Efficient decellularization of human fetal kidneys through optimized SDS exposure – Scientific Reports

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 technology^4,9.

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 procedures^14,15,16. 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-100^4,23. 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.

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 valves^37,38, dermal regenerative templates^39,40, synthetic bladders^41,42, 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 growth^44,45. 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 gestation^46,47. 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 remedies^48,49.

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 medicine^48,51. 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 failure^49,50. 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 stakeholders^49,50,52. 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.

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• Source: https://www.nature.com/articles/s41598-024-71973-4