microscopy (SEM) (VEGA, TESCAN, Czech Republic) at 5000 × and 30,000 × magnifications for further evaluation of the kidney’s ultrastructure.

Histopathological investigations

Samples prepared from the scaffolds were fixed in 10% formalin and transferred to the pathology laboratory. After washing the samples in sterile saline, we used Neutral Buffer Formalin (NBF; 10%) to preserve the specimens. The samples were then dehydrated with ethanol and underwent four to five sterile water washes. The dehydrated ingredients were put into hot paraffin wax to form blocks. We then cut 5–10 µm thick sections using a tissue microtome (Leica) and placed them on glass slides. We used a bright field microscope to image the general morphological findings and evaluated the injured tissue microscopically. Subsequently, sections were stained with Hematoxylin and Eosin (HE), Manson-Trichrome (MT), and Alcian-Blue (AB) according to the manufacturer's instructions and studied under the microscope. We investigated the cell survival rate to ensure the decellularization process succeeded. This was calculated by dividing the number of cells in the treated tissue by the number of cells in the untreated tissue and multiplying by 100. We then counted the remaining cells in eight random high-power fields (400×) and found the average number.

Immunofluorescent staining

The samples were subject to 4′,6-diamidino-2-phenylindole (DAPI) staining (Sigma-Aldrich©) to verify the presence of intact cells and remnant nuclear contents. The seeded scaffolds were washed with PBS, fixed in 4% paraformaldehyde for 30 min, and then rinsed to eliminate the PFA. Next, the samples were washed in a 10 mg/mL DAPI solution for 10 min in the dark. Following numerous PBS washes, the samples were examined under a fluorescent microscope to assess the adequacy of removing the unbound DAPI.

IHC staining

The ECM of the human fetal kidney was analyzed using elastin, laminin, fibronectin, and collagens I and IV specific antigens for further IHC staining to verify the retention of major ECM components. To prepare the tissue sections for processing, they were temporarily deparaffinized, moistened with distilled water, and subjected to inhibition of endogenous peroxidase activity with 10% hydrogen peroxide for 10 min. Antigen retrieval was carried out by boiling the tissue slices at 95 °C with sodium citrate buffer (10 mM, pH = 6) for 30 min to dissolve the protein cross-links. The slides were then PBS-washed before the primary antibodies were applied, including Rabbit polyclonal to Anti-CD31 antibody ab28364 and Rabbit monoclonal Anti-CD34 antibody [EP373Y] ab81289. Following this, the slides were cleaned with PBS before applying the secondary antibody (MAD-000237QK-125) for 45 min at room temperature. Diaminobenzene tetrahydrochloride (DAB) was used to stain the section nucleus. The kidneys' collagen and glycosaminoglycan (GAGs) content were quantified using the Sircol and Blyscan assay kits, respectively.

Statistical analysis

A one-way analysis of variance (ANOVA) and post hoc Tukey's analysis were used to examine the results. The mean and standard error (SE) from at least three subsequent experiments is presented. If P-values were 0.05 or lower, all experiments were performed at least thrice.

Author contributions

M.H.K. and A.-M.K.: conception of the study, project administration, development and supervision, and critical review. P.T., A.A., S.B.T., A.-M.K.: data collection, investigation, methodology, critical review, manuscript drafting, and editing. All figures were organized and designed by P.T.

Funding

This research work was financially supported by the Vice-Chancellor for Research of Tehran University of Medical Sciences (1401-4-468-64142). The authors appreciate the support of the Vice-Chancellor for Research of Tehran University of Medical Sciences and Pediatric Urology and Regenerative Medicine Research Center of Tehran University of Medical Sciences, the Iran Silk Research Center.

Data availability

All data generated or analyzed during this study are included in this article. Further inquiries can be directed to the corresponding author.

Competing interests

The authors declare no competing interests.

Ethical approval

All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards. All applicable international, national, and/or institutional guidelines for caring for and using human tissues were followed (Approval ID: IR.TUMS.REC.1401.781). Informed consent was obtained from parents and/ or legal guardians. Animal experiments were performed in accordance with relevant guidelines and regulations. All methods are reported in accordance with ARRIVE guidelines.

Publisher's note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

These authors contributed equally: Mohamad Hossein Khosropanah and Parham Torabinavid.
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