Current strategies on kidney regeneration using tissue engineering approaches: a systematic review

Abstract

Introduction

Over the past two decades, there has been a notable rise in the number of individuals afflicted with End-Stage Renal Disease, resulting in an increased demand for renal replacement therapies. While periodic dialysis is beneficial, it can negatively impact a patient’s quality of life and does not fully replicate the secretory functions of the kidneys. Additionally, the scarcity of organ donors and complications associated with organ transplants have underscored the importance of tissue engineering. Regenerative medicine is revolutionized by developing decellularized organs and tissue engineering, which is considered a cutting-edge area of study with enormous potential. Developing bioengineered kidneys using tissue engineering approaches for renal replacement therapy is promising.

Method and materials

We aimed to systematically review the essential preclinical data to promote the translation of tissue engineering research for kidney repair from the laboratory to clinical practice. A PubMed search strategy was systematically implemented without any linguistic restrictions. The assessment focused on complete circumferential and inlay procedures, thoroughly evaluating parameters such as cell seeding, decellularization techniques, recellularization protocols, and biomaterial types.

Results

Of the 1,484 studies retrieved from the following primary searches, 105 were included. Kidneys were harvested from eight different species. Nine studies performed kidney decellularization from discarded human kidneys. Sixty-four studies performed whole organ decellularization. Some studies used acellular scaffolds to produce hydrogels, sheets, and solutions. Decellularization is achieved through physical, chemical, or enzymatic treatment or a combination of them. Sterilization of acellular scaffolds was also thoroughly and comparatively evaluated. Lastly, different recellularization protocols and types of cells used for further cell seeding were demonstrated.

Conclusion

A comprehensive review of the existing literature about kidney tissue engineering was conducted to evaluate its effectiveness in preclinical investigations. Our findings indicate that enhancements in the design of preclinical studies are necessary to facilitate the successful translation of tissue engineering technologies into clinical applications.

Introduction

Over two decades in the USA, there has been a significant rise in the incidence of end-stage renal disease (ESRD), with approximately 135,000 individuals being diagnosed with this condition annually on a global scale. This represents a substantial increase of 42.8% in incidence and a 107% rise in total prevalence, culminating in a twofold increase in the number of patients receiving dialysis and a rate of 3.1% for kidney transplants. The most common available treatment for those with ESRD is renal replacement therapy, which is achieved by performing dialysis or organ donation. Despite satisfying outcomes, dialysis is associated with negative impacts on quality of life and subsequent events, including accelerated heart disease and the development of further infections [1]. In addition to preserving body homeostasis by controlling osmolality, acid-base balance, and electrolyte concentrations of body fluids, secretion of essential hormones including renin (maintains blood pressure) and erythropoietin (stimulates hematopoiesis), activation of vitamin D (essential in bone homeostasis), and production of prostaglandins are among crucial kidney functions which are not compensated during dialysis. Therefore, transplantation is considered a favorable approach for restoring organ function in patients with ESRD. However, the lack of transplantable organ donors and the number of candidates on the waiting list are significant obstacles. As a result, at the end of 2020, almost 75,000 candidates in the USA were waiting for organ transplants. Of these, 23,853 have undergone organ transplants (31.8% overall transplant rate). Statistics are slightly different in Europe, with 29.6 per million people receiving organ transplants in 2010 and 34.7 per million people receiving organs by the end of 2018, reporting a higher overall transplant rate [2, 3]. Additionally, despite improvements in immunosuppressive therapies, allograft function and long-term results are adversely affected by drug-specific side effects in patients undergoing transplantation.

Recent advances in regenerative medicine and stem cell therapy have introduced bioengineered organs as a potential organ to expand the number of available organs for transplantation [4]. Decellularization makes removing cells from healthy tissues possible while leaving the extracellular matrix (ECM) structure intact. The leftover ECM, after decellularization, can be employed as scaffolds in tissue engineering and regenerative medicine. Kidney decellularization and preparation of ECM are the main steps of developing a bioengineered kidney. Production of renal scaffolds from rodents, porcine, monkeys, and humans has been described in numerous studies, and whole-organ decellularization has created new possibilities for tissue engineering [5]. Development of an acellular kidney while preserving the main architecture of the kidney and minimizing the amount of remnant DNA and other molecules that provoke the recipient’s immune system are among the cornerstones of the decellularization process. Acellular scaffolds have the potential to be populated with cells before being implanted. Recellularization is also a crucial step in organ regeneration. At the same time, the proliferation of seeded cells promotes the integration of cells within the surrounding tissues, initiating tissue remodeling through the secretion of cytokines and growth factors. Moreover, remnant growth factors of the scaffolds are also shown to play a vital role in promoting further cell differentiation and proliferation following recellularization [6, 7].

Although kidney tissue engineering has demonstrated great potential in preclinical studies, its clinical application remains limited. In order to enhance the clinical application of bioengineered kidneys, it is crucial to adopt an evidence-based approach, such as performing systematic reviews. This extensive systematic review aims to offer a comprehensive overview of studies that investigate the development, characterization, and outcomes of bioengineered kidneys. Therefore, unnecessary duplication of studies can be avoided, and researchers and clinicians can be guided toward identifying the most optimal experimental design and model by comparing different decellularization strategies, specific characteristics of the scaffolds, and the results obtained in vivo. The main objective of performing this systematic review is to facilitate the translation of kidney tissue engineering from laboratory settings to clinical practice.

Methods

Search strategy

This systematic review adhered to the PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-analysis) 2020 statement to identify all relevant kidney tissue engineering studies published until December 5, 2023, without any limitation. The search strategy was designed after consultation and consensus on the definition of PICO as P: Kidneys undergoing decellularization, I: decellularization ± recellularization ± implantation/ transplantation, C: native kidneys, kidneys not undergo recellularization ± implantation/ transplantation, O: biophysical, biochemical, structural, and biocompatibility properties, and amount of remnant ECM molecules. The search strategy was conducted in PubMed as follows:

(“Decellular*“[Title/Abstract] OR “Acellular*“[Title/Abstract] OR “Scaffold“[Title/Abstract]) AND (“Kidney“[MeSH Terms] OR “Renal“[Title/Abstract] OR “Kidney“[Title/Abstract]).

Screening and study selection

For the primary review, two reviewers independently reviewed all the titles and abstracts of recorded studies using EndNote (Version X7.2, Thomson Reuters, PA, USA) according to the Exclusion criteria. Our applied exclusion criteria were (1) no kidney (2), complete in-vitro study without any patient or animal model (3), review articles (4), published as a commentary or case report (5), published prior to January 1, 2000 (6), not performing kidney decellularization. We applied no restriction and used Google Translate for studies to retrieve the variables. After primary review, the full text of all remaining articles was evaluated using the same exclusion criteria. Besides, articles with no available full text were excluded. Any disagreement between the reviewers was finalized by consulting the corresponding author.

Data extraction

Data extraction was primarily performed by reviewer one and cross-checked by reviewer 2. Extracted data were (1) Name of first author (2), year of publication (3), source of whole organ/ kidney sample (4), type of sample (Whole kidney or kidney slices) (5), Temperature which tissue/ organ decellularized at (6), Physical, chemical, and enzymes procedures performed for organ/ tissue preparation, decellularization, and sterilization (7), Technique of decellularization (8), Cell type used for in-vitro studies or further recellularization (9), the route used for cell seeding (renal artery, vein, ureter, or multiple injections to scaffold) (10), aim of the study, and (11) main findings which are comprehensively listed in Table 1.

Table 1 Study characteristics of retrieved studies

Results

Literature search and screening

Figure 1 represents the results obtained after the literature search and screening and provides details of exclusion reasons. Briefly, 1,484 studies were retrieved after an electronic search, two were excluded due to duplication, 1,262 studies were excluded after analysis of titles and abstracts according to exclusion criteria, and 234 articles were selected for further screening by full-text evaluation that led to the final inclusion of 94 studies. Furthermore, 12 studies were yielded via manual search in the references list of included articles, and one was removed. At the same time, full-text was unavailable [8], resulting in 105 articles being evaluated for final review and subsequent data extraction. Although the full text of four articles was unavailable, we included the findings from their abstracts [9,10,11,12].

Fig. 1

PRISMA-based flow diagram of the search and screening process of studies retrieved from the electronic database with the aforementioned search strategy. PICO is defined as P: Kidneys undergoing decellularization, I: decellularization ± recellularization ± implantation/ transplantation, C: native kidneys, kidneys not undergoing recellularization ± implantation/ transplantation, O: biophysical, biochemical, structural, and biocompatibility properties, and amount of remnant ECM molecules

Study characteristics

Kidneys were harvested from eight species: human, monkey, rat, pig, sheep, goat, rabbit, and dog. Sixty-seven studies did not report the number of harvested kidneys. Nine studies performed kidney decellularization from human samples (Fig. 2). Sixty-four studies used whole kidneys, 34 used kidney slices, and four used both for further decellularization. Five studies developed hydrogels from acellular kidney ECM. Three studies produced mixed matrixes of acellular ECM and another synthetic compound, making artificial scaffolds (Fig. 3).

Fig. 2

A summary of protocols of studies decellularized discarded human kidneys

Fig. 3

Schematic representation for the regeneration of bioengineered kidneys. Starting from the top left corner of the figure, native kidneys of eight species, including rats, monkeys, pigs, humans, goats, rabbits, sheep, and dogs, are utilized for the next steps as a whole organ or slices of kidneys. Acellular scaffolds can be achieved through physical, chemical, or enzymatic methods to retain the structural integrity and ECM components. Physical methods from the top left corner to the right bottom include a peristaltic pump, freeze-drying, osmotic shock, stirrer, FTC, and sonication. Chemicals administered are categorized into five sections, including (none)ionic (from top to bottom: SDS, Triton X-100, Tween 20, SDC, and SLES): left, chelators (EDTA, EGTA): middle top, acid-base (from top to bottom: PAA, NaOH): middle bottom, or zwitterionic detergent (CHAPS (3-[(3-Cholamidopropyl)dimethylammonio]−1-propanesulfonate)): right top, hyper/hypo-osmotic solutions: right bottom. Enzymes administered were Trypsin and DNase I (from left to right). Next, acellular scaffolds undergo sterilization using UV/ Gamma irradiation, antibiotic-containing solutions, ethylene oxide gas, or ethanol. Furthermore, various cultured cells were reseeded on scaffolds using (un)differentiated stem cells or specific cell lines. Lastly, recellularized scaffolds are being utilized for multiple purposes. First, regenerated whole kidneys are used for transplantation in cases that underwent nephrectomy or suffering from chronic kidney diseases with fibrotic kidneys. Second, tissues were minced and grinded to produce hydrogels, bio-ink for three-dimensional bioprinting, or raw material to be used alone or in combination with other chemicals for the synthesis of scaffolds using electrospinning. Finally, kidney slices are used for further research investigating drug safety, cancer treatment, the pathophysiology of kidney diseases, and being implanted in kidneys undergoing partial nephrectomy

Discussion

Species comparisons

Various animals are sacrificed to harvest the kidney for further application in kidney bioengineering, inclusive of eight species of mammals in published literature, including rats, pigs, rabbits, sheep, dogs, monkeys, goats, and humans.

Rats

Ross and Liu were among the first to develop acellular kidneys, harvested from rats (Sprague-Dawley and Wistar, respectively) with perfusion technique by infusion of detergents through a cannulated renal artery [13, 14]. Thirty-three studies used Sprague Dawley rats with a cumulative reported number of 400 rats, 11 used Wistar rats with a total number of 228, six used Mice (NMRI, Bulb-c), and two used Lewis rats. Despite incomplete reports regarding the number of rats, the cumulative reported number is 154 and 324 for female and male rats, respectively.

Repeated cycles of freezing and thawing showed optimal outcomes after the decellularization of rats’ kidneys compared to the application of other physical pathways of high-pressure or ultrasonic irrigation [15]. Simultaneous use of freeze-thawing and chemical protocols reduced the total time needed to decellularize rat kidneys [16]. Moreover, a temperature of −20 °C showed better results than − 80 °C for freeze-thaw cycles, providing a promising vascular integrity at lower concentrations of cryoprotectants [17]. Nonetheless, Feng introduced ice-free cryopreservation as a safer physical method than freeze-thawing [18]. Moreover, regarding the technique used for decellularization, magnetic stirring is found more effective than rotatory shaker approaches in rats’ kidneys [19].

Various ionic and non-ionic detergents are used for decellularization of rats’ kidneys. Application of SDS (Sodium Dodecyl Sulfate) with lower concentrations (3% Versus 0.66%) for rats’ kidneys showed better outcomes given architecture, ECM proteins, and growth factors preservations, providing the optimal environment for further cell adhesion, proliferation, and induction of differentiation [20]. He et al. investigated optimal SDS concentration and flow time, showing the best preservation of ECM growth factors at lower concentrations with a cut-off of 0.125% and for four hours [21]. To evaluate the adequacy of rat kidneys for kidney regeneration, Haeublein utilized three distinct methods and reseeded the scaffolds with human endothelial cells, introducing SDS 1% as the best detergent with the lowest remnant DNAs. However, SDS 0.01% or EDTA (Ethylenediaminetetraacetic Acid) culminated in better results after transplantation of recellularized kidneys, being able to produce urine within 60 min [22]. Shahraki also advocated that SDS 1% was more successful than Triton X-100 1% in decellularization, providing an appropriate environment for human adipose-derived mesenchymal stem cell differentiation [23]. However, Keshvari yielded better outcomes with SLES 1% (Sodium laureth sulfate) regarding preservation of ECM integrity of rats’ acellular kidney compared with SDS 1% [24]. Caralt supported simultaneous perfusion of detergents, introducing SDS 0.1% with Triton X-100 1% as the optimal combination for rat kidney decellularization [25].

To investigate the feasibility of fibrotic kidneys for decellularization, Zhang et al. decellularized the fibrotic kidneys of rats. They reseeded the scaffolds with endothelial progenitor cells, showing promising outcomes and optimal biocompatibility after cell seeding [26]. Moreover, Guan et al. showed the superiority and better outcomes of transplanting a recellularized kidney, compared with acellular kidneys being transplanted with the aim of subsequent recellularization [27].

Pigs

Orlando and colleagues were among the first to perfuse kidneys for decellularization. They also reseeded the scaffolds with Murine stem cells with subsequent transplantation of the regenerated organs being matched for weight and age to the organ donors [28]. Yorkshire pigs were sacrificed in 17 studies, while 13 did not mention the breed’s name, mix-breed pigs were sacrificed in two studies, and Bama miniature pigs were used in one study. Of these, most studies used female pigs. Pigs’ kidneys are similar to humans’ size, making them an appropriate model for further uses.

Chae compared the outcomes following two distinct methods for pig kidney decellularization, SDS 1% or Triton X-100 1% each for 3–4 weeks, indicating optimal biomechanical properties with SDS application, and optimal ECM proteins, growth factors, and biocompatibility with Triton X-100 [29]. Guan also simultaneously perfused Triton X-100 1% with SDS 1% into porcine kidneys, culminating in excellent ECM integrity preservation and similar growth factors with native kidneys [30]. Moreover, Wang performed a comparative analysis of decellularization outcomes in porcine kidneys after using various detergents, indicating best preservation of ECM properties and minimal immunogenicity with SDS 1% [31]. Poornejad et al. also evaluated the role of osmotic shock in porcine acellular scaffolds, showing excellent preservation of ECM properties and GAGs (Glycosaminoglycans) and substantial reduction in time to complete decellularization [32]. Later, they compared the efficacy of three chemical protocols for porcine kidney decellularization, advocating detergents as promising compounds for preserving ECM integrity [33]. Hussein could successfully decellularize porcine kidneys using 0.1% SDS and PAA (Peroxyacetic acid), achieving hemocompatibility after transplantation and minimal immunogenicity and cytotoxicity to human cells [34]. Bongolan and colleagues showed the substantial effect of submicellar SDS application on enhanced preservation of growth factors and ECM molecular properties [35]. Moreover, cross-linking of acellular porcine kidney by glycation enhanced the biomechanical properties of scaffolds [36].

Fischer evaluated the role of ambient temperature and detergent during decellularization on porcine acellular scaffolds, introducing 4 °C as the optimal temperature and SDS 1% as the optimal detergent. They also advocated that ECM integrity was most affected by ambient temperature, while ECM molecular properties (GAGs, Collagens, and cytokines) were strongly associated with the type and concentration of used detergent [37]. Corridon also evaluates the impact of blood flow rate after kidney transplantation. Although promising outcomes were achieved with physiologic blood flow (650 ml/min), maximal microvascular disturbance was also observed at this rate [38]. Another study also showed the substantial positive effect of combining detergents with sonication, achieving better cell removal in a shorter time [39]. To evaluate the feasibility of tubular basement membrane development, the recellularization of acellular porcine kidneys with human stem cells yielded promising outcomes [40]. The effectiveness of using bioactive components in acellular porcine kidneys has been investigated lately, indicating positive effects of bioactive compounds on the induction of angiogenesis and cell proliferation and the reduction of inflammation and fibrosis [41].

Human

Human discarded kidneys are used as the most compatible sources of kidney scaffold for further decellularization, with the first time being decellularized by Orlando et al. using SDS 0.5% for 48 h, yielded in cell removal and preservation of ECM integrity and biomechanical properties [42]. Nine studies used Human discarded kidneys as the source of ECM for further decellularization, with almost 102 patients who were candidates to undergo nephrectomy. Peloso perfused SDS 0.5% and DNase through the renal artery and ureters, achieving well-preserved vasculature and growth factors essential for further angiogenesis and induction of cell differentiation [7]. Bongolan also showed the positive effect of submicellar administration of SDS in human kidneys for decellularization on retention of growth factors, cytokines, and proteins, culminating in further enhancement of cell biocompatibility and differentiation [35]. Shahraki compared two distinct detergents for human kidney decellularization, finding Triton X-100 1% more efficient than SDS 0.5% in cell adhesion and proliferation along with optimal cell differentiation potential [43]. In order to induce stem cell differentiation, numerous protocols have been established lately, including the administration of VEGF (Vascular Endothelial Growth Factor) on decellularized human kidneys by Ullah, indicating significant induction of cell differentiation toward endothelial cells along with cell adhesion and proliferation promotion [44]. Moreover, they also performed a similar study, evaluating the role of heparan-sulfate on stem cell differentiation, indicating a positive role of heparan-sulfate administration on human acellular kidney on induction of cell differentiation and biocompatibility of the scaffold [45].

Sheep and goats

Goats and sheep possess the potential to serve as the optimal resource for the advancement of organ scaffolds, facilitating the engineering of bioartificial organ support. This is primarily due to the minimal risk of zoonotic diseases associated with sheep and goats. Furthermore, goat organs’ structural composition and dimensions closely resemble those of humans. Goats’ kidneys were first harvested by Vishwakarma for further decellularization, using detergents (SDS 0.1, 0.5%, and Triton X-100 0.1%) and enzymes (DNase I) [46].

Almelkar also decellularized sheep kidneys for five days through the renal artery by simultaneous perfusion of detergents and enzymes [47]. Kajbafzadeh and colleagues compared transplantation outcomes after two distinct methods of decellularization, using SDS 0.5% + Triton X-100 1% or SDS 1%, indicating better transplantation and decellularization outcomes while Triton applied prior to SDS [48]. Moreover, according to a recently published article, cross-linking of the acellular sheep kidneys enhanced the biomechanical properties of the scaffold along with optimal preservation of ECM integrity and biocompatibility [49].

Monkeys

Nakayama was among the first, used Rhesus monkey kidneys for decellularization by using immersion and agitation technique with two distinct methods (Triton X-100 1% or SDS 1%) for kidney slices of monkeys with different ages, indicating the age of organ donor as a crucial factor affecting acellular kidney and transplantation outcomes [50]. Furthermore, they performed a similar study to investigate the association between age and recellularization outcomes, advocating substantial role of young age on effectiveness of recellularization [51]. Later, they performed another study on recellularized Rhesus monkeys, investigating the factors associated with induction of differentiation by seeding the scaffolds with human stem cells, indicating the preservation of growth factors, stress proteins, and complement components as influential factors in acellular scaffolds inducing further differentiation of seeded cells [52]. Besides, Batchelder evaluated the differentiation of human stem cells seeded on acellular Rhesus monkey kidneys, indicating remnant growth factors of acellular scaffolds as an important factor in the induction of cell differentiation after recellularization with human stem cells [53].

Rabbits and dogs

According to the literature, Kajbafzadeh and colleagues harvested the kidneys of a rabbit for the first time, decellularizing the kidneys through a cannulated renal vein with Triton X-100 and SDS 2%, investigating the effectiveness of different sterilization protocols, indicating PAA as the most efficient sterilizer while gamma irradiation was known as the most devastating [54]. They also performed another study on rabbit kidneys, evaluating the amount of residual SDS after decellularization and introducing methylene blue as an excellent calorimetric agent for evaluating residual SDS on acellular scaffolds [55]. Tajima used kidneys discarded from three beagle dogs for decellularization, using a combination of freeze-thawing and detergent perfusion through the renal artery [56].

Comparison of the sources

Better understanding of human kidney development can provide new insights into the processes involved in normal development and disease. It can also give valuable insights into the similar and different aspects of development between animal and humans. Additionally, it can help establish important benchmarks for analyzing in vitro techniques used to create functional kidneys. Investigating the physiological and anatomical differences in kidneys among different species is essential before conducting preclinical studies and drawing general conclusions. The most noticeable difference is the variation in length, weight, and thickness of the kidneys. Kumar et al. comprehensively compared such variables between human and 10 species [57]. Besides, Lindstorm indicated the differences and similarities between human and mouse kidneys [58]. The rodent model is a cost-effective and manageable option in laboratory settings, but its applicability in studies is limited due to challenges associated with extrapolating data to humans, primarily stemming from anatomical differences in kidney structure between the two species. According to structural differences, rodent, dog, and rabbit kidneys are different from human and porcine kidneys, while their kidneys are unilobular and unipapillary [59]. In regards to physical dimensions, pigs and sheep exhibit similarities to humans. The sample blood volume present in these animals enables frequent blood sampling. Utilizing male subjects significantly eases the process of collecting 24-hour urine samples to monitor during renal function more precisely. Pig and sheep model are valuable tools for researching renal transplantation. Anesthesia and surgical procedures closely resemble those used in human conditions, making it an excellent training model for aspiring surgeons. Their renal anatomy closely resembles that of humans, featuring multipapillary and multilobular kidneys, making it stand out among other species. However, their size poses limitations, requiring dedicated space, expensive food, drugs, and surgical materials.

In view of vasculature diversity, sheep, rabbit and pigs have one renal artery. The absence of multiple medullary pyramids in dogs and rodents leads to the bypassing of segmental arteries, in contrast to the intricate system of interlobar and segmental arteries found in humans, pigs, and sheep that serve to supply the numerous kidney lobes [60]. However, the distribution of renal artery segments, the relative volume of each segment, and the occurrence of arterial injuries following cranial pole resection in sheep kidneys differ from those observed in human kidneys. Sheep kidneys exhibit a primary division into anterior and posterior segments, resembling human kidneys but differing from pigs [61]. Hence, these anatomical distinctions should be considered when using sheep as an animal model for renal experimental or training procedures. The way the renal artery divides into segmental arteries in pigs is different from human kidneys. Also, the relationship between the posterior segmental artery and cranial infundibulum varies between the two species. Porcine kidneys heal differently than human kidneys after partial nephrectomies. Because of these differences, the porcine model is not suitable for studying hemostatic methods in kidney procedures [62, 63]. Simoes evaluated the intrarenal anatomy and histological features of the porcine kidney, highlighting the similarities and differences compared to the human kidney [64]. On the other hand, while the pig kidney does not feature a retropielic artery, it is still regarded as the most appropriate animal model for examining partial upper pole nephrectomy, especially when vascular injury is a focal point of the investigation [65].

In view of biochemistrical and physiologic properties, porcine kidneys have been nominated as a model with closest proximity to human kidneys. The assessment of more than 100 physiological variables in pigs under basal conditions led to the conclusion that the majority of porcine values were similar to those documented in humans under similar conditions [66]. On the other hand, cross-species immunological incompatibilities have posed obstacles for xenotransplantation, however, recent progress in porcine genome engineering has allowed for the first successful experiments. Nevertheless, the immune response following the transplantation of pig kidneys into human recipients remains a topic of limited understanding. Meanwhile, two studies advocated promising cyto-compatibility between human cells and porcine acellular matrix after recellularization [35]. 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.

Decellularization

Decellularization of the kidney is a complicated procedure due to the vast number of differentiated cells and complex micro and macrostructure of the kidney. Detergents, enzymes, or other cell-lysing solutions are introduced to the whole organ or tissue to solubilize membranes and remove cells to create acellular kidney scaffolds. To replace the excretory and metabolic activities of the kidney, it is essential to preserve two components: the glomerulus and the tubule. Two of the most promising methods for organ bioengineering are the decellularization of organ-specific ECM and the use of three-dimensional bioprinting technology to arrange cells and materials precisely [67]. A method called decellularization makes it possible to remove cells from healthy tissue while leaving the ECM structure intact. The leftover ECM, after decellularization, can be employed as scaffolds for tissue engineering and regenerative medicine purposes. Effective elimination of the cellular components is mandatory to minimize possible adverse reactions on the composition, biological activity, and mechanical integrity of the ECM. The effectiveness of cell removal depends on the tissue properties and the method used for decellularization [68]. Decellularization involves physical, chemical, enzymatic treatment, or a combination. Detergents are introduced to the renal organ/ tissue, and cells are lysed and eliminated. The ensuing acellular ECM is a bioscaffold with a white, translucent appearance [69]. Techniques for whole organ decellularization are based on conventional decellularization methods. However, due to the thickness of larger organs and their intricate intrinsic structures, decellularization may require more complex processing than the straightforward tissue agitation and immersion-only decellularization methods employed for thin and organized tissues. Therefore, perfusion-based decellularization techniques are mainly used for solid organs, which enable the efficient removal of all native cells through organ’s vasculature perfusion of detergents, leading to structural integrity preservation [5]. Following variables are crucial during organ decellularization (a) the cellular density of that tissue or organ, (b) specific density, (c) lipid content, (d) thickness, and (e) the characteristics of the decellularization agent (s) that have been chosen. The choice of detergents, concentration, duration, ambient temperature, and route of administration are essential steps. Various chemical detergents with specific properties have been investigated to accomplish this goal [70]. Table 2 provides a comprehensive categorization of decellularizing agents with summarizing their mechanism of action, merits, and drawbacks. The two main requirements for a successful decellularization are maintaining the architecture of the ECM and removing as few ECM components as possible. Hence, we performed a thorough evaluation of studies that compared the effectiveness of different detergents with regarding their outcomes and summarized the findings in Table 3.

Table 2 Comparison of the methods used for kidney decellularization

Table 3 Outcomes of studies comparing the findings after different agent utilization

Physical techniques

Decellularization protocols initiate by lysing the cell membrane with physical or ionic treatments, separating the cellular components from the ECM with enzymatic processes, solubilizing the cytoplasm and nuclear cellular components with detergents, and removing the cellular debris from the tissue. Physical approaches are commonly used to increase the efficacy of decellularization.

Poornejad evaluated the impact of freeze-thawing on decellularization by comparing the outcomes in native and acellular kidneys, concluding that the effects of this process without administration of cryoprotectants on the native kidneys were substantially more destructive than acellular kidneys [71]. Hu also performed a study with similar objectives, advocating the simultaneous use of freeze-thaw and perfusion of chemicals as an optimal method with promising outcomes of decellularization while providing excellent preservation of ECM integrity, growth factors, and time to decellularization [12]. Besides, Chani evaluated the advantages of applying cryopreservation on decellularization outcomes; acellular kidneys that had undergone cryopreservation had better biomechanical properties. However, they observed no difference regarding the biocompatibility of scaffolds [72]. Feng also designed an investigation with similar aims by comparing outcomes following three physical treatments (Freeze-thaw + cryopreservation versus freeze-thaw without cryopreservation versus freeze-thaw with VS83 versus no freeze-thaw). They observed severe alteration in ECM and vascular integrity of acellular kidneys without cryopreservation. Cryoprotectant solutions can be perfused throughout the kidney scaffold to minimize the detrimental impact of freezing on the vascular architectures. Utilizing the vitrification solution VS83, which enables ice-free cryopreservation, the integrity of the vasculature within the entire kidney matrix can be effectively maintained even after freeze-thawing [18]. Furthermore, Yang conducted a similar study, showing that preserving vascular integrity was most effectively achieved when the temperature was at −20 °C. The utilization of cryoprotectants at low concentrations proved to be the most suitable approach to strike a balance between preserving vascular integrity and promoting decellularization [11]. According to a recently published article, the efficiency of various physical treatments on decellularization outcomes was evaluated comparatively. Harvested kidneys were aligned into three distinct groups according to the type of physical treatment, including high hydrostatic pressure (HHP), freezing–thawing cycles (FTC), and ultrasonic bath system (UBS). In the HHP group, kidneys were inserted into high-pressure chambers, undergoing three distinct pressures (50, 100, and 200 MPa). In the FTC group, kidneys underwent four freeze-thaw cycles between − 80 °C and 4 °C for 60 and 30 min, respectively. Lastly, in the UBS group, kidneys underwent chemical protocol within two parallel bathes of the ultrasonic generator at three distinct powers of 28, 30, or 60 W, constantly perfused with a cool solution to prevent further disruption by ultrasonically induced heat. After physical treatment, chemical treatment with CHEM protocol was performed. The optimal outcomes were achieved by combining the physical FTC and chemical decellularization techniques [15].

Regardless of the application of freeze-thawing as a physical treatment, other physical treatments are also investigated in the literature. Poornejad evaluated the role of osmotic shock on outcomes following decellularization of porcine kidneys, finding a significant reduction in total decellularization time and exposure to SDS, providing better cell removal and preservation of ECM integrity along with promising biocompatibility. Besides, they showed that applying osmotic shock resulted in optimal preservation of ECM interactions [32]. The decellularization method is another crucial factor that induces physical disruption; the most commonly utilized techniques are perfusion or immersion/agitation. According to our findings, 71 studies applied perfusion decellularization and 31 used immersion/ agitation. Perfusion parameters, such as flow rate, detergent concentration, and decellularization time, have been modified in numerous studies to optimize the entire organ decellularization protocol. He et al. aimed to investigate the optimal decellularization time, showing optimal preservation of ECM components, vascular integrity, growth factors, and GAGs at 4-hour approaches [21]. Manalastas also evaluate the role of flow rate and application of sonication with different powers on outcomes of decellularization of porcine kidneys. Sonication causes cell membrane damage by microbubble production, reducing decellularization time. Despite the positive relation between the increase of flow rate and sonication power with total decellularization time, they observed the point at which a gradual increase in sonication did not affect the total decellularization time, and that was the minimal flow rate of 15 ml/min. High sonicator power can lead to disruptive effects in detergent micelle formation. This is primarily due to breaking down the monomers responsible for micelle formation, which decreases the detergent’s ability to solubilize cell membranes. As a consequence, longer decellularization times are observed. Hence, they concluded that simultaneous application of sonication with perfusion reduced total decellularization time from 19 h (flow rate of 15 ml/min and sonication power of 0 W) to 2 h (flow rate of 45 ml/min and sonication power of 120 W), however, caused some damages to ECM integrity [39]. Sullivan first established a novel technique for kidney perfusion called a high-throughput decellularization system [73]. Liu also developed acellular kidney scaffolds using a novel micro-peristaltic pump, providing optimal cell removal with an appropriate environment for further cell seeding [74]. Recently, Taylor comprehensively investigated the feasibility of whole animal organ decellularization via perfusion decellularization, proving optimal biomechanical properties and preserving ECM components [75]. Fischer also investigated the optimal ambient temperature for kidney decellularization, proving that the integrity of the ECM was primarily linked to the ambient temperature [37].

Mallis and colleagues compared the outcomes following two distinct agitation techniques: the rotary shaker and the magnetic stirrer. Both strategies proved to be effective in achieving decellularized status. However, the magnetic stirrer demonstrated superior performance in terms of biochemical and histologic properties, resulting in successful decellularization and preservation of the ECM composition [19].

Detergents

Currently, the most effective and resilient decellularization approach involves using chemical infusion. By employing chemical reagents, specifically detergents or acids, the native cells are eliminated through the disruption of cell membranes, thereby isolating the cellular constituents of the ECM. According to our findings, numerous detergents and acids used in the literature for kidney decellularization, including SDS (91 studies with concentrations ranging from 0.1 to 3%), Triton X-100 (58 studies with concentrations ranging from 0.1 to 3%), DNase I (Deoxyribonuclease) (21 studies mostly at 0.0025% concentrations), NaOH (four studies with 0.1% concentration), Trypsin (five studies), PAA (three studies), Tween 20 (two studies), SDC (four studies), EDTA (seven studies). Moreover, eight studies also used hypertonic solutions. EGTA, SLES, and hypotonic solutions were each used once in the literature.

A combination of Triton X-100, NaCl, and DNase I solutions under constant pressure was used to obtain an intact extracellular matrix, partially preserving laminin and collagen type IV. This step is of great importance since it is well known that the extracellular matrix’s composition and structure must be preserved to be suitable for the recellularization process, and even the presence of DNA remnants of organ donor cells may develop further adverse reactions [13]. In the same year, a combination of Triton 1% and SDS 1% was administered [14], and both achieved cell removal and preservation of ECM integrity [13, 14]. Subsequently, detergents using the novel method of high-throughput system perfused, comparing outcomes following two distinct detergents, (a) SDS 0.5 + 0.25%, and (b) Triton X-100 1% + NaOH 0.1%, both yielded in ECM preservation and cell removal [73]. Orlando perfused SDS 0.5% for 48 h at 6 ml/min to decellularize the human kidneys for the first time. By utilizing SDS 1% alone as a lysis buffer for 17 h through the renal artery of rats at a rate of 0.4 ml/min, Bonandrini et al. could avoid the time-consuming perfusion approach [76]. However, Chae agitated porcine kidneys for 3–4 weeks in either SDS 1% or Triton 1% solution. By increasing the Triton-treated scaffold’s porosity, its water uptake capacity was significantly enhanced. This improvement in water absorption facilitated the preservation of crucial ECM proteins and growth factors while ensuring excellent biocompatibility. Additionally, the biomechanical properties of the scaffolds treated with SDS were optimal [29]. With the use of three methods utilizing Triton X-100 1%, Triton X-100 1% + SDS 0.1%, or Triton X-100 1% + Trypsin/EGTA, Caralt aimed to assess the biochemical properties of decellularized rat kidneys. The grading system assessed matrix-bound basic fibroblasts, vascular endothelial growth factors, and intact renal microarchitecture. Optimal outcomes were successfully obtained by implementing the second protocol in kidney decellularization. Following the second protocol, the subsequent transplantation of recellularized kidneys effectively facilitated re-perfusion with blood, thereby establishing a conducive environment for the proliferation of cells [25]. To compare the effectiveness of two decellularizing agents in porcine kidneys, Choi compared the 14 days’ use of Triton X-100 1% with the same duration of SDS 1%. The optimal detergent was determined to be Triton X-100, offering improved biomechanical and biochemical properties and enhanced biocompatibility. Furthermore, Triton X-100 could preserve higher levels of growth factors while causing minimal disruption to the structure of the kidney [77]. To develop an efficient and accelerated method for kidney decellularization, Guan perfused SDS 0.5% through the renal artery at a rate of 2 ml/min for 4 h [27]. Peloso suggested advantages over simultaneous administration of Triton X-100 1% with SDS 1% for enhancement of cell removal along with ECM components preservation [78]. In the same year, they decellularized human kidneys by simultaneous perfusion of SDS 0.5% and DNase I through the renal artery and ureter at the rate of 12 ml/min, indicating the well-preserved integrity of the structural and functional vasculature and growth factors [7]. Zambon also compared outcomes following two combination therapies (SDS 0.5% + DNase versus Triton X-100 1% + SDS 0.5%), supporting the advantages of the combination therapy of Peloso [79]. Moreover, the highest level of ECM integrity preservation, cell removal, and elimination of xenoantigens was achieved by employing an SDS 1% solution., while Wang and colleagues compared the outcomes following administration of four distinct chemicals, including SDS 1%, Triton X-100 1%, PAA, or SDC (Sodium deoxycholate). Poornejad also conducted a comparative investigation of five chemical reagents for porcine kidney decellularization, including Triton X-100 3%, SDS 1%, Trypsin/EDTA 0.05%, PAA 1%, or NaOH 0.1 N. All procedures were performed at room temperature, except Trypsin/EDTA at 37 °C. Protease enzymes demonstrated limited effectiveness in eliminating cells while causing significant damage to the collagenous structure. On the other hand, NaOH exhibited the highest speed in decellularization and optimal interactions with the ECM. However, it also led to the most significant ECM disruption and collagen integrity disruption. Although detergents had a lesser impact on ECM integrity, they did not offer the same advantages as NaOH regarding cell removal [80]. Fischer compared the outcomes of decellularization following the use of SDS 1% + DNase I, Triton X-100 1% + DNase I, or SDC 1% + DNase I. The preservation of GAGs, cytokines, and collagens is strongly associated with the detergent used, particularly when employing SDS 1% at 4 °C. The viability of reseeded cells was found to be highest when using SDC 1%, which is closely related to the preservation of cytokines. Therefore, SDS 1% at 4 °C yielded the most favorable structural and composition scores. In contrast, 1% SDC at the same temperature resulted in lower scores for structural and composition but showed a significantly improved cell performance score [37]. SDS was also shown to be a more effective detergent than Triton X-100, given cell removal [23]. However, Triton X-100 1% showed better biocompatibility than SDS 0.5%, especially for decellularizing human discarded kidneys [43]. Keshvari first utilized SLES for further kidney decellularization by comparing the outcomes following using 1% SDS. Primary findings showed similar biocompatibility and cell removal between the two agents. However, SLES culminated in better preservation of ECM integrity [24]. In order to determine the optimal concentration and decellularization time for SDS, He and colleagues compared various SDS concentrations 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 results [21]. Similarly, Manalastas aimed to determine the optimal concentration of SDS, showing a decrease of decellularization time with the increase of SDS concentration; however, they advocated that increasing the concentration of SDS from a higher concentration of 0.245% or 0.0085 mol/L does not lead to an increase in the reaction rate, nor does it lead to an increase in the rate by lowering the rate and raising the concentration beyond the determined limit, while excessive presence of detergent monomers in the solution can result in the creation of detergent micelles rather than mixed detergent-lipid micelles, which are essential for penetrating the cell membrane and eliminating cells [39].

Regardless of the chemicals used to decellularize the kidneys, some reagents are also used to enhance the biocompatibility, biomechanical properties, or induction of cell differentiation. Ko was among the first to conjugate the acellular scaffolds with CD-31 antibodies, culminating in enhanced endothelial cell attachment and retention, improving further vascular patency [81]. Rafighdoost also treated the acellular kidney scaffolds with Chondroitin sulfate and evaluated the biocompatibility of the further scaffolds, showing the enhanced ability of scaffolds for cell adhesion, migration, and proliferation after cell seeding [82]. Wang also investigates the impact of immobilization of heparin on acellular scaffolds, indicating significantly lower rates for the formation of thrombosis, providing an excellent environment for further endothelial cell seeding [83]. Similarly, Xie conducted heparinization of acellular kidneys, proving heparin’s anti-inflammatory and anti-coagulative effects on scaffolds along with optimization of recellularization and biocompatibility [84]. Likewise, Zhou’s heparin perfusion led to enhanced preservation of morphology, improved anticoagulation and biomechanical properties, enhanced release of EGF, and improved angiogenesis [85]. Besides, Ullah treated acellular scaffolds with VEGF to increase angiogenesis after recellularization, providing promising outcomes [44]. Sant also performed advanced glycation of the acellular scaffolds via cross-linking, which enhanced the scaffold’s biomechanical properties [36]. Cross-linking of acellular scaffolds with 1-(3-Dimethylaminopropyl)−3-ethyl carbodiimide hydrochloride (EDC), N-Hydroxysuccinimide (NHS), and 2-(N-Morpholine) ethyl sulfonic acid (MES) showed similar findings of greatly enhanced mechanical properties of biocompatible scaffolds while keeping the molecular structure of the kidneys intact [49].

Taken together, techniques used to evaluate the adequacy and effectiveness of decellularization process is summarized in Table 4.

Table 4 Techniques used to evaluate the adequacy of kidney decellularization

Sterilization

As the ECM needs to be decontaminated, thoroughly cleaning the tissue is the next step. After decellularization, all lingering compounds must be eliminated to avoid an adverse host-tissue reaction to the chemicals. According to Crapo et al.‘s findings, effective decellularization requires less than 50 ng/mg of DNA content in tissue According to the present study, numerous sterilizers used in the literature, including UV and gamma irradiation (13 studies); infusion of PBS (Phosphate-Buffered Saline), NaCl, DW (Distilled water), Ethanol 70% (16 studies), PAA (10 studies), and antibiotic-containing solutions (30 studies); Ethylene oxide gas (Four studies). Most common sterilizers include antibiotic-containing solutions. Poornejad and colleagues were among the first to compare outcomes after four sterilization methods in acellular kidneys. Acellular kidneys underwent sterilization by one of the following: gamma irradiation (1–10 kGy), ethanol 70% in DI water, PAA 0.2% in NaCl, or PAA 0.2% in ethanol 4%. Gamma irradiation has been identified as the most detrimental technique, significantly reducing cell adhesion, proliferation, and mechanical properties. Additionally, it has been determined that PAA 0.2% in NaCl solution is the most effective sterilizer, yielding satisfactory results in preserving the integrity of renal ECM while facilitating optimal interactions within the ECM [80]. Moreover, Kajbafzadeh also aimed to investigate the effectiveness of various sterilization techniques in an acellular rabbit kidney, comparing antibiotic-containing solutions, PAA, and gamma irradiation. PAA 0.5% has been established as the most efficient approach. Conversely, radiation has emerged as the most disruptive method [54]. Moreover, according to the previous literature, utilization of gamma irradiation, ethylene oxide, and ethanol 70% seems harmful for ECM components, including GAGs and collagen.

Cell seeding

The most complicated step of organ bioengineering with the most complexity is the recellularization of the ECM scaffolds. The type and source of cells employed to repopulate the three-dimensional ECM scaffold are essential for bioengineered organs to be functional. The parenchyma, vascular, and other components need to be repopulated to regenerate a new organ [86]. Successful recellularization of scaffolds and induction of differentiation to stem cells are associated with various factors.

Delivery and distribution

Successful cellular delivery to the acellular scaffold, homogenous distribution of the cells through the scaffold, and effective cellular delivery to a designated point in the kidney are important challenges, being meticulously evaluated in different studies. Hence, route of cell introduction is among important factors that should be addressed in all studies with recellularization process [80]. Starting with simplest cell delivery approach, Guan used multiple cell injections to the scaffold punctures for cell delivery [30]. Remuzzi evaluated simultaneous scaffold repopulation after transplantation, indicating that implanting acellular scaffolds didn’t culminate in the repopulation of the scaffolds with host cells. Thus, it becomes indispensable to execute in vitro recellularization [81]. Moreover, another study used simple contact between cultured cells and kidney slices, observing that nephron and tubular structures were not identified in histologic evaluations. They justified such failure and inability for optimal cellular organization and migration within substructures of the kidney with the type of cells (human adult primary renal epithelial cells) they used for repopulation [35]. However, we assume that the type of cell introduction in such failure is not negligible. According to a recently published article, the application of roller bottles for recellularizing scaffolds has improved cellular penetration [87]. using such techniques doesn’t seem much effective for solid organs and thick tissues.

With recent advancement in perfusion-recellularization devices, access to the whole organ and perfusion of cells through its vasculature made great progress lately. According to our systematic review, 44 studies thorough artery, seven studies through artery and ureter, three studies through artery and vein, two studies through vein, and one study through artery, vein, and ureter, recellularized the scaffolds. Song et al. introduced HUVECs (Human Umbilical Vein Endothelial Cells) into the renal artery, resulting in cell seeding within 70% of the glomeruli. Ciampi accessed renal artery for recellularization. They observed cell distribution to the cortical sections, vascular, and of glomerular structure. However, only 40% of glomeruls reached by cells were maximally repopulated partially. Thereafter, they accessed both renal artery and vein for simultaneous cell perfusion. When cells administered through the renal artery and vein, the endothelial cells effectively entered all blood vessel areas, leading to the restoration of about 89% of the glomeruli (13% partial). Additionally, these cells were evenly spread throughout the peritubular capillaries, covering the cortical and medullar regions of the kidney. This perfusion protocol enabled the cells to access not only the arterial vessels up to the glomerular capillaries, but also the renal veins and peritubular capillaries. It’s important to note that a single layer of cells was seen surrounding the inner wall of the small arterioles, however, tubular structures remained untouched [88]. Similarly, Song showed that isolated access to renal arteries repopulated almost 70% of the glumeruls without substantial tubular seeding. Therefore, by injecting neonatal rat kidney cells through the ureter, they showed that the cells migrated to the tubular compartments [89]. Hence, researches tried to increase the effectiveness of cell delivery by accessing renal vein or ureters in combination with the arterial route. Another study showed promising cell repopulation of the glomeruls and tubuls by simultaneous arterial and ureteral cell perfusion along with differentiation toward specific endothelial and tubular cells [90]. Poornejad compared the outcomes of recellularization regarding the route of cell seeding (artery or ureter) by utilizing a moderate vacuum pressure of 40 mmHg to perfuse the cells through the ureter, resulted in a noticeably uniform distribution of cells in the recellularized kidneys [91]. Similarly, Hu et al. perfused the cells via the same route and under vacuume pressure. They indicated that the introduction of cells via the renal artery solely led to a significant presence of cells in the glomerular zone, with a less dense and scattered presence in the renal tubule area. Besides, when cells were only perfused through the ureter, there was a notable presence of cells in the tubular sections, leading to the development of tubule-like structures, while the number of cells in the glomerular region was minimal. Lastly, simultaneous perfusion of cells using both routes showed optimal repopulation in tubular and vascular structures [16]. Zhang also with similar routes, showed promising cell distribution through glomeruls and tubular structures [92]. Another study used artery, vein, and ureter for recellularization and compared the outcomes. They supported the perevious findings, showing better outcomes after concomitant arterial and venous cell perfusion compared with each alone.

Taken together, cellular delivery to the vascular compartments is reachable by developing arterial and venous access and parenchymal and tubular delivery is yielded by ureteral perfusion. However, reaching the mentioned compartments is not all the thing that must be investigated for an optimal recellularization, while homogenous distribution of the cells is another factor for optimal cell delivery. Remuzzi and colleagues used three routes for kidney recellularization, isolated or in combination; however, they observed hetrogenous and focal cell distribution throughout the organ. They could enhance their outcomes by increasing concentration and flow-rate of the seeded cells [92]. Hence, there are a few challenges that needs to be addressed in the future studies. First, the number of cells perfused to the kidney must be optimized to prevent further cell leakage or hetrogenous distribution. Poornejad advocated that the lower the cell concentration, the more homogenous the cell distribution, introducing 4–5 million cells/mL as the optimal concentration for all recellularization methods [91]. Second, the pressure and flow-rate used to perfuse the cells is another factor that should be also adjusted according to the tissue and organ vasculature resistance to prevent further cell leakage or vascular rupture. Caralt showed more cellular leakage and more hetrogenous distribution by increasing the pressure [25]. Similarly, Poornejad supported their findings and showing more cellular depletion with increased pressure, observing no cells in medullary region. Moreover, with increasing the pressure during ureteral perfusion, substantial tubular damages achieved. Beside they showed that vacuum pressure is a promising force to induce cell migration with 40 mmHg being the optimal vacuum pressure for porcine ureter [91]. Third, inorder to achieve optimal cell delivery and further differentiation compatible with the region they reached, the type of administered cells is also among hurdles. For instance, Remuzzi observed satisfactory differentiation potential of embryonic stem cells to produce various kidney-specific phenotypes, stimulated by direct contact with region-specific components of the ECM [92]. Last but not least, cell delivery process is a complex step in tissue engineering facing various hardles, requiring more studies, especillay being optimizing a specific approach for human kidneys.

Influential factors

Preservation of ECM components and retention of growth factors play a crucial role in outcomes after cell seeding. Collagen preservation facilitates cellular adhesion and proliferation after recellularization. Elastin also showed significant role in maintaining mechanical properties of the scaffold. GAGs also are important for retention of GFs as they can bind to such factors [33]. Ross first reseeded the scaffolds with murine pluripotent embryonic stem cells (SC), cellular differentiation was observed, and the formation of lumens resulted from apoptosis in cells that were not in direct contact with the basement membrane matrix. These significant findings strongly suggest that the ECM plays a vital role in directing the regeneration of the kidney [13]. Moreover, the evidence from another study by Rosso has brought to the forefront a signaling process between the matrix and cells within acellular whole organ scaffolds. This signaling pathway is pivotal in directing pluripotent precursor cells toward differentiation into the endothelial lineage. Furthermore, they indicated that regeneration of the mouse basement membrane aids in the induction of remodeling of scaffolds [93]. Similarly, numerous studies found that the ECM can regulate the differentiation and maintenance of stem cells and their differentiated progeny, indicating that remnant elements in the ECM microenvironment significantly impact how seeded cells differentiate [6, 52, 53, 78, 94]. Using proteomic analysis, Nakayama proved the positive role of remnant growth factors, antimicrobial proteins, stress proteins, and complement components in enhancing kidney-related gene expression [52]. Besides, intermediate mesoderm cells derived from ADSCs demonstrated greater differentiation into tubular and podocyte-like cells than the original stem cells. This enhanced differentiation efficiency suggests that utilizing scaffolds to induce stem cell differentiation could offer a viable approach to renal regeneration. This remarkable outcome was made possible by utilizing human pluripotent stem-cell‐derived ECs. Furthermore, the recellularized kidney demonstrated the remarkable capability of being perfused with human whole blood [95]. Furthermore, the implementation of albumin-coated scaffolds has resulted in preventing blood clot development and enhancing cell proliferation. Therefore, combining dynamic culture and albumin coating is an efficient technique [87].

Rejection prevention

In view of successfulness of recellularization and lowering the risk of rejection, Nakayama evaluated the role of age in outcomes after recellularization and advocated that the age of the donor significantly influences the efficiency and outcomes of recellularization, while scaffolds from younger donors showed the most pronounced repopulation of cells [50, 51]. Du demonstrated that the recellularization of scaffolds with patient-specific cells reduced the risk of rejection and immunogenicity along with an increase in the long-term function of regenerated kidneys [96].

Differentiation

To evaluate the feasibility of induction of differentiation on reseeded scaffolds, Finesilver used kidney-derived serum-free media to induce differentiation on acellular scaffolds seeded with human embryonic stem cells (HES) [97]. Ko also used antibody conjugation (CD31) to induce angiogenesis, explained above more comprehensively [81]. Batcheldar treated recellularized scaffolds with supplements (cytokines and growth factors) for induction of differentiation, indicating a substantial positive simultaneous impact of ECM components and supplements on induction of HES differentiation by enhancement of expression of genes related with renal progenitor, proximal tubule, endothelial, and collecting duct cells [53]. Rafighdoust showed the positive impact of chondroitin sulfate on the enhancement of cell proliferation, adhesion, and migration after recellularization [82]. The function of adult kidney stem cells was shown to be impacted by decellularized ECM from three distinct kidney areas (cortex, medulla, and papilla), with varying structural and compositional impacts. Compared to cells grown on the cortex and medulla ECM, cells cultured on the papilla ECM consistently showed less proliferation, more metabolic activity, and variations in cell morphology, alignment, and structure creation [94]. Similarly, Nakayama et al. demonstrated that human ESCs planted onto decellularized rhesus monkey kidneys produced thick, symmetrical epithelial (Cytokeratin1) tubules and displayed several specific kidney tubule cells [52]. Ullah used VEGF-treated scaffolds for recellularization, culminating in a significant transformation of stem cells into endothelial cells. Thus, it was concluded that by conditioning the scaffold with specific growth factors, the possibility arises to generate functional niches through the selective promotion of cell attachment, survival, and differentiation [44]. Furthermore, they showed that the cells underwent a targeted differentiation process upon seeding, resulting in the formation of nephron cells. They concluded that the absence of heparan-sulfate proteoglycans hindered further cell differentiation, thereby establishing heparan-sulfate proteoglycans within the adult ECM as the primary factor inducing subsequent cell differentiation [45]. Moreover, by incorporating bioactive components into acellularized kidneys, there has been a notable improvement in cell proliferation and the initiation of angiogenesis, experiencing a decrease in fibrosis and inflammation while also demonstrating an increase in glomerular filtration rate. It is essential to highlight that the combination of PDRN/TI-EVs has exhibited an enhanced therapeutic effect, promoting angiogenesis, reducing renal fibrosis, and activating pro-reparative macrophages [41]. The differentiation of stem cells into kidney progenitor cells was shown as a prerequisite for the successful regeneration of kidney structures with the aid of the ECM. This crucial step must be completed before introducing the acellular organ [98].

Adequacy and functionality

Some studies investigated cell proliferation, adhesion, and viability adequacy after recellularization through different protocols. Ofenbauer evaluated the effectiveness of paraffin-embedded scaffolds in evaluating scaffolds. They demonstrated that formerly paraffin-embedded decellularized ECMs can remarkably influence the differentiation of stem cells. This method provides novel opportunities for determining the optimal decellularization protocols required for repopulating three-dimensional tissue scaffolds with embryonic stem cells and other cell types that exhibit tissue-specific characteristics [99]. Uzarski and colleagues recently developed a novel dual-purpose bioreactor to monitor scaffold recellularization adequacy. This study introduces an advanced kidney bioreactor design that outperforms previous models by integrating ureteral and arterial pathways for recellularization. Notably, the utilization of arterial (high-pressure) recellularization exhibited more favorable outcomes than other approaches. As decellularization progresses, the resistance to fluid flow through the scaffold, referred to as ΔP, diminishes due to cell loss. Conversely, upon the recellularization of the kidney, ΔP rises to nearly normal levels observed in intact organs [100]. Moreover, they performed another study in the same year, evaluating Resazurin for evaluation of the adequacy of cell proliferation and viability [101]. Similarly, they introduced Resazurin as an affordable and harmless agent that offers a non-destructive approach to assess cell population within recellularized scaffolds. This method allows for a comprehensive evaluation of cell viability and proliferation, granting researchers a clearer understanding of the scaffold’s cellular composition without sacrificing tissue [102]. Abolbashari evaluated the efficacy of recellularized kidneys by evaluating sodium uptake, EPO (Erythropoietin) production, and activity of hydrolases [103]. Poornejad used iron oxide to label the cells before seeding and MRI to detect seeded cells [91]. Finally, Uzarski introduced the glucose consumption rate (GCR) to serve as a reliable parameter for assessing the biocompatibility and functionality of regenerated kidneys. A minimum threshold of 20 mg/h has been identified for GCR, indicating the optimal metabolic activity. Notably, during days characterized by peak GCR, nearly complete endothelial coverage has been achieved [104]. Moreover, intravital microscopy used by Corridon has proven to be an effective technique in investigating transplanted decellularized kidneys [105, 106]. Several studies have demonstrated that the regenerated kidneys were able to produce urine. Song asserted that urine production was achieved within 30 to 60 min. Additionally, other studies highlighted the kidney’s capability to generate urine through the ureter by excreting solutes incrementally and reabsorbing them tubularly. However, a key question is whether the observed excretory function could have been primarily facilitated by the passive transport of creatinine, facilitated by the permeable ECM of blood capillaries and the tubular membrane [22, 27, 89, 92]. Therefore, a comprehensive assessment of the functionality and structure of the regenerated kidney, including sequential imaging such as an intravenous pyelogram and dimercaptosuccinic acid (DMSA) scan, is essential, particularly in human kidney studies. Moreover, the long-term durability of the regenerated kidney functions must be assessed through continuous follow-up.

Type of cells

The adult kidney is made up of around 30 different types of specialized cells that are dispersed throughout numerous unique renal compartments. Each cell type has distinct roles and is architecturally structured into the complex architectural renal vasculature, interstitia, glomeruli, and tubules. Therefore, finding and creating trustworthy and efficient cell sources is a requirement for renal tissue engineering. Various cell sources have been identified, obtained, and cultured for the treatment of kidney diseases regarding recent developments in stem cell biology and cell culture methods [5]. After seeding, cells lose their embryonic characteristics after ten days and begin expressing Ksp-cadherin and Pax-2, typically expressed in the ureteric bud. During the late embryonic stages, they stimulated the distal nephron tubular cells and the metanephric mesenchyme. However, the primary method used to evaluate ESC differentiation and proliferation was an in vitro culture of thin slices of the scaffolds under static conditions; only preliminary feasibility evaluation studies using whole kidneys undergoing pulsatile growth media perfusion were published [93]. In a follow-up study, the same team demonstrated that mouse ESCs infused into the renal artery reached the center of the vessels, where they experienced apoptosis and formed lumens. The endothelial marker BSLB4 lectin and the vascular endothelial growth factor receptor-2 were expressed by the remaining cells, which lined the scaffold basement membranes and took on an endothelial look [86, 92]. Moreover, to evaluate the ability of Nephrosphere (NS) cells to restore different sections of the nephron using renal ECM scaffolds derived from decellularized human renal tissue slices, Bombelli showed that the successful differentiation of NS cells into proximal and distal tubes, as well as endothelial tissue, highlights their potential as a suitable cell type for driving the differentiation of recellularized scaffolds towards nephron regeneration [107].

The successful incorporation of more differentiated cells, including human proximal tubule epithelial cells (PTECs) [41, 108, 109], human kidney cell line (HK-2) [41, 97], primary human renal cells [21, 29, 35, 73, 110], renal cortical tubular epithelial cells (RCTE) [25, 32, 33, 71, 80, 101, 111, 112], primary human renal papillae derived CD133/1 + cells [100], human glomerular endothelial cells [95, 113], and human umbilical vein endothelial cells (HUVEC) [40, 44, 83,84,85, 89, 104, 114, 115]onto scaffolds has been demonstrated. However, it is essential to note that these cell sources only provide some of the necessary cell types required for the comprehensive repopulation of the entire scaffold.

Pluripotent stem cells

Pluripotent SCs gradually run out in mammals, including humans, throughout embryonic development, most likely transforming into tissue-specific stem/progenitor cells, also known as committed stem/progenitor cells [116]. Pluripotent SCs can be grown in vitro long-term based on their functional characteristics. As a result, they produce embryoid bodies in vitro while implanting into an animal host with a compromised immune system [105]. Embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) are examples of human pluripotent SCs. It has been clearly shown that these two cell types possess the characteristics of pluripotent SCs. These cells can be helpful resources for therapeutic and research goals [117]. Embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) are examples of human pluripotent SCs. It has been clearly shown that these two cell types possess the characteristics of pluripotent SCs. These cells can be helpful resources for both therapeutic and research goals [118]. Application of endothelial cells differentiated from human pluripotent SCs for cell seeding culminated in complete re-endothelialization of glomeruli and arterioles after supplementation of the scaffold with VEGF and angiopoietin 1 [95]. Besides, studies recently used kidney and endothelium progenitor cells for further cell seeding [9, 26, 119,120,121].

Embryonic stem cells (ESCs)

Pluripotent SCs are primary cultures of embryonic cells taken from the blastocyst’s inner cell mass around five days after fertilization. After being derived from embryos, isolated, cultured, and can be stimulated to develop into various cell types, potentially appropriate cells for renal regenerative treatments that differentiate into many mature kidney cell types [27, 30, 99, 122]. Investigations have also been made on differentiating ESCs into renal lineages [53, 92, 98, 99, 123], being first used by Ross for cell seeding [13, 93]. Nakayama also used human ESCs, indicating increased kidney-associated gene expression after recellularization [52]. The impact of extrinsic/ intrinsic growth factors showed that ESCs could be differentiated into renal epithelial cells and endothelial cells successfully integrated into a developing kidney [52, 53, 93]. Remnant ECM growth factors also induced differentiation toward renal lineage [53]. Finesilver advocated substantial upregulation of NPHS-1, REN, and EPO after supplementing human ESCs with kidney-derived serum-free conditioned media [97]. However, Sambi demonstrated that the differentiation of stem cells into kidney progenitor cells is a crucial step before introducing the acellular organ. This process is necessary to facilitate the regeneration of kidney structures with the assistance of the ECM components [98].

Induced pluripotent stem cells (iPSCs)

Priming and naïve iPSCs are two different categories. While primed iPSCs mimic cells originating from post-implantation epiblasts, naive iPSCs model the inner cell mass of the pre-implantation blastocyst. Naive iPSCs require chemically defined circumstances to be created, but they are often accessible to survive and differentiate. The distinction between naive and primed iPSCs and their species of origin is of great importance, while specific therapeutic applications of each may differ subsequently [124]. Although reprogramming is not very effective, iPSCs can vastly give rise to numerous varieties of cells and tissues. Like ESCs, iPSCs can be introduced into pre-implantation embryos and develop into derivatives of all three germlines [125]. After in vitro development, iPSCs produce podocytes and tubular epithelial cells, mimicking renal progenitors and their offspring. Though iPSCs share the donor’s genetic makeup, they can be utilized for further investigation of genetic variations affecting disease pathogenesis [126, 127]. Accumulation of somatic mutations leading to immunogenicity and tumorigenicity are the primary risks of using iPSCs. According to a recent investigation, oncogenes in iPSCs were crucially activated in relation to malignant changes [128]. However, given the clonal nature of cellular reprogramming, it seems logical to attribute malignant transformation to genetic changes that are mainly inherent and occur with low frequency in the initial population. Patient-derived iPSCs, on the other hand, lack immunogenicity by nature and accumulate genetic mutations that must be considered as potentially confusing events about immunological tolerance [118]. According to the current study, numerous studies used iPSCs or derivatives for the recellularization of kidney scaffolds. Some studies investigated the outcomes following the seeding of endothelial cells derived from iPSCs, evaluating reendothelialization of capillaries and tubules [25, 37, 44, 45, 88, 129]. Du evaluated to determine the adequacy of employing each individual’s specific iPSCs as renal progenitor cells and endothelial cells within a decellularized kidney to regenerate kidney function and generate a functional whole kidney. These cells would be used to repopulate decellularized native kidneys, presenting a promising approach for restoring kidney function and addressing the needs of patients requiring renal replacement therapy. By employing patient-specific cells, the risk of rejection or immune response can be minimized, thereby enhancing the probability of successful transplantation and long-term functionality of the regenerated kidney [96].

Adipose-derived stem cells (ADSCs)

The organism’s adult stem or progenitor cells remain present throughout life and are responsible for tissue homeostasis and repair. Normal conditions cause stem cells to remain dormant in the niche until they are activated by tissue damage or the need for more cells to keep the tissue healthy. These cells are believed to be the main repair components for the associated organs. Because they are less proliferative, more mature, and a safer resource than ESCs, adult stem cells differ significantly from ESCs in this regard [130]. Many adult tissues have been found to contain stem cell populations. Adult renal stem cells have been identified in the Bowman’s capsule, glomeruli, pericytes, proximal tubules, and renal papilla of the kidney. These cell types displayed stem cell markers such as Pax-2, CD24, CD133, CD146, PECAM-1, and ECDH [43, 131]. Human adipose-derived mesenchymal stem cells (ADSCs) were extensively used for cell seeding in kidney acellular scaffolds [11, 23, 43, 49, 54, 82, 90, 132]. ASDCs have effectively undergone proliferation, adhesion, and differentiation, forming endothelial and tubular structures. This highlights the potential of ADSCs as a valuable cell source for kidney regeneration [11, 23, 43, 49, 54, 90, 132]. Zhang also used a Wnt agonist to induce the differentiation of ADSCs toward the development of an intermediate mesoderm. Intermediate mesoderm cells derived from ADSCs demonstrated greater differentiation into tubular and podocyte-like cells compared to the original stem cells. This enhanced differentiation efficiency suggests that utilizing scaffolds to induce stem cell differentiation could offer a viable approach to renal regeneration [132].

Applications

Bioengineered kidneys were also shown to have various growth factors after decellularization, providing kidney whole organ or slices as a potential ECM for further uses, including induction of cell differentiation [98], tissue model for in-vitro and in-vivo studies regarding anatomical [40, 133], drug-safety [17, 109], cancer [17], pathophysiologic [40, 119, 134] investigations [6], and whole organ development for further transplantation. Yu transplanted acellular kidney slices (1/3 of native kidney size) into kidneys and underwent partial nephrectomy; after the excision, the cut-end was supplemented with scaffolds of similar dimensions, which were securely attached to the external capsules through suturing, reperfused the regenerated kidneys with animal blood. An increase in kidney size was observed in the study, accompanied by the presence of regenerated renal parenchyma cells within the repaired area containing the grafted scaffold. Moreover, there was a notable elevation in the number of nestin-positive renal progenitor cells in the kidneys that underwent scaffold grafting compared to the control group. Furthermore, the analysis of radionuclide scans revealed a significant recovery of renal functions at the six-week postimplantation stage [135]. Zhang also evaluated the feasibility of decellularizing fibrotic kidneys and evaluated the outcomes, showing that the utilization of decellularized fibrotic kidneys as bioengineered organs is comparable to that of normal kidneys [26].

Novel applications

Some studies are using novel applications from acellular kidney ECM. O’Neill was among the first to produce hydrogel, sheet, and solution from acellular ECM, indicating all three forms of the ECM have an impact on the regulation of reseeded stem cells, with each form exerting unique effects on the structure and composition [128]. Similarly, to assess the effectiveness of renal decellularized ECM as a hydrogel, coating, and scaffold, in comparison to hyaluronic acid hydrogel and collagen scaffold, its efficiency and cellular responses were examined by Lee [108]. Another study developed hydrogel from acellular ECM. Cells cultured on hydrogel gene expression patterns differed significantly from those cultured on Collagen-I. Notably, the upregulation of genes associated with cellular quiescence and maturation was observed, while the expression of genes related to proteolytic activity and cell surface activation was downregulated. These findings suggest that the hydrogel culture environment promotes cellular quiescence and maturation while inhibiting proteolytic activity and cell surface activation [115]. Another study similarly produced hydrogels to evaluate cell culture feasibility. The survival and proliferation of encapsulated or cultured cells on hydrogels were highly successful within a week. However, it exhibited a significant decrease in the expression levels of several genes [113]. Kim also aimed to evaluate the potential impact of utilizing an acellular kidney as a hydrogel on the maturation of kidney organoids derived from stem cells. The implementation of hydrogel allowed for the extensive vascularization and maturation of kidney organoids to take place [129]. Moreover, acellular kidney ECM was used to develop bio-ink for administration in bioprinting. The bioink containing bioprinted kidneys exhibited a notable degree of viability and underwent a process of maturation. Moreover, the resulting bioprinted renal constructs successfully mimicked the structural and functional properties inherent to the native renal tissue [110]. Besides, patches of acellular ECM were applied on fibrotic kidneys. By diminishing fibrosis, the implementation of recellularized kidney patches has shown to be advantageous in restoring kidney function. This pioneering technique presents a viable alternative for treating renal fibrosis [16]. The impact of transplantation of acellular articulated kidneys on restoring kidney function in an injured kidney was also evaluated. The particles acquired from the grinding of decellularized kidneys exhibited tremendous potential. By reducing the necessity for surgeries, they can effectively reverse small injuries at a localized level, ultimately preventing the progression of chronic renal dysfunction [119].

Lih developed a mixed scaffold by administration of a synthetic nano-agent, PLGA (Poly(lactic-co-glycolic acid)). Cell proliferation was observed to be enhanced with the escalation in ECM concentration. The effectiveness of a PLGA scaffold containing 10% ECM has been demonstrated as a suitable framework for the regeneration and reconstruction of the glomerulus and blood vessels [111]. Similarly, they developed an advanced mixed scaffold of PLGA/ decellularized ECM/ Mg(OH)2, transplanted in kidneys that had undergone partial nephrectomy, observing renal glomeruli regeneration and subsequent restoration of renal function after the application of the advanced scaffold, with minimal inflammatory response, in comparison to the control group [112]. The advent of bioinks and hydrogels offers significant advancements in kidney regeneration. These materials enhance ECM biocompatibility and support vascularization, addressing key limitations in current approaches.

Translational aspects

Tissue engineering (TE) has the potential to improve patient outcomes by offering alternative treatments for organ transplants, addressing diseases without curative therapy, and providing personalized treatment options. The process of moving TE treatments from the lab to helping patients typically involves undergoing clinical trials to gather evidence, or being offered as a new therapy to enhance patient access through methods such as off-label and compassionate usage. The TE debate now concentrates on defining criteria for responsible progress and anticipating future consequences. Therefore, it is essential to provide a brief overview of ethical considerations, scalability, and potential for clinical uses in this review.

Ethical considerations

In recent years, tissue engineering has made significant progress, leading to its first clinical applications. However, there are emerging ethical concerns, particularly related to induced pluripotent stem cells (iPSCs), the integration of animal scaffolds into humans, and the use of recellularized scaffolds from donors. A recently published systematic review have addressed these ethical challenges and highlighted the numerous obstacles to translating research from the laboratory to practical medical applications [136]. The widespread use of animal organs for the development of acellular scaffolds and the conduction of xenotransplantation have raised numerous religious, cultural, moral, and technical conflicts [137]. Studies have indicated that the use of computational and/or in-vitro models, whenever feasible, can significantly reduce the reliance on animal testing [138]. In cases where animal models are unavoidable, it is recommended to use a single large animal instead of multiple small animals and to conduct all experiments on the same animal, with continuous monitoring of its well-being [139]. 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 a more detailed informed consent, including the advantages and possible risks, importance of long-term follow-up, and alternative treatments in case of transplant failure [136, 140].

Challenges for the application of recellularized kidney in clinical trials

The unique and personalized nature of TE presents challenges to traditional clinical trial pathways, emphasizing the importance of providing guidance on how to advance the clinical translation of this field. The need for appropriate comparators and the patient-specific aspects of tissue engineering interventions makes it difficult to conduct conventional randomized controlled trials (RCTs) or to generalize results from one patient to another. Therefore, it is crucial to establish comprehensive guidelines for the use of recellularized organs and to identify suitable candidates for transplantation in order to effectively utilize regenerated kidneys in clinical settings. Additionally, the social impacts of using recellularized kidneys in clinical settings should not be overlooked, as the social understanding of such interventions has extensive effects on the acceptance of this novel technique. Hence, choosing suitable patients and offering a comprehensive social context is essential. Therefore, utilizing healthy patients with favorable prognoses in clinical trials is not justifiable. Prioritizing end-stage patients who may benefit most from innovative therapies, based on comprehensive ethical and clinical assessments, could be a reasonable initial criterion for patient selection. Collecting long-term data from clinical trials and follow-up studies on the safety and effectiveness of TE interventions is crucial. Maintaining clinical trial data is essential as they allow for tracking adverse events and contribute to establishing a solid evidence. By integrating unbiased and transparent publishing practices and employing scientifically rigorous research methods, we can enhance reproducibility and facilitate the translation of TE products into clinical settings. In order to ensure proper management, it is crucial to establish clear guidelines and strong oversight mechanisms use of human tissues. It is also important to set consistent standards for tissue engineering approaches.

Future directions

To bridge the translational gap, future research should optimize scaling protocols and improve techniques for uniform cellular distribution in recellularization. Advanced bioreactors and multi-route cell delivery systems offer promising solutions. Further studies are needed to establish bioengineered kidneys more broadly available and accessible, as the incidence of chronic kidney diseases is rising. It has been proposed that kidney tissue engineering is a promising strategy for renal replacement therapy. Natural scaffolds, biochemistrical properties, vasculature, architecture, and the ability to promote stem cell differentiation into organ-specific phenotypes are among the advantages of the acellular-ECM model achieved by recellularization techniques. Promising progress has been made in healing and restoring damage caused by renal failure in tissue engineering and regenerative medicine. These methods include using endogenous renal cells and niches for in situ renal regeneration, cell therapy employing autologous and “off-the-shelf” allogeneic cell sources, and creating cell-based renal structures. These experimental investigations have introduced novel approaches to kidney diseases, presenting practical applications. The development of acellular scaffolds and their reconstitution are essential processes in the establishment of a bioengineered organ. Although the recellularization of acellular scaffolds demonstrated encouraging results, more preclinical and clinical evaluations are required to maximize all parameters and get around any potential drawbacks. Additionally, ethical oversight and regulatory controls should be put in place to guide the assessment of potential risks. Lastly, global regulations are needed to govern the use of experimental tissue engineering and regenerative medicine treatments as innovative therapies outside the scope of clinical trials.

Conclusion

In this systematic review, we have comprehensively examined current strategies in kidney regeneration using tissue engineering approaches, focusing on decellularization and recellularization techniques. These foundational methodologies represent critical advancements toward addressing the global shortage of transplantable kidneys. Decellularization has enabled the creation of acellular kidney scaffolds that preserve the ECM’s structural and biochemical integrity, while recellularization efforts have demonstrated the potential for cellular integration, vascularization, and functionality. Despite these advancements, several barriers must be addressed to translate these technologies into clinical practice. The most promising strategies for kidney regeneration include optimizing decellularization protocols to ensure complete cell removal while preserving ECM bioactivity, enhancing recellularization through patient-specific stem cells to minimize immunogenicity, and advancing bioreactor technologies for uniform cellular distribution and maturation. Emerging applications such as hydrogels, bioinks, and mixed scaffolds derived from kidney ECM offer additional avenues to address challenges like vascularization, reinnervation, and tissue-specific differentiation.

Future research should prioritize:

• 1. Developing scalable and reproducible decellularization protocols for human-sized kidneys.

• 2. Investigating strategies to achieve functional revascularization and reinnervation in bioengineered kidneys.

• 3. Exploring the integration of advanced biomaterials, such as ECM-derived hydrogels and bioinks, to enhance recellularization outcomes.

• 4. Conducting long-term preclinical and clinical studies to evaluate the safety, functionality, and durability of regenerated kidneys.

• 5. Addressing regulatory and ethical challenges, particularly regarding the use of xenogeneic scaffolds and patient-specific cells.

By focusing on these actionable goals, kidney tissue engineering holds immense promise in revolutionizing the treatment of end-stage renal disease, offering patients viable alternatives to dialysis and transplantation. Continued interdisciplinary collaboration and innovation are essential to overcome current limitations and bring bioengineered kidneys closer to clinical application.